Microbiology, 2000 May, 146 ( Pt 5), 1071 - 83 Era GTPase of Escherichia coli: binding to 16S rRNA and modulation of GTPase activity by RNA and carbohydrates; Meier TI et al.; Era, an essential GTPase, appears to play an important role in the regulation of the cell cycle and protein synthesis of bacteria and mycoplasmas . In this study, native Era, His-tagged Era (His-Era) and glutathione S-transferase (GST)-fusion Era (GST-Era) proteins from Escherichia coli were expressed and purified . It was shown that the GST-Era and His-Era proteins purified by 1-step affinity column chromatographic methods were associated with RNA and exhibited a higher GTPase activity . However, the native Era protein purified by a 3-step column chromatographic method had a much lower GTPase activity and was not associated with RNA which had been removed during purification . Purified GST-Era protein was shown to be present as a high- and a low-molecular-mass forms . The high-molecular-mass form of GST-Era was associated with RNA and exhibited a much higher GTPase activity . Removal of the RNA associated with GST-Era resulted in a significant reduction in the GTPase activity . The RNA associated with GST-Era was shown to be primarily 16S rRNA . A purified native Era protein preparation, when mixed with total cellular RNA, was found to bind to some of the RNA . The native Era protein isolated directly from the cells of a wild-type E . coli strain was also present as a high-molecular-mass form complexed with RNA and RNase treatment converted the high-molecular-mass form into a 32 kDa low-molecular-mass form, a monomer of Era . Furthermore, a C-terminally truncated Era protein, when expressed in E . coli, did not bind RNA . Finally, the GTPase activity of the Era protein free of RNA, but not the Era protein associated with the RNA, was stimulated by acetate and 3-phosphoglycerate . These carbohydrates, however, failed to activate the GTPase activity of the C-terminally truncated Era protein . Thus, the results of this study establish that the C-terminus of Era is essential for the RNA-binding activity and that the RNA and carbohydrates modulate the GTPase activity of Era possibly through a similar mechanism. Free Radic Biol Med, 2000 Apr 1, 28(7), 1132 - 6 Oxygen-insensitive nitroreductases of Escherichia coli do not reduce 3-nitrotyrosine; Lightfoot RT et al.; The oxygen-insensitive nitroreductases nfsA and nfsB are known to reduce para-nitrated aromatic compounds . We tested the hypothesis that these nitroreductases are capable of reducing 3-nitrotyrosine in proteins and peptides, as well as in free amino acids using wild-type and nfsA nfsB mutant strains of Escherichia coli . E . coli homogenates were incubated with nitrated proteins and the level of 3-nitrotyrosine immunoreactivity was assayed by Western blotting . Assay conditions that allow the nitroreductases to rapidly reduce nitrofurantoin did not result in the modification of 3-nitrotyrosine in protein, peptide, or free amino acid . Stimulation of nfsA nfsB activity with paraquat had no effect on 3-nitrotyrosine reduction . Nonlethal exposure of E . coli to peroxynitrite/CO(2) resulted in the reproducible nitration of tyrosine residues in endogenous proteins . The degree of 3-nitrotyrosine immunoreactivity over the 2-h postexposure period did not differ between mutant and wild-type strains . These results indicate that the nfsA and nfsB enzymes do not reduce 3-nitrotyrosine. Free Radic Biol Med, 2000 Apr 1, 28(7), 1009 - 16 Effects of menadione and hydrogen peroxide on glutathione status in growing Escherichia coli; Smirnova GV et al.; Menadione (MD) and H2O2 caused distinct effects on glutathione status in growing Escherichia coli . Treatment of E . coli AB1157 with 1-25 mM H2O2 did not result in an appreciable decrease in intracellular total glutathione (reduced glutathione {GSH} + oxidized glutathione {GSSG}) . Only when cells were treated with 25 mM H2O2 an increase in GSSG and a decrease in the GSH:GSSG ratio were observed . In cells deficient in catalase HPI, such effect was observed even at 10 mM H2O2 . The exposure of E . coli AB1157 to MD caused a dose-dependent decrease in intracellular total glutathione, an increase in GSSG, and a decrease in the ratio of GSH:GSSG . In E . coli deficient in cytosolic superoxide dismutase activity, a decrease in total glutathione after incubation with 0.2 mM MD was not accompanied by an increase in GSSGin, and the ratio of GSHin:GSSGin was three times higher than in the wild-type cells . The changes in the redox status of extracellular glutathione under the action of both oxidants were similar . Although the catalase activity increased several times after exposure to both oxidants, there were little or no changes in the activity of enzymes related to glutathione metabolism . A possible role of changes in redox status of glutathione under oxidative stress is discussed. Gene, 2000 May 16, 249(1-2), 153 - 60 Isolation and characterization of the epothilone biosynthetic gene cluster from Sorangium cellulosum; Julien B et al.; The epothilone biosynthetic gene cluster was isolated from Sorangium cellulosum strain SMP44 . The gene cluster contains seven genes and spans approx . 56kb . The genes encoding the PKS, epoA, epoC, epoD, epoE, and epoF, are divided into nine modules . The EpoB protein is a non-ribosomal peptide synthetase (NRPS) that catalyzes formation of the thiazole found in the epothilones . EpoK is a P450 enzyme responsible for the epoxidation of epothilones C and D to epothilones A and B, respectively . EpoK was expressed in Escherichia coli, and the purified protein was shown to convert epothilone D to epothilone B in vitro. J Cell Biol, 2000 May 29, 149(5), 1143 - 56 The cysteine-rich domain of human ADAM 12 supports cell adhesion through syndecans and triggers signaling events that lead to beta1 integrin-dependent cell spreading; Iba K et al.; The ADAMs (a disintegrin and metalloprotease) family of proteins is involved in a variety of cellular interactions, including cell adhesion and ecto- domain shedding . Here we show that ADAM 12 binds to cell surface syndecans . Three forms of recombinant ADAM 12 were used in these experiments: the cys-teine-rich domain made in Escherichia coli (rADAM 12-cys), the disintegrin-like and cysteine-rich domain made in insect cells (rADAM 12-DC), and full-length human ADAM 12-S tagged with green fluorescent protein made in mammalian cells (rADAM 12-GFP) . Mesenchymal cells specifically and in a dose-dependent manner attach to ADAM 12 via members of the syndecan family . After binding to syndecans, mesenchymal cells spread and form focal adhesions and actin stress fibers . Integrin beta1 was responsible for cell spreading because function-blocking monoclonal antibodies completely inhibited cell spreading, and chondroblasts lacking beta1 integrin attached but did not spread . These data suggest that mesenchymal cells use syndecans as the initial receptor for the ADAM 12 cysteine-rich domain-mediated cell adhesion, and then the beta1 integrin to induce cell spreading . Interestingly, carcinoma cells attached but did not spread on ADAM 12 . However, spreading could be efficiently induced by the addition of either 1 mM Mn(2+) or the beta1 integrin-activating monoclonal antibody 12G10, suggesting that in these carcinoma cells, the ADAM 12-syndecan complex fails to modulate the function of beta1 integrin. J Cell Biol, 2000 May 29, 149(5), 1039 - 52 PNG1, a yeast gene encoding a highly conserved peptide:N-glycanase; Suzuki T et al.; It has been proposed that cytoplasmic peptide:N-glycanase (PNGase) may be involved in the proteasome-dependent quality control machinery used to degrade newly synthesized glycoproteins that do not correctly fold in the ER . However, a lack of information about the structure of the enzyme has limited our ability to obtain insight into its precise biological function . A PNGase-defective mutant (png1-1) was identified by screening a collection of mutagenized strains for the absence of PNGase activity in cell extracts . The PNG1 gene was mapped to the left arm of chromosome XVI by genetic approaches and its open reading frame was identified . PNG1 encodes a soluble protein that, when expressed in Escherichia coli, exhibited PNGase activity . PNG1 may be required for efficient proteasome-mediated degradation of a misfolded glycoprotein . Subcellular localization studies indicate that Png1p is present in the nucleus as well as the cytosol . Sequencing of expressed sequence tag clones revealed that Png1p is highly conserved in a wide variety of eukaryotes including mammals, suggesting that the enzyme has an important function. J Biol Chem, 2000 Aug 18, 275(33), 25460 - 4 Modulation of the oligomerization state of the bovine F1-ATPase inhibitor protein, IF1, by pH; Cabezon E et al.; Bovine IF(1), a basic protein of 84 amino acids, is involved in the regulation of the catalytic activity of the F(1) domain of ATP synthase . At pH 6.5, but not at basic pH values, it inhibits the ATP hydrolase activity of the enzyme . The oligomeric state of bovine IF(1) has been investigated at various pH values by sedimentation equilibrium analytical ultracentrifugation and by covalent cross-linking . Both techniques confirm that the protein forms a tetramer at pH 8, and below pH 6.5, the protein is predominantly dimeric . By covalent cross-linking, it has been found that at pH 8.0 the fragment of IF(1) consisting of residues 44-84 forms a dimer, whereas the fragment from residues 32-84 is tetrameric . Therefore, some or all of the residues between positions 32 and 43 are necessary for tetramer formation and are involved in the pH-sensitive interconversion between dimer and tetramer . One important residue in the interconversion is histidine 49 . Mutation of this residue to lysine abolishes the pH-dependent activation-inactivation, and the mutant protein is active and dimeric at all pH values investigated . It is likely from NMR studies that the inhibitor protein dimerizes by forming an antiparallel alpha-helical coiled-coil over its C-terminal region and that at high pH values, where the protein is tetrameric, the inhibitory regions are masked . The mutation of histidine 49 to lysine is predicted to abolish coiled-coil formation over residues 32-43 preventing interaction between two dimers, forcing the equilibrium toward the dimeric state, thereby freeing the N-terminal inhibitory regions and allowing them to interact with F(1). Appl Environ Microbiol, 2000 Jun, 66(6), 2668 - 72 trans-o-Hydroxybenzylidenepyruvate hydratase-aldolase as a biocatalyst; Eaton RW; The hydratase-aldolase-catalyzed conversion of trans-o-hydroxybenzylidenepyruvate to salicylaldehyde and pyruvate is an intermediate reaction in the conversion of naphthalene to salicylate by bacteria . Here, a variety of aromatic aldehydes and some nonaromatic aldehydes together with pyruvate have been shown to be substrates for aldol condensations catalyzed by this enzyme in extracts of the recombinant strain Escherichia coli JM109(pRE701) . Some of the products of these reactions were also compared as substrates in the opposite (hydration-aldol cleavage) reaction. Appl Environ Microbiol, 2000 Jun, 66(6), 2484 - 90 Characterization of a bifunctional enzyme fusion of trehalose-6-phosphate synthetase and trehalose-6-phosphate phosphatase of Escherichia coli; Seo HS et al.; To test the effect of the physical proximity of two enzymes catalyzing sequential reactions, a bifunctional fusion enzyme, TPSP, was constructed by fusing the Escherichia coli genes for trehalose-6-phosphate (T6P) synthetase (TPS) and trehalose-6-phosphate phosphatase (TPP) . TPSP catalyzes the sequential reaction in which T6P is formed and then dephosphorylated, leading to the synthesis of trehalose . The fused chimeric gene was overexpressed in E . coli and purified to near homogeneity; its molecular weight was 88,300, as expected . The K(m) values of the TPSP fusion enzyme for the sequential overall reaction from UDP-glucose and glucose 6-phosphate to trehalose were smaller than those of an equimolar mixture of TPS and TPP (TPS/TPP) . However, the k(cat) values of TPSP were similar to those of TPS/TPP, resulting in a 3.5- to 4.0-fold increase in the catalytic efficiency (k(cat)/K(m)) . The K(m) and k(cat) values of TPSP and TPP for the phosphatase reaction from T6P to trehalose were quite similar . This suggests that the increased catalytic efficiency results from the proximity of TPS and TPP in the TPSP fusion enzyme . The thermal stability of the TPSP fusion enzyme was quite similar to that of the TPS/TPP mixture, suggesting that the structure of each enzyme moiety in TPSP is unperturbed by intramolecular constraint . These results clearly demonstrate that the bifunctional fusion enzyme TPSP catalyzing sequential reactions has kinetic advantages over a mixture of both enzymes (TPS and TPP) . These results are also supported by the in vivo accumulation of up to 0.48 mg of trehalose per g of cells after isopropyl-beta-D-thiogalactopyranoside treatment of cells harboring the construct encoding TPSP. Nature, 2000 May 18, 405(6784), 372 - 6 The crystal structure of the photoprotein aequorin at 2.3 A resolution; Head JF et al.; Aequorin is a calcium-sensitive photoprotein originally obtained from the jellyfish Aequorea aequorea . Because it has a high sensitivity to calcium ions and is biologically harmless, aequorin is widely used as a probe to monitor intracellular levels of free calcium . The aequorin molecule contains four helix-loop-helix 'EF-hand' domains, of which three can bind calcium . The molecule also contains coelenterazine as its chromophoric ligand . When calcium is added, the protein complex decomposes into apoaequorin, coelenteramide and CO2, accompanied by the emission of light . Apoaequorin can be regenerated into active aequorin in the absence of calcium by incubation with coelenterazine, oxygen and a thiol agent . Cloning and expression of the complementary DNA for aequorin were first reported in 1985 (refs 2, 6), and growth of crystals of the recombinant protein has been described; however, techniques have only recently been developed to prepare recombinant aequorin of the highest purity, permitting a full crystallographic study . Here we report the structure of recombinant aequorin determined by X-ray crystallography . Aequorin is found to be a globular molecule containing a hydrophobic core cavity that accommodates the ligand coelenterazine-2-hydroperoxide . The structure shows protein components stabilizing the peroxide and suggests a mechanism by which calcium activation may occur. Nature, 2000 May 18, 405(6784), 368 - 72 Translocation step size and mechanism of the RecBC DNA helicase; Bianco PR et al.; DNA helicases are ubiquitous enzymes that unwind double-stranded DNA . They are a diverse group of proteins that move in a linear fashion along a one-dimensional polymer lattice--DNA--by using a mechanism that couples nucleoside triphosphate hydrolysis to both translocation and double-stranded DNA unwinding to produce separate strands of DNA . The RecBC enzyme is a processive DNA helicase that functions in homologous recombination in Escherichia coli; it unwinds up to 6,250 base pairs per binding event and hydrolyses slightly more than one ATP molecule per base pair unwound . Here we show, by using a series of gapped oligonucleotide substrates, that this enzyme translocates along only one strand of duplex DNA in the 3'-->5' direction . The translocating enzyme will traverse, or 'step' across, single-stranded DNA gaps in defined steps that are 23 (+/-2) nucleotides in length . This step is much larger than the amount of double-stranded DNA that can be unwound using the free energy derived from hydrolysis of one molecule of ATP, implying that translocation and DNA unwinding are separate events . We propose that the RecBC enzyme both translocates and unwinds by a quantized, two-step, inchworm-like mechanism that may have parallels for translocation by other linear motor proteins. Nature, 2000 May 18, 405(6784), 364 - 8 LAG-3 is a putative transcriptional activator in the C . elegans Notch pathway; Petcherski AG et al.; Notch signalling controls growth, differentiation and patterning during normal animal development; in humans, aberrant Notch signalling has been implicated in cancer and stroke . The mechanism of Notch signalling is thought to require cleavage of the receptor in response to ligand binding, movement of the receptor's intracellular domain to the nucleus, and binding of that intracellular domain to a CSL (for CBF1, Suppressor of Hairless, LAG-1) protein . Here we identify LAG-3, a glutamine-rich protein that forms a ternary complex together with the LAG-1 DNA-binding protein and the receptor's intracellular domain . Receptors with mutant ankyrin repeats that abrogate signal transduction are incapable of complex formation both in yeast and in vitro . Using RNA interference, we find that LAG-3 activity is crucial in Caenorhabditis elegans for both GLP-1 and LIN-12 signalling . LAG-3 is a potent transcriptional activator in yeast, and a Myc-tagged LAG-3 is predominantly nuclear in C . elegans . We propose that GLP-1 and LIN-12 promote signalling by recruiting LAG-3 to target promoters, where it functions as a transcriptional activator. J Inorg Biochem, 2000 Apr, 79(1-4), 365 - 70 NMR studies of metal ion binding to the Zn-finger-like HNH motif of colicin E9; Hannan JP et al.; The 134 amino acid DNase domain of colicin E9 contains a zinc-finger-like HNH motif that binds divalent transition metal ions . We have used 1D 1H and 2D 1H-15N NMR methods to characterise the binding of Co2+, Ni2+ and Zn2+ to this protein . Data for the Co2+-substituted and Ni2+-substituted proteins show that the metal ion is coordinated by three histidine residues; and the NMR characteristics of the Ni2+-substituted protein show that two of the histidines are coordinated through their N(epsilon2) atoms and one via its N(delta1) . Furthermore, the NMR spectrum of the Ni2+-substituted protein is perturbed by the presence of phosphate, consistent with an X-ray structure showing that phosphate is coordinated to bound Ni2+, and by a change in pH, consistent with an ionisable group at the metal centre with a pKa of 7.9 . Binding of an inhibitor protein to the DNase does not perturb the resonances of the metal site, suggesting there is no substantial conformation change of the DNase HNH motif on inhibitor binding . 1H-15N NMR data for the Zn2+-substituted DNase show that this protein, like the metal-free DNase, exists as two conformers with different 1H-15N correlation NMR spectra, and that the binding of Zn2+ does not significantly perturb the spectra, and hence structures, of these conformers beyond the HNH motif region. Lab Invest, 2000 May, 80(5), 719 - 24 Plasma Escherichia coli beta-galactosidase as a marker of tumor burden and response to experimental anti-neoplastic therapy in nude mice xenografted with lacZ transduced human tumor cells; Holst-Hansen C et al.; Genetic labeling of tumor cells with the Escherichia coli lacZ reporter gene, encoding the enzyme beta-galactosidase, is widely used for histochemical detection of micrometastases in mice . Recently, we have developed a novel, highly sensitive and specific immunocapture chemiluminescence assay for the quantitation of E . coli beta-galactosidase . This assay achieved a detection limit of 0.01 mU of E . coli beta-galactosidase per milliliter, and 97% signal recovery of purified enzyme added to mouse plasma . LacZ transduced MDA-MB-231 BAG human breast cancer cells grown in vitro released soluble beta-galactosidase into the culture medium, and the concentration found correlated with cell density . Growth of the same cells in nude mice produced readily measurable levels of E . coli beta-galactosidase enzyme activity in host plasma and a highly significant correlation could be demonstrated between the size of primary tumor xenografts and the host plasma level of E . coli beta-galactosidase activity . When mice bearing MDA-MB-231 BAG tumor xenografts were treated intravenously with a single injection of doxorubicin (5 mg/kg), the mean tumor volume after 16 days was reduced 4-fold in the group of doxorubicin-treated mice compared with saline-treated control mice, and the mean level of plasma E . coli beta-galactosidase was correspondingly reduced 3.8-fold in the doxorubicin-treated mice compared with control mice . Sensitive and specific measurement of soluble E . coli beta-galactosidase in blood, using an immunocapture chemiluminescence assay, thus provides objective assessment of tumor burden in mice xenografted with lacZ transduced human tumors . This assay may have important applications as a tool for determining the efficacy of new experimental anti-tumor agents. Cancer Gene Ther, 2000 May, 7(5), 721 - 31 Expression of Escherichia coli B nitroreductase in established human tumor xenografts in mice results in potent antitumoral and bystander effects upon systemic administration of the prodrug CB1954; Djeha AH et al.; Expression of the Escherichia coli enzyme nitroreductase (NTR) in mammalian cells enables them to activate the prodrug 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954), leading to interstrand DNA cross-linking and apoptosis in both proliferating and quiescent cells . In the work reported here, we used human hepatocellular carcinoma and squamous carcinoma cell lines constitutively expressing NTR to demonstrate that the ntr/CB1954 system results in potent, long-lasting antitumoral effects in mice . We also demonstrate that this enzyme/prodrug combination results in antitumoral effects in vivo when only a minority of tumor cells express the enzyme, using either cells constitutively expressing NTR or ntr gene delivery in situ. Biosci Biotechnol Biochem, 2000 Apr, 64(4), 799 - 807 Deletion of the yhhP gene results in filamentous cell morphology in Escherichia coli; Ishii Y et al.; The Escherichia coli yhhP gene was predicted to encode a small hypothetical protein of 81 amino acids, the cellular function of which is not known . To gain insight into the function of this uncharacterized YhhP protein, genetic and biochemical studies were done . We first tried to express and purify the YhhP protein to prepare an anti-YhhP antiserum . Western blotting showed that the hypothetical yhhP gene is indeed transcribed and translated as a minor cytoplasmic protein . YhhP-deficient (delta yhhP) cells formed colonies poorly on a rich medium (e.g., Luria-Bertani medium) containing a relatively low concentration of NaCl, while they can grow normally either in LB containing 3% NaCl or in a synthetic medium (e.g., M9-glucose) . During exponential growth in rich medium, an early step of cell division was inhibited in delta yhhP cells, forming filaments . For the YhhP-deficient filamentous cells, the FtsZ-ring formation was analyzed with immunofluorescence microscopy . The FtsZ-ring formation did not occur normally in the delta yhhP filaments, although the filamentous cells contained the FtsZ protein at a certain level comparable to that in the wild-type cells . The ftsZ gene was found to function as a multicopy suppressor of the delta yhhP mutant . Another multicopy suppressor gene was identified as the dksA gene . Provided that either the ftsZ or dksA gene was introduced into the mutant cells with its multicopy state, the resulting transformants were capable of growing in rich medium, formed wild-type short rods . These results are discussed with regard to the presumed function of this ubiquitous protein. Biosci Biotechnol Biochem, 2000 Apr, 64(4), 746 - 50 Enzymatic production of trans-4-hydroxy-L-proline by regio- and stereospecific hydroxylation of L-proline; Shibasaki T et al.; A proline 4-hydroxylase gene, which was cloned from Dactylosporangium sp . RH1, was overexpressed in Escherichia coli W1485 on a plasmid under a tryptophan tandem promoter after the codon usage of the 5' end of the gene was optimized . The proline 4-hydroxylase activity was l600-fold higher than that in Dactylosporangium sp . RH1 . trans-4-Hydroxy-L-proline(Hyp) was produced and accumulated to 41 g/L (87% yield from L-proline) in 100 h when the recombinant E . coli was cultivated in a medium containing L-proline and glucose . 2-Oxoglutarate, which is necessary for the hydroxylation of L-proline by proline 4-hydroxylase, was apparently supplied from glucose through the cellular metabolic pathway . The putA mutant of W1485, which is not able to degrade L-proline, has allowed the quantitative conversion of L-proline to Hyp . The formation of other isomers of hydroxyproline was not observed . Productivity of Hyp was almost the same in a larger-scale culture . The method of manufacturing Hyp from L-proline was established. Proc Natl Acad Sci U S A, 2000 Jun 6, 97(12), 6646 - 51 The SOS response regulates adaptive mutation; McKenzie GJ et al.; Upon starvation some Escherichia coli cells undergo a transient, genome-wide hypermutation (called adaptive mutation) that is recombination-dependent and appears to be a response to a stressful environment . Adaptive mutation may reflect an inducible mechanism that generates genetic variability in times of stress . Previously, however, the regulatory components and signal transduction pathways controlling adaptive mutation were unknown . Here we show that adaptive mutation is regulated by the SOS response, a complex, graded response to DNA damage that includes induction of gene products blocking cell division and promoting mutation, recombination, and DNA repair . We find that SOS-induced levels of proteins other than RecA are needed for adaptive mutation . We report a requirement of RecF for efficient adaptive mutation and provide evidence that the role of RecF in mutation is to allow SOS induction . We also report the discovery of an SOS-controlled inhibitor of adaptive mutation, PsiB . These results indicate that adaptive mutation is a tightly regulated response, controlled both positively and negatively by the SOS system. Proc Natl Acad Sci U S A, 2000 Jun 6, 97(12), 6791 - 6 Ordered intracellular RecA-DNA assemblies: a potential site of in vivo RecA-mediated activities; Levin-Zaidman S et al.; The inducible SOS response increases the ability of bacteria to cope with DNA damage through various DNA repair processes in which the RecA protein plays a central role . Here we present the first study of the morphological aspects that accompany the SOS response in Escherichia coli . We find that induction of the SOS system in wild-type bacteria results in a fast and massive intracellular coaggregation of RecA and DNA into a lateral macroscopic assembly . The coaggregates comprise substantial portions of both the cellular RecA and the DNA complement . The structural features of the coaggregates and their relation to in vitro RecA-DNA networks, as well as morphological studies of strains carrying RecA mutants, are all consistent with the possibility that the intracellular assemblies represent a functional entity in which RecA-mediated DNA repair and protection activities occur. J Biol Chem, 2000 Aug 18, 275(33), 25180 - 7 Molecular basis for differential substrate specificity in class IV alcohol dehydrogenases: a conserved function in retinoid metabolism but not in ethanol oxidation; Crosas B et al.; Mammalian class IV alcohol dehydrogenase enzymes are characteristic of epithelial tissues, exhibit moderate to high K(m) values for ethanol, and are very active in retinol oxidation . The human enzyme shows a K(m) value for ethanol which is 2 orders of magnitude lower than that of rat class IV . The uniquely significant difference in the substrate-binding pocket between the two enzymes appears to be at position 294, Val in the human enzyme and Ala in the rat enzyme . Moreover, a deletion at position 117 (Gly in class I) has been pointed out as probably responsible for class IV specificity toward retinoids . With the aim of establishing the role of these residues, we have studied the kinetics of the recombinant human and rat wild-type enzymes, the human G117ins and V294A mutants, and the rat A294V mutant toward aliphatic alcohols and retinoids . 9-cis-Retinol was the best retinoid substrate for both human and rat class IV, strongly supporting a role of class IV in the generation of 9-cis-retinoic acid . In contrast, 13-cis retinoids were not substrates . The G117ins mutant showed a decreased catalytic efficiency toward retinoids and toward three-carbon and longer primary aliphatic alcohols, a behavior that resembles that of the human class I enzyme, which has Gly(117) . The K(m) values for ethanol dramatically changed in the 294 mutants, where the human V294A mutant showed a 280-fold increase, and the rat A294V mutant a 50-fold decrease, compared with those of the respective wild-type enzymes . This demonstrates that the Val/Ala exchange at position 294 is mostly responsible for the kinetic differences with ethanol between the human and rat class IV . In contrast, the kinetics toward retinoids was only slightly affected by the mutations at position 294, compatible with a more conserved function of mammalian class IV alcohol dehydrogenase in retinoid metabolism. J Biol Chem, 2000 Sep 1, 275(35), 27094 - 9 A conserved negatively charged cluster in the active site of creatine kinase is critical for enzymatic activity; Eder M et al.; Creatine kinase catalyzes the reversible transphosphorylation of creatine by MgATP . From the sequence homology and the molecular structure of creatine kinase isoenzymes, we have identified several highly conserved residues with a potential function in the active site: a negatively charged cluster (Glu(226), Glu(227), Asp(228)) and a serine (Ser(280)) . Mutant proteins E226Q, E226L, E227Q, E227L, D228N, and S280A/S280D of human sarcomeric mitochondrial creatine kinase were generated by in vitro mutagenesis, expressed in Escherichia coli, and purified to homogeneity . Their overall structural integrity was confirmed by CD spectroscopy and gel filtration chromatography . The enzymatic activity of all proteins mutated in the negatively charged cluster was extremely low (0.002-0.4% of wild type) and showed apparent Michaelis constants (K(m)) similar to wild type, suggesting that most of the residual activity may be attributed to wild-type revertants . Mutations of Ser(280) led to higher residual activities and altered K(m) values; S280A showed an increase of K(m) for phosphocreatine (65-fold), creatine (6-fold), and ATP (6-fold); S280D showed a decrease of K(m) for creatine (6-fold) . These results, together with the transition state structure of the homologous arginine kinase (Zhou, G., Somasundaram, T., Blanc, E., Parthasarathy G., Ellington, W . R., and Chapman, M . S . (1998) Proc . Natl . Acad . Sci . U . S . A . 95, 8449-8454), strongly suggest a critical role of Glu(226), Glu(227), and Asp(228) in substrate binding and catalysis and point to Glu(227) as a catalytic base. J Biol Chem, 2000 Aug 18, 275(33), 25633 - 40 Theoretical calculation of pKa reveals an important role of Arg205 in the activity and stability of Streptomyces sp . N174 chitosanase; Fukamizo T et al.; Based on the crystal structure of chitosanase from Streptomyces sp . N174, we have calculated theoretical pK(a) values of the ionizable groups of this protein using a combination of the boundary element method and continuum electrostatics . The pK(a) value obtained for Arg(205), which is located in the catalytic cleft, was abnormally high (>20.0), indicating that the guanidyl group may interact strongly with nearby charges . Chitosanases possessing mutations in this position (R205A, R205H, and R205Y), produced by Streptomyces lividans expression system, were found to have less than 0.3% of the activity of the wild type enzyme and to possess thermal stabilities 4-5 kcal/mol lower than that of the wild type protein . In the crystal structure, the Arg(205) side chain is in close proximity to the Asp(145) side chain (theoretical pK(a), -1.6), which is in turn close to the Arg(190) side chain (theoretical pK(a), 17.7) . These theoretical pK(a) values are abnormal, suggesting that both of these residues may participate in the Arg(205) interaction network . Activity and stability experiments using Asp(145)- and Arg(190)-mutated chitosanases (D145A and R190A) provide experimental data supporting the hypothesis derived from the theoretical pK(a) data and prompt the conclusion that Arg(205) forms a strong interaction network with Asp(145) and Arg(190) that stabilizes the catalytic cleft. J Biol Chem, 2000 Aug 18, 275(33), 25781 - 90 The receptor for advanced glycation end products is induced by the glycation products themselves and tumor necrosis factor-alpha through nuclear factor-kappa B, and by 17beta-estradiol through Sp-1 in human vascular endothelial cells; Tanaka N et al.; The binding of advanced glycation end products (AGE) to the receptor for AGE (RAGE) is known to deteriorate various cell functions and is implicated in the pathogenesis of diabetic vascular complications . Here we show that AGE, tumor necrosis factor-alpha (TNF-alpha), and 17beta-estradiol (E(2)) up-regulated RAGE mRNA and protein levels in human microvascular endothelial cells and ECV304 cells, with the mRNA stability being essentially invariant . Transient transfection experiments with human RAGE promoter-luciferase chimeras revealed that the region from nucleotide number -751 to -629 and the region from -239 to -89 in the RAGE 5'-flanking sequence exhibited the AGE/TNF-alpha and E(2) responsiveness, respectively . Site-directed mutation of an nuclear factor-kappaB (NF-kappaB) site at -671 or of Sp-1 sites at -189 and -172 residing in those regions resulted in an abrogation of the AGE/TNF-alpha- or E(2)-mediated transcriptional activation . Electrophoretic mobility shift assays revealed that ECV304 cell nuclear extracts contained factors which retarded the NF-kappaB and Sp-1 elements, and that the DNA-protein complexes were supershifted by anti-p65/p50 NF-kappaB and anti-Sp-1/estrogen receptor alpha antibodies, respectively . These results suggest that AGE, TNF-alpha, and E(2) can activate the RAGE gene through NF-kappaB and Sp-1, causing enhanced AGE-RAGE interactions, which would lead to an exacerbation of diabetic microvasculopathy. J Biol Chem, 2000 Aug 18, 275(33), 25188 - 93 Quantitative analysis of TIP47-receptor cytoplasmic domain interactions: implications for endosome-to-trans Golgi network trafficking; Krise JP et al.; TIP47 (tail-interacting protein of 47 kDa) binds to the cytoplasmic domains of the cation-independent and cation-dependent mannose 6-phosphate receptors and is required for their transport from late endosomes to the trans Golgi network in vitro and in vivo . We report here a quantitative analysis of the interaction of recombinant TIP47 with mannose 6-phosphate receptor cytoplasmic domains . Recombinant TIP47 binds more tightly to the cation-independent mannose 6-phosphate receptor (K(D) = 1 microm) than to the cation-dependent mannose 6-phosphate receptor (K(D) = 3 microm) . In addition, TIP47 fails to interact with the cytoplasmic domains of the hormone-processing enzymes, furin, phosphorylated furin, and metallocarboxypeptidase D, as well as the cytoplasmic domain of TGN38, proteins that are also transported from endosomes to the trans Golgi network . Although these proteins failed to bind TIP47, furin and TGN38 were readily recognized by the clathrin adaptor, AP-2 . These data suggest that TIP47 recognizes a very select set of cargo molecules . Moreover, our data suggest unexpectedly that furin, TGN38, and carboxypeptidase D may use a distinct vesicular carrier and perhaps a distinct route for transport between endosomes and the trans Golgi network. J Biol Chem, 2000 Aug 4, 275(31), 23769 - 73 Escherichia coli NifS-like proteins provide selenium in the pathway for the biosynthesis of selenophosphate; Lacourciere GM et al.; Selenophosphate synthetase (SPS), the selD gene product from Escherichia coli, catalyzes the biosynthesis of monoselenophosphate, AMP, and orthophosphate in a 1:1:1 ratio from selenide and ATP . Kinetic characterization revealed the K(m) value for selenide approached levels that are toxic to the cell . Our previous demonstration that a Se(0)-generating system consisting of l-selenocysteine and the Azotobacter vinelandii NifS protein can replace selenide for selenophosphate biosynthesis in vitro suggested a mechanism whereby cells can overcome selenide toxicity . Recently, three E . coli NifS-like proteins, CsdB, CSD, and IscS, have been overexpressed and characterized . All three enzymes act on selenocysteine and cysteine to produce Se(0) and S(0), respectively . In the present study, we demonstrate the ability of each E . coli NifS-like protein to function as a selenium delivery protein for the in vitro biosynthesis of selenophosphate by E . coli wild-type SPS . Significantly, the SPS (C17S) mutant, which is inactive in the standard in vitro assay with selenide as substrate, was found to exhibit detectable activity in the presence of CsdB, CSD, or IscS and l-selenocysteine . Taken together the ability of the NifS-like proteins to generate a selenium substrate for SPS and the activation of the SPS (C17S) mutant suggest a selenium delivery function for the proteins in vivo. J Biol Chem, 2000 Aug 18, 275(33), 25672 - 80 A novel human tocopherol-associated protein: cloning, in vitro expression, and characterization; Zimmer S et al.; Vitamin E (alpha-tocopherol) is an essential dietary nutrient for humans and animals . The mechanisms involved in cellular regulation as well as in the preferential cellular and tissue accumulation of alpha-tocopherol are not yet well established . We previously reported (Stocker, A., Zimmer, S., Spycher, S . E., and Azzi, A . (1999) IUBMB Life 48, 49-55) the identification of a novel 46-kDa tocopherol-associated protein (TAP) in the cytosol of bovine liver . Here, we describe the identification, the molecular cloning into Escherichia coli, and the in vitro expression of the human homologue of bovine TAP, hTAP . This protein appears to belong to a family of hydrophobic ligand binding proteins, which have the CRAL (cis-retinal binding motif) sequence in common . By using a biotinylated alpha-tocopherol derivative and the IASys resonant mirror biosensor, the purified recombinant protein was shown to bind tocopherol at a specific binding site with K(d) 4.6 x 10(-7) m . Northern analyses showed that hTAP mRNA has a size of approximately 2800 base pairs and is ubiquitously expressed . The highest amounts of hTAP message are found in liver, brain, and prostate . In conclusion, hTAP has sequence homology to proteins containing the CRAL_TRIO structural motif . TAP binds to alpha-tocopherol and biotinylated tocopherol, suggesting the existence of a hydrophobic pocket, possibly analogous to that of SEC14. Biochemistry, 2000 Jun 6, 39(22), 6732 - 42 Investigation of the DsbA mechanism through the synthesis and analysis of an irreversible enzyme-ligand complex; Couprie J et al.; Approaching the molecular mechanism of some enzymes is hindered by the difficulty of obtaining suitable protein-ligand complexes for structural characterization . DsbA, the major disulfide oxidase in the bacterial periplasm, is such an enzyme . Its structure has been well characterized in both its oxidized and its reduced states, but structural data about DsbA-peptide complexes are still missing . We report herein an original, straightforward, and versatile strategy for making a stable covalent complex with a cysteine-homoalanine thioether bond instead of the labile cystine disulfide bond which normally forms between the enzyme and polypeptides during the catalytic cycle of DsbA . We substituted a bromohomoalanine for the cysteine in a model 14-mer peptide derived from DsbB (PID-Br), the membrane partner of DsbA . When incubated in the presence of the enzyme, a selective nucleophilic substitution of the bromine by the thiolate of the DsbA Cys(30) occurred . The major advantage of this strategy is that it enables the direct use of the wild-type form of the enzyme, which is the most relevant to obtain unbiased information on the enzymatic mechanism . Numerous intermolecular NOEs between DsbA and PID could be observed by NMR, indicating the presence of preferential noncovalent interactions between the two partners . The thermodynamic properties of the DsbA-PID complex were measured by differential scanning calorimetry . In the complex, the values for both denaturation temperature and variation in enthalpy associated with thermal unfolding were between those of oxidized and reduced forms of DsbA . This progressive increase in stability along the DsbA catalytic pathway strongly supports the model of a thermodynamically driven mechanism. Biochemistry, 2000 Jun 6, 39(22), 6726 - 31 CP1 domain in Escherichia coli leucyl-tRNA synthetase is crucial for its editing function; Chen JF et al.; The amino acid discrimination by aminoacyl-tRNA synthetase is achieved through two sifting steps; amino acids larger than the cognate substrate are rejected by a "coarse sieve", while the reaction products of amino acids smaller than the cognate substrate will go through a "fine sieve" and be hydrolyzed . This "double-sieve" mechanism has been proposed for IleRS, a class I aminoacyl-tRNA synthetase . In this study, we created LeuRS-B, a mutant leucyl-tRNA synthetase from Escherichia coli with a duplication of the peptide fragment from Met328 to Pro368 (within its CP1 domain) . This mutant has 50% of the leucylation activity of the wild-type enzyme and has the same ability to discriminate noncognate amino acids in the first step of the reaction . However, LeuRS-B can catalyze mischarging of tRNA(Leu) by methionine or isoleucine, suggesting that it is impaired in the ability to edit incorrect products . Wild-type leucyl-tRNA synthetase can edit the mischarged tRNA(Leu) made by LeuRS-B, while a separated CP1 domain cannot . These data suggest that the CP1 domain of leucyl-tRNA synthetase is crucial to the second editing sieve and that CP1 needs the structural context in leucyl-tRNA synthetase to fulfill its editing function. Biochemistry, 2000 Jun 6, 39(22), 6669 - 78 Resonance raman studies of oxo intermediates in the reaction of pulsed cytochrome bo with hydrogen peroxide; Uchida T et al.; Cytochrome bo from Escherichia coli, a member of the heme-copper terminal oxidase superfamily, physiologically catalyzes reduction of O(2) by quinols and simultaneously translocates protons across the cytoplasmic membrane . The reaction of its ferric pulsed form with hydrogen peroxide was investigated with steady-state resonance Raman spectroscopy using a homemade microcirculating system . Three oxygen-isotope-sensitive Raman bands were observed at 805/X, 783/753, and (767)/730 cm(-)(1) for intermediates derived from H(2)(16)O(2)/H(2)(18)O(2) . The experiments using H(2)(16)O(18)O yielded no new bands, indicating that all the bands arose from the Fe=O stretching (nu(Fe)(=)(O)) mode . Among them, the intensity of the 805/X cm(-)(1) pair increased at higher pH, and the species giving rise to this band seemed to correspond to the P intermediate of bovine cytochrome c oxidase (CcO) on the basis of the reported fact that the P intermediate of cytochrome bo appeared prior to the formation of the F species at higher pH . For this intermediate, a Raman band assignable to the C-O stretching mode of a tyrosyl radical was deduced at 1489 cm(-)(1) from difference spectra . This suggests that the P intermediate of cytochrome bo contains an Fe(IV)=O heme and a tyrosyl radical like compound I of prostaglandin H synthase . The 783/753 cm(-)(1) pair, which was dominant at neutral pH and close to the nu(Fe)(=)(O) frequency of the oxoferryl intermediate of CcO, presumably arises from the F intermediate . On the contrary, the (767)/730 cm(-)(1) species has no counterpart in CcO . Its presence may support the branched reaction scheme proposed previously for O(2) reduction by cytochrome bo. Biochemistry, 2000 Jun 6, 39(22), 6634 - 44 Backbone dynamics of escherichia coli adenylate kinase at the extreme stages of the catalytic cycle studied by (15)N NMR relaxation; Shapiro YE et al.; Adenylate kinase from Escherichia coli (AKeco), consisting of a single 23.6 kDa polypeptide chain folded into domains CORE, AMPbd, and LID, catalyzes the reaction AMP + ATP --> 2ADP . Domains LID and AMPbd execute large-scale movements during catalysis . Backbone dynamics of ligand-free and AP(5)A-inhibitor-bound AKeco were studied comparatively with (15)N NMR relaxation methods . Overall diffusion with correlation times of 15.05 (11.42) ns and anisotropy D(parallel)/D(perp) = 1.25 (1.10), and fast internal motions with correlation times up to 100 ps (50 ps), were determined for AKeco (AKecoAP(5)A) . Fast internal motions affect 93% of the AKeco sites, with pronounced preference for domains AMPbd and LID, and 47% of the AKecoAP(5)A sites, with limited variability along the chain . The mean squared generalized order parameters, <S(2)>, of secondary structure elements and loops are affected by ligand binding differentially and in a domain-specific manner . Nanosecond motions predominate within AMPbd . Prominent exchange contributions, associated in particular with residue G10 of the nucleotide-binding P-loop motif, are interpreted to reflect hydrogen-bond dynamics at the inhibitor-binding site . The hypothesis of energetic counter balancing of substrate binding based on crystallographic data is strongly supported by the solution NMR results . Correlations between backbone dynamics and domain displacement are established. Biochemistry, 2000 May 30, 39(21), 6554 - 63 Multiple posttranslational modifications at distinct sites contribute to heterogeneity of the lipoprotein cytochrome bo(3); Prutsch A et al.; The heme-copper cytochrome oxidase of Escherichia coli (cytochrome bo(3)) was tagged with oligohistidine at the C-terminus of the small noncatalytic subunit IV . After detergent solubilization, the enzyme was purified by a one-step procedure with immobilized metal affinity chromatography . Using different cytochrome bo(3) constructs as reference, the products were investigated by mass spectroscopical and immunological methods . Several posttranslational modifications of subunits II, III, and IV were observed: (1) N-terminal methionines of subunits III and IV are split off . (2) Fifty percent of subunit III polypeptides are acetylated, presumably at the N-terminal alanine . (3) Lipoprotein processing of subunit II involves cleavage of the signal peptide . (4) Maturation of subunit II {Ma, J., Katsonouri, A., and Gennis, R . B . (1997) Biochemistry 36, 11298-11303} alters the structure of the N-terminal cysteine by N-palmitoylation and S-glyceryldipalmitoylation . (5) A hexapeptide is split off from the C-terminus of subunit II . This happens subsequently to the N-terminal lipoprotein processing step and is dependent on the growth state of cells. Biochemistry, 2000 May 30, 39(21), 6546 - 53 Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase: essential elements for recognition of tRNA substrates within the anticodon stem-loop; Soderberg T et al.; Escherichia coli dimethylallyl diphosphate:tRNA dimethylallyltransferase (DMAPP-tRNA transferase) catalyzes the alkylation of the exocyclic amine of A37 by a dimethylallyl unit in tRNAs with an adenosine in the third anticodon position (position 36) . By use of purified recombinant enzyme, steady- state kinetic studies were conducted with chemically synthesized RNA oligoribonucleotides to determine the essential elements within the tRNA anticodon stem-loop structure required for recognition by the enzyme . A 17-base oligoribonucleotide corresponding to the anticodon stem-loop of E . coli tRNA(Phe) formed a stem-loop minihelix (minihelix(Phe)) when annealed rapidly on ice, while the same molecule formed a duplex structure with a central loop when annealed slowly at higher concentrations . Both the minihelix and duplex structures gave k(cat)s similar to that for the normal substrate (full-length tRNA(Phe) unmodified at A37), although the K(m) for minihelix(Phe) was approximately 180-fold higher than full-length tRNA . The A36-A37-A38 motif, which is completely conserved in tRNAs modified by the enzyme, was found to be important for modification . Changing A36 to G in the minihelix resulted in a 260-fold reduction in k(cat) compared to minihelix(Phe) and a 13-fold increase in K(m) . An A38G variant was modified with a 9-fold reduction in k(cat) and a 5-fold increase in K(m) . A random coil 17-base oligoribonucleotide in which the loop sequence of E . coli tRNA(Phe) was preserved, but the 5 base pair helix stem was completely disrupted and showed no measurable activity, indicating that a helix-loop structure is essential for recognition . Finally, altering the identity of several base pairs in the helical stem did not have a major effect on catalytic efficiency, suggesting that the enzyme does not make base-specific contacts important for binding or catalysis in this region. Biochemistry, 2000 May 30, 39(21), 6521 - 8 Analysis of the role of interfacial tryptophan residues in controlling the topology of membrane proteins; Ridder AN et al.; Tryptophans have a high affinity for the membrane-water interface and have been suggested to play a role in determining the topology of membrane proteins . We investigated this potential role experimentally, using mutants of the single-spanning Pf3 coat protein, whose transmembrane topologies are sensitive to small changes in amino acid sequence . Mutants were constructed with varying numbers of tryptophans flanking the transmembrane region and translocation was assessed by an in vitro translation/translocation system . Translocation into Escherichia coli inner membrane vesicles could take place under a variety of experimental conditions, with co- or posttranslational assays and proton motive force-dependent or -independent mutants . It was found that translocation can even occur in pure lipid vesicles, under which conditions the tryptophans must directly interact with the lipids . However, under all these conditions tryptophans neither inhibited nor stimulated translocation, demonstrating that they do not affect topology and suggesting that this may be universal for tryptophans in membrane proteins . In contrast, we could demonstrate that lysines clearly prefer to stay on the cis-side of the membrane, in agreement with the positive-inside rule . A statistical analysis focusing on interfacially localized residues showed that in single-spanning membrane proteins lysines are indeed located on the inside, while tryptophans are preferentially localized at the outer interface . Since our experimental results show that the latter is not due to a topology-determining role, we propose instead that tryptophans fulfill a functional role as interfacially anchoring residues on the trans-side of the membrane. Biochemistry, 2000 May 30, 39(21), 6325 - 35 Structures of three inhibitor complexes provide insight into the reaction mechanism of the human methylenetetrahydrofolate dehydrogenase/cyclohydrolase; Schmidt A et al.; Enzymes involved in tetrahydrofolate metabolism are of particular pharmaceutical interest, as their function is crucial for amino acid and DNA biosynthesis . The crystal structure of the human cytosolic methylenetetrahydrofolate dehydrogenase/cyclohydrolase (DC301) domain of a trifunctional enzyme has been determined previously with a bound NADP cofactor . While the substrate binding site was identified to be localized in a deep and rather hydrophobic cleft at the interface between two protein domains, the unambiguous assignment of catalytic residues was not possible . We succeeded in determining the crystal structures of three ternary DC301/NADP/inhibitor complexes . Investigation of these structures followed by site-directed mutagenesis studies allowed identification of the amino acids involved in catalysis by both enzyme activities . The inhibitors bind close to Lys56 and Tyr52, residues of a strictly conserved motif for active sites in dehydrogenases . While Lys56 is in a good position for chemical interaction with the substrate analogue, Tyr52 was found stacking against the inhibitors' aromatic rings and hence seems to be more important for proper positioning of the ligand than for catalysis . Also, Ser49 and/or Cys147 were found to possibly act as an activator for water in the cyclohydrolase step . These and the other residues (Gln100 and Asp125), with which contacts are made, are strictly conserved in THF dehydrogenases . On the basis of structural and mutagenesis data, we propose a reaction mechanism for both activities, the dehydrogenase and the cyclohydrolase. Int Arch Allergy Immunol, 2000 Apr, 121(4), 292 - 9 Skin prick test reactivity to recombinant latex allergens; Yip L et al.; BACKGROUND: Allergy to latex has become a serious and increasingly common health problem, particularly for healthcare workers and patients who undergo frequent surgical procedures . Testing for latex allergy currently involves in vitro tests and skin prick testing using crude preparations of natural rubber latex (NRL) . To date, 10 latex proteins have received designation as allergens (Hev b 1 to Hev b 10) and, except for Hev b 4, have been cloned as recombinant proteins . Our aim was to compare the skin prick test (SPT) reactivity of six recombinant latex allergens with SPT reactivity to natural rubber latex proteins in known latex-allergic individuals . METHODS: Six recombinant proteins were expressed in Escherichia coli, and tested as the intact fusion proteins (Hev b 2, 5, 6, 8) or as purified proteins (Hev b 3 and 7) . SPT with the six recombinant latex allergens was performed using 10-fold serial dilutions on 31 latex-allergic subjects to determine the level of reactivity to each recombinant allergen . Latex-specific IgE was determined using the AlaSTAT assay . RESULTS: All six recombinant allergens were reactive by SPT in at least 1 latex-allergic patient but not in any of the control patients . The frequency of sensitization to the various recombinant allergens was similar to previous studies using the native proteins isolated from NRL . The minimal level of protein for a positive skin test was 70 pg/ml for NRL and 1 ng/ml for one recombinant allergen (Hev b 7) . In our patients, the use of a combination of recombinant latex allergens Hev b 5, 6 and 7 diagnosed latex allergy with 93% sensitivity and 100% specificity . CONCLUSION: Recombinant latex allergens are clinically reactive, can be produced in a standardized manner, and could potentially provide safe, sensitive and specific reagents for the diagnosis of latex allergy . Thromb Res, 2000 Jun 1, 98(5), 435 - 43 PLATSAK, a potent antithrombotic and fibrinolytic protein, inhibits arterial and venous thrombosis in a baboon model; van Zyl WB et al.; Antiplatelet-antithrombin-staphylokinase (PLATSAK) is a chimeric protein that was recombinantly produced in Escherichia coli cells . The protein was designed to target haemostasis at three different levels . It consists of staphylokinase for activation of fibrinolyis, the Arg-Gly-Asp sequence for the prevention of platelet aggregation, and an antithrombotic peptide for the inhibition of thrombin . The in vivo activity of PLATSAK was evaluated by assessing its effect on platelet deposition in a baboon model of arterial and venous thrombosis . Dacron vascular graft segments and expansion chambers, inserted as extensions into permanent femoral arteriovenous shunts, were used to simulate arterial and venous thrombosis, respectively . PLATSAK (3.68 mg/kg) was administered as a bolus 10 minutes before placement of the thrombogenic devices . Platelet deposition onto the graft surface and in the expansion chamber was imaged in real time with a scintillation camera as the deposition of 111In-labeled platelets . After 2 hours, platelet deposition in the graft segments and expansion chambers was inhibited by 50% and 85%, respectively, when compared to control studies . The activated partial thromboplastin time was lengthened to greater than 120 seconds . Interestingly, the level of fibrinogen degradation products in plasma did not increase after administration of PLATSAK . These results demonstrate that PLATSAK effectively inhibited platelet deposition in both arterial- and venous-type thrombosis in an animal model. Thromb Res, 2000 Jun 1, 98(5), 403 - 10 Human platelet aggregation is not altered by Shiga toxins 1 or 2; Viisoreanu D et al.; The hemolytic uremic syndrome involves the presence of Shiga toxin producing strains of Escherichia coli and is associated with thrombocytopenia, platelet activation, and microthrombi formation . We have, therefore, investigated the ability of Shiga toxin isotypes 1 and 2 to cause or enhance platelet aggregation under resting or arterial-flow conditions using a sensitive quenched-flow system and single-particle counting . Incubation of platelets with Shiga toxins 1 or 2 at 10(-10) M or 10(-9) M for 0.5-2 hours failed to induce platelet aggregation under static or physiological flow conditions, either by themselves or in the presence of ADP or thrombin . Thus, these Shiga toxins do not appear to be able to influence platelet function directly, and their ability to cause platelet thrombi in vivo must result from indirect mechanisms. FEBS Lett, 2000 May 26, 474(1), 116 - 9 Chaperone-like activity of synucleins; Souza JM et al.; Synucleins are a family of small proteins that are predominantly expressed in neurons . The functions of the synucleins are not entirely understood, but they have been implicated in the pathogenesis of several neurodegenerative diseases . Our data show that alpha-, beta- or gamma-synuclein suppresses the aggregation of thermally denatured alcohol dehydrogenase and chemically denatured insulin . The A53T but not the A30P mutant alpha-synuclein was able to inhibit the aggregation of insulin and the chaperone-like activity of alpha-synuclein was lost upon removal of its C-terminal residues 98-140 . These results demonstrate that synucleins with the exception of the A30P mutant possess chaperone-like activity. FEBS Lett, 2000 May 26, 474(1), 87 - 92 Successful mimicry of a complex viral antigen by multiple peptide insertions in a carrier protein; Feliu JX et al.; The antigenic properties of a viral peptide from the surface of foot-and-mouth disease virus particles have been successfully mimicked by multiple insertion in solvent-exposed regions of Escherichia coli beta-galactosidase . By increasing the number of viral peptides per enzyme monomer, the average IC(50) of hybrid proteins in a competitive enzyme-linked immunosorbent assay) have decreased to values close to that presented by natural virions . Moreover, the antigenic diversity of these new recombinant enzymes when measured with different anti-virus antibodies has also been largely reduced, indicating a better presentation of the epitopes located in the viral peptide . Although bivalent antibody binding could have been favoured by multiple presentation, conformational modifications of the viral peptide, due to the presence of other insertions or a cooperative antibody binding cannot be excluded . In addition, a multidimensional antigenic analysis have grouped together the multiple-inserted proteins with the native virus, suggesting that increasing the number of insertions could be a good strategy to reproduce the antigenic properties of an immunoreactive peptide in a natural multimeric disposition. FEBS Lett, 2000 May 26, 474(1), 48 - 52 14-3-3 proteins interact with a 13-lipoxygenase, but not with a 9-lipoxygenase; Holtman WL et al.; Associations between lipoxygenases (Lox) and 14-3-3 proteins were demonstrated by two different methods . First, immunoprecipitation experiments, using isoenzyme-specific monoclonal Lox antibodies, showed that 14-3-3 proteins co-precipitate with 13-Lox, but not with the 9-Lox from barley . Second, interactions between 13-Lox and 14-3-3 were established by surface plasmon resonance studies, showing that 13-Lox binds with 14-3-3 proteins in a concentration-dependent manner . The interactions between 14-3-3 proteins and 13-Lox may reveal their role during plant development. FEMS Microbiol Lett, 2000 Jun 1, 187(1), 1 - 7 Control of division gene expression in Escherichia coli; Dewar SJ et al.; Duplication of the Escherichia coli bacterial cell culminates in the formation of a division septum that splits the progenitor cell into two identical daughter cells . Invagination of the cell envelope is brought about by the co-ordinated interplay of a family of septation-specific proteins that act locally at mid-cell at a specific time in the cell cycle . The majority of the genes known to be required for septum formation are found within the large mra cluster located at 2 min on the E . coli genetic map (nucleotides 89552-107474) . Examination of the controls exerted on the mra operon shows that E . coli uses an extraordinary range of strategies to co-ordinate the expression of the cell division genes with respect to each other and to the cell cycle. Biochim Biophys Acta, 2000 Jul 3, 1475(2), 119 - 24 A silent antifungal protein (AFP)-like gene lacking two introns in the mould Trichoderma viride; Hao JJ et al.; In cells of the mould Trichoderma viride, the existence of an antifungal protein (AFP)-like gene consisting of 285 bp was confirmed by Southern analysis that genomic DNA of T . viride could hybridize with the cDNA of mature AFP of Aspergillus giganteus MDH 18894 . Except for the absence of two introns, the nucleotide sequence of the AFP-like gene was identical to that of the AFP gene of A . giganteus in positions 336-479, 568-649, and 706-765 . The AFP-like gene could not be transcribed into its mRNA in T . viride cells as examined by RT-PCR using total RNAs of T . viride as template . Furthermore, AFP could not be detected either directly from the culture medium of T . viride or by Western analysis . However, the AFP-like gene could be actively expressed like the cDNA of AFP in Escherichia coli cell . Recombinant AFP exhibited similar antifungal activity as native AFP. Biol Neonate, 2000 May, 77(4), 236 - 42 The efficacy of pentoxifylline as an anti-inflammatory agent in experimental Escherichia coli meningitis in the newborn piglet; Park WS et al.; This study was done to evaluate the anti-inflammatory effect and the ensuing neuroprotective effect of pentoxifylline in neonatal experimental bacterial meningitis . Newborn piglets were divided into three groups: 10 in the control group (CG), 13 in the meningitis group (MG), and 13 in the meningitis with pentoxifylline group (PG) . Meningitis was induced by intracisternal injection of 10(8) colony-forming units of Escherichia coli in 100 microl of saline . In PG, 20 mg/kg of pentoxifylline was given as a bolus intravenous injection 30 min before induction of meningitis and 6 mg/kg/h was given continuously throughout the experiment . In PG, the increase of CSF TNF-alpha level observed in MG was abolished . Reduced brain glucose and ATP concentrations observed in MG were significantly increased in PG . However, other parameters of inflammatory responses such as increased intracranial pressure, reduced glucose and increased lactate concentrations in the CSF observed in MG were not significantly down-modulated . The extent of CSF leukocytosis was even higher in PG than in MG . Increased cerebral cortical cell membrane lipid peroxidation products and decreased Na(+),K(+)-ATPase activity observed in MG, indicative of meningitis-induced brain cell membrane dysfunction, tended to improve without statistical significance in PG . In summary, although some anti-inflammatory effects have been observed, the overall anti-inflammatory effects of pentoxifylline was very weak, and it failed to significantly reduce the brain damage in experimental neonatal bacterial meningitis . FEMS Microbiol Lett, 2000 Jun 1, 187(1), 9 - 14 Two forms of DNA-dependent RNA polymerase alpha subunit in streptomycetes; Najmanova L et al.; We demonstrated two different DNA-dependent RNA polymerase (RNAP) alpha subunits in spores of Streptomyces granaticolor with apparent molecular masses of 40 and 43 kDa . The 43-kDa subunit was also found in vegetative cells . These two proteins are highly similar to each other as well as to other bacterial RNAP alpha subunits . The 40-kDa subunit is shortened from its C-terminus, in the portion of the protein, required for binding of DNA and transcription regulators . The gene for RNAP alpha from S . granaticolor was cloned and sequenced and the corresponding protein was overproduced in Escherichia coli . In vitro experiments using purified RNAP alpha showed that the cell free extract from spores of S . granaticolor exhibits proteolytic activity responsible for the alpha subunit shortening, whereas that from vegetative cells does not . This modification of alpha subunit might point to a novel mechanism of transcriptional control in streptomycetes. J Biol Chem, 2000 Jun 2, 275(22), 16918 - 24 Generating a high affinity scorpion toxin receptor in KcsA-Kv1.3 chimeric potassium channels; Legros C et al.; The crystal structure of the bacterial K(+) channel, KcsA (Doyle, D . A., Morais, C . J., Pfuetzner, R . A., Kuo, A., Gulbis, J . M., Cohen, S . L., Chait, B . T., and MacKinnon, R . (1998) Science 280, 69-77), and subsequent mutagenesis have revealed a high structural conservation from bacteria to human (MacKinnon, R., Cohen, S . L., Kuo, A., Lee, A., and Chait, B . T . (1998) Science 280, 106-109) . We have explored this conservation by swapping subregions of the M1-M2 linker of KcsA with those of the S5-S6 linker of the human Kv-channel Kv1.3 . The chimeric K(+) channel constructs were expressed in Escherichia coli, and their multimeric state was analyzed after purification . We used two scorpion toxins, kaliotoxin and hongotoxin 1, which bind specifically to Kv1.3, to analyze the pharmacological properties of the KcsA-Kv1.3 chimeras . The results demonstrate that the high affinity scorpion toxin receptor of Kv1.3 could be transferred to KcsA . Our biochemical studies with purified KcsA-Kv1.3 chimeras provide direct chemical evidence that a tetrameric channel structure is necessary for forming a functional scorpion toxin receptor . We have obtained KcsA-Kv1.3 chimeras with kaliotoxin affinities (IC(50) values of approximately 4 pm) like native Kv1.3 channels . Furthermore, we show that a subregion of the S5-S6 linker may be an important determinant of the pharmacological profile of K(+) channels . Using available structural information on KcsA and kaliotoxin, we have developed a structural model for the complex between KcsA-Kv1.3 chimeras and kaliotoxin to aid future pharmacological studies of K(+) channels. Blood, 2000 Jun 1, 95(11), 3396 - 402 Binding and transfer of verocytotoxin by polymorphonuclear leukocytes in hemolytic uremic syndrome; te Loo DM et al.; The hemolytic uremic syndrome (HUS) is the most common cause of acute renal failure in children . The role of a verocytotoxin (VT)-producing Escherichia coli has been strongly implicated in the epidemic form of HUS . Although direct toxicity of VT on glomerular endothelial cells has been demonstrated, it remained still unclear how the VT is transported from the intestine to the target organs . In this study we demonstrate that VT, when incubated in whole blood, binds rapidly and completely to human polymorphonuclear leukocytes (PMNs) and not to other components of blood . Binding studies with (125)I-VT-1 showed a single class of binding sites on freshly isolated, nonstimulated human PMNs . The K(d) of VT-binding to PMNs was 10(-8) mol/L, 100-fold less than that of the VT-receptor globotriaosylceramide . On incubation of VT-preloaded PMNs with human glomerular microvascular endothelial cells (GMVECs), transfer of VT-1 to the endothelial cells occurred . Incubation of nonstimulated GMVECs with VT-preloaded PMNs, but not with PMNs or VT-1 alone, caused inhibition of protein synthesis and cell death . Our data are in concert with a role of PMNs in the transfer of VT from the intestine to the kidney endothelium . This transfer occurs by selective binding to a specific receptor on PMNs and subsequent passing of the ligand VT to the VT-receptor on GMVECs, which causes cell damage . This new mechanism further underpins the important role of PMNs in HUS. J Biol Chem, 2000 Aug 18, 275(33), 25155 - 62 Dual function of the propeptide of prouroguanylin in the folding of the mature peptide: disulfide-coupled folding and dimerization; Hidaka Y et al.; Guanylyl cyclase activating peptide II (GCAP-II), an endogenous ligand of guanylyl cyclase C, is produced via the processing of the precursor protein (prepro-GCAP-II) . We have previously shown that the propeptide in pro-GCAP-II functions as an intramolecular chaperone in the proper folding of the mature peptide, GCAP-II (Hidaka, Y., Ohno, M., Hemmasi, B., Hill, O., Forssmann, W.-G., and Shimonishi, Y . (1998) Biochemistry 37, 8498-8507) . Here, we report an essential region in pro-GCAP-II for the correct disulfide pairing of the mature peptide, GCAP-II . Five mutant proteins, in which amino acid residues were sequentially deleted from the N terminus, and three mutant proteins of pro-GCAP-II, in which N-terminal 6, 11, or 17 amino acid residues were deleted, were overproduced using Escherichia coli or human kidney 293T cells, respectively . Detailed analysis of in vivo or in vitro folding of these mutant proteins revealed that one or two amino acid residues at the N terminus of pro-GCAP-II are critical, not only for the chaperone function in the folding but also for the net stabilization of pro-GCAP-II . In addition, size exclusion chromatography revealed that pro-GCAP-II exists as a dimer in solution . These data indicate that the propeptide has two roles in proper folding: the disulfide-coupled folding of the mature region and the dimerization of pro-GCAP-II. J Biol Chem, 2000 Aug 11, 275(32), 24984 - 92 HEDJ, an Hsp40 co-chaperone localized to the endoplasmic reticulum of human cells; Yu M et al.; Hsp40 co-chaperones, characterized by the presence of a highly conserved J domain, are involved in nearly all aspects of protein synthesis, folding, and secretion . Within the lumen of the endoplasmic reticulum, these chaperones are also involved in reverse translocation and degradation of misfolded proteins . We describe here the cloning and characterization of a novel Hsp40 chaperone, which we named HEDJ . Epitope-tagged HEDJ was demonstrated by confocal microscopy to be localized to the endoplasmic reticulum . Protease susceptibility, glycosidase treatment, and detergent solubility assays demonstrated that the molecule was luminally oriented and membrane-associated . In vitro experiments demonstrated that the J domain interacted with the endoplasmic reticulum-associated Hsp70, Bip, in an ATP-dependent manner and was capable of stimulating its ATPase activity . HEDJ mRNA expression was detected in all human tissues examined . Highly homologous sequences were found in mouse, Drosophila, and Caenorhabditis elegans data bases . These results suggest potential roles for HEDJ in protein import, folding, or translocation within the endoplasmic reticulum. Am J Physiol Endocrinol Metab, 2000 Jun, 278(6), E1133 - 43 Endotoxin-induced decrease in muscle protein synthesis is associated with changes in eIF2B, eIF4E, and IGF-I; Lang CH et al.; The present study examined potential mechanisms contributing to the inhibition of protein synthesis in skeletal muscle after administration of endotoxin (LPS) . Rats implanted with vascular catheters were injected intravenously with a nonlethal dose of Escherichia coli LPS, and samples were collected at 4 and 24 h thereafter; pair-fed control animals were also included . The rate of muscle (gastrocnemius) protein synthesis in vivo was reduced at both time points after LPS administration . LPS did not alter tissue RNA content, but the translational efficiency was consistently reduced at both time points . To identify mechanisms responsible for regulating translation, we examined several eukaryotic initiation factors (eIFs) . The content of eIF2alpha or the amount of eIF2alpha in the phosphorylated form did not change in response to LPS . eIF2B activity was decreased in muscle 4 h post-LPS but activity returned to control values by 24 h . A decrease in the relative amount of eIF2Balpha protein was not responsible for the LPS-induced reduction in eIF2B activity . LPS also markedly altered the distribution of eIF4E in muscle . Compared with control values, LPS-treated rats demonstrated 1) a transient increase in binding of the translation repressor 4E-binding protein-1 (4E-BP1) with eIF4E, 2) a transient decrease in the phosphorylated gamma-form of 4E-BP1, and 3) a sustained decrease in the amount of eIF4G associated with eIF4E . LPS also decreased insulin-like growth factor (IGF) I protein and mRNA expression in muscle at both times . A significant linear relationship existed between muscle IGF-I and the rate of protein synthesis or the amount of eIF4E bound to eIF4G . In summary, these data suggest that LPS impairs muscle protein synthesis, at least in part, by decreasing translational efficiency, resulting from an impairment in translation initiation associated with alterations in both eIF2B activity and eIF4E availability. Chem Biol Interact, 2000 Apr 14, 126(1), 15 - 31 Transcriptional blockages in a cell-free system by sequence-selective DNA alkylating agents; Ferguson LR et al.; There is considerable interest in DNA sequence-selective DNA-binding drugs as potential inhibitors of gene expression . Five compounds with distinctly different base pair specificities were compared in their effects on the formation and elongation of the transcription complex from the lac UV5 promoter in a cell-free system . All were tested at drug levels which killed 90% of cells in a clonogenic survival assay . Cisplatin, a selective alkylator at purine residues, inhibited transcription, decreasing the full-length transcript, and causing blockage at a number of GG or AG sequences, making it probable that intrastrand crosslinks are the blocking lesions . A cyclopropylindoline known to be an A-specific alkylator also inhibited transcription, with blocks at adenines . The aniline mustard chlorambucil, that targets primarily G but also A sequences, was also effective in blocking the formation of full-length transcripts . It produced transcription blocks either at, or one base prior to, AA or GG sequences, suggesting that intrastrand crosslinks could again be involved . The non-alkylating DNA minor groove binder Hoechst 33342 (a bisbenzimidazole) blocked formation of the full-length transcript, but without creating specific blockage sites . A bisbenzimidazole-linked aniline mustard analogue was a more effective transcription inhibitor than either chlorambucil or Hoechst 33342, with different blockage sites occurring immediately as compared with 2 h after incubation . The blockages were either immediately prior to AA or GG residues, or four to five base pairs prior to such sites, a pattern not predicted from in vitro DNA-binding studies . Minor groove DNA-binding ligands are of particular interest as inhibitors of gene expression, since they have the potential ability to bind selectively to long sequences of DNA . The results suggest that the bisbenzimidazole-linked mustard does cause alkylation and transcription blockage at novel DNA sites . in addition to sites characteristic of untargeted mustards. Exp Clin Endocrinol Diabetes, 2000, 108(2), 146 - 50 Pneumocystis carinii pneumonia associated with a rapid reduction of cortisol level in a patient with ectopic ACTH syndrome treated by octreotide and ketoconazole; Kim DS et al.; A case is herein reported of pneumocystis carinii pneumonia in a 60-year-old female patient with ectopic production of ACTH at a position 2 cm superior to her right clavicle, revealed in an octreotide scan . Her extremely high plasma ACTH and cortisol levels (460 pg/ml and 80 microg/dl, respectively) were markedly decreased with the combined treatment of octreotide (300 microg/d) and ketoconazole (600 mg/d) . As her serum cortisol concentration decreased, pneumocystis carinii pneumonia occurred on the third day of treatment . A secondary E . coli infection was superimposed and the patient died of disseminated intravascular coagulation and adult respiratory distress syndrome . This case suggests that primary prophylaxis for pneumocystis carinii infection should be initiated before cortisol lowering therapy, especially when the plasma cortisol concentration is excessively high, and that early adjunctive glucocorticoid therapy can reduce the acute mortality in patients with endogenous Cushing's syndrome and Pneumocystis carinii pneumonia . This case study would also like to point out that plasma ACTH and cortisol levels were decreased effectively by the combination of octreotide and ketoconazole in this instance of ectopic ACTH syndrome. Aviakosm Ekolog Med, 2000, 34(2), 61 - 6 {A model system for the assessment of the stability of eukaryotic genes and expressed proteins in extended space flight}; Kalinin IuT et al.; Evidence was obtained that the changed gravity, tension profiles of the magnetic fields inside the orbital station and other spaceflight factors (SFF) substantially influence the cell genome and synthesis of recombinant proteins . The authors proposed a technique of fixation of possible alterations in genes and expressed recombinant proteins in strains-producers of human alpha-interferon: HuIFN-alpha 2b, HuIFN-alpha 8a, HuIFN-alpha 10a, and HuIFN-alpha 14a . Spaceflight factors were simulated by way of gamma-irradiation at 10 Gy and 50 Gy by a cobalt unit, and centrifugation of samples at 10 g and 50 g . Cultivation of the strains-producers during the SFF simulation yielded HuIFN-alpha proteins with altered functional characteristics . Following exposure to simulated SFF, strains-producers expressed HuIFN-alpha that preserved a high titre of antiviral activity (AVA) but suppressed the antiproliferative activity (APA) inferring some structural/functional shifts in the HuIFN-alpha molecule due to, presumably, mutations in the active center responsible for APA . Determination of species specificity of the HuIFN-alpha recombinant proteins following exposure to SFF revealed dissociation of cross-AVA in homologous and heterologous cell cultures which can be also attributed to the structural/functional shifts in the HuIFN-alpha molecule . These changes can be localized in the receptor cluster of molecule or consequent to modification of the center defining HuIFN-alpha species specificity . The proposed simulation system allows fixation of shifts in the HuIFN-alpha structural/functional characteristics and investigations of the stability of eucaryotic genes in long-term space flights. Org Lett, 1999 Oct 7, 1(7), 1071 - 3 Biosynthesis of isoprenoids in Escherichia coli: stereochemistry of the reaction catalyzed by farnesyl diphosphate synthase; Leyes AE et al.; {formula: see text} Farnesyl diphosphate (FPP) synthase from Escherichia coli catalyzes the condensation of isopentenyl diphosphate (IPP) and geranyl diphosphate (GPP) with selective removal of the pro-R hydrogen at C2 of IPP, the same stereochemistry observed for the pig liver, yeast, and avian enzymes. Acta Virol, 1999 Dec, 43(6), 341 - 7 A ribozyme targeted to RNA polymerase gene of infectious bursal disease virus effectively cleaves and inhibits expression of the viral gene product; Akin A et al.; Five hammerhead-type ribozymes were designed and cloned to cleave infectious bursal disease virus (IBDV) RNA and inhibit protein synthesis from cloned full-length viral cDNA genes . Two ribozymes (R1 and R2) were directed to the large viral RNA segment gene and three ribozymes (R3, R4, and R5) were directed to the small viral RNA segment gene . Targets for the ribozymes were produced from cloned full-length coding regions of both small and large viral RNA segment genes . Ribozymes and their corresponding targets were synthesized as in vitro transcripts . Despite several attempts at different temperatures, no cleavage of viral RNA transcripts with four of the ribozymes (R1, R2, R3, and R5) was observed . One of the ribozymes, R4, was effective in cleaving the viral RNA polymerase gene transcripts in a magnesium-dependent manner . Ribozyme R4 caused 82% reduction in the synthesis of the viral RNA polymerase gene product . The inhibition was specific since there was no change in the rate of synthesis of the Escherichia coli beta-galactosidase protein . The results suggest that ribozyme R4 can be used as potential anti-IBDV agent . It was also demonstrated that the hammerhead-type ribozymes can cleave sites other than conventional GUC sequence motif as the cleavage site of ribozyme R4 had GUU motif which conformed to the NUX consensus motif. Vaccine, 2000 Jul 1, 18(26), 2970 - 8 Failure of female baboons (Papio anubis) to conceive following immunization with recombinant non-human primate zona pellucida glycoprotein-B expressed in Escherichia coli; Govind CK et al.; Progress in the development of an immunocontraceptive vaccine based on zona pellucida glycoproteins has been hampered due to observed ovarian dysfunction associated with immunization using these as immunogens . In this study four female baboons (Papio anubis) were immunized with recombinant bonnet monkey (Macaca radiata) zona pellucida glycoprotein-B (r-bmZPB) expressed in Escherichia coli and conjugated to diphtheria toxoid (DT) using Arlacel-A and Squalene as adjuvants . All the immunized animals elicited a good antibody response against r-bmZPB, continued to have ovulatory cycles and showed no disturbance in the cyclicity . In presence of high titres of circulating anti-bmZPB antibodies (>2x10(3) antibody units), the immunized animals failed to conceive following mating with males of proven fertility . Pregnancy was observed in the immunized animals subsequent to the decline in anti-r-bmZPB antibody titres . These results, though preliminary, suggest that immunization with ZPB may be used for immunocontraception without obvious ovarian dysfunction. Biochim Biophys Acta, 2000 May 23, 1478(2), 341 - 54 Dimerization of signalling modules of the EvgAS and BvgAS phosphorelay systems; Perraud AL et al.; Biophysical and biochemical properties of signalling proteins or domains derived from the unorthodox EvgAS and BvgAS two-component phosphorelay systems of Escherichia coli and Bordetella pertussis were investigated . Oligomerization of the effector proteins EvgA and BvgA and of truncated EvgS and BvgS derived signalling proteins containing the receiver and histidine containing phosphotransfer (HPt) domains or comprising only the HPt domains were characterized by native gel electrophoresis, gel permeation experiments and analytical ultracentrifugation . The results obtained by the different methods are consistent with non-phosphorylated EvgA and BvgA proteins being dimers in solution with a dissociation constant significantly below 1 microM . In contrast, all sensor derived domains of EvgS and BvgS were observed to be monomers in vitro . No indications for a phosphorylation induced stimulation of oligomerization of the C-terminal histidine kinase domains could be detected . In agreement with these data, surface plasmon resonance studies revealed a 2:1 stoichiometry in the interaction of EvgA with the immobilized EvgS HPt domain and an affinity constant of 1 . 24x10(6) M(-1). Biochim Biophys Acta, 2000 May 23, 1478(2), 318 - 24 Inhibition of serine proteinases from human blood clotting system by squash inhibitor mutants; Grzesiak A et al.; A series of six CMTI I variants mutated in the P(2)-P(4)' region of the canonical binding loop were used to probe the role of single amino acid substitutions on binding to the following human proteinases involved in blood clotting: plasmin, plasma kallikrein, factors X(a) and XII(a) . The mutants were expressed as fusion proteins with the LE1413 hydrophobic polypeptide in Escherichia coli, purified from inclusion bodies, followed by cyanobromide cleavage and refolding . The mutants inhibited the proteinases with the association constants in the range 10(3)-10(9) M(-1) . Inhibition of plasma kallikrein and factors X(a) and XII(a) could be improved up to 30-fold by single mutations . In contrast, neither of the introduced mutations increased inhibitory properties of CMTI I against plasmin . Additionally, using two inhibitors of natural origin, CMTI I (P(1) Arg) and CPTI II (P(1) Lys), we determined the effect of Lys-->Arg on binding to four proteinases . With the exception of plasmin (no effect), P(1) Arg resulted in up to 30-fold stronger binding than P(1) Lys. Biochim Biophys Acta, 2000 May 23, 1478(2), 257 - 66 Effect of ligand binding on the flexibility of dihydrofolate reductase as revealed by compressibility; Kamiyama T et al.; The partial specific volume, v, and adiabatic compressibility, beta(s), of Escherichia coli dihydrofolate reductase were measured at 30 degrees C in the presence of various ligands (folate, dihydrofolate, tetrahydrofolate, NADPH, NADP, methotrexate, and KCl) . Binding of these ligands (binary and ternary complexes) brought about large changes of v (0.734-0.754 cm(3) g(-1)) and beta(s) (6 . 6x10(-6)-9.8x10(-6) bar(-1)), keeping a linear relationship between the two parameters . The values of v and beta(s) increased with an increase in internal cavity, V(cav), and a decrease in accessible surface area, ASA, which were calculated from the X-ray crystal structures of the complexes . A large variation of V(cav) relative to ASA by ligand binding suggested that the cavity is a dominant factor and the effect of hydration might be small for the ligand-induced changes of v and beta(s) . The beta(s) values of the binary and ternary complexes suggested a characteristic conformational flexibility of the kinetic intermediates in the enzyme reaction coordinate . Comparison of beta(s) with the cavity distribution in the crystal structures revealed that the flexibility of the intermediates was mainly determined by the total cavity volume with minor contributions of the number, position, and size of cavities . These results demonstrate that the compressibility is a useful measure of the conformational flexibility of the intermediates in the enzyme reaction and that the combined study of compressibility and X-ray crystallography gives new insight into the protein dynamics through the behavior of the cavities. Biochim Biophys Acta, 2000 Jun 1, 1466(1-2), 87 - 104 Molecular dynamics study on lipid A from Escherichia coli: insights into its mechanism of biological action; Frecer V et al.; Structural properties of the Escherichia coli lipid A moiety were analysed by means of molecular mechanics and molecular dynamics simulations and compared to synthetic monophospho and dephospho analogues with different biological activities in the Limulus assay . The conformation of glucosamine disaccharide headgroup, order and packing of fatty acid chains, solvation of phosphate groups, coordination by water molecules, sodium counterions and models of cationic amino acid side chains were described in terms of mean values, mean residence times, radial distribution functions, coordination numbers, solvation and interaction energies . Solvation and polar interactions of the phosphate groups were correlated to known biological activities the lipid A variants . The observed relationship between the biological effect and the number and position of the phosphate groups were explained with the help of simple mechanistic models of lipid A action . The possible mechanism of action involving specific binding of lipid A disaccharide headgroup to cationic residues of a receptor model was compared with an alternative mechanism, which assumes a relationship between the ability to adopt non-lamellar supramolecular structures and the biological activity . Conclusions are drawn about the probable mode of lipid A action . Implications for rational drug design of endotoxin-neutralising agents are discussed. J Cell Sci, 2000 Jun, 113 ( Pt 12), 2285 - 97 Functional analysis of CLIP-115 and its binding to microtubules; Hoogenraad CC et al.; Cytoplasmic linker proteins (CLIPs) bind to microtubules and are proposed to link this cytoskeletal network to other intracellular structures . We are interested in CLIP-115, since this protein is enriched in neuronal dendrites and may operate in the control of brain-specific organelle translocations . Each CLIP monomer is characterized by two microtubule-binding (MTB) motifs, surrounded by basic, serine-rich regions . This head domain is connected to the C-terminal tail through a long coiled-coil structure . The MTB domains are conserved as a single domain in other proteins involved in microtubule based transport and dynamics, such as p150(Glued) . Here we provide evidence that efficient binding of CLIP-115 to microtubules is sensitive to phosphorylation and is not mediated by the conserved MTB domains alone, but requires the presence of the basic, serine rich regions in addition to the MTB motifs . In transfected COS-1 cells, CLIP-115 initially accumulates at the distal ends of microtubules and coincides with CLIP-170, indicating that both proteins mark growing microtubule ends . However, when expressed at higher levels, CLIP-115 and -170 affect the microtubule network differently . This might be partly due to the divergent C-termini of the two proteins . We demonstrate that, similar to CLIP-170, CLIP-115 forms homodimers, which, at least in vitro, are linked by disulfide bridges . Cysteine(391) of CLIP-115, however, is specific in that it controls the microtubule bundling capacity of certain mutant CLIP-115 molecules . Therefore, both similar and specific mechanisms appear to regulate the conformation of CLIPs as well as their binding to microtubules. J Biol Chem, 2000 Aug 4, 275(31), 23654 - 60 Molecular characterization of the yeast vacuolar H+-ATPase proton pore; Powell B et al.; The Saccharomyces cerevisiae vacuolar ATPase (V-ATPase) is composed of at least 13 polypeptides organized into two distinct domains, V(1) and V(0), that are structurally and mechanistically similar to the F(1)-F(0) domains of the F-type ATP synthases . The peripheral V(1) domain is responsible for ATP hydrolysis and is coupled to the mechanism of proton translocation . The integral V(0) domain is responsible for the translocation of protons across the membrane and is composed of five different polypeptides . Unlike the F(0) domain of the F-type ATP synthase, which contains 12 copies of a single 8-kDa proteolipid, the V-ATPase V(0) domain contains three proteolipid species, Vma3p, Vma11p, and Vma16p, with each proteolipid contributing to the mechanism of proton translocation (Hirata, R., Graham, L . A., Takatsuki, A., Stevens, T . H., and Anraku, Y . (1997) J . Biol . Chem . 272, 4795-4803) . Experiments with hemagglutinin- and c-Myc epitope-tagged copies of the proteolipids revealed that each V(0) complex contains all three species of proteolipid with only one copy each of Vma11p and Vma16p but multiple copies of Vma3p . Since the proteolipids of the V(0) complex are predicted to possess four membrane-spanning alpha-helices, twice as many as a single F-ATPase proteolipid subunit, only six V-ATPase proteolipids would be required to form a hexameric ring-like structure similar to the F(0) domain . Therefore, each V(0) complex will likely be composed of four copies of the Vma3p proteolipid in addition to Vma11p and Vma16p . Structural differences within the membrane-spanning domains of both V(0) and F(0) may account for the unique properties of the ATP-hydrolyzing V-ATPase compared with the ATP-generating F-type ATP synthase. Poult Sci, 2000 May, 79(5), 743 - 7 Effect of dietary concentration of xylitol on growth in male broiler chicks during immunological stress; Takahashi K et al.; Two experiments were conducted to determine the effect of dietary xylitol concentration on growth performance, plasma (alpha1 acid glycoprotein (AGP), nitrite, and Fe concentration in male broiler chicks during immunological stress . Ten-day-old chicks were fed a corn-soybean diet containing 15% glucose and 6% xylitol or 15% xylitol with identical metabolizable energy and crude protein content for 12 d in Experiment 1 . In Experiment 2, 12-d-old chicks were fed either the 15% glucose or 6% xylitol diet for 7 d . During the final 6 d of each experiment, half of the birds fed each diet were injected intraperitoneally with Escherichia coli lipopolysaccharide (LPS; 0127:B8) on Days 1, 3, and 5 and with Sephadex-G50 superfine on Days 2 and 4 to stimulate the immune system . The xylitol diets partially prevented reductions in body weight gain, feed intake, and feed efficiency caused by LPS and Sephadex injections, but the glucose diet did not . The injections of LPS and Sephadex increased plasma AGP and nitrite concentrations . Plasma AGP concentration on Days 2 and 6 in chicks fed the xylitol diets did not differ from that of chicks fed the glucose diet in both experiments . Nitric oxide production estimated by plasma nitrite concentration following immunological stress did not differ due to dietary treatments in Experiment 2 . The LPS and Sephadex resulted in decreased plasma Fe concentration on Day 6 in Experiment 1 in chicks fed glucose but not xylitol . These results indicate that a beneficial effect of dietary xylitol on growth is obtained with 6% xylitol given to chicks 1 d before stimulating the immune system. Poult Sci, 2000 May, 79(5), 672 - 9 The effect of vitamin D3 on resistance to stress-related infection in an experimental model of turkey osteomyelitis complex; Huff GR et al.; Male turkeys immunosuppressed by injection with dexamethasone (DEX) were given supplemental vitamin D3 in their drinking water in two experiments . In Experiment 1, vitamin D3 was supplemented at a dosage of either 2,064 IU/kg (low level) or 4,128 IU/kg (high level) in drinking water provided ad libitum only from Days 1 through 5 after hatch . In Experiment 2, vitamin D3 was provided at the low dosage for the first 5 d after hatch, followed by treatment with the high dosage for 12 h before and 12 h after each stressful event, which included weekly weighings and two DEX treatments . In both experiments, at 5 wk of age half of the birds were given intramuscular injections of 2 mg/kg DEX on 3 alternating d . In Experiment 1, 100 cfu of Escherichia coli was inoculated into the left thoracic airsac at the time of the third DEX injection . All mortalities were examined, and 10 birds per pen were necropsied 2 wk after treatment and examined for lesions of airsacculitis and turkey osteomyelitis complex (TOC) . Four birds per pen were bled before necropsy, and white blood cell total counts, differential white blood cell counts, and clinical chemistry values were determined . In Experiment 2, healthy surviving birds were grown for an additional 5-wk period, after which the DEX-treated birds were given a second series of DEX injections and were bled and necropsied 2 wk later . There were no significant effects of vitamin D3 treatment in combined general linear models analysis of Experiment 1; however, when birds not treated with DEX or E . coli were compared with those treated with both DEX and E . coli, supplementation with the low level of vitamin D3 significantly decreased TOC incidence . There were no significant effects of vitamin D3 treatment in birds treated with DEX at 5 wk of age in Experiment 2 . However, when surviving birds were given a second DEX treatment at 12 wk, vitamin D3 treatment resulted in significantly lower incidence of mortality, TOC, green liver, isolation of bacteria from tissues, and lower airsacculitis scores and heterophil-to-lymphocyte ratios than controls . Vitamin D3 also improved BW, relative weights of the liver and heart, and serum levels of glucose and alanine aminotransferase (ALT) of birds receiving two treatments with DEX . The ability of vitamin D3 supplementation to protect turkeys from the immunosuppressive effects of multiple DEX treatments emphasizes the role of vitamin D3 as a prohormone that affects health and disease resistance in turkeys. Bull Math Biol, 2000 Mar, 62(2), 247 - 92 Modeling transcriptional control in gene networks--methods, recent results, and future directions; Smolen P et al.; Mathematical models are useful for providing a framework for integrating data and gaining insights into the static and dynamic behavior of complex biological systems such as networks of interacting genes . We review the dynamic behaviors expected from model gene networks incorporating common biochemical motifs, and we compare current methods for modeling genetic networks . A common modeling technique, based on simply modeling genes as ON-OFF switches, is readily implemented and allows rapid numerical simulations . However, this method may predict dynamic solutions that do not correspond to those seen when systems are modeled with a more detailed method using ordinary differential equations . Until now, the majority of gene network modeling studies have focused on determining the types of dynamics that can be generated by common biochemical motifs such as feedback loops or protein oligomerization . For example, these elements can generate multiple stable states for gene product concentrations, state-dependent responses to stimuli, circadian rhythms and other oscillations, and optimal stimulus frequencies for maximal transcription . In the future, as new experimental techniques increase the ease of characterization of genetic networks, qualitative modeling will need to be supplanted by quantitative models for specific systems. Eur J Biochem, 2000 Jun, 267(11), 3315 - 22 Structure and chromosomal localization of human and mouse genes for hematopoietic prostaglandin D synthase . Conservation of the ancestral genomic structure of sigma-class glutathione S-transferase; Kanaoka Y et al.; Hematopoietic prostaglandin D synthase (H-PGDS) is the key enzyme for the production of the D and J series of prostanoids, and the first recognized vertebrate homolog of sigma-class glutathione S-transferase (GST) . We isolated the genes and cDNAs for human and mouse H-PGDSs . The human and mouse cDNAs contained a coding region corresponding to 199 amino-acid residues with calculated molecular masses of 23 343 and 23 226, respectively . Both H-PGDS proteins recombinantly expressed in Escherichia coli showed bifunctional activities for PGDS and GST, and had almost the same catalytic properties as the rat enzyme . Northern analyses demonstrated that the H-PGDS genes were expressed in a highly species-specific manner . Whereas the human gene was widely distributed, in contrast, the mouse gene was detected only in samples from oviduct and skin . By fluorescence in situ hybridization, the chromosomal localization of the human and mouse H-PGDS genes were mapped to 4q21-22 and 3D-E, respectively . The human and mouse H-PGDS genes spanned approximately 41 and 28 kb, respectively, and consisted of six exons divided by five introns . The exon/intron boundaries of both genes were completely identical to those of the sigma-class GST subfamily, although the amino-acid sequences of the latter were only 17.0-21.5% identical to those of either H-PGDS . These findings suggest that the H-PGDS genes evolved from the same ancestral gene as the members of the sigma-class GST family. Eur J Biochem, 2000 Jun, 267(11), 3301 - 8 Molecular cloning and characterization of murine and human N-acetylglucosamine kinase; Hinderlich S et al.; N-Acetylglucosamine is produced by the endogenous degradation of glycoconjugates and by the degradation of dietary glycoconjugates by glycosidases . It enters the pathways of aminosugar metabolism by the action of N-acetylglucosamine kinase . In this study we report the isolation and characterization of a cDNA clone encoding the murine enzyme . An open reading frame of 1029 base pairs encodes 343 amino acids with a predicted molecular mass of 37.3 kDa . The deduced amino-acid sequence contains matches of the sequences of eight peptides derived from tryptic cleavage of rat N-acetylglucosamine kinase . The recombinant murine enzyme was functionally expressed in Escherichia coli BL21 cells, where it displays N-acetylglucosamine kinase activity as well as N-acetylmannosamine kinase activity . The complete cDNA sequence of human N-acetylglucosamine kinase was derived from the nucleotide sequences of several expressed sequence tags . An open reading frame of 1032 base pairs encodes 344 amino acids and a protein with a predicted molecular mass of 37.4 kDa . Similarities between human and murine N-acetylglucosamine kinase were 86.6% on the nucleotide level and 91.6% on the amino-acid level . Amino-acid sequences of murine and human N-acetylglucosamine kinase show sequence similarities to other sugar kinases, and all five sequence motifs necessary for the binding of ATP by sugar kinases are present . Tissue distribution of murine N-acetylglucosamine kinase revealed an ubiquitous occurrence of the enzyme and a very high expression in testis . The size of the murine mRNA was 1.35 kb in all tissues investigated, with the exception of testis, where it was 1.45 kb mRNA of the murine enzyme was continuously expressed during mouse development . mRNA of the human enzyme was expressed in all investigated human tissues, as well as in cancer cell lines . In both the tissues and the cancer cell lines, the human mRNA was 1.35 kb in size. Eur J Biochem, 2000 Jun, 267(11), 3281 - 8 Interactions between the soluble domain I of nicotinamide nucleotide transhydrogenase from Rhodospirillum rubrum and transhydrogenase from Escherichia coli . Effects on catalytic and H+-pumping activities; Bizouarn T et al.; Nicotinamide nucleotide transhydrogenase from Escherichia coli is composed of two subunits, the alpha and the beta subunits, each of which contains a hydrophilic domain, domain I and III, respectively, as well as several transmembrane helices, collectively denoted domain II . The interactions between domain I from Rhodospirillum rubrum (rrI) and the intact or the protease-treated enzyme from E . coli was investigated using the separately expressed and purified domain I from R . rubrum, and His-tagged intact and trypsin-treated E . coli transhydrogenase . Despite harsh treatments with, e.g . detergents and denaturing agents, the alpha and beta subunits remained tightly associated . A monoclonal antibody directed towards the alpha subunit was strongly inhibitory, an effect that was relieved by added rrI . In addition, rrI also reactivated the trypsin-digested E . coli enzyme in which domain I had been partly removed . This suggests that the hydrophilic domains I and III are not in permanent contact but are mobile during catalysis while being anchored to domain II . Replacement of domain I of intact, as well as trypsin-digested, E . coli transhydrogenase with rrI resulted in a markedly different pH dependence of the cyclic reduction of 3-acetyl-pyridine-NAD+ by NADH in the presence of NADP(H), suggesting that the protonation of one or more protonable groups in domain I is controlling this reaction . The reverse reaction and proton pumping showed a less pronounced change in pH dependence, demonstrating the regulatory role of domain II in these reactions. Eur J Biochem, 2000 Jun, 267(11), 3270 - 80 Prolyl isomerases in a minimal cell . Catalysis of protein folding by trigger factor from Mycoplasma genitalium; Bang H et al.; Peptidyl-prolyl cis/trans isomerases (PPIases) catalyze the isomerization of prolyl peptide bonds . Distinct families of this class of enzymes are involved in protein folding in vitro, whereas their significance in free living organisms is not known . Previously, we inspected the smallest known genome of a self-replicating organism and found that Mycoplasma genitalium is devoid of all known PPIases except the trigger factor . Despite the extensive sequence information becoming available, most genes remain hypothetical and enzyme activities in many species have not been assigned to an open reading frame . Therefore, we studied the PPIase activity in crude extracts of M . genitalium . We showed that this is solely attributed to a single enzyme activity, the trigger factor . Characterization of this enzyme revealed that its PPIase activity resides in a central 12-kDa domain . Only the complete trigger factor is able to cis/trans isomerize extended peptide substrates, while the PPIase domain alone can not . The N- and the C-terminal domains of the trigger factor seem to function in binding of proteins as substrates, as demonstrated by protein refolding experiments, in which the complete trigger factor catalyzed protein refolding towards a model protein 500-fold more efficiently than the isolated central PPIase domain . Protein modeling studies suggest that the PPIase domain can fold in a similar way as the PPIase domain of FK506 binding proteins (FKBPs), one class of PPIases, despite only very limited sequence homology . Differences at the active site explain why this enzyme is not inhibited by FK506 in contrast with FKBPs . Trigger factor expressed in Escherichia coli confirms its additional chaperone functions, as shown by its association with chaperones GroEL and GroES after induction of misfolding . In contrast, the isolated PPIase-domain lacks any association with chaperones from E . coli . In summary, trigger factor of M . genitalium is the single folding isomerase of this organism, which harbors an enzymatically active PPIase domain with structural homology to FKBPs . Its additional domains confer its ability to be an efficient catalyst of protein folding . The protein folding machinery is conserved and shows a dual function as a chaperone and a prolyl isomerase. Eur J Biochem, 2000 Jun, 267(11), 3101 - 15 Site-directed mutation of noncatalytic residues of Thermobifida fusca exocellulase Cel6B; Zhang S et al.; Fifteen mutant genes in six loop residues and eight mutant genes in five conserved noncatalytic active site residues of Thermobifida fusca Cel6B were constructed, cloned and expressed in Escherichia coli or Streptomyces lividans . The mutant enzymes were assayed for catalytic activity on carboxymethyl cellulose (CMC), swollen cellulose (SC), filter paper (FP), and bacterial microcrystalline cellulose (BMCC) as well as cellotetraose, cellopentaose, and 2, 4-dinitrophenyl-beta-D-cellobioside . They were also assayed for ligand binding, enzyme processivity, thermostability, and cellobiose feedback inhibition . Two double Cys mutations that formed disulfide bonds across two tunnel forming loops were found to significantly weaken binding to ligands, lower all activities, and processivity, demonstrating that the movement of these loops is important but not essential for Cel6B function . Two single mutant enzymes, G234S and G284P, had higher activity on SC and FP, and the double mutant enzyme had threefold and twofold higher activity on these substrates, respectively . However, synergism with endocellulase T . fusca Cel5A was not increased with these mutant enzymes . All mutant enzymes with lower activity on filter paper, BMCC, and SC had lower processivity . This trend was not true for CMC, suggesting that processivity in Cel6B is a key factor in the hydrolysis of insoluble and crystalline cellulose . Three mutations (E495D, H326A and W329C) located near putative glycosyl substrate subsites -2, +1 and +2, were found to significantly increase resistance to cellobiose feedback inhibition . Both the A229V and L230C mutations specifically decreased activity on BMCC, suggesting that BMCC hydrolysis has a different rate limiting step than the other substrates . Most of the mutant enzymes had reduced thermostability although Cel6B G234S maintained wild-type thermostability . The properties of the different mutant enzymes provide insight into the catalytic mechanism of Cel6B. Proc Natl Acad Sci U S A, 2000 Jun 6, 97(12), 6415 - 20 Unpaired terminal nucleotides and 5' monophosphorylation govern 3' polyadenylation by Escherichia coli poly(A) polymerase I; Feng Y et al.; In bacteria, most mRNAs and certain regulatory RNAs are rapidly turned over, whereas mature tRNA and ribosomal RNA are highly stable . The selective susceptibility of unstable Escherichia coli RNAs to 3' polyadenylation by the pcnB gene product, poly(A) polymerase I (PAP I), in vivo is a key factor in their rapid degradation by 3' to 5' exonucleases . Using highly purified His-tagged recombinant PAP I, we show that differential adenylation of RNA substrates by PAP I occurs in vitro and that this capability resides in PAP I itself rather than in any ancillary protein(s) . Surprisingly, the efficiency of 3' polyadenylation is affected by substrate structure at both termini; single-strand segments at either the 5' or 3' end of RNA molecules and monophosphorylation at an unpaired 5' terminus dramatically increase the rate and length of 3' poly(A) tail additions by PAP I . Our results provide a mechanistic basis for the susceptibility of certain RNAs to 3' polyadenylation . They also suggest a model of "programmed" RNA decay in which endonucleolytically generated RNA fragments containing single-stranded monophosphorylated 5' termini are targeted for poly(A) addition and further degradation. Proc Natl Acad Sci U S A, 2000 Jun 6, 97(12), 6475 - 80 Mutations in the molybdenum cofactor biosynthetic protein Cnx1G from Arabidopsis thaliana define functions for molybdopterin binding, molybdenum insertion, and molybdenum cofactor stabilization; Kuper J et al.; The molybdenum cofactor (Moco), a highly conserved pterin compound coordinating molybdenum (Mo), is required for the enzymatic activities of molybdoenzymes . In all organisms studied so far Moco is synthesized by a unique and evolutionary old multistep pathway that requires the activities of at least six gene products . In eukaryotes, the last step of Moco synthesis, i.e., transfer and insertion of Mo into molybdopterin (MPT), is catalyzed by the two-domain proteins Cnx1 in plants and gephyrin in mammals . Both domains (E and G) of these proteins are able to bind MPT in vitro . Here, we show the identification and mutational dissection of functionally important regions within the Cnx1 G domain that are essential for MPT binding, the conversion of MPT to Moco, and Moco stabilization . By functional screening for mutants in the Cnx1 G domain that are no longer able to complement Escherichia coli mogA mutants, we found two classes of mutations in highly conserved amino acid residues . (i) The first class affects in vitro binding of MPT to the protein and the stabilization of Moco, the product of the G domain . (ii) The second class is represented by two independent mutations in the aspartate 515 position that is not affected in MPT binding and Moco stabilization; rather the conversion of MPT to Moco by using bound MPT and a yet unknown form of Mo is completely abolished . The results presented here provide biochemical evidence for a purified Cnx1 G domain catalyzing the insertion of Mo into MPT. Sangre (Barc), 1999 Dec, 44(6), 438 - 42 {Escherichia coli L-asparaginase induces phosphorylation of endogenous polypeptides in human immune cells}; Mercado L et al.; PURPOSE: To detect patterns of endogenous polypeptide phosphorylation in monocyte, lymphocyte, and polymorphonuclear leukocyte populations, induced by the products of the catalytic action of L-asparaginase (EcA) . MATERIALS AND METHODS: Monocytes, polymorphonuclear cells and lymphocytes were isolated from heparinized blood from healthy, voluntary donors . The samples were incubated in 0.4 mCi/ml of {gamma-32P}H3PO4, with: 1 microgram/microliter of EcA, EcA and the substrate or with the products of EcA's catalytic activity: NH4+ and aspartate . The cells were lysated and electrophoresed using denaturing polyacrylamide gels that were then exposed on radiographic plates . The levels of polypeptide phosphorylation were quantified by computer densitometric analysis . RESULTS: The autoradiographs and the densitometric quantification of the electrophoretic profiles of monocytes, polymorphonuclear leukocytes, and lymphocytes revealed an increase in polypeptide phosphorylation when the cells were incubated with the enzyme and its substrate, ammonium and aspartate, or ammonium, which demonstrates that the NH4+ triggers intracellular phosphotransferase activity . A 58 kDa phosphoprotein outstood, it being common to the three cell populations studied . There were also specific phosphorylable polypeptides in monocytes, polymorphonuclear leukocytes, and lymphocytes . CONCLUSIONS: Escherichia coli L-asparaginase, binds the plasma membrane in normal human immune cells, catalyzing the L-asparagine substrate . The products of its activity: aspartate and NH4+ modify the extracellular environment, particularly the latter since it could diffuse into the cytosol and modify t