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B Magasanik

Publications and source records attributed to B Magasanik.

At least 73 records · Page 4Linked to original sources
PubMed1983

Purification and properties of glutamine synthetase from Saccharomyces cerevisiae.

We have purified glutamine synthetase over 130-fold from Saccharomyces cerevisiae. The enzyme exhibits a Km for glutamate of 6.3 mM and a Km for ATP of 1.3 mM in the biosynthetic reaction, with a pH optimum from 6.1 to 7.0. Ten to twelve 43,000 molecular weight subunits comprise the active enzyme of 470,000 molecular weight. Rabbit antibodies prepared against the purified enzyme were used to show that induction of enzyme activity correlates with de novo synthesis of the enzyme subunit.

Glutamate-Ammonia Ligase↗
PubMed1983

Isolation of the nitrogen assimilation regulator NR(I), the product of the glnG gene of Escherichia coli.

The product of the glnG gene, a member of the complex glnALG operon, is an essential component in the response of Escherichia coli K-12 and other enteric bacteria to nitrogen-limited growth. We have purified this protein which we propose to call "NR(I)," for nitrogen regulator I, to about 95% purity from an overproducing strain. Purified NR(I) was identified as a dimer by gel filtration. NR(I) specifically inhibited initiation of transcription from a DNA fragment containing the glnL promoter but was without effect on lacZ transcription. We determined the intracellular concentration of NR(I) under different growth conditions by using immunological techniques. The ratio of glutamine synthetase polypeptides, the product of the glnA gene, to NR(I) polypeptides was about 80:1. NR(I) was not rapidly degraded after ammonia shock, even though the ability to activate nitrogen-controlled systems was lost.

Journal Article↗
PubMed1983

Nitrogen regulation system of Klebsiella aerogenes: the nac gene.

In Klebsiella aerogenes, the product of a his-linked gene, nac, appears to play a crucial role in tying the synthesis of enzymes activated or repressed by ammonia deprivation, such as histidase and glutamate dehydrogenase, to the known regulators of nitrogen assimilation, the products of glnG and glnF.

Ammonia↗
PubMed1983

Ammonia regulation of amino acid permeases in Saccharomyces cerevisiae.

The activities of the proline-specific permease (PUT4) and the general amino acid permease (GAP1) of Saccharomyces cerevisiae vary 70- to 140-fold in response to the nitrogen source of the growth medium. The PUT4 and GAP1 permease activities are regulated by control of synthesis and control of activity. These permeases are irreversibly inactivated by addition of ammonia or glutamine, lowering the activity to that found during steady-state growth on these nitrogen sources. Mutants altered in the regulation of the PUT4 permease (Per-) have been isolated. The mutations in these strains are pleiotropic and affect many other permeases, but have no direct effect on various cytoplasmic enzymes involved in nitrogen assimilation. In strains having one class of mutations (per1), ammonia inactivation of the PUT4 and GAP1 permeases did not occur, whereas glutamate and glutamine inactivation did. Thus, there appear to be two independent inactivation systems, one responding to ammonia and one responding to glutamate (or a metabolite of glutamate). The mutations were found to be nuclear and recessive. The inactivation systems are constitutive and do not require transport of the effector molecules per se, apparently operating on the inside of the cytoplasmic membrane. The ammonia inactivation was found not to require a functional glutamate dehydrogenase (NADP). These mutants were used to show that ammonia exerts control of arginase synthesis largely by inducer exclusion. This may be the primary mode of nitrogen regulation for most nitrogen-regulated enzymes of S. cerevisiae.

Amino Acid Transport Systems↗
PubMed1982

Complex glnA-glnL-glnG operon of Escherichia coli.

The glnG gene product is both a positive regulator and a negative regulator of the expression of glnA, the structural gene for glutamine synthetase, as well as a positive regulator of the expression of a number of genes whose products are involved in the uptake and degradation of nitrogen-containing compounds. The regulation of beta-galactosidase in various strains containing a Mu d1 (lac bla) insertion within glnG leads to the following conclusions regarding the expression of this gene: (i) like the synthesis of glutamine synthetase, the synthesis of the glnG product is regulated in response to the nitrogen source; (ii) high-level expression of glnG under nitrogen-limiting growth conditions depends on transcription initiated at the glnA promoter; and (iii) there is a second, glnA-distal promoter for glnG, whose activity is negatively controlled by the glnG product. Thus, the glnG product regulates the synthesis of the glnG product at two distinct promoters (positively and negatively at the glnA promoter and negatively at the glnA-distal promoter). In addition, a high level of glnG product, corresponding to the level produced by initiation of transcription at the glnA promoter under nitrogen-limiting conditions, is necessary for activation of histidase synthesis. The lower level of glnG product originating from transcription initiated at the glnA-distal promoter is not sufficient to activate histidase synthesis, but is sufficient to activate fully and to repress glnA expression.

Bacteriophage mu↗
PubMed1982

Characterization of a gene, glnL, the product of which is involved in the regulation of nitrogen utilization in Escherichia coli.

DNA was prepared from a strain of Escherichia coli bearing a mutation which confers the GlnC phenotype (inability to reduce the expression of glnA and other nitrogen-regulated operons in response to ammonia in the growth medium). A fragment of this DNA carrying glnA, the structural gene for glutamine synthetase, was cloned on plasmid pBR322. By using recombination in vitro, we mapped the GlnC mutation to a region between glnA and glnG. This region defines a gene, glnL, which codes for a trans-acting product; the GlnC mutant produces an altered product. The glnL product plays a key role in the communication of information concerning the quality and abundance of the nitrogen source in the growth medium to a destination responsible for the regulation of glnA and other genes for enzymes responsible for nitrogen utilization.

Ammonia↗
PubMed1982

Role of glnA-linked genes in regulation of glutamine synthetase and histidase formation in Klebsiella aerogenes.

We isolated mutants of Klebsiella aerogenes with insertions of transposon Tn5 linked to the structural gene for glutamine synthetase, glnA. We found that K. aerogenes, like Escherichia coli and Salmonella typhimurium, contains a regulatory gene, glnG, which is closely linked to but distinct from glnA. The product of glnG is essential for normal regulation of the synthesis of glutamine synthetase and histidase. We analyzed two mutations which affected the regulation of the formation of these enzymes and appeared to be outside glnG. The results of our studies suggest the presence in K. aerogenes of at least two regulatory genes located in the glnA region, namely, glnG and another gene, the transcription of which can be initiated at the promoter of glnA.

Ammonia-Lyases↗
PubMed1982

Effects of glnL and other regulatory loci on regulation of transcription of glnA-lacZ fusions in Klebsiella aerogenes.

Mutants of Klebsiella aerogenes containing genetic fusions of glnA to lacZ were isolated by using Mu dl (lac, bla) bacteriophage and a Mu Kmr helper phage with the host range of bacteriophage P1. Synthesis of beta-galactosidase in these strains is regulated in response to nitrogen metabolites and regulatory gln loci and is rendered constitutive by a mutation in the linked glnL gene. Complementation studies indicated that glnL is a separate locus from glnA and glnG and that insertions in glnA are partially polar on glnL expression. These results support the hypothesis that glnA, glnL, and glnG are organized in an operon with multiple promoters.

DNA, Recombinant↗
PubMed1982

Asparagine synthetases of Klebsiella aerogenes: properties and regulation of synthesis.

We isolated pleiotropic mutants of Klebsiella aerogenes with the transposon Tn5 which were unable to utilize a variety of poor sources of nitrogen. The mutation responsible was shown to be in the asnB gene, one of two genes coding for an asparagine synthetase. Mutations in both asnA and asnB were necessary to produce an asparagine requirement. Assays which could distinguish the two asparagine synthetase activities were developed in strains missing a high-affinity asparaginase. The asnA and asnB genes coded for ammonia-dependent and glutamine-dependent asparagine synthetases, respectively. Asparagine repressed both enzymes. When growth was nitrogen limited, the level of the ammonia-dependent enzyme was low and that of the glutamine-dependent enzyme was high. The reverse was true in a nitrogen-rich (ammonia-containing) medium. Furthermore, mutations in the glnG protein, a regulatory component of the nitrogen assimilatory system, increased the level of the ammonia-dependent enzyme. The glutamine-dependent asparagine synthetase was purified to 95%. It was a tetramer with four equal 57,000-dalton subunits and catalyzed the stoichiometric generation of asparagine, AMP, and inorganic pyrophosphate from aspartate, ATP, and glutamine. High levels of ammonium chloride (50 mM) could replace glutamine. The purified enzyme exhibited a substrate-independent glutaminase activity which was probably an artifact of purification. The tetramer could be dissociated; the monomer possessed the high ammonia-dependent activity and the glutaminase activity, but not the glutamine-dependent activity. In contrast, the purified ammonia-dependent asparagine synthetase, about 40% pure, had a molecular weight of 80,000 and is probably a dimer of identical subunits. Asparagine inhibited both enzymes. Kinetic constants and the effect of pH, substrate, and product analogs were determined. The regulation and biochemistry of the asparagine synthetases prove the hypothesis strongly suggested by the genetic and physiological evidence that a glutamine-dependent enzyme is essential for asparagine synthesis when the nitrogen source is growth rate limiting.

Ammonia↗
PubMed1981

Physical and genetic characterization of the glnA--glnG region of the Escherichia coli chromosome.

We have cloned and characterized a fragment of the Escherichia coli chromosome spanning glnA, the structural gene for glutamine synthetase (L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2]. The fragment also carriers glnG, whose product is necessary for regulation of glnA expression, and a previously unidentified gene whose function we have not discovered. Transcription of glnA and the newly identified gene occurs divergently from a region between the two genes. Transcription of glnA proceeds toward glnG, which is transcribed in the same direction. A region of DNA between glnA and glnG contains genetic information whose loss may result in the inability to reduce expression of glnA and other operons in response to ammonia (the GlnC phenotype).

Bacteriophage lambda↗
PubMed1981

Tryptophan metabolism in Klebsiella aerogenes: regulation of the utilization of aromatic amino acids as sources of nitrogen.

Klebsiella aerogenes utilized aromatic amino acids as sole sources of nitrogen but not as sole sources of carbon. K. aerogenes abstracted the alpha-amino group of these compounds by transamination and excreted the arylpyruvate portions into the medium. When tryptophan was utilized as the sole source of nitrogen by K. aerogenes, indolepyruvate was excreted into the medium, where it polymerized non-enzymatically to form a brick red pigment. At least four separate aromatic aminotransferase activities were found in K. aerogenes. One activity (aromatic aminotransferase I) appeared to be solely responsible for the aminotransferase reaction necessary for the growth of K. aerogenes when tryptophan was the source of nitrogen; the loss of this activity by mutation (tut) prevented the growth of cells on media containing this and other aromatic amino acids. None of the other aminotransferase activities in the cells could substitute for aromatic aminotransferase in this regard. Tryptophan-dependent pigment formation in K. aerogenes was positively controlled by the intracellular level of glutamine synthetase. Nevertheless, the aromatic aminotransferase activity in cells varied less than 2-fold in response to 10-fold or greater changes in the levels of glutamine synthetase. Glutamine synthetase affected the ability of the cells to take up tryptophan from the medium.

Amino Acids↗
PubMed1981

Purification and properties of aromatic amino acid aminotransferase from Klebsiella aerogenes.

We describe the complete purification of aromatic aminotransferase I, the enzyme responsible for the ability of Klebsiella aerogenes to use tryptophan and phenylalanine as sole sources of nitrogen, as well as the partial purification of aromatic aminotransferase IV. An examination of the properties of these enzymes revealed that aminotransferase I had much greater affinity for the aromatic amino acids than aminotransferase IV, explaining the essential role of aminotransferase I in the utilization of exogenously supplied aromatic amino acids. The properties of aminotransferase IV suggest that this enzyme is actually an aspartate aminotransferase (EC 2.6.1.1), corresponding to the product of the aspC gene of Escherichia coli.

Amino Acids↗
PubMed1981

Physical maps of Klebsiella aerogenes and Salmonella typhimurium hut genes.

The recognition sites for several restriction endonucleases were mapped within deoxyribonucleic acid coding for histidine utilization (hut) genes of Salmonella typhimurium and Klebsiella aerogenes. Deoxyribonucleic acid fragments containing the two hut promoters were identified by ribonucleic acid polymerase binding.

DNA Restriction Enzymes↗
PubMed1981

Subcellular compartmentation in control of converging pathways for proline and arginine metabolism in Saccharomyces cerevisiae.

Enzymes of proline biosynthesis and proline degradation which act on the same compound, delta 1-pyrroline-5-carboxylate, are physically separated in yeast cells. The enzyme responsible for the final step in proline biosynthesis, pyrroline-5-carboxylate reductase, converts pyrroline-5-carboxylate to proline and is located in the cytoplasm. The last enzyme in the proline degradative pathway, pyrroline-5-carboxylate dehydrogenase, converts pyrroline-5-carboxylate to glutamate and is found in the particulate fraction of the cell, presumably in the mitochondrion. By subcellular compartmentation, yeast cells avoid futile cycling between proline and pyrroline-5-carboxylate.

1-Pyrroline-5-Carboxylate Dehydrogenase↗
PubMed1981

Serine utilization by Klebsiella aerogenes.

Klebsiella aerogenes was found to contain a specific L-serine dehydrase that was induced by threonine, glycine or leucine, but not by its substrate. Cellular concentrations were sensitive to carbon rather than nitrogen sources in the growth medium. A nonspecific isoleucine-sensitive L-threonine dehydrase supplemented the specific L-serine dehydrase activity. K. aerogenes also contains a leucine-inducible L-threonine dehydrogenase which probably initiated a threonine-utilization pathway in which the serine-specific dehydrate participated. Strains that were altered in their ability to metabolize serine differed in either L-serine dehydrase or L-threonine dehydrase activity. Thus, K. aerogenes growing on L-serine as a sole nitrogen source relies upon two enzymes that metabolize the amino acid as subsidiary functions.

Enzyme Induction↗