Deamination Of Glutamine Research Articles

Inhibitor of protein degradation formed during incubation of isolated rat hepatocytes in a cell culture medium: Its identification as ammonia

Isolated rat hepatocytes, incubated as a suspension in a tissue culture medium (Dulbecco's), are in negative nitrogen balance and exhibit a continuous net release of amino acids and/or urea. The cells also generate ammonia and/or urea by the deamination of medium glutamine. Under normoxic conditions, ammonia is rapidly converted to urea, but under hypoxic conditions, arising, e.g., if the cells are allowed to sediment, ammonia accumulates. By interference with the analytical procedures and the calculation of nitrogen balance, the accumulation of ammonia may give a false impression of cellular hyper-catabolic activity during the first hour of incubation. When this effect has subsided (or been corrected for), it becomes evident that the accumulated ammonia exerts an inhibitory influence on protein degradation, reducing both the total nitrogen release and the liberation of [ 14C]valine from pre-labelled liver cell protein. The fact that protein degradation is inhibited by ammonia may be of considerable importance for the growth of cells under tissue culture conditions, where ammonia is so readily generated by the spontaneous or metabolic deamination of glutamine.

Regulation of Renal Glutamine Deamination

Regulation of Renal Glutamine Deamination

Effects of glutamine deamination on glutamine deamidation in rat kidney slices.

Glutamate is known to inhibit the activity of isolated glutaminase I; however, its actual physiologic importance in regulating renal ammoniagenesis has not been established. To determine the regulatory role of glutamate on the metabolism of glutamine by rat kidney slices, we followed the effects on glutamine (2 mM) deamidation of increased removal of glutamate via augmented deamination. Three agents (malonate, 2,4-dinitrophenol, and methylene blue) were known to and shown here to hasten exogenous glutamate deamination. In slices from 10 control rats, 21.5+/-1.7 (SEM) mumol/g of ammonia were formed from amide nitrogen and 9.3+/-0.5 (SEM) mumol/g from the amino nitrogen of glutamine in vitro. Over 90% of the glutamine deamidated formed glutamate at one point in its catabolism. After addition of malonate (10 mM), 2,4-dinitrophenol (0.1 mM), or methylene blue (0.5 mM), the production of ammonia from the amino group rose to 29.3+/-6.0 (SEM) mumol/g, 20.0+/-1.8 (SEM) mumol/g, and 15.5+/-4.2 (SEM) mumol/g, respectively; ammonia production from the amide nitrogen rose also, 45.1+/-7.3 (SEM) mumol/g, 39.7+/-2.6 (SEM) mumol/g, and 41.9+/-3.7 (SEM) mumol/g. In the case of the former two, a minimum of 99% and 75% of the glutamine catabolized formed glutamate. Despite increased glutamine catabolism, there was no build up of glutamate in the media. A correlation between the formation of ammonia from the amino and amide nitrogen was apparent. Since none of the three agents selected affected phosphate activated glutaminase I activity directly or appeared to affect glutamine transport, we interpret the increase in deamidation as an expression of deinhibition of glutaminase I activity secondary to lowered glutamate concentrations at the deamidating sites through more rapid removal of glutamate via hastened deamination. Interestingly, slices removed from acidotic rats produced more ammonia from both the amino 29.1+/-3.8 (SEM) and amide nitrogens 45.9+/-4.3 (SEM) of glutamine, without a buildup of glutamate in the medium. At least 90% of the glutamine deamidated formed glutamate. A common mechanism is proposed to explain these results and the previous ones.

Effects of ischaemia on metabolite concentrations in rat liver.

1. Changes in the concentrations of ammonia, glutamine, glutamate, 2-oxoglutarate, 3-hydroxybutyrate, acetoacetate, alanine, aspartate, malate, lactate, pyruvate, NAD(+), NADH and adenine nucleotides were measured in freeze-clamped rat liver during ischaemia. 2. Although the concentrations of most of the metabolites changed rapidly during ischaemia the ratios [glutamate]/[2-oxoglutarate][NH(4) (+)] and [3-hydroxybutyrate]/[acetoacetate] changed equally and the value of the expression [3-hydroxybutyrate][2-oxoglutarate][NH(4) (+)]/[acetoacetate][glutamate] remained approximately constant, indicating that the 3-hydroxybutyrate dehydrogenase and glutamate dehydrogenase systems were at near-equilibrium with the mitochondrial NAD(+) couple. 3. The value of the expression [alanine][oxoglutarate]/[pyruvate][glutamate] was about 0.7 in vivo and remained fairly constant during the ischaemic period of 5min, although the concentrations of alanine and oxoglutarate changed substantially. No explanation can be offered why the value of the ratio differed from that of the equilibrium constant of the alanine aminotransferase reaction, which is 1.48. 4. Injection of l-cycloserine 60min before the rats were killed increased the concentration of alanine in the liver fourfold and decreased the concentration of the other metabolites measured, except that of pyruvate. During ischaemia the concentration of alanine did not change but that of aspartate almost doubled. 5. After treatment with l-cycloserine the value in vivo of the expression [alanine][oxoglutarate]/[pyruvate][glutamate] rose from 0.7 to 2.4. During ischaemia the value returned to 0.8. 6. The effects of l-cycloserine are consistent with the assumption that it specifically inhibits alanine aminotransferase. 7. Most of the alanine formed during ischaemia is probably derived from pyruvate and from ammonia released by the deamination of adenine nucleotides and glutamine. The alanine is presumably formed by the combined action of glutamate dehydrogenase and alanine aminotransferase. 8. The rate of anaerobic glycolysis, calculated from the increase in the lactate concentration, was 1.3mumol/min per g fresh wt. 9. Although the concentrations of the adenine nucleotides changed rapidly during ischaemia, the ratio [ATP][AMP]/[ADP](2) remained constant at 0.54, indicating that adenylate kinase established near-equilibrium under these conditions.

Small intestine glutaminase deficiency in celiac disease.

Human small intestine contains glutaminase I, an enzyme responsible for the deamination of glutamine. Glutaminase activity was assayed in the jejunal mucosa of patients with celiac disease and of control subjects. Enzymatic activity was markedly diminished in the jejunal biopsy specimens from these patients at the time the diagnosis of celiac disease was made, compared with glutaminase I levels from control subjects. With successful treatment on a gluten-free diet, small-intestinal glutaminase I values returned toward normal as the biopsy specimens improved histologically. Patients with celiac disease who continued to have some symptoms had biopsy evidence of moderately severe mucosal damage, and they maintained low enzyme levels. The patients with abnormal biopsies due to other small-bowel diseases also had diminished glutaminase I activity. This study suggests that patients with untreated celiac disease have a secondary deficency of this enzyme.

Diffusion equilibrium for ammonia in the kidney of the acidotic dog.

Inflow of preformed ammonia in arterial blood, renal production of ammonia, outflow of ammonia in renal venous blood, and urinary excretion of ammonia were measured during the infusion of (15)NH(4)Cl into one renal artery of dogs with chronic metabolic acidosis. Our results show that the specific activity of ammonia measured in the urine and that calculated in the renal pool agree within 95%. Pool specific activity is obtained by dividing the rate of infusion of isotope by the pool turnover rate, i.e., the sum of the rate of ammonia output in the urine and that in renal venous blood. An average of 35% of urinary ammonia is derived from arterial ammonia in these experiments. We conclude that ammonia is distributed evenly throughout all phases of the kidney within a period less than the transit time of blood through the kidney. Furthermore, from the proportion of urinary ammonia we found to be derived from preformed arterial ammonia (35%), and from our previous demonstration that 73% of urinary ammonia derives from deamidation and/or deamination of plasma glutamine, alanine, glycine, and glutamate, we can account for all of the ammonia that leaves the kidney in renal venous blood and in urine.

Pathways of glutamine deamination and their control in the rat kidney.

Pathways of glutamine deamination and their control in the rat kidney.

Kinetics of ammonia production and excretion in the acidotic dog.

The Chinard technique of rapid injection into one renal artery of a glomerular marker (creatinine) and a compound whose utilization or excretion is under investigation has been employed in a study of the time courses of production and excretion of ammonia. Following the injection of N15H4 Cl or amide-N15-labeled glutamine, the label appeared in the urinary ammonia within a partially corrected time interval of 11 sec or less, suggesting that, in both instances, the labeled ammonia entered the urine as far along the nephron as the convoluted portion of distal tubules and cortical collecting ducts. Creatinine appeared in the urine after a delay of 60–100 sec. Following injections of amino-N15-labeled glutamine, appearance of label in urinary ammonia was delayed about 20 sec relative to that observed following injection of amide-N15-labeled glutamine. The time course of disappearance of label from the urine suggests that deamidation of glutamine is not rate-limiting for ammonia production and excretion, whereas deamination of glutamine may well be.

Source of ammonia excreted by the gills of the marine teleost, Myoxocephalus scorpius.

Measurement of ammonia concentration differences in blood entering and leaving gills showed that only about 12% of branchially excreted ammonia could be accounted for by the disappearance of blood ammonia. Gill tissue homogenates contained significant glutaminase I and glutamic acid dehydrogenase activities. The combined activity of both enzymes was more than sufficient to account for the branchially excreted ammonia. Furthermore, there was a significant deamination of blood glutamine by gill tissue. Thus, it is likely that the ammonotelism of teleosts is due to the enzymatic deamination of glutamine and other amino acids in the gill.

642. The steric course of lactonisation following the deamination of glutamic acid, glutamine, and γ-aminovaleric acid

642. The steric course of lactonisation following the deamination of glutamic acid, glutamine, and γ-aminovaleric acid