scholarly journals The distinction between γ-glutamylhydroxamate synthetase and l-glutamine–hydroxylamine glutamyltransferase activities in rat tissues. Studies in vitro

1973 ◽  
Vol 133 (1) ◽  
pp. 49-57 ◽  
Author(s):  
Annemarie Herzfeld
Keyword(s):  
Glutamine Synthetase ◽  
Enzyme Activities ◽  
Adenine Nucleotides ◽  
Water Extract ◽  
Rat Tissues ◽  
Synthetase Activity ◽  
Specific Measure ◽  
Glutamine Synthesis ◽  
Methionine Sulphoximine

Two common ways of measuring the potential for glutamine synthesis in a tissue are the rates of formation of γ-glutamylhydroxamate either by synthesis from glutamate (the glutamylhydroxamate synthetase reaction) or by transfer from glutamine (the glutamyltransferase reaction); it has not been established, however, that either reaction is a specific measure of glutamine synthetase. By differential extraction of glutamylhydroxamate synthetase and glutamyltransferase activities from water homogenates of several rat tissues I obtained an extract, rich in glutamylhydroxamate synthetase activity but nearly devoid of glutamyltransferase activity, and a fraction, solubilized by deoxycholate from the pellet, which contained virtually no glutamylhydroxamate synthetase activity but most of the original glutamyltransferase activity. Synthesis of glutamine, quantitatively similar to the γ-glutamylhydroxamate formed by glutamylhydroxamate synthetase, is catalysed in the water extract but not in the particulate fraction. γ-Glutamylhydroxamate formation by glutamylhydroxamate synthetase and glutamyltransferase shows discrepant substrate and metal specificities and can be differentially inhibited by l-methionine sulphoximine, phosphate and adenine nucleotides. The concordance between the formation of glutamine and γ-glutamylhydroxamate by glutamylhydroxamate synthetase but not by glutamyltransferase and the different solubilities of the glutamylhydroxamate synthetase and glutamyltransferase enzyme activities demonstrate that these two activities are not inextricably associated; they therefore cannot be catalysed exclusively by the same protein.

scholarly journals Glutamine synthesis from aspartate in guinea-pig renal cortex

1990 ◽  
Vol 268 (2) ◽  
pp. 437-442 ◽  
Author(s):  
G Baverel ◽  
G Martin ◽  
C Michoudet
Keyword(s):  
Glutamine Synthetase ◽  
Guinea Pig ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Tricarboxylic Acid Cycle ◽  
Kidney Cortex ◽  
Carbon And Nitrogen ◽  
Ammonia Release ◽  
Glutamine Synthesis ◽  
Methionine Sulphoximine ◽  
Main Carbon

1. Glutamine was found to be the main carbon and nitrogen product of the metabolism of aspartate in isolated guinea-pig kidney-cortex tubules. Glutamate, ammonia and alanine were only minor products. 2. Carbon-balance calculations and the release of 14CO2 from [U-14C]aspartate indicate that oxidation of the aspartate carbon skeleton occurred. 3. A pathway involving aspartate aminotransferase, glutamate dehydrogenase, glutamine synthetase, phosphoenolpyruvate carboxykinase, pyruvate kinase, pyruvate dehydrogenase and enzymes of the tricarboxylic acid cycle is proposed for the conversion of aspartate into glutamine. 4. Evidence for this pathway was obtained by: (i) inhibiting aspartate removal by amino-oxyacetate, an inhibitor of transaminases, (ii) the use of methionine sulphoximine, an inhibitor of glutamine synthetase, which induced a large increase in ammonia release from aspartate, (iii) the use of quinolinate, an inhibitor of phosphoenolpyruvate carboxykinase, which inhibited glutamine synthesis from aspartate, (iv) the use of alpha-cyano-4-hydroxycinnamate, an inhibitor of the mitochondrial transport of pyruvate, which caused an accumulation of pyruvate from aspartate, and (v) the use of fluoroacetate, an inhibitor of aconitase, which inhibited glutamine synthesis with concomitant accumulation of citrate from aspartate.

scholarly journals Control of glutamine synthesis in rat liver

1971 ◽  
Vol 124 (3) ◽  
pp. 653-660 ◽  
Author(s):  
P Lund
Keyword(s):  
Glutamine Synthetase ◽  
De Novo ◽  
Synthetic Medium ◽  
Linear Rate ◽  
Rapid Release ◽  
Initial Release ◽  
Glutamine Synthesis ◽  
Glutamine Production

1. On perfusion of livers from fed rats with a semi-synthetic medium containing no added amino acids there is a rapid release of glutamine during the first 15min (15.6±0.8μmol/h per g wet wt.; mean±s.e.m. of 35 experiments), followed by a low linear rate of production (3.6±0.3μmol/h per g wet wt.; mean±s.e.m. of three experiments). The rapid initial release can be accounted for as wash-out of preexisting intracellular glutamine. 2. Addition of readily permeating substrates, or substrate combinations, giving rise to intracellular glutamate or ammonia, or both, did not appreciably increase the rate of glutamine production over the endogenous rate. The maximum rate obtained was from proline plus alanine; even then the rate represented less than one-fortieth of the capacity of glutamine synthetase measured in vitro. 3. Complete inhibition of respiration in the perfusions [no erythrocytes in the medium; 1mm-cyanide; N2%CO2 (95:5) in the gas phase] or perfusion with glutamine synthetase inhibitors [l-methionine dl-sulphoximine; dl-(%)-allo-δ-hydroxylysine] abolishes the low linear rate of glutamine synthesis, but not the initial rapid release of glutamine. 4. In livers from 48h-starved rats initial release (0–15min) of glutamine was decreased (10.6±1.1μmol/h per g wet wt.; mean±s.e.m. of 11 experiments) and the subsequent rate of glutamine production was lower than in livers from fed rats. Again proline plus alanine was the only substrate combination giving an increase significantly above the endogenous rate. 5. The rate of glutamine synthesis de novo by the liver is apparently unrelated to the tissue content of glutamate or ammonia. 6. The blood glutamine concentration is increased by 50% within 1h of elimination of the liver from the circulation of rats in vivo. 7. There is an output of glutamine by the brain under normal conditions; the mean arterio-venous difference for six rats was 0.023μmol/ml. 8. The high potential activity of liver glutamine synthetase appears to be inhibited by some unknown mechanism: the function of the liver under normal conditions is probably the disposal of glutamine produced by extrahepatic tissues.

scholarly journals Inhibition of glutamine synthetase activity by manganous ions in a cytosol extract of rat liver

1979 ◽  
Vol 184 (2) ◽  
pp. 477-480 ◽  
Author(s):  
S K Joseph ◽  
N M Bradford ◽  
J D McGivan
Keyword(s):  
Glutamine Synthetase ◽  
Rat Liver ◽  
Isolated Hepatocytes ◽  
Synthetase Activity ◽  
Glutamine Synthetase Activity ◽  
Perfused Liver ◽  
Glutamine Synthesis

Glutamine synthetase activity in a cytosol extract of liver was inhibited non-competitively by Mn2+ ions. The apparent Ki for Mn2” in the presence of phosphate was 8 micro M. Inhibition of glutamine synthetase by intracellular Mn2+ may contribute to the very low rates of glutamine synthesis observed in perfused liver and isolated hepatocytes.

Hormonal Regulation of Rat Mammary Gland Enzyme Activities and Metabolite Patterns

1972 ◽  
Vol 50 (4) ◽  
pp. 377-385 ◽  
Author(s):  
G. O. Korsrud ◽  
R. L. Baldwin
Keyword(s):  
Mammary Gland ◽  
Enzyme Activities ◽  
Hormonal Regulation ◽  
Adenine Nucleotides ◽  
High Energy ◽  
Glycerol Phosphate ◽  
Glucose Levels ◽  
Stage Of Lactation ◽  
Udpglucose Pyrophosphorylase

Activities of nine enzymes and the levels of 15 metabolites were determined in mammary glands from normal (N) and adrenalectomized–ovariectomized (AO) rats in midlactation. Mammary gland weights and mammary DNA did not change between day 11 and day 19 of lactation in N animals and were not affected appreciably by AO. Four days after AO the activities of citrate cleavage enzyme, malic enzyme, UDPglucose pyrophosphorylase, the A protein of the lactose synthetase complex, and glucose-6-phosphate dehydrogenase were decreased to 24, 59, 53, 52, and 56% of day 11 N. These same enzymes require cortisol therapy to increase in activity after adrenalectomy on the 5th day of lactation. Activities of phosphoglucomutase, 6-phosphogluconate dehydrogenase, fructose-1,6-diphosphate aldolase, and isocitrate dehydrogenase were not affected by AO. These enzymatic changes are discussed in relation to systems regulating milk synthesis. Mammary glucose levels increased 1.9-fold in AO animals. Levels of triose phosphate, α-glycerol phosphate, and aspartate in AO animals were decreased to 52, 49, and 62% of N. The proportion of high-energy phosphate bonds to total adenine nucleotides was not affected by AO. Ratios of [free NADP]/[free NADPH2] were not significantly affected by AO or stage of lactation. Metabolite patterns are discussed in relation to changes in enzyme activities and in vitro tracer data.

scholarly journals The distinction between γ-glutamylhydroxamate synthetase and l-glutamine–hydroxylamine glutamyltransferase activities in rat tissues. Studies in vivo

1973 ◽  
Vol 133 (1) ◽  
pp. 59-66 ◽  
Author(s):  
Annemarie Herzfeld ◽  
Nathan A. Estes
Keyword(s):  
Tissue Distribution ◽  
Enzyme Activities ◽  
Differential Response ◽  
Rat Tissues ◽  
Synthetase Activity ◽  
Assay Condition ◽  
Foetal Liver ◽  
Males And Females ◽  
Neonatal Kidney

The formation of γ-glutamylhydroxamate by homogenates under optimum assay condition showed an inconstancy in the ratios of the enzyme activities utilizing l-glutamate and ATP (γ-glutamylhydroxamate synthetase) and l-glutamine and ADP (l-glutamine–hydroxylamine glutamyltransferase) in a number of normal and neoplastic rat tissues. Although γ-glutamylhydroxamate synthetase activities in adult livers and kidneys were identical in males and females, l-glutamine–hydroxylamine glutamyltransferase activities in the organs of females were significantly lower. The developmental formations of the two activities in liver, kidney, brain and muscle were not simultaneous. The l-glutamine–hydroxylamine glutamyltransferase activity in foetal liver or neonatal kidney could be prematurely evoked by thyroxine, but the γ-glutamylhydroxamate synthetase activity remained unchanged. Injections of cortisol also had dissimilar effects on the two activities in thymus and hepatomas. The discrepant tissue distribution, asynchronous developmental formation and differential response to several hormonal stimuli provide evidence in vivo that the two activities are not catalysed by the same protein.

Glutamine metabolism in rat skeletal muscle wounded with lambda-carrageenan

1987 ◽  
Vol 252 (1) ◽  
pp. E49-E56
Author(s):  
J. E. Albina ◽  
W. Henry ◽  
P. A. King ◽  
J. Shearer ◽  
B. Mastrofrancesco ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Glutamine Synthetase ◽  
Inflammatory Infiltrate ◽  
Marked Decrease ◽  
Glutamine Metabolism ◽  
Synthetase Activity ◽  
Glutamine Synthetase Activity ◽  
Rat Skeletal Muscle ◽  
Glutamine Synthesis ◽  
Cellular Components

Wounding with lambda-carrageenan results in a marked decrease in the intracellular-free glutamine content of rat skeletal muscle. The potential mechanisms for this finding, including alterations in glutamine release, glutamine utilization, and glutamine synthesis, were investigated in rats under pentobarbital anesthesia. Wounding did not increase glutamine release from muscle during incubation or isolated hindlimb perfusion. Wounded muscle utilized more glutamine than nonwounded muscle, as measured both by the production of [14C]O2 and of -glutamate from labeled glutamine. Maximal glutamine synthetase activity was increased by wounding. The increase in glutamine synthetase activity in wounded muscle was prevented by adrenalectomy and restored by replacement doses of corticosterone in wounded adrenalectomized animals. The decrease in muscle free glutamine induced by wounding is therefore not mediated by an increase in the release of this amino acid, nor by a reduction in the tissue capacity for glutamine synthesis, but by an increase in glutamine utilization at the site of injury. This difference is apparently determined by the utilization of glutamine by the cellular components of the inflammatory infiltrate, which were shown to be capable of active glutaminolysis.

Renal ammoniagenesis and acid excretion in the dogfish, Squalus acanthias

1983 ◽  
Vol 245 (4) ◽  
pp. R581-R589 ◽  
Author(s):  
P. A. King ◽  
L. Goldstein
Keyword(s):  
Glutamine Synthetase ◽  
Acid Output ◽  
Ammonia Excretion ◽  
Squalus Acanthias ◽  
Ammonia Production ◽  
Acid Excretion ◽  
Titratable Acid ◽  
Acid Load ◽  
Glutamine Synthesis

Renal ammoniagenesis and acid excretion were investigated in normal dogfish (Squalus acanthias) and dogfish made acidotic by HCl injection (0.65 meq X kg-1). After acid loading, renal ammonia excretion doubled, rising from 0.11 to 0.25 mueq X h-1, and titratable acid output increased from 28.6 to 44.9 mueq X h-1. Trimethylamine excretion averaged 23.2 mueq X h-1 and did not change in response to the acidosis. During the first 24 h postinjection, the increase in renal acid excretion accounted for the elimination of 15% of the acid load. In vitro studies with kidney slices demonstrated that the dogfish kidney has the capacity to synthesize ammonia from a number of amino acids including glutamine, glutamate, alanine, aspartate, and glycine, with the greatest ammonia production resulting from glutamine. The relatively high glutamine concentration in the kidney, compared with the blood, suggested a high renal capacity for glutamine synthesis. In renal homogenates, enzymatic activities for both the deamidation (glutaminase) and synthesis (glutamine synthetase) of glutamine were investigated. The Michaelis constant (Km) values for the two enzymes were found to be almost equal (4.48 mM glutamine and 4.33 mM glutamate) and at the same levels as the substrate concentrations in the kidney (3.69 mM glutamine and 3.36 mM glutamate). Subcellular localization revealed that both enzymes occur predominantly in the mitochondria. The activities of glutaminase and glutamine synthetase in the renal mitochondria suggest the presence of a substrate cycle that could be modulated to increase ammonia production during acidosis.

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