Biochem Biophys Res Commun, 1997 Feb 24, 231(3), 645 - 50 Cloning and expression of the adenosine kinase gene from rat and human tissues; McNally T et al.; Adenosine kinase is ubiquitous in eukaryotes and is a key enzyme in the regulation of the intracellular levels of adenosine, an important physiological effector of many cells and tissues . In this paper we report the cloning of cDNAs encoding adenosine kinase from both rat and human tissues . Two distinct forms of adenosine kinase mRNA were identified in human tissues . Sequence variation between the two forms is restricted to the extreme 5'-end of the adenosine kinase mRNA, including a portion of the coding region, and is consistent with differential splicing of a single transcriptional product . We have expressed both forms in E . coli and produced soluble active enzyme which catalyzes the phosphorylation of adenosine with high specific activity in vitro and is susceptible to known adenosine kinase inhibitors. Biochem Pharmacol, 1997 Feb 21, 53(4), 493 - 500 Repression of inducible nitric oxide synthase and cyclooxygenase-2 by prostaglandin E2 and other cyclic AMP stimulants in J774 macrophages; Pang L et al.; The enhanced nitric oxide (NO) and prostaglandin (PG) generation of activated macrophages is controlled by glucocorticoid-sensitive inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), respectively . Negative feedback regulation of iNOS expression by the products of both pathways has been suggested, but their effects on COX-2 expression have not been examined . We hae investigated the effect of E- and l-series prostaglandins that activate adenylate cyclase (AC), forskolin (a direct activator of AC), and other agents that influence the cyclicAMP/cyclicGMP systems on the ability of E . coli endotoxin (lipopolysaccharide, LPS) to induce iNOS and COX-2 in the murine macrophage cell line J774 . After a 2-hr pretreatment before adding endotoxin, PGE2, PGI2, forskolin, IBMX (isobutylmethylxanthine, a cyclicAMP/cyclicGMP phosphodiesterase inhibitor), 8-bromo cyclicAMP, and arachidonic acid itself all inhibited the expression of both iNOS and COX-2 (as shown by Western blotting) and reduced NO release and COX activity, whereas PGF2 alpha and 8-bromo cyclic GMP were only weakly effective . The effects of PGE2, PGI2, and forskolin were enhanced by cotreatment with IBMX . The suppression of LPS-induced iNOS induction by PGE2 was functionally significant, in that it protected against the mild cytotoxicity of the NO generated in response to endotoxin . These results provide the first direct evidence for the feedback regulatory suppression of COX-2 induction by a PG-driven cAMP-mediated process, and show that the modulation of iNOS and COX-2 induction shares common features . They also suggest that such modulation is normally held in check by high phosphodiesterase activity within these cells. Biochim Biophys Acta, 1997 Feb 21, 1324(1), 69 - 75 Fluorescence studies on the interaction of a synthetic signal peptide and its analog with liposomes; Wang Q et al.; The N-terminal signal sequence of glucitol permease of Escherichia coli (Gut22: MIETITPGAVWFIGLFQKGGEC) and its analog (Gut22Ana: MIETITHGAEWFIGLFQKGGEC) were synthesized . The analog had a Pro residue substituted for the His at the 7th position of Gut22 and a Val residue substituted for the Glu at the 10th position . Previous studies indicated that due to its structural rigidity, the interaction of Gut22Ana with lipid bilayer was much weaker than that of Gut22 (Wang, Q.D., Cui, D.F . and Lin, Q.S . (1996) Science in China (Series C) 39, 395-405) . To further probe the location of the tryptophan residues of the peptides in lipid bilayer, the membrane penetration depth of the tryptophan residue of Gut22 was measured using spin-labeled phospholipids, and fluorescence quenching of the peptides by iodide and acrylamide in the presence and absence of phosphatidylserine/phosphatidylcholine liposomes were also studied . Fluorescent labeling of the peptides enabled the study of their association with membrane by fluorospectrophotometry . In the presence of liposomes, the peptides were protected from reaction with chymotrypsin, indicating that the peptide incorporated into the membrane . However, dithionite, which acts external to the membrane, reacted with the peptide, showing that the peptides did not translocate across lipid bilayer spontaneously. J Mol Biol, 1997 Feb 21, 266(2), 381 - 99 Structure and mechanism of a sub-family of enzymes related to N-acetylneuraminate lyase; Lawrence MC et al.; We describe here a sub-family of enzymes related both structurally and functionally to N-acetylneuraminate lyase . Two members of this family (N-acetylneuraminate lyase and dihydrodipicolinate synthase) have known three-dimensional structures and we now proceed to show their structural and functional relationship to two further proteins, trans-o-hydroxybenzylidenepyruvate hydratase-aldolase and D-4-deoxy-5-oxoglucarate dehydratase . These enzymes are all thought to involve intermediate Schiff-base formation with their respective substrates . In order to understand the nature of this intermediate, we have determined the three-dimensional structure of N-acetylneuraminate lyase in complex with hydroxypyruvate (a product analogue) and in complex with one of its products (pyruvate) . From these structures we deduce the presence of a closely similar Schiff-base forming motif in all members of the N-acetylneuraminate lyase sub-family . A fifth protein, MosA, is also confirmed to be a member of the sub-family although the involvement of an intermediate Schiff-base in its proposed reaction is unclear. J Mol Biol, 1997 Feb 21, 266(2), 343 - 56 Direct localization of the tRNAs within the elongating ribosome by means of neutron scattering (proton-spin contrast-variation); Wadzack J et al.; A new technique for neutron scattering, the proton-spin contrast-variation, improves the signal-to-noise ratio more than one order of magnitude as compared to conventional techniques . The improved signal enables small RNA ligands within a large deuterated ribonucleic acid-protein complex to be measured . We used this technique to determine the positions of the two tRNAs within the elongating ribosome before and after translocation . Using a four-sphere model for each of the L-shaped tRNAs, unequivocal solutions were found for the localization of the mass centre of both tRNAs . The centre of gravity is located in the interface cavity separating the ribosomal subunits near the neck of the 30 S subunit . It moves during translocation by 12(+/-4) A towards the head of the 30 S subunit and slightly towards the L1 protuberance of the 50 S subunit. J Mol Biol, 1997 Feb 21, 266(2), 317 - 30 A 29 residue region of the sarcomeric myosin rod is necessary for filament formation; Sohn RL et al.; Myosin is a motor protein whose functional unit in the sarcomere is the thick filament . The myosin molecule is capable of self-assembly into thick filaments through its alpha-helical coiled-coil rod domain . To define more precisely the sequence requirements for this assembly, segments of the human fast IId skeletal myosin rod were expressed in Escherichia coli and examined differential solubility and the formation of ordered paracrystals . We show that both properties appear to require a 29 residue sequence (residues 1874 to 1902) near the C terminus of the rod region . To test further the role of this region in assembly, a protein was constructed which consisted of this assembly competence domain (ACD) fused to the carboxy terminus of an assembly-incompetent myosin rod fragment . This chimeric fragment exhibited myosin's characteristic solubility properties and formed ordered paracrystals . To complement these in vitro experiments, both a full-length myosin heavy chain (MYH) and one from which the 29 residues were deleted were transfected into cultured mammalian cells . While the full-length construct formed the spindle-shaped structures characteristic of arrays of thick filaments, the deleted MYH showed only diffuse staining throughout the cytoplasm by light microscopy . Thus, there appears to be a specific sequence in the C-terminal region of the myosin heavy chain rod which is necessary for ordered paracrystal formation and is sufficient to confer assembly properties to an assembly-incompetent rod fragment. J Mol Biol, 1997 Feb 21, 266(2), 297 - 305 Short-homology-independent illegitimate recombination in Escherichia coli: distinct mechanism from short-homology-dependent illegitimate recombination; Shimizu H et al.; We have shown elsewhere that there is no, or very little, homology at the recombination sites in DNA gyrase-mediated illegitimate recombination in vitro . On the other hand, many reports have indicated that illegitimate recombination takes place between sequences with a short homology . To clarify this contradiction, we analyzed the mechanism of DNA gyrase-mediated illegitimate recombination in vivo, by isolating a temperature-sensitive gyrA mutant (gyrAhr1) that causes spontaneous illegitimate recombination at a higher frequency than that of the wild-type . This mutant also causes spontaneous induction of lambda prophage . It is therefore suggested that the gyrAhr1 mutation induces strand breaks in the chromosome, resulting in the formation of illegitimate recombinants . Analysis of the recombination junctions of lambdabio transducing phages formed spontaneously in the gyrAhr1 mutant revealed that the Escherichia coli bio and lambda recombination sites have an average homologous sequence of only 1.3 base pairs . This is the first indication that homology in vivo is not required for illegitimate recombination . On the other hand, a short homology of 8.4 bp, on average, was found in the junctions of lambdabio transducing phages formed spontaneously in the wild-type bacteria . When the gyrAhr1 mutant was irradiated with UV, short homologies were also detected in the junctions . We concluded that illegitimate recombination, which takes place spontaneously in the gyrAhr1 mutants, is distinguishable from spontaneous recombination in the wild-type and from UV-induced recombination in the mutant with regard to the requirement for short homology . We propose that short-homology-independent illegitimate recombination is mediated by subunit exchange between DNA gyrase, while short-homology-dependent recombination is triggered by double-strand breaks and completed by processing, annealing, and ligation of DNA ends. J Biol Chem, 1997 Feb 21, 272(8), 5335 - 41 NH2-terminal proline acts as a nucleophile in the glycosylase/AP-lyase reaction catalyzed by Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) protein; Zharkov DO et al.; Formamidopyrimidine-DNA glycosylase (Fpg) protein plays a prominent role in the repair of oxidatively damaged DNA in Escherichia coli . The protein possesses three enzymatic activities, hydrolysis of the N-glycosidic bond (DNA glycosylase), beta-elimination (AP lyase), and delta-elimination; these functions act in a concerted manner to excise oxidized deoxynucleosides from duplex DNA . Schiff base formation between the enzyme and substrate has been demonstrated (Tchou, J., and Grollman, A . P . (1995) J . Biol . Chem . 270, 11671-11677); this protein-DNA complex can be trapped by reduction with sodium borohydride . By digesting the stable, covalently linked intermediate with proteases and determining the accurate mass of the products by negative electrospray ionization-mass spectrometry, we show that the N-terminal proline of Fpg protein is linked to DNA and, therefore, is identified as the nucleophile that initiates the catalytic excision of oxidized bases from DNA . This experimental approach may be applicable to the analysis of other protein-DNA complexes. J Biol Chem, 1997 Feb 21, 272(8), 5305 - 12 Lysine 207 as the site of cross-linking between the 3'-end of Escherichia coli initiator tRNA and methionyl-tRNA formyltransferase; Gite S et al.; The specific formylation of initiator methionyl-tRNA by methionyl-tRNA formyltransferase (MTF) is important for initiation of protein synthesis in Escherichia coli . In attempts to identify regions of MTF that come close to the 3'-end of the tRNA, we oxidized 32P-3'-end-labeled E . coli initiator methionine tRNA with sodium metaperiodate and cross-linked it to MTF . The cross-linked MTF was separated from uncross-linked MTF by DEAE-cellulose chromatography, and the tRNA in the cross-linked MTF was hydrolyzed with nuclease P1 and RNase T1, leaving behind an oxidized fragment of {32P}AMP attached to MTF . Trypsin digestion of the cross-linked MTF followed by high pressure liquid chromatography of the digest yielded two peaks of radioactive peptides, I* and II* . These peptides were characterized by N- and/or C-terminal sequencing and by matrix-assisted laser desorption ionization mass spectroscopy . Peptide I* contained amino acids Gln186-Lys210 with Lys207 as the site of the cross-link . Peptide II*, a partial digestion product, contained amino acids Gln186-Arg214 also with Lys207 as the site of the cross-link . The molecular masses of peptides I* and II* indicate that the final product of the cross-linking reaction between the periodate-oxidized AMP moiety of the tRNA and Lys207 is most likely a morpholino derivative rather than a reduced Schiff's base. J Biol Chem, 1997 Feb 21, 272(8), 5141 - 51 Molecular cloning and functional characterization of a novel mitogen-activated protein kinase phosphatase, MKP-4; Muda M et al.; Extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), and p38/RK/CSBP (p38) mitogen-activated protein (MAP) kinases are target enzymes activated by a wide range of cell-surface stimuli . Recently, a distinct class of dual specificity phosphatase has been shown to reverse activation of MAP kinases by dephosphorylating critical tyrosine and threonine residues . By searching the expressed sequence tag data base (dbEST) for homologues of known dual specificity phosphatases, we identified a novel partial human sequence for which we isolated a full-length cDNA (termed MKP-4) . The deduced amino acid sequence of MKP-4 is most similar to MKP-X/PYST2 (61% identity) and MKP-3/PYST1 (57% identity), includes two N-terminal CH2 domains homologous to the cell cycle regulator Cdc25 phosphatase, and contains the extended active site sequence motif VXVHCXAGXSRSXTX3AYLM (where X is any amino acid) conserved in dual specificity phosphatases . MKP-4 produced in Escherichia coli catalyzes vanadate-sensitive breakdown of p-nitrophenyl phosphate as well as in vitro inactivation of purified ERK2 . When expressed in COS-7 cells, MKP-4 blocks activation of MAP kinases with the selectivity ERK > p38 = JNK/SAPK . This cellular specificity is similar to MKP-3/PYST1, although distinct from hVH-5/M3-6 (JNK/SAPK = p38 >>> ERK) . Northern analysis reveals a highly restricted tissue distribution with a single MKP-4 mRNA species of approximately 2.5 kilobases detected only in placenta, kidney, and embryonic liver . Immunocytochemical analysis showed MKP-4 to be present within cytosol although punctate nuclear staining co-localizing with promyelocytic protein was also observed in a subpopulation (10-20%) of cells . Chromosomal localization by analysis of DNAs from human/rodent somatic cell hybrids and a panel of radiation hybrids assign the human gene for MKP-4 to Xq28 . The identification and characterization of MKP-4 highlights the emergence of an expanding family of structurally homologous dual specificity phosphatases possessing distinct MAP kinase specificity and subcellular localization as well as diverse patterns of tissue expression. J Biol Chem, 1997 Feb 21, 272(8), 5082 - 6 Regulation of the soxRS oxidative stress regulon . Reversible oxidation of the Fe-S centers of SoxR in vivo; Gaudu P et al.; SoxR protein, a transcriptional activator of the soxRS (superoxide response) regulon of Escherichia coli, contains autooxidizable {2Fe-2S} centers that are presumed to serve as redox sensors . In vitro transcription experiments previously demonstrated that only the oxidized form is active . Reduced SoxR was detected in overproducing strains by EPR spectroscopy of suspensions of intact cells . Oxidized Fe-S centers were determined by lysing the cells and treating them with the reducing agent sodium dithionite prior to EPR measurements . In uninduced cells, 90% of the SoxR was in the reduced form . Treatment with the redox cycling agents phenazine methosulfate or plumbagin was accompanied by reversible oxidation of the Fe-S centers . Mutant SoxR derivatives that were constitutively activated existed constitutively in an oxidized state . The results indicate the presence of a cellular pathway for countering the autooxidation of SoxR and confirm the hypothesis that induction of the regulon is mediated by a shift in the redox equilibrium of SoxR rather than by assembly of its Fe-S clusters. J Biol Chem, 1997 Feb 21, 272(8), 5000 - 6 Crystal structures of CheY mutants Y106W and T87I/Y106W . CheY activation correlates with movement of residue 106; Zhu X et al.; Position 106 in CheY is highly conserved as an aromatic residue in the response regulator superfamily . In the structure of the wild-type, apo-CheY, Tyr106 is a rotamer whose electron density is observed in both the inside and the outside positions . In the structure of the T87I mutant of CheY, the threonine to isoleucine change at position 87 causes the side chain of Tyr106 to be exclusively restricted to the outside position . In this report we demonstrate that the T87I mutation causes cells to be smooth swimming and non-chemotactic . We also show that another CheY mutant, Y106W, causes cells to be more tumbly than wild-type CheY, and impairs chemotaxis . In the structure of Y106W, the side chain of Trp106 stays exclusively in the inside position . Furthermore, a T87I/Y106W double mutant, which confers the same phenotype as T87I, restricts the side chain of Trp106 to the outside position . The results from these behavioral and structural studies indicate that the rotameric nature of the Tyr106 residue is involved in activation of the CheY molecule . Specifically, CheY's signaling ability correlates with the conformational heterogeneity of the Tyr106 side chain . Our data also suggest that these mutations affect the signal at an event subsequent to phosphorylation. J Biol Chem, 1997 Feb 21, 272(8), 4985 - 92 Mutagenesis studies of the human erythropoietin receptor . Establishment of structure-function relationships; Barbone FP et al.; Mutagenesis of the erythropoietin receptor (EPOR) permits analysis of the contribution that individual amino acid residues make to erythropoietin (EPO) binding . We employed both random and site-specific mutagenesis to determine the function of amino acid residues in the extracellular domain (referred to as EPO binding protein, EBP) of the EPOR . Residues were chosen for site-specific alanine substitution based on the results of the random mutagenesis or on their homology to residues that are conserved or have been reported to be involved in ligand binding in other receptors of the cytokine receptor family . Site-specific mutants were expressed in Escherichia coli as soluble EBP and analyzed for EPO binding in several different assay formats . In addition, selected mutant proteins were expressed as full-length EPOR on the surface of COS cells and analyzed for 125I-EPO binding in receptor binding assays . Using these methods, we have identified residues that appear to be involved in EPO binding as well as other residues, most of which are conserved in receptors of the cytokine receptor family, that appear to be necessary for the proper folding and/or stability of the EPOR . We present correlations between these mutagenesis data and the recently solved crystal structure of the EBP with a peptide ligand. J Biol Chem, 1997 Feb 21, 272(8), 4843 - 9 Cooperativity and dimerization of recombinant human estrogen receptor hormone-binding domain; Brandt ME et al.; The estrogen receptor dimerizes and exhibits cooperative ligand binding as part of its normal functioning . Interaction of the estrogen receptor with its ligands is mediated by a C-terminal hormone-binding domain (HBD), and residues within the HBD are thought to contribute to dimerization . To examine dimer interactions in the isolated HBD, a human estrogen receptor HBD fragment was expressed in high yield as a cleavable fusion protein in Escherichia coli . The isolated HBD peptide exhibited affinity for estradiol, ligand discrimination, and cooperative estradiol binding (Hill coefficient approximately 1.6) similar to the full-length protein . Circular dichroism spectroscopy suggests that the HBD contains significant amounts of alpha-helix ( approximately 60%) and some beta-strand ( approximately 7%) and that ligand binding induces little change in secondary structure . HBD dimer dissociation, measured using size exclusion chromatography, exhibited a half-life of approximately 1.2 h, which ligand binding increased approximately 3-fold (estradiol) to approximately 4-fold (4-hydroxytamoxifen) . These results suggest that the isolated estrogen receptor HBD dimerizes and undergoes conformational changes associated with cooperative ligand binding in a manner comparable to the full-length protein, and that one effect of ligand binding is to alter the receptor dimer dissociation kinetics. J Biol Chem, 1997 Feb 21, 272(8), 4820 - 7 Formation of DNA repair intermediates and incision by the ATP-dependent UvrB-UvrC endonuclease; Zou Y et al.; The Escherichia coli UvrB and UvrC proteins play key roles in DNA damage processing and incisions during nucleotide excision repair . To study the DNA structural requirements and protein-DNA intermediates formed during these processes, benzo{a}pyrene diol epoxide-damaged and structure-specific 50-base pair substrates were constructed . DNA fragments containing a preexisting 3' incision were rapidly and efficiently incised 5' to the adduct . Gel mobility shift assays indicated that this substrate supported UvrA dissociation from the UvrB-DNA complex, which led to efficient incision . Experiments with a DNA fragment containing an internal noncomplementary 11-base region surrounding the benzo{a}pyrene diol epoxide adduct indicated that UvrABC nuclease does not require fully duplexed DNA for binding and incision . In the absence of UvrA, UvrB (UvrC) bound to an 11-base noncomplementary region containing a 3' nick (Y substrate), forming a stable protein-DNA complex (Kd approximately 5-10 nM) . Formation of this complex was absolutely dependent upon UvrC . Addition to this complex of ATP, but not adenosine 5'-(beta,gamma-iminotriphosphate) or adenosine 5'-(beta, gamma-methylene)triphosphate, caused incision three or four nucleotides 5' to the double strand-single strand junction . The ATPase activity of native UvrB is activated upon interaction with UvrC and enhanced further by the addition of Y substrate . Incision of this Y structure occurs even without DNA damage . Thus the UvrBC complex is a structure-specific, ATP-dependent endonuclease. J Biol Chem, 1997 Feb 21, 272(8), 4814 - 9 Substrate specificity of hybrid modules from peptide synthetases; Elsner A et al.; Homologous modules from two different peptide synthetases were analyzed for functionally equivalent regions . Hybrids between the coding regions of the phenylalanine-activating module of tyrocidine synthetase and the valine-activating module of surfactin synthetase were constructed by combining the two reading frames at various highly conserved consensus sequences . The resulting DNA fragments were expressed in Escherichia coli as C-terminal fusions to the gene encoding for the maltose-binding protein . The fusion proteins were purified, and the amino acid specificities, the acceptance of different nucleotide analogues, and the substrate binding affinities were analyzed . We found evidence for a large N-terminal domain and a short C-terminal domain of about 19 kDa within the two modules, which are separated by the sequence motif GELCIGG . The two domains could be reciprocally transferred between the two modules, and the constructed hybrid proteins showed amino acid adenylating activity . Hybrid proteins fused at various consensus motifs within the two domains were inactive, indicating that the domains may fold independently and represent complex functional units . The N-terminal domain was found to be responsible for the amino acid specificity of the modules, and it is also involved in the recognition of the ribosyl and the phosphate moieties of the nucleotide substrate . For tyrocidine synthetase I, we could confine the sites for amino acid specificity to a region of 330 residues . The C-terminal domain is essential for the enzymatic activity and has a strong impact on the specific activity of the modules. J Biol Chem, 1997 Feb 21, 272(8), 4770 - 4 Molecular cloning of acetone cyanohydrin lyase from flax (Linum usitatissimum) . Definition of a novel class of hydroxynitrile lyases; Trummler K et al.; Acetone cyanohydrin lyase from Linum usitatissimum is a hydroxynitrile lyase (HNL) which is involved in the catabolism of cyanogenic glycosides in young seedlings of flax . We have isolated a full-length cDNA clone encoding L . usitatissimum HNL (LuHNL) from a cDNA expression library by immunoscreening . LuHNL cDNA was expressed in Escherichia coli and isolated from the respective soluble fraction in an active form which was biochemically indistinguishable from the natural enzyme . An open reading frame of 1266 base pairs encodes for a protein of 45,780 kDa . The derived amino acid sequence shows no overall homologies to the to date cloned HNLs, but has significant similarities to members of the alcohol dehydrogenase (ADH) family of enzymes . In particular, the cysteine and histidine residues responsible for coordination of an active site Zn2+ and a second structurally important Zn2+ in alcohol dehydrogenases are conserved . Nevertheless, we found neither alcohol dehydrogenase activity in LuHNL nor HNL activity in ADH . Moreover, well known inhibitors of ADHs, which interfere with the coordination of the active site Zn2+, fail to affect HNL activity of LuHNL, suggesting principally different mechanisms of cyanohydrin cleavage and alcohol oxidation . Interestingly, LuHNL like ADH and Prunus serotina (PsHNL) possesses an ADP-binding betaalphabeta unit motif, pointing to the possibility that the non-flavoprotein PsHNL and the flavoprotein LuHNL have developed from two independent lines of evolution of a common ancestor with an ADP-binding betaalphabeta unit. J Biol Chem, 1997 Feb 21, 272(8), 4671 - 9 Residues of the Rho family GTPases Rho and Cdc42 that specify sensitivity to Dbl-like guanine nucleotide exchange factors; Li R et al.; The Dbl-like guanine nucleotide exchange factor (GEF) Lbc oncoprotein specifically activates the small GTP-binding protein Rho in mammalian fibroblasts to induce transformation and actin stress fiber formation, whereas another Dbl-related molecule, Cdc24, stimulates guanine nucleotide exchange of the Rho family GTPase Cdc42 to elicit effects on both gene induction and actin-based cytoskeleton change in Saccharomyces cerevisiae . To understand the mechanism of these functional interactions, we have taken a biochemical approach to probe the sites on Rho and Cdc42 that are involved in coupling to their respective GEFs, the Lbc and Cdc24 proteins . Point mutations in the switch II region of the small G-proteins, many of which would affect the interaction with GEF in the case of Ras, or a mutation in the switch I region that was identified as a contact site between Rab3A and Rab GEF had little effect on RhoA or Cdc42Hs with regard to the ability to interact with Lbc or Cdc24, suggesting that there exists a unique mechanism of regulation of the Rho family proteins by their GEFs . Analysis of a panel of chimeras made between RhoA and Cdc42Hs, which all maintained the ability to respond to Dbl, their mutual GEF, and to GTPase-activating protein, revealed that at least two distinct sites in each of the GTPases are required for activation by the respective GEFs . Further site-directed mutagenesis studies showed that the conserved residue Tyr32 in the putative effector region of both GTPases (numbered by Cdc42Hs) is critical for binding of the GEFs and that specific recognition for Lbc or Cdc24 is achieved at least in part through residues Lys27 of Rho and Gln116 of Cdc42 . Moreover, the loss of GEF responsiveness of a RhoA mutation (D76Q) was found to be caused by the impaired GEF catalysis, not by a change in the GEF binding affinity . Together, these results indicate that multiple sites of the Rho GTPases are involved in the regulation by GEFs, contributing to GEF binding or GEF catalysis, and raise the possibility that activation of each Rho family G-protein by a specific GEF may engage in a distinct mechanism. Carbohydr Res, 1997 Feb 20, 298(1-2), 65 - 73 Transglycosylation in a two-phase aqueous-organic system with catalysis by a lipid-coated beta-D-galactosidase; Mori T et al.; A lipid-coated beta-D-galactosidase was prepared in which the enzyme surface is covered with a lipid monolayer and two long alkyl lipophilic tails serve to solubilize the enzyme in organic solvents . In a two-phase aqueous-organic system, a lipid-coated enzyme exists in the organic (2-propyl ether) phase and acts as an efficient transgalactosylation catalyst for various hydrophobic alcohols with lactose in the aqueous buffer solution . When a native beta-D-galactosidase was employed in the two-phase system, neither the transgalactosylation nor the hydrolysis reaction proceeded due to denaturation of the enzyme at the interface . Effects of coating lipid molecules, origins of enzymes, reaction in organic solvents, and chemical structures of acceptor alcohols on the transgalactosylation catalyzed by the lipid-coated enzyme were studied . This system could also be applied in a large-scale synthesis on the 0.1-1 g scale. Mol Gen Genet, 1997 Feb 20, 253(5), 634 - 41 Adducts formed by the food mutagen 2-amino-3-methylimidazo(4,5-f) quinoline induce frameshift mutations at hot spots through an SOS-independent pathway; Maenhaut-Michel G et al.; The potency of 2-amino-3-methylimidazo(4,5-f)quinoline (IQ) adducts to induce -2, -1 and +1 frameshift mutations has been determined on specific target DNA sequences, namely short runs of alternating GpC sequences and short runs of guanines . The genetic control of the mutational processes has been analyzed using different Escherichia coli mutants, affected either in the control or in the mutagenesis pathway of the SOS system . We have shown that IQ adducts induce very efficiently both -1 and -2 frameshift mutations in E . coli . Both types of deletion mutations are induced in bacteria without the need of SOS induction, indicating that no LexA-controlled functions, in particular the UmuDC proteins, are required for mutation fixation . We have also shown that the frequency of IQ-induced -2 frameshift mutations in alternating GC sequences increases with the length of the repetition . The efficiency of IQ adducts to induce -1 and -2 frameshift mutations is similar to that of N-2-acetylaminofluorene (AAF) adducts . Both chemicals are potent carcinogens which form covalent adducts at the C8 position of guanines . We suggest that in both cases the adduct-induced DNA structure allows the replication complex to perform a mutagenic bypass of the lesion by a slippage mechanism . However, in contrast to AAF-induced frameshift mutagenesis, IQ-induced frameshift mutagenesis is SOS-independent. Gene, 1997 Feb 20, 186(1), 55 - 60 A new expression vector for high level protein production, one step purification and direct isotopic labeling of calmodulin-binding peptide fusion proteins; Zheng CF et al.; Calmodulin-binding peptide (CBP), a peptide of 26 amino acids derived from muscle myosin light chain kinase (MLCK), binds to calmodulin with nanomolar affinity . Proteins fused in frame with CBP can be purified from crude E . coli lysates in a single step using calmodulin affinity chromatography (Stofko-Hahn et al., 1992) . Because the binding between CBP and calmodulin is calcium-dependent, the fusion protein can be eluted from the resin with virtually any buffer containing EGTA (2 mM) and used directly for many applications . To take full advantage of this affinity purification system, we constructed the versatile CBP fusion protein expression vector pCAL-n . The CBP coding sequence was positioned for fusion at the N-terminus, an advantage that ensures consistent high level synthesis of fusion proteins due to the efficient translation of the CBP in E . coli . The production of fusion proteins from pCAL-n is controlled by the tightly regulated T7(lac)O promoter . A versatile multiple cloning site (MCS) was included to facilitate the cloning of genes of interest . The protein coding sequence for the enzyme c-Jun N-terminal kinase (JNK) was inserted into the MCS of pCAL-n, and the resulting fusion protein CBP-JNK synthesized in E . coli cells at 15-20 mg/1 culture . CBP-JNK was purified to near homogeneity in one step with calmodulin affinity resin . Purified CBP-JNK is fully active, and the CBP peptide tag can be removed by cleavage with thrombin . We also show that CBP can be efficiently phosphorylated by cAMP-dependent protein kinase . Hence, the purified fusion proteins can be labeled directly with {gamma-32P}ATP and used to probe protein-protein or protein-nucleic acid interactions. Cell Tissue Res, 1997 Feb 20, 287(3), 507 - 12 Expression and characterization of the "laminin binding protein" in hydra Keppel E, Fenger U, Schaller HC. Recently, a cDNA was isolated from hydra with extensive homology to a mammalian and invertebrate gene which codes for a protein called laminin binding protein (LBP) . In this paper we describe the protein expression of the hydra LBP in Escherichia coli . On SDS gels the recombinant hydra LBP displayed an apparent molecular mass of 43 kDa, although the calculated mass, including six additional histidines, is 33.7 kDa . Polyclonal antibodies were produced against the hydra recombinant LBP . The antiserum reacted with a 42-kDa and a 43-kDa protein from Hydra vulgaris and from a multiheaded mutant of Chlorohydra viridissima, respectively . In hydra, LBP RNA and protein were highly expressed in cells with short cell cycles, such as all cells of the interstitial cell lineage, less in slowly cycling epithelial cells, and at very reduced levels or not at all in differentiated cells . Higher expression in the multiheaded mutant of C . viridissima than in H . vulgaris, the cells of which differ in doubling time, hint at a function in cell proliferation . This is supported by the finding that in vitro hydra LBP is a substrate for the cell-cycle-specific kinase CDC2. Biochemistry, 1997 Feb 18, 36(7), 1943 - 52 {125I}Iberiotoxin-D19Y/Y36F, the first selective, high specific activity radioligand for high-conductance calcium-activated potassium channels; Koschak A et al.; Iberiotoxin (IbTX), a selective peptidyl ligand for high-conductance Ca2(+)-activated K+ (maxi-K) channels cannot be radioiodinated in biologically active form due to the importance of Y36 in interacting with the channel pore . Therefore, an IbTX double mutant (IbTX-D19Y/Y36F) was engineered, expressed in Escherichia coli, purified to homogeneity, and radiolabeled to high specific activity with 125I . IbTX-D19Y/Y36F and {127I}IbTX-D19Y/Y36F block maxi-K channels expressed in Xenopus laevis oocytes with equal potency as wild-type IbTX (Kd approximately 1 nM) . Under low ionic strength conditions, {125I}IbTX-D19Y/Y36F binds with high affinity to smooth muscle sarcolemmal maxi-K channels (Kd of 5 pM as determined by either equilibrium binding or kinetic binding analysis), and with a binding site density of 0.45 pmol/mg of protein . Competition studies with wild-type IbTX, IbTX-D19Y/Y36F or charybdotoxin (ChTX) result in complete inhibition of binding whereas toxins selective for voltage-gated K+ channels (margatoxin (MgTX) or alpha-dendrotoxin (alpha-DaTX) do not have any effect on IbTX binding . Indole diterpene alkaloids, which are selective inhibitors of maxi-K channels, and potassium ions both modulate {125I}IbTX-D19Y/Y36F binding in a complex manner . This pattern is also reflected during covalent incorporation of the radiolabeled toxin into the 31 kDa beta-subunit of the maxi-K channel in the presence of a bifunctional cross-linking reagent . In rat brain membranes, IbTX-D19Y/Y36F does not displace binding of {125I}MgTX or {125I}-alpha-DaTX to sites associated with voltage-gated K+ channels, nor do these latter toxins inhibit {125I}IbTX-D19Y/Y36F binding . Taken together, these results demonstrate that {125I}IbTX-D19Y/Y36F is the first selective radioligand for maxi-K channels with high specific activity. Biochemistry, 1997 Feb 18, 36(7), 1900 - 5 Role of Glu51 for cofactor binding and catalytic activity in pyruvate decarboxylase from yeast studied by site-directed mutagenesis; Killenberg-Jabs M et al.; We investigated the importance of the interaction between the Nl'-atom of the cofactor thiamine diphosphate and glutamic acid residue 51 in pyruvate decarboxylase (EC 4.1 . 1.1) . The yeast wild type gene PDCl and the respective mutant genes (E51Q and E51A) were expressed in Escherichia coli . The three enzymes were purified to homogeneity . They comigrated as a single band during silver-stained SDS/PAGE with a molecular mass of 60 000 Da . A molecular mass of 61 200 +/- 200 Da was determined by mass spectrometry for the subunit . The native enzyme is a homotetramer as demonstrated by gel filtration experiments . Near- and far-UV CD spectra showed no significant differences for the apoenzyme of the wild type and the mutants . Slight differences in the rate of thiamine diphosphate binding to the apoprotein component were observed between the wild type and the E51Q PDC by CD spectroscopy . Compared to the wild type enzyme, thiamine diphosphate binding at the E51A mutant apoprotein is very slow . Only 0.04% of the catalytic activity of the wild type enzyme was observed for the E51Q mutant; the E51A mutant has no detectable catalytic activity . The S0.5 value for the substrate pyruvate is increased 33-fold for the E51Q mutant . Substrate activation was observed for both the wild type and the E51Q mutant . The interaction between the N1'-atom of the coenzyme and glutamic acid 51 strongly influences the catalytic activity but only moderately the binding of the cofactor to the apoenzyme and the substrate activation rate. Biochemistry, 1997 Feb 18, 36(7), 1755 - 65 Miscoding properties of model estrogen-DNA adducts in reactions catalyzed by mammalian and Escherichia coli DNA polymerases; Shibutani S et al.; The miscoding properties of the model estrogen-derived DNA adducts, N2-{3-methoxyestra-1,3,5(10)-trien-6-yl}-2'-deoxyguanosine (dG-N2-3MeE) and N6-{3-methoxyestra-1,3,5(10)-trien-6-yl}-2'- deoxyadenosine (dA-N6-3MeE), have been explored, using an in vitro experimental system to quantify base substitutions and deletions . Site-specifically modified oligodeoxynucleotides containing a single dG-N2-3MeE or dA-N6-3MeE were prepared postsynthetically and used as templates in primer extension reactions catalyzed by Escherichia coli and mammalian DNA polymerases . When the 3'-->5' exonuclease free (exo-) Klenow fragment of DNA polymerase I was used, dG-N2-3MeE promoted mostly one- and two-base deletions, along with small amounts of incorporation of dAMP, dGMP, and dCMP opposite the lesion . dA-N6-3MeE promoted the incorporation of dTMP opposite the lesion as well as two-base deletions, accompanied by the incorporation of dAMP . Using pol alpha, primer extension reactions were blocked at dG-N2-3MeE; however, dA-N6-3MeE promoted preferential incorporation of dTMP opposite the lesion with small amounts of incorporation of dCMP and deletions . Primer extension reactions catalyzed by pol delta were blocked at these lesions . When pol beta was used, dG-N2-3MeE produced small amounts of incorporation of dAMP and deletions . dA-N6-3MeE promoted preferential incorporation of dTMP, along with incorporation of dCMP and two-base deletions . The miscoding specificities and frequencies varied depending on the DNA polymerase used . These results indicate that estrogen-DNA adducts have miscoding potential. Biochemistry, 1997 Feb 18, 36(7), 1748 - 54 Comparative analysis of the interactions of Escherichia coli sigma S and sigma 70 RNA polymerase holoenzyme with the stationary-phase-specific bolAp1 promoter; Nguyen LH et al.; We have investigated the interactions of Escherichia coli sigma 70 and sigma S holoenzyme RNA polymerases (E sigma S and E sigma 70) with the stationary-phase-specific bolAp1 promoter by various footprinting methods in vitro . E sigma S and E sigma 70 have been shown to transcribe the bolApl promoter in vitro . We have determined the effects of salt and holoenzyme concentrations on E sigma S and E sigma 70 open complex formation at the bolAp1 promoter in vitro . We have obtained a high-resolution hydroxyl radical (OH.) footprint of E sigma S and E sigma 70 on the bolApl promoter . The OH . footprinting data show remarkable similarities between the footprints of the heparin-resistant transcription complexes of the two holoenzymes which have the same +1 transcription start site . However, there are distinctive differences in the protection patterns in the region between -20 and -10 of the bolAp1 promoter . KMnO4 reactivity assays reveal that, at 37 degrees C, both holoenzymes produced similar but not identical patterns of reactivities. Biochemistry, 1997 Feb 18, 36(7), 1730 - 9 Dihydrodipicolinate synthase from Escherichia coli: pH dependent changes in the kinetic mechanism and kinetic mechanism of allosteric inhibition by L-lysine; Karsten WE; Dihydrodipicolinate synthase (DHDPS) catalyzes the formation of dihydrodipicolinate from pyruvate and L-aspartate beta-semialdehyde (ASA) . A parallel initial velocity pattern that displays competitive substrate inhibition by ASA and dead-end inhibition patterns obtained at pH 8 are consistent with a ping pong kinetic mechanism for DHDPS . The results suggest that pyruvate binds to free enzyme with subsequent formation of a Schiff base with an enzymic lysine residue followed by binding of ASA to the F enzyme form to initiate the second half-reaction . At low pH (5.7) the initial velocity and dead-end inhibition patterns are consistent with a sequential steady state ordered kinetic mechanism with pyruvate binding to enzyme prior to ASA . The irreversible step in the reaction, leading to the ping pong kinetic mechanism at high pH, is proposed to be loss of a proton from the methyl group of pyruvate in Schiff base with enzyme to form an enamine intermediate . Consistent with this proposal is the change to a sequential steady state ordered kinetic mechanism at low pH at or below the pK of the enamine intermediate . L-Lysine is an allosteric inhibitor of the DHDPS reaction that causes partial inhibition (approximately 90%) at saturating concentrations . Inhibition patterns for L-lysine vs pyruvate and ASA suggest that lysine binds to the F enzyme form at pH 8 with a Ki value of about 0.3 mM . An examination of the effects of different L-lysine concentrations on the kinetic parameters V/Kpyruvate, V/KASA, and V indicate that L-lysine decreases only the values of V/KASA and Vmax, which is consistent with the inhibitory effects of lysine manifested on the second half-reaction . In contrast at low pH the data suggest L-lysine binds to free enzyme with an inhibition constant of about 5 mM. Biochemistry, 1997 Feb 18, 36(7), 1608 - 20 Catalytic mechanism of the quinoenzyme amine oxidase from Escherichia coli: exploring the reductive half-reaction; Wilmot CM et al.; The crystal structure of the complex between the copper amine oxidase from Escherichia coli (ECAO) and a covalently bound inhibitor, 2-hydrazinopyridine, has been determined to a resolution of 2.0 A . The inhibitor covalently binds at the 5 position of the quinone ring of the cofactor, 2,4,5-trihydroxyphenylalaninequinone (TPQ) . The inhibitor complex is analogous to the substrate Schiff base formed during the reaction with natural monoamine substrate . A proton is abstracted from a methylene group adjacent to the amine group by a catalytic base during the reaction . The inhibitor, however, has a nitrogen at this position, preventing proton abstraction and trapping the enzyme in a covalent complex . The electron density shows this nitrogen is hydrogen bonded to the side chain of Asp383, a totally conserved residue, identifying it as the probable catalytic base . The positioning of Asp383 is such that the pro-S proton of a substrate would be abstracted, consistent with the stereospecificity of the enzyme determined by 1H NMR spectroscopy . Site-directed mutagenesis and in vivo suppression have been used to substitute Asp383 for 12 other residues . The resulting proteins either lack or, in the case of glutamic acid, have very low enzyme activity consistent with an essential catalytic role for Asp383 . The O4 position on the quinone ring is involved in a short hydrogen bond with the hydroxyl of conserved residue Tyr369 . The distance between the oxygens is less than 2.5 A, consistent with a shared proton, and suggesting ionization at the O4 position of the quinone ring . The Tyr369 residue appears to play an important role in stabilizing the position of the quinone/inhibitor complex . The O2 position on the quinone ring is hydrogen bonded to the apical water ligand of the copper . The basal water ligand, which lies 2.0 A from the copper in the native structure, is at a distance of 3.0 A in the complex . In the native structure, the active site is completely buried, with no obvious route for entry of substrate . In the complex, the tip of the pyridine ring of the bound inhibitor is on the surface of the protein at the edge of the interface between domains 3 and 4, suggesting this as the entry point for the amine substrate. Proc Natl Acad Sci U S A, 1997 Feb 18, 94(4), 1556 - 61 Interconversion of red opsin isoforms by the cyclophilin-related chaperone protein Ran-binding protein 2; Ferreira PA et al.; Ran-binding protein 2 (RanBP2) (type II) is a retinal cyclophilin-related protein that binds Ran-GTPase . Type I cyclophilin is a shorter, alternatively spliced isoform of RanBP2 . Recently, we showed that the Ran-binding domain 4 (RBD4)/cyclophilin (CY) supradomain of RanBP2 acts both in vitro and in vivo as a specific chaperone for bovine red/green opsin (R/G opsin) . R/G opsin undergoes a stable modification of its electrophoretic mobility upon binding to RanBP2 . This modification is likely due to cis-trans isomerization of one or more proline residues in the opsin protein . Here, we show that expression of human red opsin in Escherichia coli and COS cells results in the production of still a third electrophoretic variant of this protein . This variant was converted to the RBD4 binding-competent form of opsin through direct interaction with RBD4/CY, both in vivo and in vitro . We suggest that these distinct opsin species may represent kinetically or thermodynamically trapped prolyl conformers that can be interconverted by concerted action of the RBD4 and CY domains of RanBP2 . We also show that the C-terminal half of RBD4 is the binding domain for bovine R/G opsin and that coexpression of human red opsin with type I cyclophilin in vivo enhances the production of functional visual pigment . These observations imply that prolyl isomerization may have importance beyond its role in protein folding, possibly as a molecular switch modulated by cyclophilin for the loading of opsin onto RanBP2 during visual pigment processing in cones. Proc Natl Acad Sci U S A, 1997 Feb 18, 94(4), 1304 - 9 High-resolution functional mapping of a cloned gene by genetic footprinting; Singh IR et al.; We describe an efficient method for introducing and analyzing a comprehensive set of mutations in a cloned gene to map its functional organization . The technique, genetic footprinting, uses a retroviral integrase to generate a comprehensive library of mutants, each of which bears a single insertion of a defined oligonucleotide at a random position in the gene of interest . This mutant library is selected for gene function en masse . DNA samples are isolated from the library both before and after selection, and the mutations represented in each sample are then analyzed . The analysis is designed so that a mutation at a particular location gives rise to an electrophoretic band of discrete mobility . For the whole library, this results in a ladder of bands, each band representing a specific mutation . Mutants in which the inserted sequence disrupts a feature that is required for the selected function, ipso facto, fail the selection . The corresponding bands are therefore absent from the ladder of bands obtained from the library after selection, giving rise to a footprint representing features of the gene that are essential for the selected function . Because the sequence of the inserted oligonucleotide is known, and its position can be inferred precisely from the electrophoretic mobility of the corresponding band, the precise location and sequence of mutations that disrupt gene function can be determined without isolating or sequencing individual mutants . This method should be generally applicable for saturation mutagenesis and high-resolution functional mapping of cloned DNA sequences. Proc Natl Acad Sci U S A, 1997 Feb 18, 94(4), 1107 - 12 The effect of macromolecular crowding on chaperonin-mediated protein folding; Martin J et al.; The cylindrical chaperonin GroEL and its cofactor GroES mediate ATP-dependent protein folding in Escherichia coli . Recent studies in vitro demonstrated that GroES binding to GroEL causes the displacement of unfolded polypeptide into the central volume of the GroEL cavity for folding in a sequestrated environment . Resulting native protein leaves GroEL upon GroES release, whereas incompletely folded polypeptide can be recaptured for structural rearrangement followed by another folding trial . Additionally, each cycle of GroES binding and dissociation is associated with the release of nonnative polypeptide into the bulk solution . Here we show that this loss of substrate from GroEL is prevented when the folding reaction is carried out in the presence of macromolecular crowding agents, such as Ficoll and dextran, or in a dense cytosolic solution . Thus, the release of nonnative polypeptide is not an essential feature of the productive chaperonin mechanism . Our results argue that conditions of excluded volume, thought to prevail in the bacterial cytosol, increase the capacity of the chaperonin to retain nonnative polypeptide throughout successive reaction cycles . We propose that the leakiness of the chaperonin system under physiological conditions is adjusted such that E . coli proteins are likely to complete folding without partitioning between different GroEL complexes . Polypeptides that are unable to fold on GroEL eventually will be transferred to other chaperones or the degradation machinery. Proc Natl Acad Sci U S A, 1997 Feb 18, 94(4), 1096 - 100 Catalysis of protein folding by symmetric chaperone complexes; Sparrer H et al.; The GroE chaperones of Escherichia coli assist protein folding under physiological and heat shock conditions in an ATP-dependent way . Although a number of details of assisted folding have been elucidated, the molecular mechanism of the GroE cycle remains unresolved . Here we present an experimental system that allows the direct analysis of the GroE-mediated folding cycle under stringent conditions . We demonstrate that the GroE proteins efficiently catalyze the folding of kinetically trapped folding intermediates of a mutant of maltose-binding protein (MBP Y283D) in an ATP-dependent way . GroES plays a key role in this reaction cycle, accelerating the folding of the substrate protein MBP Y283D up to 50-fold . Interestingly, catalysis of the folding reaction requires the formation of symmetrical football-shaped GroEL x GroES2 particles and the intermediate release of the nonnative protein from the chaperone complex . Our results show that, in the presence of GroES, the complex architecture of the GroEL toroids allows maintenance of two highly interregulated rings simultaneously active in protein folding. Proc Natl Acad Sci U S A, 1997 Feb 18, 94(4), 1069 - 73 Assembly of an active enzyme by the linkage of two protein modules; Nixon AE et al.; The feasibility of creating new enzyme activities from enzymes of known function has precedence in view of protein evolution based on the concepts of molecular recruitment and exon shuffling . The enzymes encoded by the Escherichia coli genes purU and purN, N10-formyltetrahydrofolate hydrolase and glycinamide ribonucleotide (GAR) transformylase, respectively, catalyze similiar yet distinct reactions . N10-formyltetrahydrofolate hydrolase uses water to cleave N10-formyltetrahydrofolate into tetrahydrofolate and formate, whereas GAR transformylase catalyses the transfer of formyl from N10-formyltetrahydrofolate to GAR to yield formyl-GAR and tetrahydrofolate . The two enzymes show significant homology (approximately 60%) in the carboxyl-terminal region which, from the GAR transformylase crystal structure and labeling studies, is known to be the site of N10-formyltetrahydrofolate binding . Hybrid proteins were created by joining varying length segments of the N-terminal region of the PurN gene (GAR binding region) and the C-terminal (N10-formyltetrahydrofolate binding) region of PurU . Active PurN/PurU hybrids were then selected for by their ability to complement an auxotrophic E . coli strain . Hybrids able to complement the auxotrophs were purified to homogeneity and assayed for activity . The specific activity of two hybrid proteins was within 100- to 1000-fold of the native purN GAR transformylase validating the approach of constructing an enzyme active site from functional parts of others. Virology, 1997 Feb 17, 228(2), 200 - 12 Phosphorylated states of vesicular stomatitis virus P protein in vitro and in vivo; Chen JL et al.; We have previously shown that the phosphoprotein (P) of vesicular stomatitis virus (VSV), New Jersey serotype (PNJ) is phosphorylated by casein kinase II, within the N-terminal domain I (P1 form), whereas the C-terminal domain II is phosphorylated by a protein kinase activity associated with the L protein (P2 form) (D . J . Chattopadhyay and A.K . Banerjee, Cell 49, 407, 1987; A.M . Takacs et al., J . Virol . 66, 5842, 1992) . In the present studies, we have mapped the corresponding P1 and P2 phosphorylation sites in the P protein of the well-studied Indiana serotype (PIND) and compared that with the two previously designated NS1 and NS2 forms present in vivo . The PIND expressed in Escherichia coli in an unphosphorylated form (P0) was used as substrate for recombinant casein kinase II (CKII) . By site-directed mutagenesis, the CKII-mediated phosphorylation sites in the P protein were mapped at S60, T62, and S64 within the acidic domain I in vitro . In contrast, using BHK cell extract as the source of CKII or expressing P protein in COS cells labeled with 32PI, the phosphorylation sites were mapped at S60 and S64 with no phosphorylation at T62 residue . We used a peptide mapping technique by which the phosphorylation sites within domain I and domain II were determined . Using this method we demonstrated that the P1 and P2 forms are similar, if not identical, to the previously designated NS1 and NS2 forms, respectively . The domain II phosphorylating kinase activity, associated with the L protein, is shown to be present also in the N-RNA complex, indicating that this activity is of cellular origin . By site-directed mutagenesis, we have shown that S226 and S227 are involved in phosphorylation within domain II . We also demonstrate that the P1 and P2 forms are interconvertible and arise by phosphorylation/dephosphorylation of the phosphate groups in domain II, confirming the precursor-product relationship between the two phosphorylated forms of P protein. EMBO J, 1997 Feb 17, 16(4), 889 - 95 The limited strand-separating activity of the UvrAB protein complex and its role in the recognition of DNA damage; Gordienko I et al.; The recognition by Escherichia coli Uvr nucleotide excision repair proteins of a variety of lesions with diverse chemical structures and the presence of helicase activity in the UvrAB complex which can displace short oligonucleotides annealed to single-stranded DNA led to a model in which this activity moves UvrAB along undamaged DNA to damaged sites where the lesion blocks further translocation and the protein-DNA pre-incision complex is formed . To evaluate this mechanism for damage recognition, we constructed substrates with oligonucleotides of different lengths annealed to single-stranded DNA circles and placed a single 2-(acetylamino)fluorene (AAF) lesion either on the oligonucleotide or on the circle . For the substrates with no lesion, the UvrAB complex effectively displaced a 22-mer but not a 27-mer or longer fragments . The presence of AAF on the oligonucleotide significantly increased the release of the 27-mer but oligomers of 30 or longer were not separated . Placing the lesion on the circular strand did not block the release of the fragments . Instead, the releasing activity of UvrAB was stimulated and also depended on the length of the annealed oligonucleotide . These observations do not agree with the predictions of a damage recognition mechanism that depends on helicase-driven translocation . Most likely, the strand-separating activity of UvrAB is a consequence of local changes occurring during the formation of a DNA-protein pre-incision complex at the damaged site and is not due to translocation of the protein along undamaged DNA to locate a lesion. EMBO J, 1997 Feb 17, 16(4), 880 - 8 UvrAB activity at a damaged DNA site: is unpaired DNA present? Gordienko I, Rupp WD. To study the activity of the Escherichia coli UvrA and UvrB nucleotide excision repair proteins during the formation of the pre-incision complex at a damaged DNA site, we used substrates with modifications around a single 2-(acetylamino)fluorene (AAF) lesion . Based on the release of AAF-containing oligonucleotides from a single-stranded DNA circle, we conclude that during interaction with our substrates UvrAB introduces changes in DNA which are localized at the lesion and are limited to 1-3 bp . Since these changes might include a denaturation of DNA at the lesion site and, consequently, a bubble structure might be present in a pre-incision complex, we studied incision activity of UvrABC excinuclease on substrates with 1-4 unpaired bases next to an AAF adduct . Opening more than one base on either or both sides of the lesion caused a significant decrease in the incision activity of UvrABC, but did not change the position of the incision sites . We conclude that the UvrAB action leading to a pre-incision complex does not include the formation of a bubble intermediate generated by extensive denaturation of base pairs. EMBO J, 1997 Feb 17, 16(4), 826 - 33 Evidence that translation reinitiation abrogates nonsense-mediated mRNA decay in mammalian cells; Zhang J et al.; Nonsense codons upstream of and including position 192 of the human gene for triosephosphate isomerase (TPI) have been found to reduce the abundance of TPI mRNA to approximately 25% of normal . The reduction is due to the decay of newly synthesized TPI mRNA that co-purifies with nuclei . TPI mRNA that co-purifies with cytoplasm is immune to nonsense-mediated decay . Until now, a nonsense codon at position 23 has been the 5'-most nonsense codon that has been analyzed . Here, we provide evidence that a nonsense codon at position 1, 2 or 10 reduces the abundance of nucleus-associated TPI mRNA to an average of only 84% of normal because translation reinitiates at the methionine codon at position 14 . First, converting codon 14 to one for valine increased the effectiveness with which an upstream nonsense codon reduces mRNA abundance . Second, when TPI gene sequences, including codon 14, were fused upstream of and in-frame to the translational reading frame of an Escherichia coli chloramphenicol acetyl transferase (CAT) gene that lacked an initiation codon, a nonsense codon at TPI position 1 or 2 allowed for the production of TPI-CAT that was an estimated 14 amino acids smaller than TPI-CAT produced by a nonsense-free gene, whereas a nonsense codon at TPI position 23 precluded the production of TPI-CAT . These and related findings lend credence to the concept that the nonsense-mediated reduction in the half-life of nucleus-associated TPI mRNA involves cytoplasmic ribosomes. FEBS Lett, 1997 Feb 17, 403(2), 203 - 7 Positively charged lysine at the N-terminus of the signal peptide of the Escherichia coli alkaline phosphatase provides the secretion efficiency and is involved in the interaction with anionic phospholipids; Nesmeyanova MA et al.; Positively charged amino acid residues at the N-terminus of the signal peptide (SP) have been proposed to play a significant role in the initial step of protein secretion in bacteria . To test this hypothesis, Lys(-20) of the Escherichia coli alkaline phosphatase SP was replaced by other amino acid residues, and the effect of these substitutions on protein maturation was studied . The introduction of negatively charged and hydrophobic amino acids resulted in a decrease in secretion efficiency and impaired the SP-APL interaction, whereas His and Tyr had no significant effect . A structural analysis of the SP-APL interaction suggests that the positively charged Lys(-20) determines the stability of the complex. FEBS Lett, 1997 Feb 17, 403(2), 116 - 22 Use of phage display for isolation and characterization of single-chain variable fragments against dihydroflavonol 4-reductase from Petunia hybrida; De Jaeger G et al.; To isolate specific single-chain variable (scFv) fragments against dihydroflavonol 4-reductase (DFR) from Petunia hybrida the phage display technology was used . DFR was overproduced in Escherichia coli, purified and used for immunization . From DFR-immunized mice, a phage display library was made starting from spleen mRNA using an optimized set of primers for V(H) and V(L) amplification . Several rounds of panning against recombinant DFR yielded five different scFv fragments, confirmed by subsequent DNA sequencing . They all specifically bound to recombinant DFR in ELISA and DFR in flower extracts on Western blot . These results show that phage display is a promising technology in plant molecular biology to obtain specific recombinant antibodies not only for ELISA and Western blot but also for in vivo applications in the long run. J Surg Res, 1997 Feb 15, 68(1), 79 - 86 Nitric oxide inhibition decreases neutrophil adhesion at the inflammatory site, while increasing adhesion in remote organs in peritonitis; Fukatsu K et al.; Nitric oxide (NO) is a regulator of leukocyte adhesion in the microcirculation . This study was designed to examine the effects of a NO synthase inhibitor on neutrophil adhesion in the peritoneum, lung, liver, and kidney in a rat peritonitis model using a fluorescence microscopic method . Sprague-Dawley rats were given normal saline (control) or N omega-nitro-L-arginine methyl ester (L-NAME) at dosages of 10 mg/kg (N10) or 100 mg/kg (N100) (n = 66) intraperitoneally . One hour after pretreatment fluorescein-labeled neutrophils were infused without bacterial challenge (0 hr) . Other rats received an injection of 10(7) Escherichia coli into the peritoneal cavity 1 hr after pretreatment . Labeled neutrophils were infused 1 and 5 hr after bacterial challenge . Just 2 min after neutrophil injection, blood samples were obtained and the animals were killed . Five peritoneal samples (omentum, mesentery, parietal peritoneum, colon, and ileum), both lungs, the liver, and the right kidney were harvested for counting of labeled neutrophils under epifluorescent microscopy . Combined plasma nitrite/nitrate levels were determined . In another set of rats (n = 36), an arterial catheter was inserted after L-NAME treatment and bacterial challenge . At 0, 1, 5, and 12 hr after challenge, blood pressure, heart rate, and arterial blood gas data were measured . One hour after E . coli challenge, the number of neutrophils in the peritoneum was significantly lower in both L-NAME-treated groups than in the control group . In contrast, the number of labeled neutrophils in the lungs was significantly higher in the N100 group than in the control group . Neutrophil accumulation in the lungs and peritoneum at 0 and 5 hr and in the liver and kidney at 0, 1, and 5 hr did not differ among groups, nor did combined plasma nitrite/nitrate levels . L-NAME treatment had no influence on either hemodynamic or blood gas data . In conclusion, administration of L-NAME increases neutrophil adhesion in the lung, while decreasing that in the peritoneum . NO plays an important role in neutrophil adhesion at the inflammatory site, as well as in remote organs, during peritonitis . NO inhibition may be detrimental, due to neutrophil sequestration, in this peritonitis model. Biochem J, 1997 Feb 15, 322 ( Pt 1), 241 - 4 Identification of a putative flexible loop in Arabidopsis glutathione synthetase; Wang CL et al.; Glutathione synthetase catalyses the ATP-dependent ligation of gamma-glutamylcystene with glycine to form glutathione . Amino acid sequence comparisons between the Arabidopsis and the Escherichia coli proteins suggested that a region, identified as a small flexible loop that covers the active site of the E . coli protein, might be conserved in the eukaryotic protein . Three site-directed mutations in the Arabidopsis protein were generated to test this hypothesis . Two mutations within the conserved region (Lys367/ Pro368-->Asn/Ser and Gly374-->Val) inactivated the enzyme in an in vivo assay based on cadmium resistance in S . pombe, and in an in vitro assay of the activity of the enzyme expressed in E . coli . A third mutation outside of this conserved region (Leu363-->Glu) had a smaller effect in both assays . These results are consistent with the idea that this glycine-rich loop in the Arabidopsis and E . coli proteins might serve the same function in covering the active site of the enzyme. Genomics, 1997 Feb 15, 40(1), 41 - 7 PMM (PMM1), the human homologue of SEC53 or yeast phosphomannomutase, is localized on chromosome 22q13; Matthijs G et al.; We have cloned the human homologue of SEC53 or yeast phosphomannomutase (HGMW-approved symbol PMM1) from a liver cDNA library . This cDNA encodes a protein of 262 amino acids with a predicted molecular mass of 29 kDa and 54% identity with yeast phosphomannomutase . Expression of the human cDNA in Escherichia coli yielded an active phosphomannomutase, which was purified to homogeneity . Northern blot analysis of human tissues showed strong expression in liver, heart, brain, and pancreas and a lower expression in skeletal muscle . The gene was assigned to chromosome 22q13.1 by the use of hybrid cell lines and by fluorescence in situ hybridization . Most patients presenting with carbohydrate-deficient glycoprotein syndrome type 1 (CDG1 or Jaeken disease) have a greatly reduced phosphomannomutase activity; the gene encoding this enzyme is a likely candidate for CDG1 . Since the CDG1 locus maps else where in the genome (16p13), mutations in the phosphomannomutase gene encoded by chromosome 22 are not a major cause of CDG1. Nucleic Acids Res, 1997 Feb 15, 25(4), 806 - 13 Role of proofreading and mismatch repair in maintaining the stability of nucleotide repeats in DNA; Strauss BS et al.; The role of the proofreading exonuclease in maintaining the stability of multiply repeated units in DNA was studied in Escherichia coli . Reversion of plasmids in which the beta-galactosidase alpha complementing sequence was moved +2 out of frame by inserts containing (CA)14, (CA)5, (CA)2 or (TA)6 or +1 by creating a run of 8 C was compared in mutS and mutSdnaQ strains . Proofreading corrects at least half of the frameshift errors for all the plasmids and at least 99% of the errors in the (CA)2 plasmid . The (CA)2 plasmid reverts mostly by +1 frameshifts in the restriction sites flanking the insert . With the (CA)14, (TA)6, (CA)5 and 8C plasmids, reversion is mainly by loss of a repeat unit . The data support the hypothesis that the dnaQgene product recognizes frameshifts close to the DNA growing point . Frameshifts distal to the growing point are mainly corrected by mismatch repair.We speculate that mismatches in mononucleotide repeats are susceptible to proofreading because they can either migrate to a point where they are recognized by the exonuclease or, alternatively, because single nucleotide distortions are more readily detected than dinucleotides. Eur J Biochem, 1997 Feb 15, 244(1), 235 - 41 Activity of tethered human immunodeficiency virus 1 protease containing mutations in the flap region of one subunit; Tozser J et al.; The tethered-dimer protease of human immunodeficiency virus 1 (HIV-1) {Cheng Y.-S . E., Yin, F.H., Foundling, S., Blomstrom, D . & Kettner, C . A . (1990) Proc . Natl Acad . Sci . USA 87, 9660-9664} and its mutants containing amino acid substitutions or deletions or both in only one flap region were expressed in Escherichia coli . These mutant enzymes showed various degrees of self-processing and significantly reduced catalytic activity toward oligopeptide substrates compared with the wild type . Kinetic parameters determined for one of the oligopeptide substrates showed a dramatic increase in K(m) and decrease in Kcat values . Unexpectedly, the substrate cleavage was more efficient in low salt concentration for a mutant containing a shortened hydrophilic flap . Assays with oligopeptides representing naturally occurring cleavage sites or oligopeptides containing single amino acid substitutions at the P2 and P2' substrate positions showed only moderate changes in the substrate specificity of the mutant proteases . Predicted structures for the mutants were constructed by molecular modeling and used to interpret the results of kinetic measurements . In general, the data suggest that the mutated part of the flaps does not have a major role in determining substrate specificity; rather, it provides the hydrophobic environment and hydrogen-bond interactions with the conserved water that are necessary for efficient substrate binding and catalysis. Eur J Biochem, 1997 Feb 15, 244(1), 155 - 60 Requirement for the proton-pumping NADH dehydrogenase I of Escherichia coli in respiration of NADH to fumarate and its bioenergetic implications; Tran QH et al.; In Escherichia coli the expression of the nuo genes encoding the proton pumping NADH dehydrogenase I is stimulated by the presence of fumarate during anaerobic respiration . The regulatory sites required for the induction by fumarate, nitrate and O2 are located at positions around -309, -277, and downstream of -231 bp, respectively, relative to the transcriptional-start site . The fumarate regulator has to be different from the O2 and nitrate regulators ArcA and NarL . For growth by fumarate respiration, the presence of NADH dehydrogenase I was essential, in contrast to aerobic or nitrate respiration which used preferentially NADH dehydrogenase II . The electron transport from NADH to fumarate strongly decreased in a mutant lacking NADH dehydrogenase I . The mutant used acetyl-CoA instead of fumarate to an increased extent as an electron acceptor for NADH, and excreted ethanol . Therefore, NADH dehydrogenase I is essential for NADH-->fumarate respiration, and is able to use menaquinone as an electron acceptor . NADH-->dimethylsulfoxide respiration is also dependent on NADH dehydrogenase I . The consequences for energy conservation by anaerobic respiration with NADH as a donor are discussed. Arch Biochem Biophys, 1997 Feb 15, 338(2), 157 - 64 Targeted antipeptide antibodies to cytochrome P450 2C18 based on epitope mapping of an inhibitory monoclonal antibody to P450 2C51; Richardson TH et al.; The epitope recognized by the inhibitory monoclonal antibody designated 2F5, which was raised against P450 2C5, was mapped to amino acids 237-260 by immunoblotting using a combination of recombinant antigens and chimeric and partial fusion proteins constructed from rabbit P450s 2C2, 2C4, 2C5, and 2C16, which are recognized by 2F5, and from 2C1 and 2C3, which are not . When the sequence of the epitope for 2F5 (amino acids 237-260) was compared with those of other rabbit 2C P450s, a single lysine residue at position 253 appeared to be a likely determinant of 2F5 immunoreactivity . Substitution of lysine for glutamic acid 253 in P450 2C3 (2C3E253K) conferred immunoreactivity and the ability of 2F5 to inhibit progesterone metabolism catalyzed by P450 2C3E253K . Sequence alignment revealed that this epitope lies in close proximity to the epitope identified for LKM-1 autoantibodies to P450 2D6 . Based on these results, an antipeptide antibody was raised to the corresponding region (amino acids 252-263) of human P450 2C18 . The resulting antipeptide antiserum recognizes P450 2C18 but not P450 2C8, 2C9, or 2C19 . However, the antipeptide 2C18 antiserum did not inhibit 2C18-catalyzed diazepam N-demethylation . Human 2C P450s were also quantitated by immunoblot analysis in a panel of six human liver microsomes using Escherichia coli expressed P450s as standards . Analysis of immunoblots indicated that, if present, P450 2C18 was expressed at very low levels (<2.5 pmol/mg), whereas P450s 2C8, 2C9, and 2C19 were easily detected. Nucleic Acids Res, 1997 Feb 15, 25(4), 781 - 6 Libraries for genomic SELEX; Singer BS et al.; An increasing number of proteins are being identified that regulate gene expression by binding specific nucleic acidsin vivo . A method termed genomic SELEX facilitates the rapid identification of networks of protein-nucleic acid interactions by identifying within the genomic sequences of an organism the highest affinity sites for any protein of the organism . As with its progenitor, SELEX of random-sequence nucleic acids, genomic SELEX involves iterative binding, partitioning, and amplification of nucleic acids . The two methods differ in that the variable region of the nucleic acid library for genomic SELEX is derived from the genome of an organism . We have used a quick and simple method to construct Escherichia coli, Saccharomyces cerevisiae, and human genomic DNA PCR libraries that can be transcribed with T7 RNA polymerase . We present evidence that the libraries contain overlapping inserts starting at most of the positions within the genome, making these libraries suitable for genomic SELEX. Nucleic Acids Res, 1997 Feb 15, 25(4), 764 - 8 Flavin adenine dinucleotide as a chromophore of the Xenopus (6-4)photolyase; Todo T et al.; Two types of enzyme utilizing light from the blue and near-UV spectral range (320-520 nm) are known to have related primary structures: DNA photolyase, which repairs UV-induced DNA damage in a light-dependent manner, and the blue light photoreceptor of plants, which mediates light-dependent regulation of seedling development . Cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidone photoproducts {(6-4)photoproducts} are the two major photoproducts produced in DNA by UV irradiation . Two types of photolyases have been identified, one specific for CPDs (CPD photolyase) and another specific for (6-4)photoproducts {(6-4)photolyase} . (6-4)Photolyase activity was first found in Drosophila melanogaster and to date this gene has been cloned only from this organism . The deduced amino acid sequence of the cloned gene shows that (6-4)photolyase is a member of the CPD photolyase/blue light photoreceptor family . Both CPD photolyase and blue light photoreceptor are flavoproteins and bound flavin adenine dinucleotides (FADs) are essential for their catalytic activity . Here we report isolation of a Xenopus laevis(6-4)photolyase gene and show that the (6-4)photolyase binds non- covalently to stoichiometric amounts of FAD . This is the first indication of FAD as the chromophore of (6-4)photolyase. Nucleic Acids Res, 1997 Feb 15, 25(4), 719 - 26 Characterization of a RecA/RAD51 homologue from the hyperthermophilic archaeon Pyrococcus sp . KOD1; Rashid N et al.; The Pk-rec gene, encoding a RecA/RAD51 homologue from the hyperthermophilic archaeon Pyrococcussp . KOD1, was expressed in Escherichia coli . The recombinant Pk-REC was purified to homogeneity and was shown to be in a dimeric form . A striking property of the purified recombinant Pk-REC was the unusual DNase activity on both single- and double-stranded DNAs along with the ATPase activity . The reaction product of this DNase activity was mononucleotides . The optimum temperature and pH for the DNase activity were 60 degrees C and 8-8.5, respectively . In addition, the metal ion requirement for DNase activity was different from that for the ATPase activity . The protein exhibited no DNase activity in the presence of Zn2+ion, which was one of the most preferable divalent cations for ATPase activity . Another unique characteristic of the recombinant protein was that the reaction product of ATPase activity was AMP instead of ADP.Pk-REC may represent a common prototype of the RecA family proteins with high RecA-like activity. Nucleic Acids Res, 1997 Feb 15, 25(4), 713 - 8 DNA flexibility of the UP element is a major determinant for transcriptional activation at the Escherichia coli acetate promoter; Negre D et al.; The specific interaction of the upstream element-containing promoter of the Escherichia coli acetate operon with either the RNA polymerase holoenzyme or its alpha subunit has been analyzed by the base removal method . Our results indicate that: (i) direct and specific base contacts can be detected in the acetate promoter-alpha subunit complex; (ii) base elimination in the upstream element of the acetate promoter enhances the binding of RNA polymerase . A similar effect is observed when studying the interactions between RNA polymerase and the rrnB ribosomal operon P1 promoter. Mutat Res, 1997 Feb 14, 388(2-3), 239 - 48 Somatic and germ cell mutagenesis in lambda lacZ transgenic mice treated with acrylamide or ethylnitrosourea; Krebs O et al.; The transgenic Muta Mouse in vivo mutagenesis assay was employed to determine the activity of acrylamide and ethylnitrosourea in liver and germ cells after 3, 10 and 100 days following treatment . Each cell of the Muta Mouse carries 80 copies of the lambda gt10 phage including the bacterial lacZ gene, which act as the target gene for the mutagenesis assay . Groups of Muta Mice were given a single intraperitoneal injection of 80 or 160 mg/kg ethylnitrosourea or 50 or 100 mg/kg acrylamide . The tissues were prepared 3, 10 or 100 days post treatment . The liver genomic DNA was extracted with the manufacturer's standard protocol, while the genomic germ cell DNA was extracted with 4 different methods due to problems encountered in DNA yields and packaging efficiency . The mutation analysis of the lacZ gene was carried out by the positive selective assay method {Gossen et al . (1989) Proc . Natl . Acad . Sci . USA, 86, 7971-7975; Dean and Myhr (1994) Mutagenesis, 9, 183-185} . There was a slight increase due to treatment of the observed mutation frequencies in the acrylamide liver group for all three assay times . From the day 3 group to the day 100 group a time dependent decrease in all the absolute mutant frequencies was detectable . The ethylnitrosourea liver group showed a time- and dose-dependent increase in the mutant frequencies from day 3 to day 100 . No meaningful results were obtained for the germ cell tissue assays due to the low amount of genomic DNA extracted which was not packageable in the lambda lacZ assay . At present for the mutagenesis assay of isolated spermatozoa in our laboratory we would be forced to pool tissues from animals to obtain enough DNA for an assay . Since 'jackpot'-animals may exist {Heddle et al . (1992) Mutation Res., 272, 195-203} the individual animals of such a pooled analysis group must be tested before pooling. Mutat Res, 1997 Feb 14, 388(2-3), 229 - 37 Mutations induced in male germ cells after treatment of transgenic mice with ethylnitrosourea; Katoh M et al.; The present study was undertaken to clarify whether the transgenic mouse mutagenesis assay system can be used instead of dominant lethals or specific locus test after treatment of male germ cells in mouse with ethylnitrosourea (ENU) . Male Big Blue transgenic mice (BB) carrying a lacI target gene were given a single intraperitoneal injection of 150 mg/kg ENU . Vasa deferential sperm, caudal epididymal sperm or whole testes were assayed for mutation at 3, 14, 22 and 93 days after treatment with ENU . The average of background lacI- mutant frequencies was 2.05 x 10(-5) . The MF observed in post spermatogonial stage after treatment with ENU were slightly increased over background . On the other hand, ENU induced high MF in the spermatogonial stage . MF detected after treatment of BB male germ cells with ENU were lower than those detected in the mouse visible specific-locus mutations in previous reports . Nevertheless, it is clear that this assay is a practical alternative to the specific locus test for detecting mutations induced in spermatogonial stage. Mutat Res, 1997 Feb 14, 388(2-3), 223 - 8 Germ cell mutagenesis in lacZ transgenic mice treated with methyl methanesulfonate; Itoh S et al.; Mutagenesis induced by methyl methanesulfonate (MMS), a germ cell mutagen, in the testis and the sperm isolated from epididymis and vas deferens have been investigated using lacZ transgenic mice (Muta Mouse) . Male Muta Mice were injected intraperitoneally with MMS at a dose of 80 mg/kg, a potent dominant lethal dose . Animals were killed on days 3 and 7 (Experiment 1) or days 10 and 14 (Experiment 2) after the treatment . Mutant frequencies (MFs) in the testis, sperm and spleen (Experiment 2 only) were analyzed by the positive selection system using E . coli C (GalE-) strain and phenyl beta-D-galactoside . The spontaneous MFs in the testis and sperm were 2.0-3.1 x 10(-5) . No induction of mutation in the testis or sperm of the MMS-treated groups was observed at any sampling point . In the spleen, the spontaneous MF was approximately twice as high as that in the germ cells although the MF at each sampling point was almost the same as the spontaneous MF . MMS is known as a potent clastogen from the results of the dominant lethal assay and the micronucleus assay . The reason for the discrepancy between the results of these assays and the present results may have been insensitivity of the in vitro packaging to large deletion due to the failure to rescue the large deleted gene . It is suggested that the transgenic mouse assay using the in vitro packaging can not replace the dominant lethal assay in the case of MMS. Mutat Res, 1997 Feb 14, 388(2-3), 219 - 22 The detection of gene mutation in the tubular sperm of Muta Mice following a single intraperitoneal treatment with methyl methanesulphonate or ethylnitrosourea; Brooks TM et al.; Transgenic mouse assays, such as Muta Mouse, provide a method to predict the potential target organ carcinogenicity of chemical compounds . As part of a collaborative study, the effects of the direct-acting mutagens, methyl methanesulphonate (MMS) and ethylnitrosourea (ENU), were investigated for gene mutation in the tubular sperm of Muta Mice testes after a single intraperitoneal exposure . Groups of male Muta Mice were dosed intraperitoneally with either 1/15 M phosphate buffer, pH 6.0 (vehicle control), 40 mg/kg methyl methanesulphonate (MMS) or 150 mg/kg ethylnitrosourea (ENU) . The animals were sacrificed 14 days after the single dose . Mutation frequencies were determined in tubular sperm DNA . The results showed a mean mutation frequency (MF) of 2.1 x 10(5) (64 mutants per 3.05 x 10(6) PFU) for the 10 vehicle-treated mice, a mean MF of 2.8 x 10(5) (78 mutants per 2.75 x 10(6) PFU) for the 10 MMS-treated mice and a mean MF of 9.1 x 10(5) (194 mutants per 2.14 x 10(6) PFU) for the 8 ENU-treated mice; this latter value representing a 4.5-fold increase over the vehicle control values. Mutat Res, 1997 Feb 14, 388(2-3), 187 - 95 Evaluation of lacI mutation in germ cells and micronuclei in peripheral blood after treatment of male lacI transgenic mice with ethylnitrosourea, isopropylmethane sulfonate or methylmethane sulfonate; Gorelick NJ et al.; Male C57B1/6 lacI transgenic mice were used to evaluate germ cell mutagenesis in vivo as part of a collaborative study . Groups of 10 mice were administered single intraperitoneal doses of ethylnitrosourea (ENU; 150 mg/kg), isopropyl methanesulfonate (IPMS; 200 mg/kg), methyl methanesulfonate (MMS; 40 mg/kg) or vehicle . Epididymal spermatozoa and testes were recovered 3 days later and DNA isolated subsequently from epididymal spermatozoa and seminiferous tubules were analyzed for lacI mutations . The mutant frequency in seminiferous tubules (average +/- SEM) increased significantly compared with untreated controls (7.2 +/- 0.7 x 10(-5) following treatment with ENU (11.7 +/- 0.8 x 10(-5), p = 0.003) or with IPMS (9.6 +/- 0.5 x 10(-5), p = 0.018) but not following treatment with MMS (8.1 +/- 0.8 x 10(-5), p = 0.213) . Group mutant frequencies were not determined for epididymal spermatozoa from MMS- or IPMS-treated mice because of poor DNA recoveries . As another indicator of the genotoxicity of these alkylating agents, the frequencies of micronuclei were determined in the peripheral blood 48 h after carcinogen administration in the same transgenic mice . The micronuclei frequencies were elevated significantly (p < 0.05) by each treatment (IPMS: 1.0%; MMS: 0.94%) compared to vehicle controls (0.3%) . In a separate experiment, 40 mg/kg ENU was previously found to increase the frequency of micronuclei in peripheral blood of lacI transgenic mice 48 h after treatment (3.2%; Gibson et al., 1995) . These results demonstrate that the lacI transgenic mouse male germ cells are sensitive to some, but not all, mutagens under the conditions used in this experiment . Investigation of other experimental designs would offer additional perspective on the usefulness of this transgenic model for routine mutagenicity testing in germ cells. Mutat Res, 1997 Feb 14, 388(2-3), 155 - 63 Ethyl nitrosourea and methyl methanesulfonate mutagenicity in sperm and testicular germ cells of lacZ transgenic mice (Muta Mouse); Suzuki T et al.; The germ cell mutagens ethyl nitrosourea (ENU) and methyl methanesulfonate (MMS), were tested for their genotoxicity in sperm cells and testicular germ cells using lacZ transgenic mice (Muta Mouse) . Eight- to 10-week-old Muta mice were treated with ENU (150 mg/kg) or MMS (40 mg/kg) by intraperitoneal injection . Three and 14 days after treatment, testes and sperm were collected for lacZ mutation analysis . Sperm were isolated from the epididymis and vas deferens by washing out the minced tissue . Germ cell DNA was isolated from testicular germ cells and sperm with the help of 2-mercaptoethanol, and the target lacZ gene, which is integrated into a lambda shuttle vector, was recovered by in vitro packaging . The resultant phages were allowed to infect to E . coli C (galE), and the lacZ mutant plaques were dominantly selected on a plate containing phenyl-beta-D-galactoside . Spontaneous mutant frequencies (MF) in vehicle-treated control mice were approximately 1 x 10(-5) and 3 x 10(-5) in testicular germ cells and sperm, respectively, at both sampling times . ENU treatment increased the MF in the testicular germ cells to 5 x 10(-5) on days 3 and 14, but did not affect sperm MF . MMS was not mutagenic in either tissue . The peripheral blood micronucleus assay was performed on the same animals 48 h after treatment, and strong inductions of micronucleated reticulocytes (MNRETs) were observed in both ENU- and MMS-treated mice . These data suggest that agents mutagenic to premeiotic germ cells, e.g., ENU, can be detected by transgenic mutation assay system using germ cells isolated from the testis . On the other hand, those mutagenic to postmeiotic cells, e.g., MMS, are insensitive in the assay system. Mutat Res, 1997 Feb 14, 388(2-3), 137 - 43 Evaluation of spontaneous and chemical-induced lacI mutations in germ cells from lambda/lacI transgenic mice; Putman DL et al.; The spontaneous mutant frequency in germ cells isolated from seminiferous tubules of two lambda/lacI transgenic mouse strains, C57BL/6 and B6C3F1 was evaluated . At least 500 000 phage were screened for mutation at lacI for each animal using standardized assay procedures . The germ cell spontaneous lacI mutant frequency was 17.8 +/- 8.1 x 10(-6) in C57BL/6 mice and 17.0 +/- 10.0 x 10(-6) in B6C3F1 mice . The induction of germ cell mutations by three well characterized alkylating agents were also evaluated in C57BL/6 mice on day 3 after a single dose administration . The lacI mutant frequencies were significantly elevated in transgenic mice dosed with ENU at 150 mg/kg (2-fold increase above control) and iPMS at 200 mg/kg (3-fold increase above control) but not in those receiving MMS at 40 mg/kg . These findings suggest that single dose studies using the lambda/lacI transgenic system may be capable of detecting germ mutations induced by chemicals characterized either by point mutations or small, intragenic deletions but not those characterized by a predominance of multi-locus deletions. Mutat Res, 1997 Feb 14, 388(2-3), 129 - 36 Evaluation of the transgenic Lambda/LacI mouse model as a short-term predictor of heritable risk; Provost GS et al.; Transgenic male C57BL/6 lambda/lacI mice were used to assess the mutagenic response in seminiferous tubules and epididymal spermatozoa 3 days after exposure to ethylnitrosourea (ENU), iso-propyl methanesulfonate (iPMS) and methyl methanesulfonate (MMS) . No significant mutagenic response was observed in epididymal spermatozoa for all three compounds, as expected 3 days after treatment . However, ENU and iPMS treated samples demonstrated significant mutagenic inductions relative to controls in seminiferous tubules while MMS treated samples did not . The failure of MMS to induce a mutagenic response in lambda/lacI transgenic mice is likely due to a combination of the low dose used, the short expression time after exposure and the reduced sensitivity to large deletion events in transgenic lambda/lacI shuttle vectors . In addition, ex vivo mutations were measured in control samples and iPMS treated samples, where 33% of mutants from control samples and 35% of mutants from iPMS treated samples were mosaic. J Mol Biol, 1997 Feb 14, 266(1), 122 - 34 The resolving enzyme CCE1 of yeast opens the structure of the four-way DNA junction; White MF et al.; Junction-resolving enzymes exhibit structure-selective binding to DNA, but may also manipulate the DNA structure . CCE1 is a junction-resolving enzyme found in the yeast mitochondrion . To facilitate the analysis of the CCE1-junction interaction, we have exploited the sequence dependence of the cleavage reaction to devise a junction that is refractory to cleavage by this enzyme, even in the presence of magnesium ions . On binding to four-way DNA junctions, pure recombinant CCE1 opens the global structure into a 4-fold symmetrical configuration of arms with an open, chemically reactive centre . The structure of the CCE1-junction complex is independent of the sequence of the junction, and of the presence or absence of magnesium or other ions . This and other functional properties of CCE1 are strikingly similar to those of RuvC resolving enzyme of Escherichia coli. J Mol Biol, 1997 Feb 14, 266(1), 76 - 92 Characterization of the mycobacteriophage L5 attachment site, attP; Pena CE et al.; Lysogenization of mycobacteriophage L5 involves integration of the phage genome into the Mycobacterium smegmatis chromosome . Integration occurs by a site-specific recombination event between a phage attachment site, attP, and a bacterial attachment site, attB, which is catalyzed by the phage-encoded integrase protein . DNase I footprinting reveals that L5 integrase binds to two types of sites within attP which span an unexpectedly large region of 413 bp: seven arm-type sites (P1 to P7) each of which correspond to a consensus sequence 5'-TGCaaCtcYy, and core-type sites at the points of strand exchange . Mutational analyses indicate that not all of the arm-type sites are required for integration, and that the P3 site and the rightmost pair of sites (P6 and P7) are dispensable for integration . We show that a 252 bp segment of attP DNA is sufficient for efficient integrative recombination and that int can be provided in trans for simple and efficient transformation of the mycobacteria. J Mol Biol, 1997 Feb 14, 266(1), 51 - 65 Filamentous phage IKe mRNAs conserve form and function despite divergence in regulatory elements; Stump MD et al.; As a means of determining whether there has been selection to conserve the basic pattern of filamentous phage mRNAs, the major mRNAs representing genes II to VIII have been defined for a phage distantly related to the Ff group specific for Escherichia coli hosts bearing F pili . Phage IKe has a genome with 55% identity with the Ff genome and infects E . coli strains bearing N pili . The results reveal a remarkably similar pattern of overlapping polycistronic mRNAs with a common 3' end and unique 5' ends . The IKe mRNAs, like the Ff phage mRNAs, represent a combination of primary transcripts and processed RNAs . However, examination of the sequences containing the RNA endpoint positions revealed that effectively the only highly conserved regulatory element is the rho-independent terminator that generates the common 3' end . Promoters and processing sites have not been maintained in identical positions, but frequently are placed so as to yield RNAs with similar coding function . By conserving the pattern of transcription and processing despite divergence in the regulatory elements and possibly the requirements for host, endoribonucleases, the results argue that the pattern is not simply fortuitous. J Mol Biol, 1997 Feb 14, 266(1), 40 - 50 Mutations at nucleotides G2251 and U2585 of 23 S rRNA perturb the peptidyl transferase center of the ribosome; Green R et al.; Previous experiments have shown that the phylogenetically conserved G2252 of 23 S rRNA forms a Watson-Crick base-pair with C74 of peptidyl-tRNA . In the studies presented here, site-directed mutations were introduced at two other conserved positions in 23 S rRNA, G2251 and U2585, that were previously implicated in interaction of the CCA acceptor end of tRNA with the 50 S subunit P site . The mutant 23 S rRNAs were characterized by determining (1) the in vivo phenotypes, (2) the ability of mutant ribosomes to bind tRNA oligonucleotide fragments in vitro, using footprinting with allele-specific primer extension and (3) the ability of mutant ribosomes to catalyze peptide bond formation using a chimeric reconstitution approach . Mutations at either position confer a dominant lethal phenotype when the mutant 23 S rRNA is coexpressed with the endogenous wild-type 23 S rRNA . Mutations at 2585 disrupt binding of the wild-type (CCA) tRNA oligonucleotide fragment and cause a modest decrease in the peptidyl transferase activity of reconstituted ribosomes . By contrast, mutations at 2251 abolish both binding of the wild-type (CCA) tRNA fragment and peptidyl transferase activity using the wild-type tRNA fragment . In neither case was the loss of binding or peptidyl transferase activity suppressed by mutations in the tRNA oligonucleotide fragment . Chemical modification analysis revealed that mutations at 2251 perturb the reactivity of bases 2584 to 2586, providing further evidence that the 2250 loop of 23 S rRNA interacts, either directly or indirectly, with the 2585 region in the central loop of domain V of 23 S rRNA. J Biol Chem, 1997 Feb 14, 272(7), 4516 - 21 Identification of a novel gene encoding the yeast mitochondrial dicarboxylate transport protein via overexpression, purification, and characterization of its protein product; Kakhniashvili D et al.; A gene encoding the mitochondrial dicarboxylate transport protein (DTP) has been identified for the first time from any organism . Our strategy involved overexpression of putative mitochondrial transporter genes, selected based on analysis of the yeast genome, followed by purification and functional reconstitution of the resulting protein products . The DTP gene from the yeast Saccharomyces cerevisiae encodes a 298-residue basic protein which, in common with other mitochondrial anion transporters of known sequence and function, displays the mitochondrial transporter signature motif, three homologous 100-amino acid sequence domains, and six predicted membrane-spanning regions . The product of this gene has been abundantly expressed in Escherichia coli where it accumulates in inclusion bodies . Upon solubilization of the overexpressed DTP from isolated inclusion bodies with Sarkosyl, 28 mg of DTP was obtained per liter of E . coli culture at a purity of 75% . The purified, overexpressed DTP was then reconstituted in phospholipid vesicles where both its kinetic properties (i.e . Km = 1 . 55 mM and Vmax = 3.0 micro;mol/min/mg protein) and its substrate specificity were determined . The intraliposomal substrates malonate, malate, succinate, and phosphate effectively supported {14C}malonate uptake, whereas other anions tested did not . External substrate competition studies revealed a similar specificity profile . Inhibitor studies indicated that the reconstituted transporter was sensitive to inhibition by n-butylmalonate, p-chloromercuribenzoate, mersalyl, and to a lesser extent pyridoxal 5'-phosphate but was insensitive to N-ethylmaleimide and selective inhibitors of other mitochondrial anion transporters . In combination, the above findings indicate that the identified gene encodes a mitochondrial transport protein which upon overexpression and reconstitution displays functional properties that are virtually identical to those of the native mitochondrial dicarboxylate transport system . In conclusion, the present investigation has resulted in identification of a gene encoding the mitochondrial DTP and thus eliminates a major impediment to molecular studies with this metabolically important transporter . Based on both structural and functional considerations, the yeast DTP is assignable to the mitochondrial carrier family . Additionally, the development of a procedure that enables the expression and isolation of large quantities of functional DTP provides the foundation for comprehensive investigations into the structure/function relationships within this transporter via site-directed mutagenesis, as well as for the initiation of crystallization trials. J Biol Chem, 1997 Feb 14, 272(7), 4398 - 403 Mutational analysis of the properties of caveolin-1 . A novel role for the C-terminal domain in mediating homo-typic caveolin-caveolin interactions; Song KS et al.; Caveolin is a principal structural component of caveolae membranes in vivo . Recently, a family of caveolin-related proteins has been identified; caveolin has been retermed caveolin-1 . Caveolin family members share three characteristic properties: (i) detergent insolubility at low temperatures; (ii) self-oligomerization; and (iii) incorporation into low density Triton-insoluble fractions enriched in caveolae membranes . Here, we have used a deletion mutagenesis approach as a first step toward understanding which regions of caveolin-1 contribute to its unusual properties . Two caveolin-1 deletion mutants were created that lack either the C-terminal domain (Cav-1DeltaC) or the N-terminal domain (Cav-1DeltaN); these mutants were compared with the behavior of full-length caveolin-1 (Cav-1FL) expressed in parallel . Our results show that the N-terminal domain and membrane spanning segment are sufficient to form high molecular mass oligomers of caveolin-1 . However, a complete caveolin-1 molecule is required for conveying detergent insolubility and incorporation into low density Triton-insoluble complexes . These data indicate that homo-oligomerization and an intact transmembrane are not sufficient to confer detergent insolubility, suggesting an unknown role for the C-terminal domain in this process . To better understand the role of the C-terminal domain, this region of caveolin-1 (residues 135-178) was expressed as a glutathione S-transferase fusion protein in Escherichia coli . Purified recombinant glutathione S-transferase-C-Cav-1 was found to stably interact with full-length caveolin-1 but not with the two caveolin-1 deletion mutants . These results suggest that the C-terminal domain interacts with both the N-terminal and C-terminal domains of an adjacent caveolin-1 homo-oligomer . This appears to be a specific homo-typic interaction, because the C-terminal domain of caveolin-1 failed to interact with full-length forms of caveolin-2 and caveolin-3 . Homo-typic interaction of the C-terminal domain with an adjacent homo-oligomer could provide a mechanism for clustering caveolin-1 homo-oligomers while excluding other caveolin family members . This type of lateral segregation event could promote caveolae membrane formation and contribute to the detergent insolubility of caveolins-1, -2, and -3. J Biol Chem, 1997 Feb 14, 272(7), 4384 - 90 Cloning and characterization of a wortmannin-sensitive human phosphatidylinositol 4-kinase; Meyers R et al.; Phosphatidylinositol (PtdIns) 4-kinases catalyze the synthesis of PtdIns-4-P, the immediate precursor of PtdIns-4,5-P2 . Here we report the cloning of a novel, ubiquitously expressed PtdIns 4-kinase (PI4Kbeta) . The 2.4-kilobase pair cDNA encodes a putative translation product of 801 amino acids which shows greatest homology to the yeast PIK1 gene . The recombinant protein exhibits lipid kinase activity when expressed in Escherichia coli, and specific antibodies recognize a 110-kDa PtdIns 4-kinase in cell lysates . The biochemical properties of PI4Kbeta are characteristic of a type III enzyme . Interestingly, both recombinant PI4Kbeta and the endogenous protein are inhibited by 150 nM wortmannin, suggesting that we have cloned the previously described PtdIns 4-kinase that is responsible for regulating the synthesis of agonist-sensitive pools of polyphosphoinositides (Nakanishi, S., Catt, J . K., and Balla, T . (1995) Proc . Natl . Acad . Sci . U . S . A . 92, 5317-5321). J Biol Chem, 1997 Feb 14, 272(7), 4281 - 6 Identification and characterization of a novel human matrix metalloproteinase with unique structural characteristics, chromosomal location, and tissue distribution; Pendas AM et al.; We have cloned a novel member of the matrix metalloproteinase (MMP) family of proteins from a human liver cDNA library . The isolated cDNA contains an open reading frame coding for a polypeptide of 508 amino acids, which has been tentatively called MMP-19 . This protein exhibits the domain structure characteristic of previously described MMPs, including a signal sequence, a prodomain with the cysteine residue essential for maintaining the latency of these enzymes, an activation locus with the zinc-binding site, and a COOH-terminal fragment with sequence similarity to hemopexin . However, it lacks a series of structural features distinctive of the diverse MMP subclasses, including the Asp, Tyr, and Gly residues located close to the zinc-binding site in collagenases, the fibronectin-like domain of gelatinases, the transmembrane domain of membrane-type (MT) MMPs, and the furin-activation sequence common to stromelysin-3 and MT-MMPs . In addition, the 9-residue insertion rich in hydrophobic amino acids present at the hinge region in stromelysins is replaced in MMP-19 by a longer insertion very rich in acidic residues . On the basis of these structural characteristics, we propose that MMP-19 does not belong to any of the previously defined MMP-subclasses and may represent the first member of a new MMP subfamily . Chromosomal location of the MMP-19 gene revealed that it maps to chromosome 12q14, which is also a unique location for any MMPs mapped to date . The cDNA encoding a full-length MMP-19 was expressed in Escherichia coli, and after purification and refolding, the recombinant protein was able to degrade synthetic substrates for MMPs . MMP-19 proteolytic activity was abolished by TIMP-2 and EDTA, thus providing additional evidence that the isolated cDNA codes for an authentic MMP . Northern blot analysis of polyadenylated RNAs isolated from a variety of human tissues revealed that MMP-19 is mainly expressed in placenta, lung, pancreas, ovary, spleen, and intestine, suggesting that it may play a specialized role in these tissues. J Biol Chem, 1997 Feb 14, 272(7), 4087 - 93 prl mutations in the Escherichia coli secG gene; Bost S et al.; SecG, an integral membrane component of the Escherichia coli preprotein translocase, contributes to the efficiency of the export process by undergoing cycles of topology inversion in the membrane, coupled with the insertion-deinsertion cycles of SecA . We have previously identified sec alleles of secG that cause a generalized secretion defect . In this study, by screening mutagenized secG libraries for suppressors of a malE signal sequence mutation, we isolated prl alleles of secG . By analogy with secY/prlA, secA/prlD, and secE/prlG, secG could therefore be called secG/prlH . The prlH mutations affect 13 codons distributed along the secG sequence, and none map to the codons affected by sec mutations . prlH suppressors suppress a variety of signal sequence mutations and they allow export of alkaline phosphatase lacking its entire signal sequence . Although secG was not identified in previous selections for prl mutants, several prlH alleles are as strong as the strongest known prlG alleles of secE . Some prlH alleles can also promote the export of alkaline phosphatase fused to predicted cytoplasmic domains of UhpT, an integral membrane protein . These results support the notion that SecG contributes to signal sequence recognition, and suggest that it may also contribute to the topology of integral membrane proteins. J Biol Chem, 1997 Feb 14, 272(7), 3879 - 82 Lysine 129 of CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase) participates in the binding of ATP to inhibit the cyclic ADP-ribose hydrolase; Tohgo A et al.; CD38 catalyzes not only the formation of cyclic ADP-ribose (cADPR) from NAD+ but also the hydrolysis of cADPR to ADP-ribose (ADPR), and ATP inhibits the hydrolysis (Takasawa, S., Tohgo, A., Noguchi, N., Koguma, T., Nata, K., Sugimoto, T., Yonekura, H., and Okamoto, H . (1993) J . Biol . Chem . 268, 26052-26054) . In the present study, using purified recombinant CD38, we showed that the cADPR hydrolase activity of CD38 was inhibited by ATP in a competitive manner with cADPR . To identify the binding site for ATP and/or cADPR, we labeled the purified CD38 with FSBA . Sequence analysis of the lysylendopeptidase-digested fragment of the labeled CD38 indicated that the FSBA-labeled residue was Lys-129 . We introduced site-directed mutations to change the Lys-129 of CD38 to Ala and to Arg . Neither mutant was labeled with FSBA nor catalyzed the hydrolysis of cADPR to ADPR . Furthermore, the mutants did not bind cADPR, whereas they still used NAD+ as a substrate to form cADPR and ADPR . These results indicate that Lys-129 of CD38 participates in cADPR binding and that ATP competes with cADPR for the binding site, resulting in the inhibition of the cADPR hydrolase activity of CD38. Science, 1997 Feb 14, 275(5302), 945 - 8 Chemical selection for catalysis in combinatorial antibody libraries; Janda KD et al.; For the past decade the immune system has been exploited as a rich source of de novo catalysts . Catalytic antibodies have been shown to have chemoselectivity, enantioselectivity, large rate accelerations, and even an ability to reroute chemical reactions . In many instances catalysts have been made for reactions for which there are no known natural or man-made enzymes . Yet, the full power of this combinatorial system can only be exploited if there was a system that allows for the direct selection of a particular function . A method that allows for the direct chemical selection for catalysis from antibody libraries was so devised, whereby the positive aspects of hybridoma technology were preserved and re-formatted in the filamentous phage system to allow direct selection of catalysis . This methodology is based on a purely chemical selection process, making it more general than biologically based selection systems because it is not limited to reaction products that perturb cellular machinery. Biochem Biophys Res Commun, 1997 Feb 13, 231(2), 452 - 6 The thioesterase I of Escherichia coli has arylesterase activity and shows stereospecificity for protease substrates; Lee YL et al.; A thioesterase I gene was recloned and sequenced from Escherichia coli strain JM109 . The overexpressed, matured enzyme from JM109 was purified to homogeneity . The enzyme showed broad hydrolytic activity toward three kinds of substrates including acyl-CoAs, esters, and amino acid derivatives . The enzyme had a kcat/Km value of 0.363 s-1 microM-1, for a typical thioesterase I substrate, palmitoyl-CoA . The arylesterase activity of the enzyme was observed by its ability to hydrolyze several aromatic esters including alpha-naphthyl acetate, alpha-naphthyl butyrate, phenyl acetate, benzyl acetate, and eight p-nitrophenyl esters . In kinetic studies a chymotrypsin-like substrate (an amino acid derivative), N-carbobenzoxy-L-phenylalanine p-nitrophenyl ester (L-NBPNPE), was the best substrate for the enzyme with a catalytic efficiency (kcat/Km) of 4.00 s-1 microM-1, which was 23 times higher than that of the enantiomer D-NBPNPE (0.171 s-1 microM-1) . It was concluded that the thioesterase I of E . coli had arylesterase activity and it possessed stereospecificity for protease substrates. Biochemistry, 1997 Feb 11, 36(6), 1450 - 60 Discrete backbone disorder in the nuclear magnetic resonance structure of apo intestinal fatty acid-binding protein: implications for the mechanism of ligand entry; Hodsdon ME et al.; The three-dimensional structure of the unliganded form of Escherichia coli-derived rat intestinal fatty acid-binding protein (I-FABP) has been determined using triple-resonance three-dimensional nuclear magnetic resonance (3D NMR) methods . Sequence-specific 1H, 13C, and 15N resonance assignments were established at pH 7.2 and 33 degrees C and used to determine the consensus 1H/13C chemical shift-derived secondary structure . Subsequently, an eight-stage iterative procedure was used to assign the 3D 13C- and 15N-resolved NOESY spectra, yielding a total of 3335 interproton distance restraints or 26 restraints/residue . The tertiary structures were calculated using a distance geometry/simulated annealing algorithm that employs pairwise Gaussian metrization to achieve improved sampling and convergence . The final ensemble of NMR structures exhibited a backbone conformation generally consistent with the beta-clam motif described for members of the lipid-binding protein family . However, unlike holo-I-FABP, the structure ensemble for apo-I-FABP exhibited variability in a discrete region of the backbone . This variability was evaluated by comparing the apo- and holoproteins with respect to their backbone 1H and 13C chemical shifts, amide 1H exchange rates, and 15N relaxation rates . Together, these results established that the structural variability represented backbone disorder in apo-I-FABP . The disorder was most pronounced in residues K29-L36 and N54-N57, encompassing the distal half of alpha-helix II, the linker between helix II and beta-strand B, and the reverse turn between beta-strands C and D . It was characterized by a destablization of long-range interactions between helix II and the C-D turn and a fraying of the C-terminal half of the helix . Unlike the solution-state NMR structure, the 1.2-A X-ray crystal structure of apo-I-FABP did not exhibit this backbone disorder . In solution, the disordered region may function as a dynamic portal that regulates the entry and exit of fatty acid . We hypothesize that fatty acid binding shifts the order-disorder equilibrium toward the ordered state and closes the portal by stabilizing a series of cooperative interactions resembling a helix capping box . This proposed mechanism has implications for the acquisition, release, and targeting of fatty acids by I-FABP within the cell. Biochemistry, 1997 Feb 11, 36(6), 1281 - 6 Monomer-dimer equilibrium of uncomplemented M15 beta-galactosidase from Escherichia coli; Gallagher CN et al.; A series of gel filtration, native polyacrylamide gel electrophoresis (PAGE) and sucrose density experiments showed that uncomplemented M15 beta-galactosidase is in a monomer-dimer equilibrium and that only under some specific conditions does the equilibrium strongly favor dimerization . The ratio of dimer to monomer increased as a function of the protein concentration, and a very good fit to a theoretical plot of the effect of protein concentration on an associating system of this type was found . The Kdiss (equili