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J F Kane

Publications and source records attributed to J F Kane.

At least 37 records · Page 2Linked to original sources
PubMed1981

Effect of glutamine on enzymes of nitrogen metabolism in Bacillus subtilis.

An earlier study of the regulation of glutamate synthase (GOGAT) in Bacillus subtilis (Deshpande et al., Bichem. Biophys. Res. Commun. 95:55--60, 1980) revealed an inverse relationship between the specific activity of this essential ammonia-assimilatory enzyme and the intracellular pool of glutamine: GOGAT activity decreased when the internal glutamine concentration reached or exceeded 2.5 mM. This finding prompted the present investigation of the intracellular events linking glutamine formation to the regulation of GOGAT. A growing culture of B. subtilis was shifted from glutamate plus NH+4 medium (high GOGAT activity) to glutamate medium (low GOGAT activity). At various times after the shift, the intracellular concentrations of aspartate, glutamate, glutamine, alanine, and NH+4 and the activities of GOGAT and glutamine synthetase (GS) were measured. After 30 min, the only significant pool level change was an eightfold increase in glutamine, which paralleled a 2- to 3-fold increase in GS activity. Approximately 15 min after the glutamine pool reached its peak, GOGAT activity began to decrease and eventually declined 2.5-fold. In contrast, when B. subtilis was shifted from glutamate medium to glutamate plus NH+4 medium, there was a 1- to 2-h lag before the glutamine pool and GS activity approached a steady state. As a result, GOGAT activity was low until the concentration of glutamine dropped below 2.5 mM. We propose that glutamine is an important regulatory element in the control of GOGAT activity and that one form of GOGAT regulation involves enzyme inactivation. In addition, these results indicate that glutamine is neither a corepressor nor a feedback inhibitor of GS.

Bacillus subtilis↗
PubMed1981

Regulation of glutamate dehydrogenase in Bacillus subtilis.

The activity of the nicotinamide adenine dinucleotide-dependent glutamate dehydrogenase in Bacillus subtilis was influenced by the carbon source, but not the nitrogen source, in the growth medium. The highest specific activity for this enzyme was found when B. subtilis was grown in a minimal or rich medium that contained glutamate as the carbon source. It is proposed that glutamate dehydrogenase serves a catabolic function in the metabolism of glutamate, is induced by glutamate, and is subject to catabolite repression.

Alanine Dehydrogenase↗
PubMed1980

Regulation of a glutamine amidotransferase subunit from Bacillus subtilis by a cloned trpE gene from Bacillus pumilus.

The influence of a cloned trpE (trpE+p) gene from Bacillus pumilus on the expression of the gat locus in Bacillus subtilis was examined. The trpE gene was regulated by tryptophan and the mtr locus, which specifies the presumed aporepressor. The specific activity of subunit G varied directly with the level of subunit Ep, and the heterologous EpG complex that was formed was stable to gel filtration.

Anthranilate Synthase↗
PubMed1979

Increased levels of dihydrofolate reductase in rifampin-resistant mutants of Bacillus subtilis.

Several independent, spontaneous rifampin-resistant mutants of Bacillus subtilis were isolated and found to have an increased resistance to trimethoprim, an inhibitor of dihydrofolate reductase. This increased resistance in the rif mutants was the result of a specific threefold increase in the activity of dihydrofolate reductase, since six other enzymes examined remained unchanged. This increased level of dihydrofolate reductase and the trimethoprim resistance were cotransformed (100%) with the rif marker. These results suggest that the RNA polymerase is altered in its recognition of the gene that specifies dihydrofolate reductase.

4-Aminobenzoic Acid↗
PubMed1977

Regulation of a common amidotransferase subunit.

In Bacillus subtilis the trpX locus specifies a glutamine-binding protein designated subunit X, which forms a complex with subunit E to constitute the anthranilate synthase enzyme aggregate (EX) and subunit A to constitute the p-aminobenzoate synthase enzyme aggregate (AX). Subunit X confers upon these enzyme complexes the ability to utilize glutamine as a substrate. The trpX locus has been examined to determine its map position and control. (i) The trpX locus was found to be cotransformed with the lysS and pabA loci. The results of three-factor transformation analyses suggest the following order of these markers: lysS-sul-trpX-pabA. (ii) Mutation to constitutivity of the tryptophan operon resulted in a 50- to 60-fold increase in the level of subunit X when the mutant contained functional trE and abA gene products; however, in the absence of subunit E there was only a 4- to 5-fold increase in the glutamine-binding protein. (iii) Formation of subunit X was derepressed under conditions that allow for the derepression of the trpE and/or pabA loci. (iv) Subunit X synthesis was derepressed to a greater extent in mutants that contain a functional trpE gene product than in mutants that contain a nonsense mutation in the trpE locus. These results are consistent with the hypothesis that the trpE and pabA gene products affect the expression and control of the trpX locus.

4-Aminobenzoic Acid↗
PubMed1975

Anthranilate synthase from Bacillus subtilis. The role of a reduced subunit X in aggregate formation and amidotransferase activity.

With respect to its sulfhydryl groups, subunit X can exist in at least two forms, oxidized (Xox) and reduced (Xre). The importance of the Xre form for the formation of an EX complex and for amidotransferase activity has been examined. Subunit Xre is rapidly inactivated by p-chloromercuribenzoate and bromopyruvate, whereas subunit Xox, which is not catalytically functional in amidotransferase activity, is not affected. The glutamine analogue 6-diazo-5-oxo-L-norleucine (DON) has no effect on Xre alone but rapidly inactivates the EXre complex. DON-inactivated subunit X cannot be reactivated by 2-mercaptoethanol but can be readily displaced from subunit E by free subunit Xre. The integrity of the EXre complex is maintained following gel filtration on Sephadex G-100 in the presence of glutamine and 2-mercaptoethanol, thus the binding of glutamine to the complex does not require the binding of other substrates. Subunit Xox, however, does not aggregate with subunit E since no EXox complex is found following gel filtration on Sephadex G-100 in the presence of glutamine and in the absence of 2-mercaptoethanol. Thus, a reduced sulfhydryl group(s) is not only essential for amidotransferase activity but also for the formation of the aggregate as well. The following model is proposed to explain these results. Free subunit Xre does not bind DON or glutamine to the catalytically functional sulfhydryl group. Upon aggregation with subunit E, however, the glutamine or DON binds to the glutamine catalytic site on subunit Xre and amidotransfer or alkylation occurs. An EX complex which has been alkylated by DON can be readily dissociated and it is suggested that following catalysis the EX complex may also dissociate.

Amides↗
PubMed1975

Xanthosine-5'-phosphate amidotransferase from Escherichia coli.

The purified enzyme xanthosine-5'-monophosphate (XMP) aminase from Escherichia coli strain B-96 is shown to possess catalytic activity with either glutamine or ammonia as a substrate. This enzyme, which possesses identical subunits, has the following properties: (a) a pH optimum of 8.3 for both aminase and amidotransferase; (b) an apparent K-m for both glutamine and NH3 of 1 mM; (c) an amidotransferase that is approximately 2 times more active than the aminase; (d) a linear relationship between velocity and enzyme concentrationfor both activities; (e) inhibition of both activities by the glutamine analogue 6-diazo-5-oxo-L-norleucine, but the amidotransferase is more sensitive than the aminase; and (f) inhbiition of both activities by the adenosine analogue, psicofuranine, but again the amidotransferase activity is more sensitive than the aminase. The so-called XMP aminase from the E. coli mutant B-24-1 also has been examined in both crude extracts nad ammonium sulfate fractions and the following data have been obtained: (a) both preparations of enzyme contain aminase and amidotransferase activity; (b) both activities have the same substrate requirements; (c) the pH optima for both activities in the crude extract are identical with those found with the purified enzyme preparation; and (d) the amidotransferase activity in the crude extract and the ammonium sulfate fractions is 2- to 3-fold more active than the aminase. These data demonstrate that this enzyme from E. coli is not strictly a XMP aminase but is, in fact, an amidotransferase capable of utilizing either glutamine or NH3 as a substrate.

Aminohydrolases↗
PubMed1975

Multiple mutations in cysA 14 MUTANTS OF Bacillus subtilis.

Isolates of Bacillus subtilis that had been presumed to carry the cysA14 lesion have been studied. Our data indicate that these strains contain four mutations, all of which are linked by transformation and lie in the region of the ribosomal markers. The requirement for cysteine results from a defective serine transacetylase that is coded for by the cysA locus. Therefore, these mutants grow only in the presence of cysteine but not with sulfate, sulfite, or sulfide as the sole source of sulfur. A second genetic lesion (css) can be recognized by an increased sensitivity to the amino acid L-cysteine. The inhibited enzyme(s) has not been determined but inhibition is overcome by a mixture of eight amino acids. The third mutation (hts) results in the overproduction and excretion of hydrogen sulfide. This compound appears to be produced from cysteine by the enzyme cysteine desulfhydrylase and not by an increased activity of the sulfate-reductive pathway. This locus presumably codes for a regulatory element involved in the control of cysteine desulfhydrylase. The fourth mutation (cym) is not well characterized biochemically but results in a requirement for cysteine or methionine. The following order of these mutations has been established by transformation studies: hts, cysA, css, cym. The generally poor growth of these mutants in minimal-salts glucose media supplemented with cysteine can now be explained by these observations. The cysA14 mutants not only require an amino acid that is itself inhibitory to growth but they also overproduce the highly toxic compound hydrogen sulfide.

Acetyltransferases↗
PubMed1975

p-Aminobenzoate-p-aminobenzoate.

p-Aminobenzoate (PABA) synthase from Bacillus subtilis is an aggregate composed of two nonidentical subunits and has the following properties. (i) In crude extracts this enzyme catalyzes the formation of PABA in the presence of chorismate and either glutamine (amidotransferase) or ammonia (aminase). The amidotransferase activity is about 5- to 10-fold higher than the aminase activity and is stable for at least 1 week when frozen at -70 C. (II) Although no divalent cation requirement could be demonstrated with crude extracts, 2 mM ethylene-diaminetetraacetic acid completely inhibits both activities. (iii) After ammonium sulfate fractionation both the aminase and amidotransferase activities require Mg2+ and guanosine in addition to the substrates indicated above for optimal activity. The guanosine requirement can be replaced by guanosine 5'-monophosphate, guanosine 5'-diphosphate, and guanosine 5'-triphosphate but not by guanine, adenosine 5'-triphosphate, uridine 5'-triphosphate, cytidine 5'-triphosphate, thymidine 5'-triphosphate, inorganic phosphate, and phosphoribosylpyrophosphate. Furthermore, at a pH above 7.4 or below 6.4 activity is rapidly lost a 4 C, or -60 C. (IV) The enzyme is composed of two non-identical subunits, designated subunit A and subunit X. Subunit A has an estimated molecular weight of 31,000, whereas subunit X has an estimated molecular weight of 19,000. Subunit A has aminase activity but no amidotransferase activity; a mutation at the pabA locus results in the loss of PABA synthase activity. Subunit X, which is also a component of the anthranilate synthase complex, has no PABA synthase activity itself but complexes with subunit A to give an AX aggregate that can use glutamine as a substrate. (v) The molecular weight of the AX complex has been estimated at 50,000, suggesting a 1:1 ratio of subunits. (vi) The enzyme is readily associated and dissociated.

Aminobenzoates↗
PubMed1974

Homologous and hybrid complexes of anthranilate synthase from Bacillus species.

The subunits of anthranilate synthase were separated and partially purified by Sephadex G-100 gel filtration from the following six species of Bacillus: Bacillus subtilis, Bacillus licheniformis, Bacillus alvei, Bacillus coagulans, Bacillus pumilus, and Bacillus mascerans. Our data suggest that the enzyme from B. alvei is unique among these species. First, the anthranilate synthase complexes are readily dissociated during gel filtration in the absence of glutamine into a large component (aminotransferase), subunit E, and a small component subunit X (glutamine-binding protein), whereas a higher salt concentration is required to dissociate the complex from B. alvei. Second, the aminotransferase activity from all six species is stimulated by glycerol and inhibited by tryptophan; however, only the large component from B. alvei is stimulated by 2-mercaptoethanol. Finally, the large component can be titrated with the small component to yield a complex which can utilize glutamine as a substrate (amidotransferase). The homologous complexes have an amidotransferase to aminotransferase ratio of 1.4 to 2.3, but the B. alvei complex has a ratio of 0.9. Except for complexes that involve the large component from B. alvei, hybrid complexes can be formed which have ratios as good as the homologous complexes. These data are consistent with the hypothesis that B. alvei is unique among the bacilli with respect to some enzymes in the aromatic amino acid biosynthetic pathway.

Amides↗