J Biol Chem, 1995 Mar 17, 270(11), 6081 - 7 Immunological characterization and chloroplast localization of the tryptophan biosynthetic enzymes of the flowering plant Arabidopsis thaliana; Zhao J et al.; In order to study the tryptophan biosynthetic enzymes of the plant Arabidopsis thaliana, polyclonal antibodies were raised against five of the tryptophan biosynthetic pathway proteins: anthranilate synthase alpha subunit, phosphoribosylanthranilate transferase, phosphoribosylanthranilate isomerase, and the tryptophan synthase alpha and beta subunits . Immunoblot analysis of Arabidopsis leaf protein extracts revealed that the antibodies identify the corresponding proteins that are enriched in Arabidopsis chloroplast fractions . Precursors of phosphoribosylanthranilate isomerase and tryptophan synthase alpha subunit were synthesized by in vitro translation . The precursors were efficiently imported and processed by isolated spinach chloroplasts, and the cleavage sites within the precursors were determined . These results provide the first direct evidence that the tryptophan biosynthetic enzymes from Arabidopsis are synthesized as higher molecular weight precursors and then imported into chloroplasts and processed into their mature forms. J Biol Chem, 1995 Mar 17, 270(11), 5963 - 78 Inhibition of HIV-1 replication and activation of RNase L by phosphorothioate/phosphodiester 2',5'-oligoadenylate derivatives; Sobol RW et al.; 2',5'-Oligoadenylate (2-5A) derivatives have been designed to act distal to the human immunodeficiency virus-1 (HIV-1)-induced blockade in the 2-5A synthetase/RNase L antiviral pathway . Stereochemical modification of individual internucleotide linkages of the 2-5A molecule was accomplished by phosphoramidite and phosphotriester chemical syntheses . Phosphorothioate/phosphodiester trimer and tetramer 2-5A derivatives revealed differences in the stereodynamics of activation of RNase L and inhibition of HIV-1 replication . The first and second internucleotide linkages are critical for activation of recombinant, human RNase L; A(Rp)ApA, A(Sp)ApA and ApA(Rp)A are agonists (IC50 = 2 x 10(-7), 2 x 10(-6) and 8 x 10(-6) M); ApA(Sp)A is an antagonist . The second and third internucleotide linkages are crucial for activation of murine RNase L; ApA(Rp)A, ApA(Rp)ApA, and ApApA(Rp)A are agonists (IC50 = 5 x 10(-7) M); ApA(Sp)A, ApA(Sp)ApA, and ApApA(Sp)A are antagonists . Inhibition of HIV-1-induced syncytia formation by the phosphorothioate/phosphodiester derivatives is specific for derivatives with substitution at the 2',3'-terminus . ApA(Rp)A, ApA(Sp)A, ApApA(Rp)A, and ApApA(Sp)A are potent inhibitors of HIV-1-induced syncytia formation (80-, 10-, 40-, and 15-fold more inhibitory, respectively, than solvent control) . HIV-1 infection results in enhanced uptake and accumulation of ApA(Rp)A and ApA(Sp)A (7- and 10-fold, respectively) . These stereochemically modified 2-5A derivatives are taken up preferentially by HIV-1-infected cells and show promise in anti-HIV-1 chemotherapy. J Biol Chem, 1995 Mar 17, 270(11), 5818 - 22 Inorganic polyphosphate in mammalian cells and tissues; Kumble KD et al.; Inorganic polyphosphate (polyP), a linear polymer of hundreds of orthophosphate (Pi) residues linked by high-energy, phosphoanhydride bonds, has been identified and measured in a variety of mammalian cell lines and tissues by unambiguous enzyme methods . Subpicomole amounts of polyP (0.5 pmol/100 micrograms of protein) were determined by its conversion to ATP by Escherichia coli polyphosphate kinase and, alternatively, to Pi by Saccharomyces cerevisiae exopolyphosphatase . Levels of 25 to 120 microM (in terms of Pi residues), in chains 50 to 800 residues long, were found in rodent tissues (brain, heart, kidneys, liver, and lungs) and in subcellular fractions (nuclei, mitochondria, plasma membranes, and microsomes) . PolyP in brain was predominantly near 800 residues and found at similar levels pre- and postnatally . Conversion of Pi into polyP by cell lines of fibroblasts, T-cells, kidney, and adrenal cells attained levels in excess of 10 pmol per mg of cell protein per h . Synthesis of polyP from Pi in the medium bypasses intracellular Pi and ATP pools suggesting the direct involvement of membrane component(s) . In confluent PC12 (adrenal pheochromocytoma) cells, polyP turnover was virtually complete in an hour, whereas in fibroblasts there was little turnover in four hours . The ubiquity of polyP and variations in its size, location, and metabolism are indicative of a multiplicity of functions for this polymer in mammalian systems. J Biol Chem, 1995 Mar 17, 270(11), 5805 - 11 Characterization of the Elk-1 ETS DNA-binding domain; Shore P et al.; The ETS domain family of transcription factors is comprised of several important proteins that are involved in controlling key cellular events such as proliferation, differentiation, and development . One such protein, Elk-1, regulates the activity of the c-fos promoter in response to extracellular stimuli . Elk-1 is representative of a subgroup of ETS domain proteins that utilize a bipartite recognition mechanism that is mediated by both protein-DNA and protein-protein interactions . In this study, we have overexpressed, purified, and characterized the ETS DNA-binding domain of Elk-1 (Elk-93) . Elk-93 was expressed in Escherichia coli as a fusion protein with glutathione S-transferase and purified to homogeneity from both the soluble and insoluble fractions using a two-column protocol . A combination of CD, NMR, and fluorescence spectroscopy demonstrates that Elk-93 represents an independently folded domain of mixed alpha/beta structure in which the three conserved tryptophans appear to contribute to the hydrophobic core of the protein . Moreover, DNA binding studies demonstrate that Elk-93 binds DNA with both high affinity (Kd approximately 0.85 x 10(-10)M) and specificity . Circular permutation analysis indicates that DNA binding by Elk-93 does not induce significant bending of the DNA . Our results are discussed with respect to predictive models for the structure of the ETS DNA-binding domain. J Biol Chem, 1995 Mar 17, 270(11), 5764 - 71 Protein synthesis initiation factor eIF-1A is a moderately abundant RNA-binding protein; Wei CL et al.; Eukaryotic initiation factor (eIF) 1A (formerly called eIF-4C) is a small protein that promotes dissociation of 80 S ribosomes into subunits, stabilizes methionyl-tRNA binding to 40 S ribosomal subunits, and is required for the binding of mRNA to ribosomes . The sequence of eIF-1A derived from its cloned cDNA possesses a high frequency of basic residues and acidic residues at its N and C termini, respectively . Northwestern blotting with a fragment of mRNA indicates that eIF-1A binds RNA . Overexpression of the human eIF-1A cDNA in Escherichia coli and subsequent purification enabled us to prepare large quantities of active factor . The level of eIF-1A in HeLa cells determined by Western immunoblotting is 0.01% of total protein, which corresponds to 0.2 molecules of eIF-1A/ribosome . The moderate abundance means that eIF-1A is equal to or in excess of native 40 S subunits and suggests that the factor may not be limiting for protein synthesis, a conclusion reinforced by the failure of overproduced eIF-1A to stimulate translation rates in transiently transfected COS-1 cells . S1 nuclease protection and primer extension analyses show that eIF-1A mRNA possesses an unusually long 5'-untranslated leader that is very G/C-rich (72%) . Unexpectedly, the mRNA is efficiently translated in HeLa cells as judged by polysome profile analyses. J Biol Chem, 1995 Mar 17, 270(11), 5748 - 55 The purification, cloning, and high level expression of a glutaredoxin-like protein from the hyperthermophilic archaeon Pyrococcus furiosus; Guagliardi A et al.; A protein has been purified to homogeneity from crude extracts of the hyperthermophilic archaeon Pyrococcus furiosus based on its ability to catalyze the reduction of insulin disulfides in the presence of dithiothreitol; the protein has a molecular mass of 24.8 kDa and a pI of 4.9, and it is highly heat-stable . The first 29 amino acid residues at the N terminus of the P . furiosus protein were determined by Edman degradation, and its gene was cloned in Escherichia coli . The amino acid sequence derived from the DNA sequence contains the CPYC sequence, which is typical of the active site of glutaredoxin (also called thioltransferase) . The C-terminal portion of the P . furiosus protein, containing the conserved sequence, shows sequence similarity with glutaredoxins from different sources . The P . furiosus protein can reduce disulfide bonds in L-cystine in the presence of GSH (the thioltransferase activity) with an optimum pH of 8.0 . The expression of the P . furiosus protein, with full activity, in E . coli at a very high level (21% of total soluble protein) is described; the recombinant protein was purified to homogeneity by merely two successive heat treatments and gel filtration chromatography . The features of the P . furiosus protein here described are discussed in light of the current knowledge about the ubiquitous family of protein disulfide oxidoreductases. J Med Chem, 1995 Mar 17, 38(6), 967 - 72 Quantitative structure-activity relationships of the inhibition of Pneumocystis carinii dihydrofolate reductase by 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(X-phenyl)-s-triazines; Marlowe CK et al.; The inhibitory activities of 60 4,6-diamino-1,2-dihydro-2,2-dimethyl-1- (X-phenyl)-s-triazines versus purified, recombinant Pneumocystis carinii (Pc) dihydrofolate reductase (DHFR) have been determined at pH 7.0 . Utilization of these Kiapp values has led to the formulation of appropriate quantitative structure-activity relationships (QSAR's) for both meta- and parasubstituted derivatives . The QSAR's from Pc are compared with other triazine QSAR's derived versus chicken, murine tumor, Escherichia coli, and particularly human DHFR . Selectivity indices indicate that hydrophobic triazines are particularly effective versus Pc DHFR; they have lower Ki values for Pc DHFR than for human DHFR. J Med Chem, 1995 Mar 17, 38(6), 869 - 74 1-Thiomethoxsalen and 1-thiopsoralen: synthesis, photobiological properties, and site specific reaction of thiopsoralens with DNA; Jakobs AE et al.; The sulfur analogues of psoralen and 8-methoxypsoralen (8-MOP) in the pyrone moiety were synthesized and compared to the parent compounds in terms of photoreactivity with viral M13mp19 RF DNA . The damaged viral DNA was transfected into Escherichia coli and scored for infectivity toward Ca-treated wild-type E . coli . This allowed a comparative study of the sulfur and oxygen analogues to be made in terms of photoreactivity . Furthermore, the DNA sequence specificity for the formation of monoadducts and cross-links of the four analogues was determined with 32P-labeled oligonucleotides containing thymidine in different sequences . The most site specific of the studied psoralens is 8-MOP, while 1-thiopsoralen is the most reactive analogue . This new thio analogue of psoralen leads to the efficient formation of monoadducts and cross-links in any pyrimidine-purine site. Biochem Biophys Res Commun, 1995 Mar 17, 208(2), 714 - 20 Cloning, expression and purification of full length Rep78 of adeno-associated virus as a fusion protein with maltose binding protein in Escherichia coli; Batchu RB et al.; The adeno-associated virus (AAV) Rep78 protein is required for many aspects of AAV's life cycle including its DNA replication and the regulation of its gene expression . Because of increasing utilization of AAV as a gene therapy vector and its possible use as an anti-cancer/anti-viral agent, the complete characterization of its Rep78 regulatory protein is important . In order to study various functional aspects of Rep78, we have cloned and expressed the Rep78 gene in Escherichia coli using an inducible expression plasmid . The entire Rep78 open reading frame (nt 321 to 2185) was cloned into the LacZ inducible expression vector pMALc2 . Upon induction of the Ptac promoter with isopropyl thio-beta-D-galactopyranoside (IPTG), Rep78 is produced as a fusion protein with maltose binding protein (MBP) . This recombinant MBP-Rep78 protein displayed all the biochemical activities which are described for the wild type protein including binding to the AAV terminal repeats (TR), endonuclease activity, and helicase activity . Furthermore, for the first time, ATP binding by Rep78 is demonstrated. J Biol Chem, 1995 Mar 17, 270(11), 6298 - 307 Self-coded 3'-extension of run-off transcripts produces aberrant products during in vitro transcription with T7 RNA polymerase; Triana-Alonso FJ et al.; More than 70% of the RNA synthesized by T7 RNA polymerase during run-off transcription in vitro can be incorrect products, up to twice as long as the expected transcripts . Transcriptions with model templates indicate that false transcription is mainly observed when the correct product cannot form stable secondary structures at the 3'-end . Therefore, the following hypothesis is tested: after leaving the DNA template, the polymerase can bind a transcript to the template site and the 3'-end of the transcript to the product site and extend it, if the 3'-end is not part of a stable secondary structure . Indeed, incubation of purified transcripts with the polymerase in transcription conditions triggers a 3'-end prolongation of the RNA . When two RNAs of different lengths are added to the transcription mix, both generate distinct and specific patterns of prolonged RNA products without any interference, demonstrating the self-coding nature of the prolongation process . Furthermore, sequencing of the high molecular weight transcripts demonstrates that their 5'-ends are precisely defined in sequence, whereas the 3'-ends contain size-variable extensions which show complementarity to the correct transcript . Surprisingly, a reduction of the UTP concentration to 0.2-1.0 mM in the presence of 3.5-4.0 mM of the other NTPs leads to faithful transcription and good yields, irrespective of the nucleotide composition of the template. Eur J Pharmacol, 1995 Mar 16, 292(3-4), 257 - 63 Mechanisms of interleukin-2-induced depression of hepatic cytochrome P-450 in mice; Cantoni L et al.; Interleukin-2 (15 micrograms/mouse, i.p . twice daily for 4 days and once on the 5th day) significantly lowered cytochrome P-450 and heme content and increased heme oxygenase mRNA accumulation; the activities of 7-ethoxycoumarin O-deethylase, ethoxy- and pentoxyphenoxazone O-dealkylases were decreased . The activity of the type O form of hepatic xanthine oxidase increased, but there was no increase in lipid peroxide, expressed in terms of microsomal malondialdehyde . In vivo inactivation of xanthine oxidase activity by feeding mice with tungstate did not substantially change the degree of interleukin-2-induced cytochrome P-450 depression, suggesting that the two processes are not causally linked . Induction of tolerance to endotoxin by a 4-day pretreatment with lipopolysaccharide resulted in 50% protection against this depression despite inhibition of the interleukin-2 induced formation of tumor necrosis factor . This suggests that the release of tumor necrosis factor per se does not fully account for the depression of cytochrome P-450 . Dexamethasone, already used in patients to reduce the toxicity of interleukin-2 therapy, provided full protection against the cytochrome P-450 depression. Nature, 1995 Mar 16, 374(6519), 287 - 90 Site-specific RNase E cleavage of oligonucleotides and inhibition by stem-loops; McDowall KJ et al.; The enzyme RNase E (ref . 1) cuts RNA at specific sites within single-stranded segments . The role of adjacent regions of secondary structure in such cleavages is controversial . Here we report that 10-13-nucleotide oligomers lacking any stem-loop but containing the RNase E-cleaved sequence of RNA I, the antisense repressor of replication of ColE1-type plasmids, are cut at the same phosphodiester bond as, and 20 times more efficiently than, RNA I . These findings indicate that, contrary to previous proposals, stem-loops do not serve as entry sites for RNase E, but instead limit cleavage at potentially susceptible sites . Cleavage was reduced further by mutations in a non-adjacent stem-loop, suggesting that distant conformational changes can also affect enzyme access . Modulation of RNase E cleavages by stem-loop regions and to a lesser extent by higher-order structure may explain why this enzyme, which does not have stringent sequence specificity, cleaves complex RNAs at a limited number of sites. Structure, 1995 Mar 15, 3(3), 239 - 43 Thioredoxin structure and mechanism: conformational changes on oxidation of the active-site sulfhydryls to a disulfide; Holmgren A; The recent high-resolution solution structures of human and Escherichia coli thioredoxin in their oxidized and reduced states support a catalytic model of protein disulfide reduction involving binding of a target protein and nucleophilic attack by the active-site Cys32 thiolate to form a transition state mixed disulfide. FEMS Microbiol Lett, 1995 Mar 15, 127(1-2), 35 - 9 Use of conjugative and thermosensitive cloning vectors for transposon delivery to Mycobacterium smegmatis; Gavigan JA et al.; Conjugative, thermosensitive shuttle plasmids capable of transfer from Escherichia coli to Mycobacterium smegmatis were constructed . They contain both an E . coli replicon and a thermosensitive derivative of the pAL5000 mycobacterial replicon . Using a temperature shift protocol, the conjugative plasmid, pJAZ11 was used to deliver the transposon Tn611 from E . coli into the chromosome of M . smegmatis . Analysis of transconjugants revealed the random insertion of the transposon. FEMS Microbiol Lett, 1995 Mar 15, 127(1-2), 133 - 8 Characterization of eukaryotic-like kinase activity in Escherichia coli using the gene-protein database; Sweeney ST et al.; The gene-protein database was used to obtain the two-dimensional polyacrylamide gel coordinates of proteins phosphorylated in extracts of Escherichia coli including those phosphorylated by eukaryotic-like kinase activities . These suggest that the phosphoproteins correspond to, or co-migrate with, the product of an open reading frame at 1.3 min (Orf80), Enzyme 1 of the phosphoenolpyruvate-dependent phosphotransferase system (PtsI), the tRNA synthetase for histidine (HisS), and proteins involved in the response to carbon starvation and quinone treatment. Eur J Biochem, 1995 Mar 15, 228(3), 863 - 9 Expression and characterization of Geotrichum candidum lipase I gene . Comparison of specificity profile with lipase II; Bertolini MC et al.; Despite tremendous progress in the elucidation of three-dimensional structures of lipases, the molecular basis for their observed substrate preference is not well understood . In an effort to correlate the lipase structure with its substrate preference and to clarify the contradicting reports in the literature, we have compared the enzymic characteristics of two closely related recombinant lipases from the fungus Geotrichum candidum . These enzymes were expressed in the yeast Saccharomyces cerevisiae as fusions with an N-terminal poly(His) tag and were purified in a single step by metal-affinity chromatography . Their specific activities against a series of triacylglycerol substrates were compared using a titrimetric assay . The substrates varied in fatty acyl chain length, number of double bonds and their position along the chain . G . candidum lipases I and II (GCL I and GLC II) are markedly different with respect to their substrate preferences . For unsaturated substrates having long fatty acyl chains (C18:2 cis-9, cis-12 and C18:3 cis-9, cis-12, cis-15), GCL I showed higher specific activity than GCL II, whereas GCL II showed higher specific activity against saturated substrates having short fatty acid chains (C8, C10, C12 and C14) . We have constructed a hybrid molecule containing the N-terminal portion of GCL I (including the flap covering the active site) linked to the C-terminal portion of GCL II . The hybrid molecule showed a substrate preference pattern identical to that of GCL II . These results indicate that sequence variation within the N-terminal 194 amino acids of G . candidum lipases do not contribute to the observed variation in efficiency by which the lipases hydrolyze their substrates . Moreover, it also shows that the flap region in GCL is not directly involved in substrate differentiation, even though this region is thought to be involved in recognition of the interface and in the activation of the enzyme. Eur J Biochem, 1995 Mar 15, 228(3), 745 - 52 Site-directed mutagenesis of the redox-active cysteines of Trypanosoma cruzi trypanothione reductase; Borges A et al.; The gene for trypanothione reductase from the Silvio strain of Trypanosoma cruzi has been cloned, sequenced and overexpressed in Escherichia coli using the constitutive lpp promoter on the expression plasmid pBSTNAV . Up to 13% of the total soluble protein is enzymically active trypanothione reductase with kinetic properties similar to the enzyme purified from T . cruzi . In order to assess the catalytic role of the putative active-site cysteine residues (C53 and C58), three mutant proteins have been constructed by site-directed mutagenesis substituting alanine or serine residues for cysteine; {C53A}trypanothione reductase, {C53S}trypanothione reductase and {C58S}trypanothione reductase . Although the purified, recombinant mutant proteins were catalytically inactive with NADPH and trypanothione disulphide as substrates, all showed comparable levels of transhydrogenase activity between NADPH and thio-NADP+, suggesting that the mutant proteins had correctly folded in vivo . All three mutants showed substantially different catalytic parameters for thio-NADP+ than the wild-type enzyme, presumably as a consequence of modifying the environment of the enzyme-bound flavin, thereby altering its chemical reactivity . The purified {C58S}trypanothione reductase showed spectral properties similar to the oxidised wild-type enzyme but, unlike the wild-type enzyme, did not acquire the characteristic charge-transfer complex of the EH2 form on addition of NADPH . In contrast, in the absence of NADPH both {C53A}trypanothione reductase and {C53S}trypanothione reductase showed spectral properties similar to the EH2 form of the wild-type enzyme . These data indicate that both C53 and C58 are essential for overall catalysis, with the thiolate anion of C58 interacting with the enzyme-bound FAD and C53 interacting with the disulphide substrate . These mutants should be useful in crystallographic studies of reaction intermediates which cannot be obtained with the catalytically active native enzyme. Eur J Biochem, 1995 Mar 15, 228(3), 739 - 44 The gag homologue of retrotransposon Ty1 assembles into spherical particles in Escherichia coli; Luschnig C et al.; Expression of TyA (reading frame A) of the yeast retrotransposon Ty1 in Escherichia coli is possible by using efficient transcriptional and translational initiation signals . When expressed in E . coli, the gag homologue of Ty1 assembles into spherical particles similar, but not identical to virus-like particles in the natural host of Ty1, Saccharomyces cerevisiae . Deletion analysis reveals a domain in the C-terminus of TyA that is essential for the assembly process . These findings indicate that an early step of the retroelement life cycle, assembly of the gag homologue into spherical particles, does not depend on specific host factors . The experiments also demonstrate that Ty1 Gag fusion proteins, potential tools for immunization, can be produced in E . coli, an organism that lacks endogenous retrotransposons. Eur J Biochem, 1995 Mar 15, 228(3), 531 - 50 Signal recognition particle (SRP), a ubiquitous initiator of protein translocation; Lutcke H; In higher eukaryotes, most secretory and membrane proteins are synthesised by ribosomes which are attached to the membrane of the rough endoplasmic reticulum (RER) . This allows the proteins to be translocated across that membrane already during their synthesis . The ribosomes are directed to the RER membrane by a cytoplasmic ribonucleoprotein particle, the signal recognition particle (SRP) . SRP fulfills its task by virtue of three distinguishable activities: the binding of a signal sequence which, being part of the nascent polypeptide to be translocated, is exposed on the surface of a translating ribosome; the retardation of any further elongation; and the SRP-receptor-mediated binding of the complex of ribosome, nascent polypeptide and SRP to the RER membrane which results in the detachment of SRP from the signal sequence and the ribosome and the insertion of the nascent polypeptide into the membrane . Evidence is accumulating that SRP is not restricted to eukaryotes: SRP-related particles and SRP-receptor-related molecules are found ubiquitously and may function in protein translocation in every living organism . This review focuses on the mammalian SRP . A brief discussion of its overall structure is followed by a detailed description of the structures of its RNA and protein constituents and the requirements for their assembly into the particle . Homologues of SRP components from organisms other than mammals are mentioned to emphasize the components' conserved or less conserved features . Subsequently, the functions of each of the SRP constituents are discussed . This sets the stage for a presentation of a model for the mechanism by which SRP cyclically assembles and disassembles with translating ribosomes and the RER membrane . It may be expected that similar mechanisms are used by SRP homologues in organisms other than mammals . However, the mammalian SRP-mediated translocation mechanism may not be conserved in its entirety in organisms like Escherichia coli whose SRP lack components required for the function of the mammalian SRP . Possible translocation pathways involving the rudimentary SRP are discussed in view of the existence of alternative, chaperone-mediated translocation pathways with which they may intersect . The concluding two sections deal with open questions in two areas of SRP research . One formulates basic questions regarding the little-investigated biogenesis of SRP . The other gives an outlook over the insights into the mechanisms of each of the known activities of the SRP that are to be expected in the short and medium-term future. Biochem J, 1995 Mar 15, 306 ( Pt 3), 865 - 9 Free calcium transients in chemotactic and non-chemotactic strains of Escherichia coli determined by using recombinant aequorin; Watkins NJ et al.; Intracellular Ca2+ has been previously implicated in the chemotactic response of Escherichia coli . However, no correlative measurements of intracellular free Ca2+ have been made during bacterial chemotaxis, essential if this is to be established . In order to monitor internal free Ca2+ in E . coli during challenge with chemotactic agents, the Ca(2+)-activated photoprotein aequorin was expressed in a chemotactic strain (AB1157) and a non-chemotactic strain {BL21(DE3)} of E . coli . Repellents were found to cause an increase (50-150 nM) in intracellular free Ca2+, whereas attractants caused a small but consistent decrease in intracellular free Ca2+ . These data are in agreement with the proposed model that an increase in intracellular free Ca2+ causes tumbling . The effect of increasing external Ca2+ on the regulation of intracellular free Ca2+ in both strains was monitored by using aequorin . The resting level of free Ca2+ in E . coli (AB1157) was found to be 100 nM, which agrees with previous data {Gangola and Rosen (1987) J . Biol . Chem . 262, 12570-12574} . As these results also show differences in the regulation of intracellular free Ca2+ between the two strains in the presence of high external Ca2+ concentrations, this may have implications for the effect of high-Ca2+ environments on E . coli. Biochem J, 1995 Mar 15, 306 ( Pt 3), 627 - 30 Formation in vitro of the 3,4,6-trihydroxyphenylalanine quinone cofactor; Hanlon SP et al.; An Escherichia coli K-12 2-phenylethylamine oxidase gene with a mutated leader sequence region produced a largely inactive form of the enzyme in the cytoplasm . This form of the enzyme was activated 30-50-fold on incubation at 30 degrees C in the absence of any added cofactors . After activation the enzyme contained a quinone which was not detected in the non-activated form . This is the first report of the formation in vitro of any quinoenzyme cofactor. Biochim Biophys Acta, 1995 Mar 15, 1247(2), 284 - 92 NMR studies of binding of 5-FdUDP and dCDP to ribonucleoside-diphosphate reductase from Escherichia coli; Roy B et al.; 5-Fluoro-2'-deoxyuridine-5'-diphosphate (5-FdUDP) has been synthesised using an original route, previously applied to the synthesis of natural nucleoside diphosphates . The interaction between 5-FdUDP and the enzyme ribonucleoside-diphosphate reductase (EC 1.17.4.1) has been studied with 19F-NMR . The product analogue is shown to be in fast exchange with substrate binding sites on protein subunit 1 (R1) of ribonucleoside-diphosphate (NDP) reductase . The number of binding sites is reduced to half when the complete holoenzyme R1R2 is formed . The temperature dependence of the line broadening of 5-FdUDP was studied using 19F-NMR, and of dCDP and dUDP using 1H-NMR . The temperature dependences are complex and a molecular model in which R1 is in a temperature dependent equilibrium between at least two conformations is suggested in order to explain the observed behaviour . Binding of a ligand to the substrate binding sites affects the conformational equilibrium in a ligand specific way . Formation of the holoenzyme R1R2 also affects the equilibrium. Biochim Biophys Acta, 1995 Mar 15, 1247(2), 265 - 71 Purification and characterization of the pyridoxol-5'-phosphate:oxygen oxidoreductase (deaminating) from Escherichia coli; Notheis C et al.; The E . coli gene pdxH encoding pyridoxol-5'-phosphate:oxygen oxidoreductase (deaminating) (EC 1.4.3.5, PdxH) was cloned, located to phage 20B5 of the library of Kohara et al . (Kohara, Y, Akiyama, K . and Isono K . (1987) Cell 50, 495-508) and assigned to a stretch between 36.0 and 36.1 min of the E . coli chromosome . The gene was overexpressed as a MBP/PdxH fusion protein . The fusion protein was purified by affinity chromatography on an amylose resin and hydrolyzed in the presence of protease 'factor Xa' resulting in homogeneous PdxH protein after another column chromatography . Both the MBP/PdxH fusion protein and the PdxH protein were characterized . Both enzymes are FMN-dependent enzymes which oxidize pyridoxol phosphate and pyridoxamine phosphate in the presence of oxygen to pyridoxal phosphate . Km values of both proteins were similar ranging from 350 to 400 microM for the two substrates . The enzymes did not accept non-phosphorylated substrates . Kinetic data indicate that the enzyme (MBP/PdxH) is product inhibited (Ki 8 microM) by pyridoxal phosphate as a mixed type inhibitor . As revealed by gel exclusion chromatography a minor fraction of the fusion protein formed a dimer, whereas the bulk amount of protein was a monomer . No indication was found that the PdxH protein forms a dimer . The monomer was shown to be catalytically active. Biochim Biophys Acta, 1995 Mar 15, 1247(2), 253 - 9 Avian cytosolic 3-hydroxy-3-methylglutaryl-CoA synthase: evaluation of the role of cysteines in reaction chemistry; Misra I et al.; The pH dependence of avian cytosolic HMG-CoA synthase activity is fit by a titration curve with a pK = 8.6 . The observation of optimal activity at alkaline pH and the insensitivity of pK to divalent cation concentration suggest that the pK reflects ionization of an amino-acid side chain (e.g., cysteinyl sulfhydryl) rather than substrate enolization . Upon reaction of 3-chloropropionyl-CoA with HMG-CoA synthase C129S, an enzyme variant lacking the sulfhydryl group normally targeted by this mechanism-based inhibitor, stoichiometric modification occurs . Amino-acid analysis indicates that cysteine is the principal target in C129S enzyme, demonstrating the presence of a second reactive cysteine within this enzyme . To test whether another cysteine functions in reaction chemistry, conserved cysteines were identified by sequence homology analysis . Five cysteine residues (C59, C69, C224, C232, C268), invariant in the nine sequences available for various eukaryotic HMG-CoA synthase isozymes, were individually replaced by alanine in a series of mutant enzymes . Kinetic analyses of the isolated mutant HMG-CoA synthases indicate that none of these is crucial to the chemistry that results in production of HMG-CoA . These results further distinguish the HMG-CoA synthase reaction from the related condensation of acyl-CoA substrates catalyzed by beta-ketothiolase. Biochim Biophys Acta, 1995 Mar 15, 1247(2), 179 - 84 Refolding of alpha 1-antitrypsin expressed as inclusion bodies in Escherichia coli: characterization of aggregation; Kwon KS et al.; Recombinant alpha 1-antitrypsin (alpha 1AT) produced as inclusion bodies in Escherichia coli was purified via several steps including solubilization of the inclusion bodies in 8 M urea and refolding by direct dilution of denaturant, followed by ion-exchange chromatography . The purified recombinant alpha 1AT has an activity comparable to human plasma alpha 1AT . During refolding, prolonged incubation of the alpha 1AT polypeptides at intermediate urea concentration favored production of inactive but soluble aggregates, which could regain activity after denaturation and renaturation . Nondenaturing polyacrylamide gel electrophoresis of the aggregates revealed the existence of dimers and higher oligomers . Immunological approach to characterize conformation showed that the oligomers were distinct from the native, the cleaved, or the denatured form, but was similar to the polymers induced from the native structure in mild denaturing condition . These results suggest that the oligomers are formed through specific interactions between aggregation-competent species which are stabilized at intermediate denaturant concentration. Biochim Biophys Acta, 1995 Mar 14, 1261(1), 35 - 43 Expression and mutagenesis of recombinant human and murine erythropoietins in Escherichia coli; Bill RM et al.; Expression of the polypeptide hormone erythropoietin (EPO) in Escherichia coli by four bacterial expression vectors was examined . Complementary DNAs encoding human and murine EPO were amplified by polymerase chain reaction (PCR) and cloned into the glutathione-S-transferase (GST) fusion vector, pGEX-2T . Human EPO DNA was also cloned into the vectors, pET14b, pIN III-Omp A2 and pT7/7 . Expression of human and murine EPO was obtained using constructs based on pGEX-2T . For constructs based on the other vectors, expression of EPO was absent or occurred at low levels, despite attempts to optimise conditions . Human and murine EPO, expressed as fusion proteins with GST, were partially soluble and displayed EPO bioactivity . Soluble GST-EPO fusion proteins were affinity purified on immobilised glutathione . Insoluble protein could also be purified by elution from gel slices following SDS-PAGE to yield either fusion protein or, after treatment with thrombin, unmodified EPO which was both soluble and bioactive . The pGEX expression system was evaluated as a means of analysing the structure-function relationships of EPO by in vitro mutagenesis . Three human and three murine EPO mutants were constructed and expressed as GST fusion proteins . Following purification, biological activity was evaluated using assays for bioactivity, immunoactivity and GST activity . The pGEX expression system complements eukaryotic systems described previously for expression of EPO and should provide much useful information about the structure-function relationships of the hormone. Proc Natl Acad Sci U S A, 1995 Mar 14, 92(6), 2333 - 7 HypB protein of Bradyrhizobium japonicum is a metal-binding GTPase capable of binding 18 divalent nickel ions per dimer; Fu C et al.; Bradyrhizobium japonicum hypB encodes a protein containing an extremely histidine-rich region (24 histidine residues within a 39-amino-acid stretch) and guanine nucleotide-binding domains . The product of the hypB gene was overexpressed in Escherichia coli and purified by Ni(2+)-charged metal chelate affinity chromatography (MCAC) in a single step . In SDS/PAGE, HypB migrated at 38 kDa--slightly larger than the calculated molecular mass (32.8 kDa) . Purified HypB has GTPase activity with a kcat of 0.18 min-1 and a Km for GTP of 7 microM, and it has dGTPase activity as well . HypB exists as a dimer of molecular mass 78 kDa in native solution as determined by fast protein liquid chromatography on Superose 12 . It binds 9.0 +/- 0.14 divalent nickel ions per monomer (18 Ni2+ per dimer) with a Kd of 2.3 microM; it also binds Zn2+, Cu2+, Co2+, Cd2+, and Mn2+ . In-frame deletion of the histidine-rich region (deletion of 38 amino acids including 23 histidine residues) resulted in a truncated HypB that did not bind to the MCAC column, whereas in-frame deletion of 14 amino acids including 8 histidine residues within HypB resulted in a truncated HypB that still bound to the column . The results indicate that the histidine residues within the histidine-rich region of HypB are involved in metal binding. Proc Natl Acad Sci U S A, 1995 Mar 14, 92(6), 2328 - 32 Cloning, expression, and characterization of cDNAs encoding Arabidopsis thaliana squalene synthase; Nakashima T et al.; We have isolated and characterized two overlapping cDNA clones for Arabidopsis thaliana squalene synthase . Their nucleotide sequences contained an open reading frame for a 410-amino acid polypeptide (calculated molecular mass, 47 kDa) . The deduced amino acid sequence of the Arabidopsis polypeptide was significantly homologous (42-44% identical) to the sequences of known squalene synthases of several species, from yeast to man, but it was much less homologous to that of tomato phytoene synthase . To express the Arabidopsis enzyme in Escherichia coli, the entire coding region was subcloned into an expression vector . A cell-free extract of E . coli transformed with the recombinant plasmid, in the presence of NADPH and Mg2+, efficiently converted {14C}farnesyl diphosphate into squalene . On the other hand, in the absence of NADPH and the presence of Mn2+, the cell-free extract formed dehydrosqualene as a secondary product . Another E . coli extract expressing mouse squalene synthase showed the same activity as the Arabidopsis enzyme . Therefore, both the structure and reaction mechanism of squalene synthases are markedly conserved in taxonomically remote eukaryotes. Proc Natl Acad Sci U S A, 1995 Mar 14, 92(6), 2298 - 302 Nuclear foci of mammalian Rad51 recombination protein in somatic cells after DNA damage and its localization in synaptonemal complexes; Haaf T et al.; Rad51 protein of Saccharomyces cerevisiae is a structural homolog of the Escherichia coli recombination enzyme RecA . In yeast, the Rad51 protein is required for mitotic and meiotic recombination and for repair of double-strand breaks in DNA . We have used antibodies raised against the homologous human protein, HsRad51, expressed in E . coli, to visualize the spatial distribution of the protein in mammalian somatic and meiotic cells . In cultured human cells, the HsRad51 protein is concentrated in multiple discrete foci in the nucleoplasm; it is largely absent from cytoplasm and nucleoli . After treatment of cells with methyl methanesulfonate, ultraviolet irradiation, or 137Cs irradiation, the percentage of cells with HsRad51 protein immunofluorescence increases; the same cells show unscheduled DNA synthesis . Induction of Rad51 foci is blocked by inhibitors of transcription . In mouse pachytene spermatocytes, the mouse homolog of Rad51 protein is highly enriched in synaptonemal complexes that are formed between the paired homologous chromosomes during meiotic prophase . We conclude that the mammalian proteins homologous to yeast Rad51 are involved in repair of DNA damage and recombinational repair during meiosis. Proc Natl Acad Sci U S A, 1995 Mar 14, 92(6), 2229 - 33 A region in the cytosolic domain of the epidermal growth factor receptor antithetically regulates the stimulatory and inhibitory guanine nucleotide-binding regulatory proteins of adenylyl cyclase; Sun H et al.; Epidermal growth factor (EGF) stimulates adenylyl cyclase in the heart via activation of the stimulatory GTP-binding protein Gs . Therefore, employing peptides corresponding to regions in the cytosolic domain of the EGF receptor, we have investigated the ability of sequences within the EGF receptor to activate Gs . A 13-aa peptide (EGFR-13) corresponding to the juxtamembrane region in the cytosolic domain of the EGF receptor stimulated GTP binding and GTPase activity of Gs . This peptide did not stimulate GTP binding to Gi but increased the GTPase activity of this protein . Additionally, phosphorylation of the protein kinase C site (threonine residue) within EGFR-13 decreased the ability of the peptide to stimulate Gs and increase GTPase activity of Gi . Further, in functional assays of Gs employing S49 cyc- cell membranes, EGFR-13 increased the ability of Gs to stimulate adenylyl cyclase; phospho-EGFR-13 and a 14-aa peptide corresponding to a sequence in the cytosolic domain of the EGF receptor did not alter the functional activity of Gs . Hence, the juxtamembrane region of the EGF receptor can activate Gs and, by stimulating GTPase activity of Gi, inactivates this latter G protein . Phosphorylation of the threonine residue within this region attenuates the activity of the peptide as a modulator of G-protein function. Proc Natl Acad Sci U S A, 1995 Mar 14, 92(6), 2204 - 8 Site-specific rates of excision repair of benzo{a}pyrene diol epoxide adducts in the hypoxanthine phosphoribosyltransferase gene of human fibroblasts: correlation with mutation spectra; Wei D et al.; When populations of repair-proficient diploid human fibroblasts were treated with (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo{a}pyrene (BPDE) during early S phase, just as the hypoxanthine phosphoribosyltransferase gene (HPRT) was being replicated, 5% of the induced base substitutions were found at nt 212, and 5% of the substitutions were found at nt 229 in exon 3 . However, when the population was treated in early G1 phase to allow at least 12 hr for repair before the onset of S phase, 21% of the substitutions were found at nt 212, and 10% were found at nt 229 . No such cell-cycle-dependent difference in distribution of base substitutions occurred in excision-repair-deficient cells . To test whether the increase in the relative frequency of mutations resulted from inefficient repair at these sites, we adapted ligation-mediated PCR to measure the rates of removal of BPDE adducts from individual sites in exon 3 of the HPRT gene . Cells were treated with 0.5 microM BPDE in early G1 phase and harvested immediately or after 10, 20, and 30 hr for repair . the nontranscribed strand of exon 3 was analyzed for the original distribution of adducts and those remaining after repair, using Escherichia coli UvrABC excinuclease to excise the adducts and annealing a 5' biotinylated gene-specific primer to the DNA and extending it with Sequenase 2.0 to generate a blunt end at the site of each cut . A linker was ligated to the blunt end, and the desired fragments were isolated from the rest of the genomic DNA by using magnetic beads, amplified by PCR, and analyzed on a sequencing gel . The distribution of fragments of particular lengths indicated the relative number of BPDE adducts initially formed or remaining at specific sites . The rates of repair at individual sites varied widely along exon 3 of the HPRT gene and were very slow at nt 212 and 229, strongly supporting the hypothesis that inefficient DNA repair plays an important role in the formation of mutation hotspots. Proc Natl Acad Sci U S A, 1995 Mar 14, 92(6), 2136 - 40 Partial rescue of human carbonic anhydrase II frameshift mutation by ribosomal frameshift; Hu PY et al.; A single-base-pair deletion in exon 7 of the human carbonic anhydrase II gene was found to be the molecular defect in a group of independently ascertained, clinically heterogeneous, Hispanic carbonic anhydrase II-deficient patients, all of whom had ancestors from the Caribbean islands . This mutation predicts a +1 frameshift at codon 227 and incorporation of 12 missense amino acids before an early stop codon at position 239 produces a 27-kDa truncated carbonic anhydrase II . Expression of the Hispanic mutant cDNA in bacteria produced predominantly the 27-kDa protein, which was inactive . However, a minor 29-kDa polypeptide species was also produced that had 10% the specific activity of the wild-type enzyme after affinity purification . Amino acid sequencing showed that the 29-kDa mutant protein was produced by two frameshift events: a +1 frameshift at codon 227 due to the single-base deletion and a -1 ribosomal frameshift at codon 237 that restored the original reading frame after 11 missense amino acids were incorporated . Antibody against the 11-amino acid frameshift peptide detected the 29-kDa mutant protein in lysates of transfected COS cells . These results indicate that ribosomal frameshift can partially rescue the human carbonic anhydrase II frameshift mutation and suggest a mechanism whereby a compensatory ribosomal frameshift can ameliorate the consequences of certain frameshift mutations . Whether individual differences in efficiency of ribosomal frameshift contribute to clinical heterogeneity in patients with such mutations deserves further study. Proc Natl Acad Sci U S A, 1995 Mar 14, 92(6), 2111 - 5 A method for probing the topography and interactions of proteins: footprinting of myoglobin; Zhong M et al.; We describe a procedure for mapping residues on the surface of a protein molecule to its sequence, using a scheme that is analogous to nucleic acid footprinting . The protein is end labeled radioactively and subjected to limited proteolysis, and the products are analyzed by denaturing polyacrylamide gel electrophoresis and autoradiography . The method is tested with the heme protein myoglobin and applied to mapping the (unknown) surface of the molecule lacking the heme group: apomyoglobin . Sites of protein-protein interaction can be identified, as illustrated by footprinting the association between myoglobin and an anti-myoglobin monoclonal antibody. Proc Natl Acad Sci U S A, 1995 Mar 14, 92(6), 1950 - 4 Restoration of mismatch repair to nuclear extracts of H6 colorectal tumor cells by a heterodimer of human MutL homologs; Li GM et al.; Hypermutable H6 colorectal tumor cells are defective in strand-specific mismatch repair and bear defects in both alleles of the hMLH1 gene . We have purified to near homogeneity an activity from HeLa cells that complements H6 nuclear extracts to restore repair proficiency on a set of heteroduplex DNAs representing the eight base-base mismatches as well as a number of slipped-strand, insertion/deletion mispairs . This activity behaves as a single species during fractionation and copurifies with proteins of 85 and 110 kDa . Microsequence analysis demonstrated both of these proteins to be homologs of bacterial MutL, with the former corresponding to the hMLH1 product and the latter to the product of hPMS2 or a closely related gene . The 1:1 molar stoichiometry of the two polypeptides and their hydrodynamic behavior indicate formation of a heterodimer, which we have designated hMutL alpha . These observations indicate that interactions between members of the family of human MutL homologs may be restricted. Proc Natl Acad Sci U S A, 1995 Mar 14, 92(6), 1945 - 9 GTP consumption of elongation factor Tu during translation of heteropolymeric mRNAs; Rodnina MV et al.; The stoichiometry of elongation factor Tu (EF-Tu) and GTP in the complex with aminoacyl-tRNA and the consumption of GTP during peptide bond formation on the ribosome were studied in the Escherichia coli system . The ribosomes were programmed either with two different heteropolymeric mRNAs coding for Met-Phe-Thr-Ile .. . (mMFTI) or Met-Phe-Phe-Gly .. . (mMFFG) or with poly(U) . The composition of the complex of EF-Tu, GTP, and Phe-tRNA(Phe) was studied by gel chromatography . With equimolar amounts of factor and Phe-tRNA(Phe), a pentameric complex, (EF-Tu.GTP)2.Phe-tRNA(Phe), was observed, whereas the classical ternary complex, EF-Tu.GTP.Phe-tRNA(Phe), was found only when Phe-tRNA(Phe) was in excess . Upon binding of the purified pentameric complex to ribosomes carrying fMet-tRNA(fMet) in the peptidyl site and exposing a Phe codon in the aminoacyl site, only one out of two GTPs of the pentameric complex was hydrolyzed per Phe-tRNA bound and peptide bond formed, regardless of the mRNA used . In the presence of EF-G, the stoichiometry of one GTP hydrolyzed per peptide bond formed was found on mMFTI when one or two elongation cycles were completed . In contrast, on mMFFG, which contains two contiguous Phe codons, UUU-UUC, two GTP molecules of the pentameric complex were hydrolyzed per Phe incorporated into dipeptide, whereas the incorporation of the second Phe to form tripeptide consumed only one GTP . Thus, generally one GTP is hydrolyzed by EF-Tu per aminoacyl-tRNA bound and peptide bond formed, and more than one GTP is hydrolyzed only when a particular mRNA sequence, such as a homopolymeric stretch, is translated . The role of the additional GTP hydrolysis is not known; it may be related to frameshifting of peptidyl-tRNA during translocation. Proc Natl Acad Sci U S A, 1995 Mar 14, 92(6), 1812 - 6 The N-terminal portion of domain E of retinoic acid receptors alpha and beta is essential for the recognition of retinoic acid and various analogs; Ostrowski J et al.; Utilizing a strategy involving domain exchange between retinoic acid receptors alpha and beta (RAR alpha and RAR beta) and monitoring the transcriptional activity of the resulting chimeric receptors with receptor-selective retinoids, we identified a 70-aa region within the N-terminal portion of the RAR alpha and -beta domain E which is important for an RAR alpha- or RAR beta-specific response . Two amino acid residues within this region, serine-232 (S232) and threonine-239 (T239) in RAR alpha and the corresponding alanine-225 (A225) and isoleucine-232 (I232) in RAR beta, were found to be essential for this effect . In addition, binding studies using the chimeric receptors expressed in Escherichia coli showed that the N-terminal portion of domain E was also important for the characteristic binding profile of t-RA and various retinoids with RAR alpha or RAR beta . Structural predictions of the primary amino acid sequence in this region indicate the presence of an amphipathic helix-turn-helix structure with five hydrophobic amino acids that resemble a leucine zipper motif . The amino acid residues identified by domain swapping, S232 and T239 in RAR alpha and A225 and I232 in RAR beta, were found within the hydrophobic face of an alpha-helix in close proximity to this zipper motif, suggesting that the ligand may interact with the receptor in the region adjacent to a surface involved in protein-protein interactions . This finding may link ligand binding to other processes important for transcriptional activation. Biochemistry, 1995 Mar 14, 34(10), 3430 - 7 Insertion of the polytopic membrane protein lactose permease occurs by multiple mechanisms; Zen KH et al.; The lactose permease of Escherichia coli has 12 transmembrane hydrophobic domains in probable alpha-helical conformation connected by hydrophilic loops . Previous studies {Consler, T . G., Persson, B., et al . (1993) Proc . Natl . Acad . Sci . U.S.A . 90, 6934-6938} demonstrate that a peptide fragment (the XB domain) containing a factor Xa protease site immediately upstream of a biotin acceptor domain can be engineered into the permease, thereby allowing rapid purification to a high state of purity . Here we describe the use of the XB domain to probe topology and insertion . Cells expressing permease with the XB domain at the N terminus, at the C terminus, or in loop 6 or 10 on the cytoplasmic face of the membrane catalyze active transport, although only the chimeras with the XB domain at the C terminus or in loop 6 are biotinylated . In contrast, chimeras with the XB domain in periplasmic loop 3 or 7 are inactive, but strikingly, both constructs are biotinylated . Furthermore, the XB domain in all the constructs, particularly in the loop 3 and loop 7 chimeras, is accessible from the cytoplasmic face of the membrane, as evidenced by factor Xa proteolysis or avidin binding studies with spheroplasts and disrupted membrane preparations . Finally, alkaline phosphatase fusions one loop downstream from each periplasmic XB domain exhibit high phosphatase activity . Thus, the presence of the XB domain in a periplasmic loop apparently blocks translocation of a discrete segment of the permease consisting of the loop and the two adjoining helices without altering insertion of the remainder of the protein.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1995 Mar 14, 34(10), 3386 - 95 Characterization of a Thermomonospora fusca exocellulase; Zhang S et al.; The exocellulase E3 gene was cloned on a 7.1 kb NotI fragment from Thermomonospora fusca genomic DNA into Escherichia coli and expressed in Streptomyces lividans . The E3 gene was sequenced and encoded a 596 residue peptide . The molecular masses of the native and cloned E3s were determined by mass spectrometry, and the value for E . coli E3, 59,797 Da, agreed well with that predicted from the DNA sequence, 59,646 Da . The value of 61,200 Da for T . fusca E3 is consistent with E3 being a glycoprotein . E3 is thermostable, retaining full activity after 16 h at 55 degrees C . It also has a broad pH optimum around 7-8, retaining 90% of its maximal activity between pH 6 and 10 . The cloned E3s were identical to the native enzyme in their activity, cellulose binding, and thermostability . Papain digestion produced a 45.7 kDa catalytic domain with 77% of the native activity on amorphous cellulose and 33% on crystalline cellulose . E3 belongs to cellulase family B and retains the residues that have been identified to be crucial for catalytic activity in Trichoderma reesei cellobiohydrolase II and T . fusca E2 . The E3 gene contains a 14 bp inverted repeat regulatory sequence 212 bp before the translational start codon instead of the 30-70 bp found for the other T . fusca cellulase genes . An additional copy of this sequence with one base changed is 314 bp before the translational start codon . The transcriptional start site of the E3 gene was shown to be between these two inverted repeats. Biochemistry, 1995 Mar 14, 34(10), 3344 - 51 Critical side-chain interactions at a subunit interface in the Arc repressor dimer; Milla ME et al.; In the Arc repressor dimer, the side chains of Ile37 and Val41 in alpha-helix B pack against each other and against the symmetry-related side chains of Ile37' and Val41' in alpha-helix B' to form part of the hydrophobic core and the dimer interface . Following combinatorial mutagenesis of these positions, only the wild-type combination of hydrophobic residues was recovered as a fully active protein, and only a few conservative replacements were recovered as stably folded or partially active proteins . Equilibrium and kinetic studies of the folding of purified mutants show that the delta-CH3 groups of Ile37 and Ile37' contribute approximately 2 kcal/mol of dimer to protein stability and are involved in interactions that are only partially formed in the transition state for protein folding . Alanine substitution at either position 37 or 41 results in proteins which differ from wild type in being monomeric at a concentration of 10 microM, having reduced secondary structure, having solvent-exposed tryptophans, and showing non-cooperative thermal and urea denaturation transitions . These mutants appear to exist in a physiologically denatured state that is similar in many ways to the molten globule state. Biochemistry, 1995 Mar 14, 34(10), 3319 - 24 Site-directed mutagenesis of Lys36 in human thioredoxin: the highly conserved residue affects reduction rates and growth stimulation but is not essential for the redox protein's biochemical or biological properties; Oblong JE et al.; Previous studies have demonstrated that a recombinant form of the human redox protein thioredoxin can stimulate the growth rate of Swiss 3T3 murine fibroblasts and that this ability to promote cellular proliferation was dependent upon a redox-active form . A site-directed mutagenesis study of the highly conserved Lys36 adjacent to the two active site cysteines of thioredoxin was performed to determine whether the basic residue was essential for the biochemical and mitogenic properties of human thioredoxin . Two mutants were generated in which the lysine residue was replaced with either glutamic acid (K36E) or leucine (K36L) . While K36E and K36L were both redox-active in a thioredoxin-specific assay, the mutants exhibited decreased affinities for thioredoxin reductase relative to wild-type thioredoxin since their respective KM values increased by a factor of 5 and 7 . Examination of the secondary structure of the variants by circular dichroism spectroscopy revealed that both mutants had minor variations in the overall structural content when compared to thioredoxin, with K36L being most similar to the wild-type protein . Thermal equilibrium denaturation studies of the variants showed that K36E had a TM of 69.5 degrees C . A TM value for thioredoxin and K36L could not be established because the absence of a plateau above 83 degrees C rendered it difficult to establish an upper base line and, hence, the TM . The two mutants were able to stimulate cellular proliferation, albeit with reduced efficiency when compared with wild-type thioredoxin.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1995 Mar 14, 34(10), 3310 - 8 Recombinant Desulfovibrio vulgaris rubrerythrin . Isolation and characterization of the diiron domain; Gupta N et al.; The gene encoding Desulfovibrio (D.) vulgaris rubrerythrin (Prickril, B . C., Kurtz, D . M., Jr., LeGall, J., & Voordouw, G . (1991) Biochemistry 30, 1118), a protein of unknown function containing both FeS4 and (mu-oxo)diiron sites, was cloned and overexpressed in Escherichia coli . Upon cell lysis, the overexpressed protein was found in an insoluble form deficient in iron . Iron was incorporated in vitro by dissolving the protein in 3 M guanidinium chloride, adding Fe(II) anaerobically and diluting the denaturant . This recombinant rubrerythrin was found to have properties very similar to those of rubrerythrin isolated from D . vulgaris, except that the recombinant rubrerythrin contained six rather than four (or five) iron atoms per 44 kDa homodimer . Analyses of UV-vis, Mossbauer, and EPR spectra showed that the six iron atoms in recombinant rubrerythrin are organized as two FeS4 and two (mu-oxo/hydroxo)diiron sites . In order to allow examination of the diiron sites in the absence of the FeS4 sites, a truncated gene encoding the N-terminal 152 residues of D . vulgaris rubrerythrin was also cloned and overexpressed as an insoluble protein in E . coli, and iron was incorporated by a procedure analogous to that for recombinant rubrerythrin . This so-called "chopped" rubrerythrin (CRr) was found to consist of an approximately 35 kDa homodimer containing four iron atoms . Spectroscopic characterization indicated that the four iron atoms in CRr are organized as two diiron sites, the majority of which closely resemble the (mu-oxo)diiron(III) sites in E . coli ribonucleotide reductase R2 protein, and a minor fraction of which resemble the mixed-valent diiron(II,III) site in methane monooxygenase hydroxylase.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1995 Mar 14, 34(10), 3286 - 99 Thermodynamics of denaturation of barstar: evidence for cold denaturation and evaluation of the interaction with guanidine hydrochloride; Agashe VR et al.; Isothermal guanidine hydrochloride (GdnHCl)-induced denaturation curves obtained at 14 different temperatures in the range 273-323 K have been used in conjunction with thermally-induced denaturation curves obtained in the presence of 15 different concentrations of GdnHCl to characterize the thermodynamics of cold and heat denaturation of barstar . The linear free energy model has been used to determine the excess changes in free energy, enthalpy, entropy, and heat capacity that occur on denaturation . The stability of barstar in water decreases as the temperature is decreased from 300 to 273 K . This decrease in stability is not accompanied by a change in structure as monitored by measurement of the mean residue ellipticities at both 222 and 275 nm . When GdnHCl is present at concentrations between 1.2 and 2.0 M, the decrease in stability with decrease in temperature is however so large that the protein undergoes cold denaturation . The structural transition accompanying the cold denaturation process has been monitored by measuring the mean residue ellipticity at 222 nm . The temperature dependence of the change in free energy, obtained in the presence of 10 different concentrations of GdnHCl in the range 0.2-2.0 M, shows a decrease in stability with a decrease as well as an increase in temperature from 300 K . Values of the thermodynamic parameters governing the cold and the heart denaturation of barstar have been obtained with high precision by analysis of these bell-shaped stability curves . The change in heat capacity accompanying the unfolding reaction, delta Cp, has a value of 1460 +/- 70 cal mol-1 K-1 in water . The dependencies of the changes in enthalpy, entropy, free energy, and heat capacity on GdnHCl concentration have been analyzed on the basis of the linear free energy model . The changes in enthalpy (delta Hi) and entropy (delta Si), which occur on preferential binding of GdnHCl to the unfolded state, vis-a-vis the folded state, both have a negative value at low temperatures . With an increase in temperature delta Hi makes a less favorable contribution, while delta Si makes a more favorable contribution to the change in free energy (delta Gi) due to this interaction . The change in heat capacity (delta CPi) that occurs on preferential interaction of GdnHCl with the unfolded form has a value of only 53 +/- 36 cal mol-1 K-1 M-1 . The data validate the linear free energy model that is commonly used to analyze protein stability. Biochemistry, 1995 Mar 14, 34(10), 3248 - 52 Local structural preferences in the alpha-lactalbumin molten globule; Peng ZY et al.; Molten globules have been proposed to be general intermediates in protein folding . Despite numerous studies, a detailed description of the structure of a molten globule remains elusive . Recently, we showed that the molten globule formed by the helical domain of alpha-lactalbumin (alpha-LA) has a native-like backbone topology . Here we probe local structural preferences in the helical domain of the alpha-LA molten globule by analyzing a set of native and nonnative single disulfide bond variants using a combination of circular dichroism spectroscopy and determination of the equilibrium constant for disulfide bond formation . We find that the region surrounding the 28-111 disulfide bond has a high preference to adopt a native-like structure . Formation of other native or nonnative disulfide bonds is significantly less favorable . Our results suggest that molten globules contain regions with varying degrees of specificity for native-like structure and that the core region surrounding the 28-111 disulfide bond plays an important role in alpha-LA folding by stabilizing the molten globule intermediate. Biochemistry, 1995 Mar 14, 34(10), 3239 - 47 Phosphorylation site mutants of the mannitol transport protein enzyme IImtl of Escherichia coli: studies on the interaction between the mannitol translocating C-domain and the phosphorylation site on the energy-coupling B-domain; Boer H et al.; Mannitol binding and translocation catalyzed by the C domain of the Escherichia coli mannitol transport protein enzyme IImtl is influenced by domain B . This interaction was studied by monitoring the effects of mutating the B domain phosphorylation site, C384, on the kinetics of mannitol binding to the C domain . The dissociation constants for mannitol to the C384 mutants in inside-out membrane vesicles varied from 45 nM for the wild-type enzyme to 306 nM for the mutants . The rate constants pertinent to the binding equilibrium were also altered by the mutations . The association rate of mannitol to the cytoplasmic binding site in the mutants was accelerated for all mutants . The exchange rate of bound mannitol on the wild-type enzyme was shown to be pH dependent with a pKa of approximately 8 and increasing rates at higher pH . This rate was increased for all the mutants, but the pKas differed for the various mutants . The exchange rate for binding to the isolated IICmtl, however, was not pH dependent and exhibited a low rate . Exchange measured at 4 degrees C showed that, of the two steps, binding and occlusion, involved in binding to wild-type EIImtl in inside-out vesicles, only one could be detected for the C384E and C384L mutants . This suggests that the mutations increased the rate of the occlusion step so that it was no longer separable from the initial binding step or that the mutations eliminated the occlusion step altogether . The change in the mannitol binding kinetics of the C domain indicates that the B and C domains of EIImtl influence each other's conformation.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1995 Mar 14, 34(10), 3231 - 8 Resolution of the steroid-binding and dimerization domains of human sex hormone-binding globulin by expression in Escherichia coli; Hildebrand C et al.; To determine the minimal sequence requirements for steroid binding and dimerization of human sex hormone-binding globulin (SHBG), the SHBG polypeptide and various SHBG deletion mutants were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli . Fusion proteins containing the complete SHBG sequence, or the first 177 N-terminal residues of SHBG, bound steroids with high affinity and specificity . Further deletions from the C-terminus severely compromised steroid-binding activity, as did N-terminal deletions beyond residue 18 in the SHBG sequence . Thus, residues 18-177 in SHBG encompass a region required for its steroid-binding activity, and a disulfide bridge normally present between Cys-164 and Cys-188 in SHBG is not obviously essential for steroid binding . Most of the GST/SHBG fusion proteins undergo cleavage at 4 degrees C, releasing immunoreactive polypeptides that correspond approximately in size to their respective SHBG sequences . The 23-kDa immunoreactive cleavage product released from the fusion protein containing residues 1-205 in the SHBG sequence (SHBG 1-205) has a 50-fold greater steroid-binding capacity but a 7.5-fold lower affinity than its parent fusion protein . In addition, the 22-kDa immunoreactive polypeptide released from SHBG(1-194) binds steroid, and its dimerization is promoted by steroid ligands that bind SHBG with high affinity . These data suggest that the N-terminal region of SHBG dimerizes readily in the absence of GST and in doing so acquires steroid-binding sites.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1995 Mar 14, 34(10), 3212 - 21 Thermodynamic evaluation of binding interactions in the methionine repressor system of Escherichia coli using isothermal titration calorimetry; Hyre DE et al.; The binding interactions of the methionine repressor protein, MetJ, from Escherichia coli with its cognate, metbox DNA sequence and corepressor S-adenosylmethionine were examined using calorimetric methods . A detailed thermodynamic characterization of this system which exhibits the recently reported (beta alpha alpha)2 binding motif provides values for delta G, delta H, and delta S for each step in the repressor binding cycle . These studies show that, in the presence of corepressor, MetJ binds to a single metbox operator site with delta G = -7.7 kcal.mol-1, whereas in the absence of corepressor, the free energy of interaction with a single site is -5.8 kcal.mol-1 . Cooperative interactions between two repressor molecules bound to two adjacent sites contribute an additional free energy of -1.3 kcal.mol-1 to binding at the second site . Binding is enthalpically unfavorable in the absence of the corepressor with delta H = +2.6 kcal.mol-1 but becomes exothermic with delta H = -4.6 kcal.mol-1 when corepressor is present . The heat capacity for the system decreases significantly by delta Cp = -290 cal.mol-1.K-1 on a per site basis when the protein binds to DNA, and interactions between repressor molecules bound to adjacent sites contribute a delta Cp = -800 cal.mol-1.K-1, indicating that solvent exclusion plays a significant role in binding in this system . The corepressor binds to the unbound repressor protein with a free energy of delta G = -6.0 kcal.mol-1 and to the MetJ-operator complex with delta G = -6.95 kcal.mol-1 . Repressor binding to random-sequence DNA was estimated to occur with a free energy of -5.7 kcal.mol-1 in the presence of corepressor . These data clearly indicate that MetJ repressor dimer binds specifically to the central region of its 8 bp cognate metbox operator but recognizes partial operator sequences as short as 6 bp . Cooperativity in binding of adjacent MetJ dimers to a double metbox sequence is demonstrated to be important in determining the energetics of the interaction . Finally, the corepressor S-adenosylmethionine enhances the affinity of MetJ for its recognition site DNA by a factor of 25 and contributes significantly to the net exothermicity of repressor binding. Biochemistry, 1995 Mar 14, 34(10), 3183 - 92 Electrostatic effects of surface acidic amino acid residues on the oxidation-reduction potentials of the flavodoxin from Desulfovibrio vulgaris (Hildenborough); Zhou Z et al.; The flavodoxin from Desulfovibrio vulgaris (Hildenborough) is a member of a family of small, acidic proteins that contain a single noncovalently bound flavin mononucleotide (FMN) cofactor . These proteins function as low-potential one-electron transferases in bacteria . A distinguishing feature of these flavoproteins is the dramatic decrease in the midpoint potential of the semiquinone/hydroquinone couple of the FMN upon binding to the apoprotein (-172 mV for FMN free in solution versus -443 mV when bound), a perturbation thought to be essential for physiological function . The structural basis of this phenomenon is not yet thoroughly understood . In this study, the contribution of six acidic residues (Asp62, Asp63, Glu66, Asp95, Glu99, and Asp106) to the perturbation of the redox properties of the cofactor has been investigated . These residues are clustered about the FMN binding site within 13 A of the N(1) atom of the cofactor . Using oligonucleotide-directed mutagenesis, these residues were neutralized in various combinations through the substitution of asparagine for aspartate and glutamine for glutamate . Seventeen mutant flavodoxins were generated in which one to all six acidic residues were systematically neutralized, often in various spatial configurations . There was no obvious correlation between the midpoint potentials for the oxidized/semiquinone couple and general electrostatic environment, although some differences were noted . However, the midpoint potential for the semiquinone/hydroquinone couple for each of the mutants was less negative than that of the wild type . These increases are strongly correlated with the number of acid to amide substitutions, with an average contribution of about 15 mV per substitution . Collectively, the unfavorable electrostatic environment provided by these acidic residues accounts for approximately one-third of the large midpoint potential shift for the semiquinone/hydroquinone couple that typifies the flavodoxin family, apparently through the destabilization of the flavin hydroquinone anion. Biochemistry, 1995 Mar 14, 34(10), 3172 - 82 Mechanism of adenylate kinase . The "essential lysine" helps to orient the phosphates and the active site residues to proper conformations; Byeon L et al.; Although how Lys21 interacts with the substrate MgATP of muscle adenylate kinase (AK) can now be deduced from the crystal structure of Escherichia coli AK.MgAP5A {P1,P5-bis(5'-adenosyl) pentaphosphate} {Muller, C . W., & Schulz, G . E . (1992) J . Mol . Biol . 224, 159-177}, its contribution to catalysis has not yet been demonstrated by functional studies since the proton NMR of the K21M mutant was shown to be perturbed significantly {Tian, G., Yan., H., Jiang, R.-T., Kishi, F., Nakazawa, A., & Tsai, M.-D . (1990) Biochemistry 29, 4296-4304} . We therefore undertook further structural and functional analyses of a conservative mutant K21R and a nonconservative mutant K21A . In addition to kinetic analyses, the structures of the mutants were analyzed by one- and two-dimensional proton NMR spectroscopy and (1H, 15N) heteronuclear multiple-quantum coherence (HMQC) experiments . Detailed assignments were performed in reference to the total backbone assignments of the WT AK.MgAP5A complex {Byeon, I.-J . L., Yan, H., Edison, A . S., Mooberry, E . S., Abildgaard, F., Markley, J . L., & Tsai, M.-D . (1993) Biochemistry 32, 12508-12521} . The analysis showed that the residues located near the active site (Gly15, Thr23, Arg97, Gln101, Arg128, Arg132, Asp140, Asp141, and Tyr153) exhibit greater changes in 1H-15N chemical shifts . Finally, two-dimensional 31P-31P COSY experiments were used to examine the effects of the lysine side chain on the phosphate groups in the bound AP5A . Our data have led to the following conclusions independent of the crystal structure: (i) Because the perturbations in the conformation of the mutants are not global and are mainly localized at active site residues and Tyr153, the side chain of Lys21 can be concluded to stabilize the transition state in the catalysis of AK by up to 7 kcal/mol on the basis of the 10(5)-fold decreases in the kcat/Km of mutants . (ii) The results of 31P NMR analyses suggest that Lys21 functions by orienting the triphosphate chain of MgATP to a proper conformation required for catalysis . (iii) The interaction between Lys21 and the phosphate chain in turn dictates the interactions between the substrates and the active site residues . In the K21R.MgATP complex, the NH chemical shifts of many of the active site residues are perturbed . (iv) The catalytic functions of Lys21 cannot be replaced by a conservative residue arginine . In addition, since K21A and K21R behave similarly, the catalytic function of Lys21 should not be merely a charge effect. Biochemistry, 1995 Mar 14, 34(10), 3140 - 3 A native tertiary interaction stabilizes the A state of cytochrome c; Marmorino JL et al.; Certain kinetic intermediates in protein folding are similar to the molten globule, or A state, an equilibrium state of many proteins that is populated under high salt and low pH conditions . Many A states are nearly as compact as native proteins and have native-like secondary structure, but the extent to which nonlocal interactions stabilize the A state is unclear . In this study, thermal denaturation, monitored by circular dichroism, was used to determine the free energy of denaturation of the A state (delta GA<-->D) for Saccharomyces cerevisiae iso-1-ferricytochrome c . Specifically, we examined the wild-type protein, seven variants with amino acid substitutions at the interface between the N- and C-terminal helices, and two variants with mutations at a position close to, but not involved in, the interface . A plot of delta GA<-->D versus delta GN<-->D (the free energy of denaturation of the native state) has a slope near unity, showing that the evolutionarily conserved helix-helix interaction stabilizes the A state to the same degree that it stabilizes the native state. Proc Natl Acad Sci U S A, 1995 Mar 14, 92(6), 2086 - 90 The N terminus of phosducin is involved in binding of beta gamma subunits of G protein; Xu J et al.; Phosducin is a soluble phosphoprotein found in retinal photoreceptor cells and in the pineal gland . It binds to the beta gamma subunits of guanine nucleotide-binding proteins (G proteins) (G beta gamma) and may regulate G-protein function . In this study, the ability of specific regions of phosducin to bind G beta gamma was characterized . A series of deletion mutants were made in bovine phosducin . They were tested in cotransfection assays for their ability to inhibit G beta gamma-mediated phospholipase C beta 2 isoform activation . Overexpression of the N-terminal half of phosducin showed inhibition, whereas overexpression of the C-terminal half did not . The first 63 amino acid residues were required for inhibition . A tryptophan-to-valine substitution at residue 29, which is part of a well conserved 11-amino acid sequence, severely impaired phosducin inhibitory function . Glutathione S-transferase-phosducin fusion proteins were expressed in Escherichia coli to study phosducin-G beta gamma interaction in vitro . The N-terminal 63-amino acid fragment was able to bind to G beta gamma . In contrast, the C-terminal half failed to bind to G beta gamma . The substitution mutants showed little or no binding . Furthermore, direct measurements of interaction between G beta gamma and fragments of phosducin, using surface plasmon resonance technology, confirmed the assignment of binding activity to the 63-amino acid fragment and the importance of the tryptophan residue. Proc Natl Acad Sci U S A, 1995 Mar 14, 92(6), 2003 - 7 A small RNA acts as an antisilencer of the H-NS-silenced rcsA gene of Escherichia coli; Sledjeski D et al.; The regulation of capsular polysaccharide synthesis in Escherichia coli K-12 depends on the level of an unstable positive regulator, RcsA . The amount of RcsA protein is limited both by its rapid degradation by Lon, an ATP-dependent protease, and by its low level of synthesis . We have found that the low level of expression from the rcsA promoter is due to transcriptional silencing by the histone-like protein H-NS; this silencing is sensitive to both sequence and context in a region upstream of the -35 region of the promoter . A small (85-nt) RNA, DsrA, when overproduced, activates transcription of rcsA::lacZ fusions by counteracting H-NS silencing . DsrA RNA does not show any extended homology with the rcsA promoter or other sequenced regions of E . coli . Since the stimulation of rcsA transcription by this small RNA does not depend on any sequences from within the rcsA transcript, DsrA acts, either directly or indirectly, on rcsA transcription initiation. Proc Natl Acad Sci U S A, 1995 Mar 14, 92(6), 1896 - 900 SopB protein-mediated silencing of genes linked to the sopC locus of Escherichia coli F plasmid; Lynch AS et al.; Expression of a high level of F-plasmid-encoded SopB protein in Escherichia coli is found to repress genes linked to sopC, a sequence element of F consisting of 12 tandemly joined imperfect repeats of a 43-bp motif . Repression of a gene can occur over a distance of at least 10 kb from the sopC element and is not affected by the relative orientation of sopC . In the repressed state, accessibility of intracellular DNA to cellular proteins is greatly reduced in the region containing sopC, as monitored by the trapping of the covalent intermediate between DNA and DNA gyrase and by Dam methylase-catalyzed DNA methylation . These results signify the formation of a nucleoprotein structure emanating from sopC and are discussed in terms of position-dependent silencing of genes in general and the IncG type of plasmid incompatibility in particular. Proc Natl Acad Sci U S A, 1995 Mar 14, 92(6), 1807 - 11 Polyadenylylation helps regulate mRNA decay in Escherichia coli; O'Hara EB et al.; As part of our genetic analysis of mRNA decay in Escherichia coli K-12, we examined the effect of the pcnB gene {encoding poly(A) polymerase I} on message stability . Eliminating poly(A) polymerase I (delta pcnB) dramatically stabilized the lpp, ompA, and trxA transcripts . The half-lives of individual mRNAs were increased in both a delta pcnB single mutant and a delta pcnB pnp-7 rnb-500 rne-1 multiple mutant . We also found mRNA decay intermediates in delta pcnB mutants that were not detected in control strains . By end-labeling total E . coli RNA with {32P}pCp and T4 RNA ligase and then digesting the RNA with RNase A and T1, we showed that many RNAs in a wild-type strain contained poly(A) tails ranging from 10 nt to > 50 nt long . When polynucleotide phosphorylase, RNase II, and RNase E were absent, the length (> 100 nt) and number (10- to 20-fold) of the poly(A) tails increased . After transcription initiation was stopped with rifampicin, polyadenylylation apparently continued . Deleting the structural gene for poly(A) polymerase I (pcnB) reduced the amount of 3'-terminal poly(A) sequences by > 90% . We propose a model for the role of polyadenylylation in mRNA decay. Biochemistry, 1995 Mar 14, 34(10), 3404 - 15 Sites within the 39-kDa protein important for regulating ligand binding to the low-density lipoprotein receptor-related protein; Warshawsky I et al.; A 39-kDa protein copurifies with the low-density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP) and inhibits the binding and/or cellular uptake of ligands by this receptor . We recently utilized glutathione S-transferase (GST)-39-kDa fusion protein constructs to demonstrate that constructs encoding amino-terminal residues 1-114 and carboxy-terminal residues 115-319 of the 39-kDa protein independently bind to purified LRP and to LRP on hepatoma cells with similar affinities as the full-length GST-39-kDa protein (Kd approximately 8-10 nM) . These regions, however, inhibit ligand binding to LRP differently: GST/1-114 inhibits both tissue-type plasminogen activator (t-PA) and alpha 2-macroglobulin-methylamine (alpha 2M*) binding whereas GST/115-319 only potently inhibits t-PA binding . Four domains, containing residues 18-24 and 100-107 within amino-terminal constructs and residues 200-225 and 311-319 within carboxy-terminal constructs, are required for inhibition of ligand binding . In the present study, we generated additional 39-kDa protein constructs to precisely define residues within each domain required for inhibition of t-PA and alpha 2M* binding to LRP . The potential importance of these residues in mediating direct binding both to purified LRP and to LRP on hepatoma cells was examined . Within amino-terminal residues 1-114, alanine 103 and leucine 104 are required for inhibition of t-PA and alpha 2M* binding . These residues, however, are not required for binding either to purified LRP or to LRP on hepatoma cells . Within domain 18-24, arginine 21 is required for inhibition of t-PA and alpha 2M* binding as well as for the direct binding of amino-terminal constructs to LRP . Within carboxy-terminal domains 200-225 and 311-319, leucine 222 and leucine 319 are both required for inhibition of t-PA binding . Deletion of leucine 319 changes the ligand specificity from inhibition of t-PA binding to inhibition of alpha 2M* binding . Thus, leucine 319 is not required for direct binding to LRP whereas leucine 222 is required for high-affinity binding to LRP. FEBS Lett, 1995 Mar 13, 361(1), 55 - 60 In vitro dissociation of self-assembly of three chaperonin 60s: the role of ATP; Lissin NM; A comparative study has investigated the in vitro dissociation and self-assembly of chaperonin 60 14-mers isolated from E . coli (GroEL), yeast mitochondria and pea chloroplasts . In all cases Mg2+ inhibits, and low temperature stimulates, the urea-induced dissociation . ATP or ADP in the presence of Mg2+ enhance the dissociation of the chaperonins . Re-assembly of the 14-mers from their monomers shows different efficiencies between the three proteins . In all cases, however, self-assembly is stimulated by Mg-adenine nucleotides . Surprisingly, effective self-assembly of GroEL is promoted by 20% glycerol in the absence of ATP . The role of Mg-adenine nucleotides in the dissociation and assembly of the chaperonins is discussed. FEBS Lett, 1995 Mar 13, 361(1), 25 - 8 Effects of mutations at position 36 of tRNA(Glu) on missense and nonsense suppression in Escherichia coli; Gregory ST et al.; Mutations in the anticodon of tRNA(Glu) (UUC) were isolated or constructed and characterized for their ability to suppress cognate nonsense or missense mutations in vivo . The C36-to-A36 transversion mutation was isolated as an ochre and an amber suppressor, while the G36 transversion was selected as a CAG missense suppressor . tRNA(Glu) suppressors of an AAG missense mutation could not be isolated, and a U36 transition mutation introduced into tRNA(Glu) in vitro conferred no suppressor phenotype . Over-expression of glutamyl-tRNA synthetase did not increase the activity of the U36 mutant tRNA(Glu), suggesting a defect at the level of translation rather than at the level of synthetase recognition. FEBS Lett, 1995 Mar 13, 361(1), 111 - 4 Site-directed mutagenesis of histidine residues in the delta 12 acyl-lipid desaturase of Synechocystis; Avelange-Macherel MH et al.; In the cyanobacterium Synechocystis sp . PCC 6803, there are four acyl-lipid desaturases that are, respectively, specific to the delta 6, delta 9, delta 12 and omega 3 positions of fatty acids . The desA gene for the delta 12 acyl-lipid desaturase was modified by site-directed mutagenesis, such that four of the histidine residues that are conserved in the four desaturases and one histidine residue that is not conserved were replaced by arginine, and the mutated desA genes were overexpressed in Escherichia coli . All of these mutations eliminated the delta 12 desaturase activity . These results demonstrate that the five histidine residues are essential for the activity of the delta 12 desaturase, perhaps by providing the ligands for the catalytic Fe center. J Immunol Methods, 1995 Mar 13, 180(1), 69 - 79 Immunochemical analyses of human plasma fibronectin-cytosolic transglutaminase interactions; Achyuthan KE et al.; Fibronectin is a glycoprotein involved in cell adhesion, tissue organization and wound healing . Transglutaminase binding and covalent cross-linking of fibronectin are physiologically important reactions . We describe microtiter plate-based immunochemical methods to analyze cytosolic transglutaminase-human plasma fibronectin interactions . The method was sensitive, specific, species-independent and capable of simultaneously analyzing 96 samples for binding . Binding was time-, temperature- and concentration-dependent and demonstrable with either protein immobilized to the plastic . The assay detected 1-5 ng transglutaminase or 50 pg fibronectin and was comparable in sensitivity to enzyme-linked immunosorbent assays . CaCl2 (8 mM) enhanced transglutaminase binding by two-fold . Molar concentrations of NaCl or millimolar concentrations of chloride salts of barium, copper or zinc inhibited binding by 50-60% . The binding was also competitively blocked by soluble fibronectin (IC50 = 2.3 nM) or by anti-fibronectin IgG (IC50 = 0.5 microM) . Inclusion of dithiothreitol or 2-mercaptoethanol during binding resulted in a concentration-dependent inhibition of transglutaminase-fibronectin interactions (IC50 = 1.5 mM and 20 mM, respectively) . A complex of {anti-transglutaminase IgG-transglutaminase-fibronectin-anti- fibronectin IgG} suggested that the binding sites and antibody epitopes could overlap, but are distinct and surface-exposed in the two proteins . Liver transglutaminase bound fibronectin 30-50% less compared to erythrocyte transglutaminase . Fibronectin-transglutaminase affinity was adequate for quantitating either antigen in lysates of lung fibroblasts, breast carcinomas or Escherichia coli . These immunochemical analyses will be useful for determining the affinity and mapping the domains involved in antibody recognition or protein-protein interactions using recombinant molecules of transglutaminase and fibronectin. Nucleic Acids Res, 1995 Mar 11, 23(5), 869 - 75 Characterization of a specific interaction between Escherichia coli thymidylate synthase and Escherichia coli thymidylate synthase mRNA; Voeller DM et al.; Previous studies have shown that human TS mRNA translation is controlled by a negative autoregulatory mechanism . In this study, an RNA electrophoretic gel mobility shift assay confirmed a direct interaction between Escherichia coli (E.coli) TS protein and its own E.coli TS mRNA . Two cis-acting sequences in the E.coli TS mRNA protein-coding region were identified, with one site corresponding to nucleotides 207-460 and the second site corresponding to nucleotides 461-807 . Each of these mRNA sequences bind TS with a relative affinity similar to that of the full-length E.coli TS mRNA sequence (IC50 = 1 nM) . A third binding site was identified, corresponding to nucleotides 808-1015, although its relative affinity for TS (IC50 = 5.1 nM) was lower than that of the other two cis-acting elements . E.coli TS proteins with mutations in amino acids located within the nucleotide-binding region retained the ability to bind RNA while proteins with mutations at either the nucleotide active site cysteine (C146S) or at amino acids located within the folate-binding region were unable to bind TS mRNA . These studies suggest that the regions on E.coli TS defined by the folate-binding site and/or critical cysteine sulfhydryl groups may represent important RNA binding domains . Further evidence is presented which demonstrates that the direct interaction with TS results in in vitro repression of E.coli TS mRNA translation. Nucleic Acids Res, 1995 Mar 11, 23(5), 827 - 34 Promoter determinants for Escherichia coli RNA polymerase holoenzyme containing sigma 38 (the rpoS gene product); Tanaka K et al.; Sequence determinants responsible for promoter recognition by RNA polymerase holoenzyme containing sigma 38, the rpoS gene product, were analyzed . In a previous study {Tanaka et al . (1993) Proc . Natl . Acad . Sci . USA, 90, 3511-3515}, Escherichia coli promoters were classified into three groups: promoters recognized only by RNA polymerase holoenzyme containing sigma 70 (E sigma 70); promoters recognized preferentially by that containing sigma 38 (E sigma 38); promoters recognized by both E sigma 70 and E sigma 38 . As representatives of each group of promoter, we chose the alaS, fic and lacUV5 promoters . Making use of a restriction enzyme site inserted between the -10 and -35 hexamer sequences, promoters were divided into the upstream (UE) and downstream (DE) elements . These UEs and DEs were combined in all possible combinations and used for in vitro transcription reactions . Promoters containing DE from the fic or lacUV5 promoter were found to be recognized by E sigma 38, while those containing DE from the alaS promoter were not . Moreover, fic DE alone functioned as an efficient promoter for E sigma 38 . Thus we conclude that the discrimination signal resides within the DE sequence . To test the activator response of E sigma 38, in vitro transcription reactions were also performed with the gal and lac promoters . For both CRP-responsive P1 promoters, E sigma 38 was found to be activated by the CRP-cAMP complex. Nucleic Acids Res, 1995 Mar 11, 23(5), 819 - 26 Selectivity of the Escherichia coli RNA polymerase E sigma 38 for overlapping promoters and ability to support CRP activation; Kolb A et al.; A series of gal promoter mutants has been used to compare the in vitro selectivities of the two forms of Escherichia coli RNA polymerase, E sigma 38 and E sigma 70 . In the absence of the CRP-cAMP complex, E sigma 38 shows a strong preference for the ga/P1 promoter, whereas E sigma 70 preferentially initiates transcription from the ga/P2 promoter . E sigma 38 selectivity is not affected by the nature and position of the upstream sequences or by the phasing between synthetic upstream curved sequences and the -10 regions . In fact, all effects of mutations in the extended -10 region can be accounted for without evoking strong new sequence preferences for E sigma 38 . Finally, both E sigma 38 and E sigma 70 initiate transcription from the ga/P1 promoter in the presence of CRP-cAMP complex and support direct cAMP-CRP activation at several CRP-dependent promoters. Nucleic Acids Res, 1995 Mar 11, 23(5), 785 - 7 A motif conserved among the type I restriction-modification enzymes and antirestriction proteins: a possible basis for mechanism of action of plasmid-encoded antirestriction functions; Belogurov AA et al.; Antirestriction proteins Ard encoded by some self-transmissible plasmids specifically inhibit restriction by members of all three families of type I restriction-modification (R-M) systems in E.coli . Recently, we have identified the amino acid region, 'antirestriction' domain, that is conserved within different plasmid and phage T7-encoded antirestriction proteins and may be involved in interaction with the type I R-M systems . In this paper we demonstrate that this amino acid sequence shares considerable similarity with a well-known conserved sequence (the Argos repeat) found in the DNA sequence specificity (S) polypeptides of type I systems . We suggest that the presence of these similar motifs in restriction and antirestriction proteins may give a structural basis for their interaction and that the antirestriction action of Ard proteins may be a result of the competition between the 'antirestriction' domains of Ard proteins and the similar conserved domains of the S subunits that are believed to play a role in the subunit assembly of type I R-M systems. Nucleic Acids Res, 1995 Mar 11, 23(5), 761 - 6 Misincorporation of dAMP opposite 2-hydroxyadenine, an oxidative form of adenine; Kamiya H et al.; Nucleotide incorporation opposite an oxidative form of adenine, 2-hydroxyadenine (2-OH-Ade) was investigated . When a primed template with 2-OH-Ade was treated with an exonuclease-deficient Klenow fragment of Escherichia coli DNA polymerase I (KFexo-), recombinant rat DNA polymerase beta (pol beta) or calf thymus DNA polymerase alpha (pol alpha), incorporation of dTMP and dAMP was observed . In addition, KFexo- inserted dGMP as well . A steady-state kinetic study indicated that the insertion of dAMP and dTMP opposite the DNA lesion occurred with similar frequency with KFexo- and pol beta . Insertion of dTMP opposite 2-OH-Ade was favored to that of dAMP by pol alpha . Chain extension from the A.2-OH-Ade pair is less favored than that from the T.2-OH-Ade pair by all three DNA polymerase . Analysis of full-length products of in vitro DNA synthesis showed that dTMP and dAMP were incorporated by DNA polymerases and that exonuclease-proficient and -deficient Klenow fragments also inserted dGMP opposite 2-OH-Ade . These results suggest that formation of 2-OH-Ade from A in DNA will induce A-->T and A-->C transversions in cells. Nucleic Acids Res, 1995 Mar 11, 23(5), 803 - 10 Mutational sensitivity patterns define critical residues in the palm subdomain of the reverse transcriptase of human immunodeficiency virus type 1; Chao SF et al.; We have analyzed 154 single amino acid replacement mutants within a 40 amino acid region (residues 164-203) of the reverse transcriptase (RT) from human immunodeficiency virus type 1 (HIV-1) . This region consists of two antiparallel beta-strands (strands 9 and 10) flanked by two alpha helices (E and F) . The structure of this region of the 'palm' subdomain is conserved in a variety of DNA and RNA polymerases, indicating a critical role in enzyme structure and function . Functional assays were performed by screening RT activity of mutants expressed in E . coli . A functionally important region corresponding closely to beta-strands 9 and 10 and the loop joining them was revealed by its mutational sensitivity . Structural analysis of mutants was performed by using Western blots to assay correct folding, which is required for processing to produce the mature p66 and p51 RT species . This analysis indicates that beta-strand 10 is a structurally important region . Combined analysis of these two assays revealed diagnostic patterns of mutational sensitivity which identify key positions in the RT sequence at which a specific amino acid side chain is critical, either for structure or function, as well as residues which are external to the RT structure . This work illustrates the utility of large-scale mutagenesis in relating primary sequence to significant features of protein structure and function. Life Sci, 1995 Mar 10, 56(16), 1343 - 6 Is hypothalamic prostaglandin E2 involved in avian fever? Fraifeld V, Blaicher-Kulick R, Degen AA, Kaplanski J. In chickens, the effect of Escherichia coli lipopolysaccharide (LPS) on body temperature and ex vivo hypothalamic prostaglandin E2 (PGE2) production was examined to test the possible involvement of PGE2 in mechanisms of avian fever . PGE2 is reported to be the major central mediator of fever in mammals; it has not been examined in birds . An intraperitoneal injection of LPS caused an elevation of body temperature but not an elevation of hypothalamic PGE2 production . It seems that: (a) hypothalamic PGE2 is not involved in the development of the febrile response in birds; (b) central mechanisms of avian fever differ from those in mammals. Arch Biochem Biophys, 1995 Mar 10, 317(2), 348 - 56 Enhancement of Escherichia coli H(+)-ATPase caused by binding of monoclonal antibodies is attributed to structural changes of Leu-456 and Ser-440 in the alpha subunit; Kanazawa H et al.; Five monoclonal antibodies against the alpha subunit of F1-ATPase from Escherichia coli alpha 104, alpha 105, alpha 107, alpha 109, and alpha 110 were prepared . The monoclonal antibodies alpha 104 and alpha 110 enhanced the F1-ATPase activity maximally to 1.6- and 1.7-fold that of the wild-type, respectively, while alpha 105 did not . Both antibodies bound to a peptide corresponding to the region between residues 354 and 513 . Mutations in this region which caused reduced binding of the alpha subunit to the antibodies were identified at residues Ser-440, Leu-456, Leu-471, Leu-482, Met-483, and Ser-506 for alpha 104 and residues Ser-440, Leu-456, Leu-471, Asp-476, Leu-482, Met-483, and Ser-506 for alpha 110 . These residues are possibly involved in the epitopes for the antibodies and are located close together on the surface of the alpha subunit . Among the mutations, Leu-456 to Pro and Ser-440 to Pro mutations caused increase of the F1-ATPase activity up to 1.9 and 1.2 times that of the wild-type, respectively, while Leu-471 to Pro mutation caused a defect in assembly of the F1-ATPase on the membrane . The other mutations caused no significant change in ATPase activity . These results suggested that Ser-440 and Leu-456 have an important role in regulating catalysis by the F1-ATPase, but that the neighboring residue Leu-471 has an important role in assembly of the F1-ATPase complex . It was also suggested that binding of the monoclonal antibodies alpha 104 and alpha 110 to residues Ser-440 and Leu-456 caused local conformational changes, leading to enhancing effects on F1-ATPase activity similar to the Ser-440 to Pro and Leu-456 to Pro mutations. Arch Biochem Biophys, 1995 Mar 10, 317(2), 343 - 7 The role of cytochrome b5 in the biosynthesis of androgens by human P450c17; Katagiri M et al.; Human cytochrome b5 has a profound effect on the 17,20-lyase activities catalyzed by purified, human cytochrome P450c17 . It enhances the conversion of 17 alpha-hydroxypregnenolone to dehydroepiandrosterone by 13-fold and the conversion of 17 alpha-hydroxyprogesterone to androstenedione by at least 10-fold . This latter activity is virtually undetectable in the absence of cytochrome b5 . Other activities catalyzed by P450c17 include 17 alpha-hydroxylation of progesterone and pregnenolone and are much less influenced by cytochrome b5 . The conversion of pregnenolone to 17 alpha-hydroxypregnenolone is increased by 2-fold, while that of progesterone to 17 alpha-hydroxyprogesterone is unchanged . These studies using purified systems suggest that cytochrome b5 plays a role in regulating the activities of P450c17 to optimize the balance between sex hormone synthesis and glucocorticoid synthesis . In particular, they indicate that in human testes which contains a high b5/P450 ratio, 17 alpha-hydroxyprogesterone can serve as an intermediate in testosterone production, rather than being a dead-end product, or stated another way, because of the relatively high concentration of cytochrome b5 in the human testis, both the delta 4 and the delta 5 steroidogenic pathways can lead to testosterone production. J Biol Chem, 1995 Mar 10, 270(10), 5642 - 8 Cloning and sequencing of an intronless mouse S-adenosylmethionine decarboxylase gene coding for a functional enzyme strongly expressed in the liver; Persson K et al.; A genomic clone for a mouse S-adenosylmethionine decarboxylase (AdoMetDC) gene was isolated from a cosmid library . Surprisingly, the gene proved to be intronless . With the exception of three base substitutions (changing 2 amino acids in the deduced protein), the 1002-nucleotide sequence of the open reading frame was identical to that of mouse AdoMetDC cDNA . Moreover, the gene contained a poly(dA) tract at the 3' end and was flanked by 13-base pair direct repeats . Our findings suggest that this gene has arisen by retroposition, in which a fully processed AdoMetDC mRNA has been reverse transcribed into a DNA copy and inserted into the genome . By polymerase chain reaction, we positively identified the intronless gene in the mouse genome, and, by primer extension analysis, we proved the gene to be functional . Thus, its transcripts were found in many cell lines and tissues of the mouse and were particularly abundant in the liver . When the open reading frame of the intronless gene was expressed in Escherichia coli HT551, a strain with no AdoMetDC activity, it was found to encode a 38-kDa protein, corresponding to AdoMetDC proenzyme . Although the change of methionine 70 to isoleucine was close to the cleavage site at serine 68, this protein underwent proenzyme processing, generating a 31-kDa alpha subunit and an 8-kDa beta subunit . Importantly, the protein encoded by the intronless gene was functional, i.e . it catalyzed the decarboxylation of S-adenosylmethionine, and its specific activity was comparable with that of recombinant human AdoMetDC purified according to the same procedure. J Biol Chem, 1995 Mar 10, 270(10), 5606 - 13 Escherichia coli DNA polymerase III holoenzyme subunits alpha, beta, and gamma directly contact the primer-template; Reems JA et al.; Escherichia coli DNA polymerase III holoenzyme forms a stable initiation complex with RNA-primed template in the presence of ATP . To determine the linear arrangement of the holoenzyme subunits along the primer-template duplex region, we cross-linked holoenzyme to a series of photo-reactive primers . Site-specific photo-cross-linking revealed that the alpha, beta, and gamma subunits formed ATP-dependent contacts with the primer-template . The alpha-polymerase catalytic subunit covalently attached to nucleotide positions -3, -9, and -13 upstream of the primer terminus, with the most efficient adduct formation occurring at position -9 . The gamma subunit contacted the primer at positions -13, -18, and -22, with the strongest gamma-primer interactions occurring at position -18 . The beta subunit predominated in cross-linking at position -22 . Thus, within the initiation complex, alpha contacts roughly the first 13 nucleotides upstream of the 3'-primer terminus followed by gamma at -18 and beta at -22, and the gamma subunit remains a part of the initiation complex, bridging the alpha and beta subunits . Analyses of the interaction of photo-activatible primer-templates with the preinitiation complex proteins (gamma-complex (gamma-delta-delta'-chi-psi) and beta subunit) revealed the gamma subunit within the preinitiation complex covalently attached to primer at position -3 . However, addition of core DNA polymerase III to preinitiation complex, fully reconstituting holoenzyme resulted in replacement of gamma by alpha at the primer terminus . These data indicate that assembly of holoenzyme onto a primer-template can occur in distinct stages and results in a structural rearrangement during initiation complex formation. J Biol Chem, 1995 Mar 10, 270(10), 5578 - 86 Alternate binding of actin and calmodulin to multiple sites on dystrophin; Jarrett HW et al.; Mouse dystrophin protein sequence 1-385 and various deletion mutants were expressed in Escherichia coli as fusion proteins, and the binding of actin, calmodulin, and troponin C were characterized . The fusion protein-containing sequence 1-385 bound actin with an apparent dissociation constant of 129 +/- 65 nM as measured using a solid-phase immunoassay . High affinity was also observed with ultracentrifuge cosedimentation assays and biotinylated-actin binding assays . Results with deletion mutants and analysis based upon sequence homology were consistent with two or three high affinity F-actin-binding sequences within this region of dystrophin at sequence positions 18-37 (ABS 1), 128-149 (ABS 2), and potentially at a new region called ABS 3 (86-120) . A fusion protein lacking these sequences but containing dystrophin triple-helix sequences also bound actin but with reduced affinity . Calmodulin binds to dystrophin sequence 1-385 in a Ca(2+)-dependent manner and competitively inhibits F-actin binding . Results were consistent with two Ca(2+)-calmodulin-binding sites in this region of dystrophin at approximate sequence positions 18-42 (CBS 1) and 104-125 (CBS 2) with calmodulin affinities of 2.1 +/- 1 and 1.6 +/- 1.2 microM, respectively . Troponin C can substitute for calmodulin, although it binds with about 2-fold lower affinity . These results suggest that calmodulin (or troponin C) binding alternates with and may regulate F-actin binding by dystrophin much as has been postulated for other cytoskeletal proteins which are homologous to dystrophin. J Biol Chem, 1995 Mar 10, 270(10), 5519 - 26 Product of a new gene, syd, functionally interacts with SecY when overproduced in Escherichia coli; Shimoike T et al.; A mutant form of SecY, SecY-d1, was previously suggested to sequester a component(s) of the protein translocator complex . Its synthesis from a plasmid leads to interference with protein export in Escherichia coli . SecE is a target of this sequestration, and its overproduction cancels the export interference . We now report that overexpression of another gene, termed syd, also suppresses secY-d1 . The nucleotide sequence of syd predicted that it encodes a protein of 181 amino acid residues, which has been identified by overproduction, purification, and determination of the amino-terminal sequence . Cell fractionation experiments suggested that Syd is loosely associated with the cytoplasmic surface of the cytoplasmic membrane . SecY may be involved in the membrane association of Syd since the association is saturable, the extent of which depends on the overproduction of SecY . SecY is rapidly degraded in vivo unless its primary partner, SecE, is sufficiently available . Overproduction of Syd was found to stabilize oversynthesized SecY . However, Syd cannot stabilize the SecY-d1 form of SecY . Thus, in the presence of both secY+ and secY-d1, Syd increases the effective SecY+/SecY-d1 ratio in the cell and cancels the dominant interference by the latter . We also found that overproduction of Syd dramatically inhibits protein export in the secY24 mutant cell in which SecY-SecE interaction has been weakened . These results indicate that Syd, especially when it is overproduced, has abilities to interact with SecY . Possible significance of such interactions is discussed in conjunction with the apparent lack of phenotypic consequences of genetic disruption of syd. J Biol Chem, 1995 Mar 10, 270(10), 5495 - 505 Cloning and identification of amino acid residues of human phospholipase C delta 1 essential for catalysis; Cheng HF et al.; In vitro single point mutagenesis, inositol phospholipid hydrolysis, and substrate protection experiments were used to identify catalytic residues of human phosphatidylinositide-specific phospholipase C delta 1 (PLC delta 1) isolated from a human aorta cDNA library . Invariant amino acid residues containing a functional side chain in the highly conserved X region were changed by in vitro mutagenesis . Most of the mutant enzymes were still able to hydrolyze inositol phospholipid with activity ranging from 10 to 100% of levels in the wild type enzyme . Exceptions were mutants with the conversion of Arg338 to Leu (R338L), Glu341 to Gly (E341G), or His356 to Leu (H356L), which made the enzyme severely defective in hydrolyzing inositol phospholipid . Phospholipid vesicle binding experiments showed that these three cleavage-defective mutant forms of PLC delta 1 could specifically bind to phosphatidylinositol 4,5-bisphosphate (PIP2) with an affinity similar to that of wild type enzyme . Western blotting analysis of trypsin-treated enzyme-PIP2 complexes revealed that a 67-kDa major protein fragment survived trypsin digestion if the wild type enzyme, E341G, or H356L mutant PLC delta 1 was preincubated with 7.5 microM PIP2, whereas if it was preincubated with 80 microM PIP2, the size of major protein surviving was comparable to that of intact enzyme . However, mutant enzyme R338L was not protected from trypsin degradation by PIP2 binding . These observations suggest that PLC delta 1 can recognize PIP2 through a high affinity and a low affinity binding site and that residues Glu341 and His356 are not involved in either high affinity or low affinity PIP2 binding but rather are essential for the Ca(2+)-dependent cleavage activity of PLC. J Biol Chem, 1995 Mar 10, 270(10), 5469 - 75 Carboxyl-terminal domain truncation alters apolipoprotein A-I in vivo catabolism; Schmidt HH et al.; Apolipoprotein A-I (apoA-I), the major protein of high density lipoproteins, facilitates reverse cholesterol transport from peripheral tissue to liver . To determine the structural motifs important for modulating the in vivo catabolism of human apoA-I (h-apoA-I), we generated carboxyl-terminal truncation mutants at residues 201 (apoA-I201), 217 (apoA-I217), and 226 (apoA-I226) by site-directed mutagenesis . ApoA-I was expressed in Escherichia coli as a fusion protein with the maltose binding protein, which was removed by factor Xa cleavage . The in vivo kinetic analysis of the radioiodinated apoA-I in normolipemic rabbits revealed a markedly increased rate of catabolism for the truncated forms of apoA-I . The fractional catabolic rates (FCR) of 9.10 +/- 1.28/day (+/- S.D.) for apoA-I201, 6.34 +/- 0.81/day for apoA-I217, and 4.42 +/- 0.51/day for apoA-I226 were much faster than the FCR of recombinant intact apoA-I (r-apoA-I, 0.93 +/- 0.07/day) and h-apoA-I (0.91 +/- 0.34/day) . All the truncated forms of apoA-I were associated with very high density lipoproteins, whereas the intact recombinant apoA-I (r-apoA-I) and h-apoA-I associated with HDL2 and HDL3 . Gel filtration chromatography revealed that in contrast to r-apoA-I, the mutant apoA-I201 associated with a phospholipid-rich rabbit apoA-I containing particle . Analysis by agarose gel electrophoresis demonstrated that the same mutant migrated in the pre-beta position, but not within the alpha position as did r-apoA-I . These results indicate that the carboxyl-terminal region (residue 227-243) of apoA-I is critical in modulating the association of apoA-I with lipoproteins and in vivo metabolism of apoA-I. J Biol Chem, 1995 Mar 10, 270(10), 5388 - 94 Stability of the asymmetric Escherichia coli chaperonin complex . Guanidine chloride causes rapid dissociation; Todd MJ et al.; The chaperonin proteins, GroEL14 and GroES7, inhibit protein aggregation and assist in protein folding in a potassium/ATP-dependent manner . In vitro, assays for chaperonin activity typically involve adding a denatured substrate protein to the chaperonins and measuring the appearance of correctly folded substrate protein . The influence of denaturant is generally ignored . Low concentrations of guanidinium chloride (< 100 mM) had a profound effect on the activity/structure of the chaperonins . Guanidinium decreased the ATPase activity of GroEL and attenuated the inhibition of GroEL ATP hydrolysis by GroES . The stable, asymmetric chaperonin complex which forms in the presence of GroES and ADP (GroES7.ADP7.GroEL7-GroEL7) rapidly dissociated upon addition of 80 mM guandinium chloride . Dissociation was enhanced at high ionic strength, but rapid dissociation was guanidinium-specific . Accelerated release of the GroES from the complex was also demonstrated . Unfolded proteins alone had no effect on complex stability . Residual guanidinium depressed the rate of Rhodospirillum rubrum ri