Site-directed mutagenesis of Escherichia coli succinyl-CoA synthetase. Histidine 142 alpha is a facilitative catalytic residue

There are 11 histidine residues in Escherichia coli succinyl-CoA synthetase. His-246 alpha is well established as the phosphorylation site of the enzyme. Replacement of this histidine by asparagine (Mann, C. J., Mitchell, T., and Nishimura, J. S. (1991) Biochemistry 30, 1497-1503) or by aspartic acid (Majumdar, R., Guest, J. R., and Bridger, W. A. (1991) Biochim. Biophys. Acta 1076, 86-90) through site-directed mutagenesis resulted in complete loss of enzyme activity. Chemical modification experiments suggested a second histidine at the active site (Collier, G. E., and Nishimura, J. S. (1979) J. Biol. Chem. 254, 10925-10930). In the present study, we have changed His-142 alpha to an asparagine residue using the technique of site-directed mutagenesis and have purified the mutant enzyme to homogeneity. The resulting mutant enzyme is practically devoid of enzyme activity but can be thiophosphorylated with adenosine 5'-O-(thiotriphosphate) and dethiophosphorylated with ADP at rates that are significantly faster than those with wild type enzyme. The observation that phosphorylated mutant enzyme can be dephosphorylated with succinate and with succinate plus desulfo-CoA at rates comparable with those with wild type enzyme suggests that mutant enzyme can bind succinate and CoA. Dethiophosphorylation of the enzyme in the presence of CoA plus succinate proceeds much faster with wild type than with mutant. While there was no significant change in KCoA or Ksuccinate, the turnover number for dethiophosphorylation of the mutant was 10-fold lower. These data are consistent with location of His-142 alpha at the active site and a facilitative role for this residue in catalysis.     

Preferential utilization of glutamine for amination of xanthosine 5'-phosphate to guanosine 5'-phosphate by purified enzymes from Escherichia coli

GMP synthetase was purified 180-fold from $\text{E. coli}$ B and 18-fold from the derepressed purine auxotroph, $\text{E. coli}$ B-96. The enzymes from both sources show the same preference for glutamine over ammonia as amino donor. Each is dimeric, consisting of subunits of molecular weight about 60,000. Thus the two are apparently identical. The similarities between GMP synthetase and xanthosine 5′-phosphate aminase of $\text{E. coli}$ B-96 (N. Sakamoto, G.W. Hatfield, and H.S. Moyed, J. Biol. Chem. (1972) $\text{247}$, 5880–5887) in respect to structure, state of derepression, and behavior during purification, lead us to the conclusion that the synthetase and the aminase are a single entity. We observe no loss or separation of glutamine-dependent activity upon purification of GMP synthetase and we suggest that such loss, reported by other workers, results artifactually by inactivation of an intrinsic glutamine-binding site. GMP synthetase appears not to contain a glutamine-binding subunit which is separable from the xanthosine 5′-phosphate-aminating component.     

Expression of two succinyl-CoA synthetases with different nucleotide specificities in mammalian tissues

For nearly 50 years, succinyl-CoA synthetase in animals was thought to be specific for guanine nucleotides. Recently, we purified and characterized both an ADP-forming succinyl-CoA synthetase from pigeon breast muscle and the GDP-forming enzyme from liver (Johnson, J. D., Muhonen, W. W., and Lambeth, D. O. (1998) J. Biol. Chem. 273, 27573-27579). Using the sequences of the pigeon enzymes as queries in BLAST searches, we obtained genetic evidence that both enzymes are expressed in a wide range of animal species (Johnson, J. D., Mehus, J. G., Tews, K., Milavetz, B. I., and Lambeth, D. O. (1998) J. Biol. Chem. 273, 27580-27586). Here we extend those observations by presenting data from Western and Northern blots and enzymatic assays showing that both proteins are widely expressed in mammals with the relative amounts varying from tissue to tissue. We suggest that both succinyl-CoA synthetases catalyze the reverse reaction in the citric acid cycle in which the ADP-forming enzyme augments ATP production, whereas the GDP-forming enzyme supports GTP-dependent anabolic processes. Widely accepted shuttle mechanisms are invoked to explain how transport of P-enolpyruvate across mitochondrial membranes can transfer high energy phosphate between the cytosol and mitochondrial matrix.     

Improved statistical inference from DNA microarray data using analysis of variance and a Bayesian statistical framework. Analysis of global gene expression in Escherichia coli K12

We describe statistical methods based on the t test that can be conveniently used on high density array data to test for statistically significant differences between treatments. These t tests employ either the observed variance among replicates within treatments or a Bayesian estimate of the variance among replicates within treatments based on a prior estimate obtained from a local estimate of the standard deviation. The Bayesian prior allows statistical inference to be made from microarray data even when experiments are only replicated at nominal levels. We apply these new statistical tests to a data set that examined differential gene expression patterns in IHF(+) and IHF(-) Escherichia coli cells (Arfin, S. M., Long, A. D., Ito, E. T., Tolleri, L., Riehle, M. M., Paegle, E. S., and Hatfield, G. W. (2000) J. Biol. Chem. 275, 29672-29684). These analyses identify a more biologically reasonable set of candidate genes than those identified using statistical tests not incorporating a Bayesian prior. We also show that statistical tests based on analysis of variance and a Bayesian prior identify genes that are up- or down-regulated following an experimental manipulation more reliably than approaches based only on a t test or fold change. All the described tests are implemented in a simple-to-use web interface called Cyber-T that is located on the University of California at Irvine genomics web site.     

Reaction of substrates with 35S-thiophosphorylated succinyl-CoA synthetase of pig heart. Similarities to the case of the Escherichia coli enzyme

Guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) was found to be a substrate of pig heart succinyl-CoA synthetase with Km and kcat values of 3 microM and 0.23 s-1, respectively. The corresponding values with GTP as substrate were 48 microM and 65 s-1. 35S-thiophosphorylated enzyme was prepared by incubation of pig heart succinyl-CoA synthetase with [35S]GTP gamma S. A comparison was made of thiophosphoryl group release by substrates from this alpha beta (one active site) enzyme with that of the alpha 2 beta 2 (two active sites) Escherichia coli enzyme (Wolodko, W. T., Brownie, E. R., O'Connor, M. D., and Bridger, W. A. (1983) J. Biol. Chem. 258, 14116-14119; Nishimura, J. S., and Mitchell, T. (1984) J. Biol. Chem. 259, 9642-9645). It was found, as in the case of the E. coli enzyme, that thiophosphoryl group release by GDP and by succinate plus CoA was stimulated by succinyl-CoA and GTP, respectively. The same result was observed at 1, 0.1, and 0.01 mg/ml, lending assurance that these phenomena were not exhibited by an aggregated form of the pig heart enzyme. While an alternating-sites catalytic cooperativity model is not ruled out for the E. coli enzyme, it is proposed that the NTP- and succinyl-CoA-stimulated release of thiophosphoryl groups from either enzyme involves a "same-site" mechanism, to be distinguished from an "other-site" mechanism.     

Peptide:N-glycosidase activity found in the early embryos of Oryzias latipes (Medaka fish). The first demonstration of the occurrence of peptide:N-glycosidase in animal cells and its implication for the presence of a de-N-glycosylation system in living organisms.

The recent discovery of free oligosaccharides typical for the complex type of glycan chains terminating with a free di-N-acetylchitobiosyl structure in certain fish eggs and early embryos (Ishii, K., Iwasaki, M., Inoue, S., Kenny, P. T. M., Komura, H., and Inoue, Y. (1989) J. Biol. Chem. 264, 1623-1630; Seko, A., Kitajima, K., Iwasaki, M., Inoue, S., and Inoue, Y. (1989) J. Biol. Chem. 264, 15922-15929; Inoue, S., Iwasaki, M., Ishii, K., Kitajima, K., and Inoue, Y. (1989) J. Biol. Chem. 264, 18520-18526) led us to find an enzyme responsible for detachment of N-linked glycan chains from glycoproteins by hydrolyzing the beta-aspartyl-glucosylamine linkage in Oryzias latipes embryos. The enzyme, peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine amidase or peptide:N-glycosidase (PNGase), was partially (2090-fold) purified, and the reaction site at which this enzyme acts was specified by analysis and identification of the reaction products. This is the first demonstration showing PNGase in animal sources, although the presence of PNGases was reported in a variety of plant extracts and bacteria. Thus, the commonality of this type of enzyme is now demonstrated, and the possible physiological role of PNGase in de-N-glycosylation as a basic biologic process is proposed.     

Characterization of inhibitors acting at the synthetase site of Escherichia coli asparagine synthetase B

Asparagine synthetase catalyzes the ATP-dependent formation of L-asparagine from L-aspartate and L-glutamine, via a beta-aspartyl-AMP intermediate. Since interfering with this enzyme activity might be useful for treating leukemia and solid tumors, we have sought small-molecule inhibitors of Escherichia coli asparagine synthetase B (AS-B) as a model system for the human enzyme. Prior work showed that L-cysteine sulfinic acid competitively inhibits this enzyme by interfering with L-aspartate binding. Here, we demonstrate that cysteine sulfinic acid is also a partial substrate for E. coli asparagine synthetase, acting as a nucleophile to form the sulfur analogue of beta-aspartyl-AMP, which is subsequently hydrolyzed back to cysteine sulfinic acid and AMP in a futile cycle. While cysteine sulfinic acid did not itself constitute a clinically useful inhibitor of asparagine synthetase B, these results suggested that replacing this linkage by a more stable analogue might lead to a more potent inhibitor. A sulfoximine reported recently by Koizumi et al. as a competitive inhibitor of the ammonia-dependent E. coli asparagine synthetase A (AS-A) [Koizumi, M., Hiratake, J., Nakatsu, T., Kato, H., and Oda, J. (1999) J. Am. Chem. Soc. 121, 5799-5800] can be regarded as such a species. We found that this sulfoximine also inhibited AS-B, effectively irreversibly. Unlike either the cysteine sulfinic acid interaction with AS-B or the sulfoximine interaction with AS-A, only AS-B productively engaged in asparagine synthesis could be inactivated by the sulfoximine; free enzyme was unaffected even after extended incubation with the sulfoximine. Taken together, these results support the notion that sulfur-containing analogues of aspartate can serve as platforms for developing useful inhibitors of AS-B.     

ATP-dependent and NAD-dependent modification of glutamine synthetase from Rhodospirillum rubrum in vitro

Glutamine synthetase from the photosynthetic bacterium Rhodospirillum rubrum is the target of both ATP- and NAD-dependent modification. Incubation of R. rubrum cell supernatant with [alpha-32P]NAD results in the labeling of glutamine synthetase and two other unidentified proteins. Dinitrogenase reductase ADP-ribosyltransferase does not appear to be responsible for the modification of glutamine synthetase or the unidentified proteins. The [alpha-32P]ATP- and [alpha-32P] NAD-dependent modifications of R. rubrum glutamine synthetase appear to be exclusive and the two forms of modified glutamine synthetase are separable on two-dimensional gels. Loss of enzymatic activity by glutamine synthetase did not correlate with [alpha-32P]NAD labeling. This is in contrast to inactivation by nonphysiological ADP-ribosylation of other glutamine synthetases by an NAD:arginine ADP-ribosyltransferase from turkey erythrocytes (Moss, J., Watkins, P.A., Stanley, S.J., Purnell, M.R., and Kidwell, W.R. (1984) J. Biol. Chem. 259, 5100-5104). A 32P-labeled protein spot comigrates with the NAD-treated glutamine synthetase spot when glutamine synthetase purified from H3 32PO4-grown cells is analyzed on two-dimensional gels. The adenylylation site of R. rubrum glutamine synthetase has been determined to be Leu-(Asp)-Tyr-Leu-Pro-Pro-Glu-Glu-Leu-Met; the tyrosine residue is the site of modification.     

Purification and characterization of Chlamydomonas reinhardtii chloroplast glutamyl-tRNA synthetase, a natural misacylating enzyme

Glutamyl-tRNA synthetase from Chlamydomonas reinhardtii was purified by sequential column chromatography on DEAE-cellulose, phosphocellulose, Mono Q, and Mono S. The apparent molecular mass of the protein when analyzed under both denaturing conditions (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and nondenaturing conditions (rate zonal sedimentation on glycerol gradients) was 62,000 Da; this indicates that the active enzyme is a monomer. The purified glutamyl-tRNA synthetase was identified as the chloroplast enzyme by its tRNA charging specificity. Reversed-phase chromatography of unfractionated C. reinhardtii tRNA resolved four peaks of glutamate acceptor RNA when assayed with the purified enzyme. The enzyme can also glutamylate Escherichia coli tRNA(2Glu), but not cytoplasmic tRNA(Glu) from yeast or barley. In addition, the enzyme misacylates chloroplast tRNA(Gln) with glutamate. A similar mischarging phenomenon has been demonstrated for the barley chloroplast enzyme (Sch?n, A., Kannangara, C.G., Gough, S., and S?ll, D. (1988) Nature 331, 187-190) and for Bacillus subtilis glutamyl-tRNA synthetase (Proulx, M., Duplain, L., Lacoste, L., Yaguchi, M., and Lapointe, J. (1983) J. Biol. Chem. 258, 753-759).     

Sequence of the cloned Escherichia coli K1 CMP-N-acetylneuraminic acid synthetase gene

The Escherichia coli CMP-N-acetylneuraminic acid (CMP-NeuAc) synthetase gene is located on a 3.3-kilobase (kb) HindIII fragment of the plasmid pSR23 which contains the genes for K1 capsule production (Vann, W. F., Silver, R. P., Abeijon, C., Chang, K., Aaronson, W., Sutton, A., Finn, C. W., Lindner, W., and Kotsatos, M. (1987) J. Biol. Chem. 262, 17556-17562). The CMP-NeuAc synthetase gene expression was increased 10-30-fold by cloning of a 2.7-kb EcoRI-HindIII fragment onto the vector pKK223-3 containing the tac promoter. The complete nucleotide sequence of the gene encoding CMP-NeuAc synthetase was determined from progressive deletions generated by selective digestion of M13 clones containing the 2.7-kb fragment. CMP-NeuAc synthetase is located near the EcoRI site on this fragment as indicated by the detection of an open reading frame encoding a 49,000-dalton polypeptide. The amino- and carboxyl-terminal sequences of the encoded protein were confirmed by sequencing of peptides cleaved from both ends of the purified enzyme. The nucleotide deduced amino acid sequence was confirmed by sequencing several tryptic peptides of purified enzyme. The molecular weight is consistent with that determined from sodium dodecyl sulfate-gel electrophoresis. Gel filtration and ultracentrifugation experiments under nondenaturing conditions suggest that the enzyme is active as a 49,000-dalton monomer but may form aggregates.     

Phosphorothioate analogues of 5-phosphoribosyl 1-diphosphate: synthesis, purification, and partial characterization

The 1-phosphorothioate analogues of 5-phosphoribosyl 1-diphosphate (P-Rib-PP) have been prepared enzymatically, in reactions catalyzed by P-Rib-PP synthetase from Salmonella typhimurium. 5-Phosphoribosyl 1-O-(2-thiodiphosphate) (P-Rib-PP beta S) was synthesized from ribose 5-phosphate (Rib-5-P) and the Mg2+ complex of adenosine 5'-O-(3-thiotriphosphate). The SP and RP diastereomers of 5-phosphoribosyl 1-O-(1-thiodiphosphate) (P-Rib-PP alpha S) were synthesized from Rib-5-P and the Mg2+ complex of adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) (SP diastereomer, delta-configuration) and the Cd2+ complex of ATP beta S (RP diastereomer, delta-configuration), respectively. The strategy for the synthesis and stereochemical assignment of the P-Rib-PP alpha S diastereomers was based on the specificity of P-Rib-PP synthetase for the (delta)-beta, gamma-bidentate metal-nucleotide substrate and the stereochemical course of the synthetase reaction, leading to inversion of configuration at the P beta atom of the nucleotide [Li, T. M., Mildvan, A. S., & Switzer, R. L. (1978) J. Biol. Chem. 253, 3918-3923], and the known configurations of the Mg2+ and Cd2+ beta, gamma-bidentate complexes of the ATP beta S diastereomers [Jaffe, E. K., & Cohn, M. (1979) J. Biol. Chem. 254, 10839-10845]. The P-Rib-PP analogues were purified by gradient elution from DEAE-Sephadex and characterized by chemical analysis and 31P nuclear magnetic resonance [Smithers, G. W., & O'Sullivan, W. J. (1984) Biochemistry (following paper in this issue)]. A preliminary account of their interaction with human brain hypoxanthine phosphoribosyltransferase and yeast orotate phosphoribosyltransferase (OPRTase) is described.(ABSTRACT TRUNCATED AT 250 WORDS)     

The monomeric glutamyl-tRNA synthetase of Escherichia coli. Purification and relation between its structural and catalytic properties.

The glutamyl-tRNA synthetase has been purified to homogeneity from Escherichia coli with a yield of about 50%. It is a monomer with a molecular weight of 56,000 and has the same kinetic properties as those of the alpha chain of the dimeric alphabeta-glutamyl-tRNA synthetase described previously (Lapointe, J., and S?ll, D. (1972) J. Biol. Chem. 247, 4966-4974). It is the smallest amino-acyl-tRNA synthetase purified from E. coli and contains no important sequence repetition. It is also the only monomeric aminoacyl-tRNA synthetase reported so far to contain no major sequence duplication. Considering its structural and mechanistic similarities with the glutaminyl- and the arginyl-tRNA synthetases of E. coli, we propose the existence of a relation between the true monomeric character of the glutamyl-tRNA synthetase (as opposed to monomers with sequence duplications) and its requirement for tRNA in the activation of glutamate. A single sulfhydryl group of the native enzyme reacts with 5,5'-dithiobis(2-nitrobenzoic acid) causing no loss of enzymatic activity, whereas four such groups per enzyme react in the presence of 4 M guanidine HCl.     

Site-directed mutagenesis of Escherichia coli succinyl-CoA synthetase. beta-Cys325 is a nonessential active site residue

Chemical modification experiments have shown that sulfhydryl groups play an important role in the mechanism of action of Escherichia coli succinyl-CoA synthetase. One of these sulfhydryl groups has been localized in the beta-subunit of the enzyme using the coenzyme A affinity analog, CoA disulfide-S,S-dioxide (Collier, G. E., and Nishimura, J. S. (1978) J. Biol. Chem. 253, 4938-4943). Recently, it has been shown that the reactive sulfhydryl group resides in Cys325 (Nishimura, J. S., Mitchell, T., Ybarra, J., and Matula, J. M., submitted to Eur. J. Biochem. for publication). In the present study, we have changed Cys325 to a glycine residue using the technique of site-directed mutagenesis and have purified the mutant enzyme to homogeneity. The resulting mutant enzyme is 83% as active as wild type enzyme. In contrast to wild type succinyl-CoA synthetase, the mutant is refractory to chemical modification by CoA disulfide-S,S-dioxide and methyl methanethiolsulfonate. It is also less reactive with N-ethylmaleimide. Thus, beta-Cys325 is a nonessential active site residue.     

Purification and functional characterization of glutamate-1-semialdehyde aminotransferase from Chlamydomonas reinhardtii

The formation of delta-aminolevulinic acid, the first committed precursor of chlorophyll biosynthesis, occurs in the chloroplast of plants and algae by the C5-pathway, a three-step, tRNA-dependent transformation of glutamate. Previously, we reported the purification and characterization of the first two enzymes of this pathway, glutamyl-tRNA synthetase and Glu-tRNA reductase from the green alga Chlamydomonas reinhardtii (Chen, M.-W., Jahn, D., Sch?n, A., O'Neill, G. P., and S?ll, D. (1990) J. Biol. Chem. 265, 4054-4057 and Chen, M.-W., Jahn, D., O'Neill, G. P., and S?ll, D. (1990) J. Biol. Chem. 265, 4058-4063). Here we present the purification of the third enzyme of the pathway, the glutamate-1-semialdehyde aminotransferase from C. reinhardtii. The enzyme was purified from the membrane fraction of a whole cell extract employing four different chromatographic separations. The apparent molecular mass of the protein was approximately 43,000 Da as analyzed by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, by nondenaturing rate zonal sedimentation on glycerol gradients, and by gel filtration. By these criteria, the enzyme in its active form is a monomer of 43,000 Da. In the presence of pyridoxal 5'-phosphate, purified glutamate-1-semialdehyde aminotransferase converts synthetic glutamate 1-semialdehyde to delta-aminolevulinic acid. The enzyme is inhibited by gabaculine and aminooxyacetate, both typical inhibitors of aminotransferases. The purified glutamate-1-semialdehyde aminotransferase successfully reconstitutes the whole C5-pathway in vitro from glutamate in the presence of purified glutamyl-tRNA synthetase, glutamyl-tRNA reductase, Mg2+, ATP, NADPH, tRNA, and pyridoxal 5'-phosphate.     

The monomeric glutamyl-tRNA synthetase from Bacillus subtilis 168 and its regulatory factor. Their purification, characterization, and the study of their interaction

The glutamyl-tRNA synthetase from Bacillus subtilis has been purified to homogeneity. It is a monomer of Mr = 65,500 whose NH2-terminal sequence is Met-Asn-Glu-Val-Arg-Val-Arg-Tyr-Ser-Pro-Ser-Pro-Thr-Gly-His-Leu. The number of tryptic peptides indicates the absence of a significant amount of sequence duplication. Under certain conditions, this monomeric enzyme is co-purified with a polypeptide beta of Mr = 46,000, which increases the affinity of the enzyme about 10-fold for glutamate and for ATP, and stabilizes it against heat inactivation. gamma-Globulins prepared against the monomeric enzyme can inhibit completely the glutamyl-tRNA synthetase activity of a B. subtilis extract and precipitate from this extract both the monomeric enzyme and the regulatory factor beta. These anti-alpha immunoglobulins do nt precipitate pure beta. These results show that the glutamyl-tRNA synthetase of B. subtilis has a structure similar to that of the Escherichia coli enzyme (Lapointe, J., and S?ll, D. (1972) J. Biol. Chem. 247, 4966-4974) and indicate that the beta factor has a function in the regulation of glutamyl-tRNA biosynthesis in vivo.     

Regulation of nitrogen fixation. Nitrogenase-derepressed mutants of Klebsiella pneumoniae.

1. A new procedure is described for selecting nitrogenase-derepressed mutants based on the method of Brenchley et al. (Brenchley, J.E., Prival, M.J. and Magasanik, B. (1973) J. Biol. Chem. 248, 6122-6128) for isolating histidase-constitutive mutants of a non-N2-fixing bacterium. 2. Nitrogenase levels of the new mutants in the presence of NH4+ were as high as 100% of the nitrogenase activity detected in the absence of NH4+. 3. Biochemical characterization of these nitrogen fixation (nif) derepressed mutants reveals that they fall into three classes. Three mutants (strains SK-24, 28 and 29), requiring glutamate for growth, synthesize nitrogenase and glutamine synthetase constitutively (in the presence of NH4+). A second class of mutants (strains SK-27 and 37) requiring glutamine for growth produces derepressed levels of nitrogenase activity and synthesized catalytically inactive glutamine synthetase protein, as determined immunologically. A third class of glutamine-requiring, nitrogenase-derepressed mutants (strain SK-25 and 26) synthesizes neither a catalytically active glutamine synthetase enzyme nor an immunologically cross-reactive glutamine synthetase protein. 4. F-prime complementation analysis reveals that the mutant strains SK-25, 26, 27, 37 map in a segment of the Klebsiella chromosome corresponding to the region coding for glutamine synthetase. Since the mutant strains SK-27 and SK-37 produce inactive glutamine synthetase protein, it is concluded that these mutations map within the glutamine synthetase structural gene.     

Phosphorylation of human CTP synthetase 1 by protein kinase C: identification of Ser(462) and Thr(455) as major sites of phosphorylation

Phosphorylation of human CTP synthetase 1 by mammalian protein kinase C was examined. Using purified Escherichia coli-expressed CTP synthetase 1 as a substrate, protein kinase C activity was time- and dose-dependent and dependent on the concentrations of ATP and CTP synthetase 1. The protein kinase C phosphorylation of the recombinant enzyme was accompanied by a 95-fold increase in CTP synthetase 1 activity. Phosphopeptide mapping and phosphoamino acid analyses showed that CTP synthetase 1 was phosphorylated on multiple serine and threonine residues. The induction of PKC1(R398A)-encoded protein kinase C resulted in a 50% increase for human CTP synthetase 1 phosphorylation in the Saccharomyces cerevisiae ura7Delta ura8Delta mutant lacking yeast CTP synthetase activity. Synthetic peptides that contain the protein kinase C motif for Ser(462) and Thr(455) were substrates for mammalian protein kinase C, and S462A and T455A mutations resulted in decreases in the extent of CTP synthetase 1 phosphorylation that occurred in vivo. Phosphopeptide mapping analysis of S. cerevisiae-expressed CTP synthetase 1 mutant enzymes phosphorylated with mammalian protein kinase C confirmed that Ser(462) and Thr(455) were phosphorylation sites. The S. cerevisiae-expressed and purified S462A mutant enzyme exhibited a 2-fold reduction in CTP synthetase 1 activity, whereas the purified T455A mutant enzyme exhibited a 2-fold elevation in CTP synthetase 1 activity (Choi, M.-G., and Carman, G.M. (2006) J. Biol. Chem. 282, 5367-5377). These data indicated that protein kinase C phosphorylation at Ser(462) stimulates human CTP synthetase 1 activity, whereas phosphorylation at Thr(455) inhibits activity.     

Absence of pyridoxine- (pyridoxamine-) 5'-phosphate oxidase in Morris hepatoma 7777

Morris hepatoma 7777 previously has been shown to have no detectable pyridoxine- (pyridoxamine-) 5'-phosphate oxidase activity [Thanassi, J. W., Nutter, L. M., Meisler, N. T., Commers, P., & Chiu, J.-F. (1981) J. Biol. Chem. 256, 3370-3375]. In order to determine if this enzyme was missing in the hepatoma, we purified rat liver oxidase and raised antibodies to it in rabbits. Final purification of rat liver oxidase for use as an antigen was accomplished by affinity chromatography and gel electrophoresis. The rat liver enzyme is similar to rabbit liver oxidase [Kazarinoff, M. N., & McCormick, D. B. (1975) J. Biol. Chem. 250, 3436-3442] having two noncovalently linked subunits with molecular weights in the range of 25 000-28 000. Evidence indicating that inactive enzyme was simultaneously purified with native enzyme was obtained. The IgG fraction was purified from the serum of a rabbit that had been immunized with rat liver oxidase. This was used in the development of ELISA and immunoblot analyses for the presence of antigenically active pyridoxine- (pyridoxamine-) 5'-phosphate oxidase in cytosolic preparations from normal rat liver and Morris hepatoma 7777. The results indicated that there was no immunologically detectable oxidase protein in the tumor. An alternate pathway of pyridoxal 5'-phosphate synthesis, involving oxidation of pyridoxine to pyridoxal followed by phosphorylation, was ruled out. The implications of these findings with respect to acquisition of nutrients by tumors are discussed.