Richard A. Freedland

The effect of glucagon on ureagenesis from ammonia by isolated rat hepatocytes

Abstract Glucagon, at a maximally effective concentration of 1 μM, stimulated by 35% the rate at which rat hepatocytes synthesized urea from 10 mM NH 4 Cl in the presence of 10 mM ornithine. The rate at which citrulline accumulated in the incubations was relatively unchanged by the presence of glucagon. Mitochondria isolated from glucagon treated hepatocytes were observed to synthesize citrulline from 10 mM NH 4 Cl and 10 mM ornithine more rapidly than did mitochondria isolated from untreated hepatocytes. The role of the intracellular malate concentration in the regulation of the rate of urea synthesis, and the changes observed in the cellular content of malate in response to glucagon are discussed.

Arginine uptake by isolated rat liver mitochondria

The question of arginine uptake by mitochondria is important in that arginine is an allosteric effector of N-acetylglutamate synthetase. Thus, changes in mitochondrial arginine concentration have the potential for acutely modifying levels of N-acetylglutamate, a compound necessary for maximal activity of carbamyl phosphate synthesis. Mitochondria were isolated from chow-fed rats, incubated with [guanido-14C]arginine and were centrifuged through silicon oil into perchloric acid for determination of intramitochondrial metabolites. Arginine was separated from urea by cation-exchange resin. Mitochondrial water space was determined by [14C]urea arising from arginase activity associated with the mitochondrial preparations. Extramatrix space was determined by parallel incubations with [inulin-14C]carboxylic acid or [14C]sucrose. There was considerable degradation of arginine by arginase associated with the mitochondrial preparation. This was inhibited by 7 mM ornithine and 7 mM lysine. Arginine was concentrated intramitochondrially to 4-times the extramitochondrial levels. The concentration ratio was decreased in the presence of ornithine and lysine but not with citrulline, NH4Cl, glutamate, glutamate or leucine. No uptake was observed when mitochondria were incubated at 0 degree C. Mitochondria did not concentrate citrulline.

Activities of some enzymes involved in lipogenesis, gluconeogenesis, glycolysis and glycogen metabolism in chicks (Gallus domesticus) from day of hatch to adulthood.

Abstract 1. 1. Activities of some enzymes involved in lipogenesis, gluconeogenesis, glycolysis and glycogen metabolism were studied in the chick ( Gallus domesticus ) from day 1 to adulthood. Malic enzyme and glucose-6-phosphate dehydrogenase were negligible at day 1 and increased significantly during the first week and the declined, G-6-PDH showed great variability. 2. 2. Phosphoenolpyruvate carboxykinase and glucose-6-phosphatase were high at day 1 and then decreased. Fructose diphosphatase increased significantly during the first 2 weeks and remained high. 3. 3. Glutamic oxaloacetic transminase and glutamic pyruvic transaminase were also high at day 1 and then decreased. Both GOT and phosphoenolpyruvate carboxykinase showed a rise again at 14 weeks, possibly indicating a second phase of gluconeogenesis. 4. 4. Phosphorylase and phosphoglucomutase increased during the first week and then dropped, whereas liver glycogen stayed high for 3 weeks before it started to drop. Thus possibly phosphorylase is increased before glycogenolysis starts.

Effect of Diets and Hormones on Two Urea Cycle Enzymes.

Summary All treatments increasing protein catabolism produced an increase in arginine synthetase activity. Arginase activity was increased only in cases where the increased protein breakdown was accompanied by an increase or maintenance of body weight. The changes in lactic acid dehydrogenase, used as a control enzyme, more nearly parallel those in arginase and show very little relationship to changes in arginine synthetase. It has been suggested that arginine synthetase would be a better indicator enzyme of nitrogen catabolism than arginase.

Factors affecting the activity vitro of the tryptophan- and phenylalanine-hydroxylating systems

Abstract Although phenylalanine 4-hydroxylase (EC 1.99.1.2) and tryptophan 5-hydroxylase (EC 1.99.1.4) appear to be the same enzyme, various factors have markedly different effects upon the activities in vitro of the two hydroxylases. “Induction” or suppression of one hydroxylase in vivo produces an equal change in the same direction in the other hydroxylase. The addition in vitro of ascorbic acid and/or Fe 2+ increased the tryptophan hydroxylase activity and EDTA decreased this activity. None of these additions had any effect on phenylalanine hydroxylation. Cupric ion inhibited both reactions, but the tryptophan reaction was more severely inhibited. TPN + could replace DPN + in both reactions. The possible physiological importance of the tryptophan-hydroxylating system is also discussed.

A possible method for estimating hormone effects on enzyme synthesis.

Abstract The activities of four rat liver enzymes (glucose 6-phosphate dehydrogenase, serine dehydrase, glutamic-pyruvic transaminase, and glutamic-oxaloacetic transaminase) were studied in a 90% glucose to 90% casein or a 90% casein to 90% glucose dietary change in the absence and in the presence of exogenous hydrocortisone. From the rates of increases or decreases in enzyme activities the rates of synthesis and values of half-lives of these enzymes were estimated. Comparison of these parameters in the presence and absence of exogenous hydrocortisone indicate that the hormone increases the activity of glucose 6-phosphate dehydrogenase (in the glucose to protein dietary change but not in the reverse dietary change) and glutamic-pyruvic transaminase by increasing the rates of synthesis of these enzymes without altering their rates of degradation. The half-lives of these enzymes were not altered by dietary protein. Serine dehydrase activity was increased by hydrocortisone and it appeared that while protein only increased the rate of synthesis of this enzyme, hydrocortisone increased its half-life and rate of synthesis. The activity of glutamic-oxaloacetic transaminase was increased by dietary protein and also by the hormone. The half-life of the enzyme was smaller in protein-fed or hydrocortisone-treated animals; the hormone apparently mimicking the effect of dietary protein. The utility of the method used for evaluating hormone effects on enzyme activities were discussed with respect to applicability, rapidity, and ease especially in cases when antibody precipitation is not practical.

Metabolism of serine, glycine and threonine in isolated cat hepatocytes Felis domestica.

1. Hepatocytes isolated from the cat demonstrate differences with regard to amino acid metabolism as compared to rat hepatocytes. 2. [14C]serine is metabolized via transamination in isolated cat hepatocyte, whereas its major catabolic route in the rat is via serine dehydratase. 3. [14C]glycerine, a poor gluconeogenic precursor in rat hepatocytes, appears to be equal to serine as a precursor for cat hepatocytes. 4. [14C]threonine does not compete with serine for metabolism, and may be metabolized via threonine aldolase. 5. These results illustrate a protection of amino acids from oxidative metabolism, and a high priority for their metabolism towards anabolic processes.

The role of L-serine dehydratase in the metabolism of L-serine in the perfused rat liver

Abstract 1. 1. To determine the role of L -serine dehydratase ( L -serine hydrolyase, deaminating, EC 4.2.1.13) in L -serine metabolism in the rat liver, experiments involving in situ liver perfusion, metabolite measurement in freeze-clamped tissues and enzyme activity determinations were conducted. 2. 2. Results from liver perfusion experiments showed that the maximal rate of gluconeogenesis from 10 mM L -serine was identical to that from equimolar quantity of L -lactate or pyruvate in starved animals. Combining L -serine and L -lactate in the perfusion medium did not produce glucose at a faster rate than with either L -lactate or L -serine alone. Glucose formation from L -serine or L -lactate was inhibited by 4.8 mM quinolinate which was reversible by 4.8 mM MnCl 2 . 3. 3. Metabolite measurements revealed that perfusion with L -serine increased liver concentrations of pyruvate, lactate, aspartate, malate, and glutamate. The presence of 4.8 mM quinolinate further elevated the levels of aspartate, malate and glutamate. These metabolite levels dropped after the reversal of quinolinate inhibition by MnCl 2 . 4. 4. The increase in glucogenic rate from L -serine by starvation was paralleled by similar increase in serine dehydratase activity in the supernatant fraction of liver homogenate and L -serine disappearance from the perfusion medium. 5. 5. These results strongly suggest that a major portion of L -serine was metabolized by L -serine dehydratase in the perfused rat liver.

Effect of Thyroxine Administration on Enzymes Associated with Glucose Metabolism in the Liver.

Summary The activities of 3 liver enzymes : glucose-6-phosphatase, fructose-1,6-diphosphatase, and pyruvate kinase were studied in thyroidectomized and hyperthyroid rats. Thyroidectomy considerably decreased all 3 activities. Thyroxine treatment resulted in an increase of glucose- 6-phosphatase activity but caused decrease in fructose-l?6-diphosphatase activity of normal animals and no change in pyruvate kinase activity of thyroidectomized animals. This contradictory picture has been discussed as thyroxine causing no increase in gluconeogenesis. The specific increases in glucose-6-phosphatase activity may be explained by the increased synthesis of microsomal protein on thyroxine treatment.

Bile pigments in gallbladder and freshly-secreted hepatic duct bile from fed and fasted rainbow trout, Oncorhynchus mykiss

1. Chromatographic analyses of bile pigments in rainbow trout reveal the presence of primarily unconjugated biliverdin (BV) and bilirubin (BR) glycosyl conjugates. Only trace amounts of unconjugated BR are present in hepatic duct (HD) bile: no beta-glucuronidase activity is detectable. 2. The per cent of BV and BR in HD and gallbladder biles is similar in fasted trout; however, the per cent of BV is significantly increased in HD bile from fed fish. 3. Fasting decreases the rate of choleresis but does not alter the excretory rate of endogenous BV or BR. 4. Erythrocyte life span is estimated to be approximately 500 days.