Studies on the Mechanism of Glutamine Synthesis; Isolation and Properties of the Enzyme from Sheep Brain<sup>*</sup>

Pamiljans, Vaira; Krishnaswamy, P. R.; Dumville, Georgia; Meister, Alton

doi:10.1021/bi00907a023

Cited by 202 publications

(85 citation statements)

References 15 publications

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“…Because the y-glutamyltransferase activity of the enzyme from N. europaea was protected by L-glutamine against thermal denaturation, this substrate interacts with the enzyme even in the absence of metal ions and nucleotides. These findings support the proposed random model (Wedler 1974) for the E. coli enzyme rather than sequential order of binding of the substrate in the enzyme from sheep brain (Pamiljans et al 1962). On the other hand, the failure of either ammonia or hydroxylamine to provide such protection is due either to their inability to bind to the enzyme in the absence of other substrates (Rhee et al 1976) or simply to the non-conversion of the enzyme into a more stable form.…”

Section: Discussionsupporting

confidence: 82%

Some Properties of Glutamine Synthetase From the Nitrifying Bacterium Nitrosomonas Euro Paea

Bhandari¹,

Nicholas²

1981

Aust. Jnl. Of Bio. Sci.

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Nitrosomonas europaea oxidizes ammonia to nitrite, thereby deriving energy for growth. Glutamate dehydrogenase (NADP+) (EC 1.4.1.4) is the main route for the incorporation of ammonia into glutamic acid, because glutamate synthase (NADPH) (EC 1.4.1.13) was not detected in cell-free extracts of N. europaea.Some properties of a partially purified glutamine synthetase (EC 6.3.1.2) have been determined, namely the effects of pH and metal ions, substrate requirements, Km and Ki values, based on biosynthetic and y-glutamyltransferase (EC 2.3.2.2) assays. The molecular .weight of the enzyme preparation was approximately 440000. The y-glutamlytransferase activity was markedly inhibited by alanine, lysine, glutamic acid, aspartic acid and serine and to a lesser extent by glycine, asparagine, arginine and histidine. Except for tryptophan and cystine, the y-glutamlytransferase activity was inhibited to a greater extent by these amino acids than was the biosynthetic activity. Different pairs of amino acids in various combinations resulted in a cumulative inhibition of enzyme activity determined by either method. Of the various nuc1eotides tested, the y-glutamlytransferase activity of the enzyme was inhibited to a greater extent by di-and triphosphate nuc1eotides-IDP, CDP, UDP, ITP, CTP, TTP and ATP (except GDP and GTP) than by monophosphate nuc1eotides except AMP. Saturating concentrations of pyruvate, oxalate, oxaloacetate and ",-ketoglutarate depressed enzyme activity. Various combinations of amino acids with adenine nuc1eotides exerted cumulative inhibitory effects on the transferase activity.

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Section: Discussionsupporting

confidence: 82%

Some Properties of Glutamine Synthetase From the Nitrifying Bacterium Nitrosomonas Euro Paea

Bhandari¹,

Nicholas²

1981

Aust. Jnl. Of Bio. Sci.

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“…After the reaction was stopped and neutralized as in (c), the amount of glutamate produced was measured by the DPN-glutamate dehydrogenase system (9). The results agreed with determinations of the disappearance of y-glutamylhydroxamate by the ferric chloride procedure (10). (e) Glutaminase was assayed by measurement of the formation of glutamate by the DPN-glutamate dehydrogenase system (9).…”

supporting

confidence: 59%

“…ATP was then measured by adding 0.5 ml of a solution containing 40 mM glucose, 2.5 mg of bovine serum albumin, 2 mM TPN, 100 mM Tris*HCI (pH 7.6), 1.6 units of glucose-6-phosphate dehydrogenase and 1.6 units of hexokinase; the increase in absorbance at 340 nm was determined. (d) The rate of hydrolysis of y-glutamylhydroxamate was determined in reaction mixtures (final volume, 0.3 ml) that contained 5 mM y-glutamylhydroxamate, 10 mM ATP, 10 mM MgCl2, 10 mM NaHCO3, and 50 mM potassium phosphate (pH 7.6). After the reaction was stopped and neutralized as in (c), the amount of glutamate produced was measured by the DPN-glutamate dehydrogenase system (9).…”

mentioning

confidence: 99%

Reversible Dissociation of Carbamyl Phosphate Synthetase into a Regulated Synthesis Subunit and a Subunit Required for Glutamine Utilization

Trotta

Burt

Haschemeyer

et al. 1971

Proc. Natl. Acad. Sci. U.S.A.

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153

118

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Carbamyl phosphate synthetase (from Escherichia coli) consists of a 7.3S protomeric unit that contains one heavy polypeptide chain (molecular weight about 130,000) and one light chain (molecular weight about 42,000). The heavy and light chains were separated by gel filtration in the presence of 1 M potassium thiocyanate. In contrast to the native enzyme and the reconstituted enzyme (prepared by mixing the separated heavy and light chains), the heavy chain does not catalyze glutamine-dependent carbamyl phosphate synthesis, although it does catalyze the synthesis of carbamyl phosphate from ammonia. The heavy chain also catalyzes two of the partial reactions catalyzed by the intact enzyme; i.e., the bicarbonate-dependent cleavage of ATP and the synthesis of ATP from ADP and carbamyl phosphate. Both positive (ammonia, ornithine, IMP) and negative (UMP) allosteric regulatory sites are located on the heavy chain. The only catalytic activity exhibited by the light chain is the hydrolysis of glutamine. A model is presented according to which glutamine binds to the light chain, which is followed by release of nitrogen from the amide group for use by the heavy chain. The findings suggest that glutamine-dependent carbamyl phosphate synthetase (and perhaps other glutamine amidotransferases) arose in the course of evolution by a combination of a primitive ammonia-dependent synthetic enzyme and a glutaminase; this combination may have been associated with a change from ammonia to glutamine as the principal source of nitrogen.Carbamyl phosphate, a metabolite of broad biological significance, is required for the biosynthesis of the pyrimidines and of the amino acid arginine. There are two types of carbamyl phosphate synthetases (1-3). One of these uses ammonia and requires N-acetylglutamate as a cofactor. The other can use both glutamine and ammonia and does not require N-acetylglutamate; glutamine is thought to be the "natural" substrate because it exhibits a higher affinity for the enzyme. Enzymes of the latter type resemble the other glutamine amidotransferases, which catalyze reactions in which the amide group of glutamine is used for various synthetic purposes; these enzymes can also use ammonia in place of glutamine (4).The glutamine-dependent carbamyl phosphate synthetase of Escherichia coli is subject to positive and negative feedback control by products of the purine and pyrimidine biosynthetic pathways, respectively (5), as well as by positive regulation by ornithine (6) and ammonia (unpublished It has recently been demonstrated that the enzyme can be dissociated into two kinds of polypeptide chains under extreme denaturing conditions*. We have independently made similar observationst, and now report that a relatively mild solvent perturbation promotes a reversible dissociation into nonidentical subunits. We were thus able to physically separate the two subunits and to compare their regulatory and catalytic properties with those of the recombined system and of the original enzyme.

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“…A single batch of glutamine synthetase was prepared from 400 g of acetone-extracted and powdered calf brain by the method of Pamiljans, Krishnaswamy, Dumville, and Meister (26). Purification was carried only through step 6, i.e., through differential precipitation at pH 4.8 and pH 4.2.…”

Section: Discussionmentioning

confidence: 99%

N15 Tracer Studies on the Origin of Urinary Ammonia in the Acidotic Dog, with Notes on the Enzymatic Synthesis of Labeled Glutamic Acid and Glutamines*

Pitts¹,

Pilkington²,

deHaas³

1965

J. Clin. Invest.

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Studies on the Mechanism of Glutamine Synthesis; Isolation and Properties of the Enzyme from Sheep Brain^*

Cited by 202 publications

References 15 publications

Some Properties of Glutamine Synthetase From the Nitrifying Bacterium Nitrosomonas Euro Paea

Some Properties of Glutamine Synthetase From the Nitrifying Bacterium Nitrosomonas Euro Paea

Reversible Dissociation of Carbamyl Phosphate Synthetase into a Regulated Synthesis Subunit and a Subunit Required for Glutamine Utilization

N15 Tracer Studies on the Origin of Urinary Ammonia in the Acidotic Dog, with Notes on the Enzymatic Synthesis of Labeled Glutamic Acid and Glutamines*

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Studies on the Mechanism of Glutamine Synthesis; Isolation and Properties of the Enzyme from Sheep Brain*

Cited by 202 publications

References 15 publications

Some Properties of Glutamine Synthetase From the Nitrifying Bacterium Nitrosomonas Euro Paea

Some Properties of Glutamine Synthetase From the Nitrifying Bacterium Nitrosomonas Euro Paea

Reversible Dissociation of Carbamyl Phosphate Synthetase into a Regulated Synthesis Subunit and a Subunit Required for Glutamine Utilization

N15 Tracer Studies on the Origin of Urinary Ammonia in the Acidotic Dog, with Notes on the Enzymatic Synthesis of Labeled Glutamic Acid and Glutamines*

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Studies on the Mechanism of Glutamine Synthesis; Isolation and Properties of the Enzyme from Sheep Brain^*