Carl Peraino

Ornithine-δ-transaminase in the rat I. Assay and some general properties

Abstract This report describes the assay, partial purification, and general properties of ornithine-δ-transaminase (EC 2.6.1.13) in the rat. 1. 1. The assay involves the quantitative estimation of glutamic semialdehyde, a product of the enzymic transamination, through the use of o-aminobenzaldehyde, which reacts with the glutamic semialdehyde to form a derivative with an absorption maximum at 440 mμ. 2. 2. The relative activity of ornithine-δ-transaminase in different tissues was found to be: kidney > liver > heart ≈ spleen ≈ brain > skeletal muscle 3. 3. Ornithine-δ-transaminase was found to be located almost exclusively in the mitochondrial fraction of the liver cell. 4. 4. A 7-fold purification of the enzyme was accomplished by subjecting isolated liver mitochondria to ultrasonic vibration, followed by ammonium sulfate fractionation of the solubilized protein. 5. 5. General properties of the enzyme: (a) The enzyme was specific for ornithine and α-ketoglutarate. (b) PCMB inhibited the enzyme which also required mercapto-ethanol for maximum activity. (c) A pyridoxal phosphate requirement was exhibited only by dialyzed enzyme preparations. (d) The pH optimum of ornithine-δ-transaminase was found to be approx. 7.4. .

Studies on the mechanism of carbohydrate repression in rat liver.

Abstract In earlier published studies we have shown that the amino acid catabolizing enzymes, threonine dehydrase and ornithine transaminase, can be rapidly induced in the livers of protein depleted rats force-fed casein hydrolysate or injected with glucagon. This induction, which is inhibited by the administration of gamma irradiation, actinomycin, puromycin, or fluoroorotic acid, is also inhibited by the forced feeding of glucose or fructose. The kinetic picture of the inhibition by carbohydrate feeding appears to resemble that produced by puromycin administration rather than by actinomycin or fluoroorotic acid administration, suggesting that the repressive effect of carbohydrate is exerted at the stage of protein synthesis involving peptide formation. The feeding of carbohydrates is accompanied by the rapid accumulation of large quantities of glycogen in the liver, and when glucagon is injected into rats receiving dietary glucose the resultant enzyme induction is accompanied by a decrease in liver glycogen concentration. During the course of these studies we have evolved the working hypothesis that the metabolic control phenomena which we have observed (induction and repression) arise through the interaction of enzyme forming systems with intracellular lipoprotein membranes such as the endoplasmic reticulum. Agents which alter the configuration of these membranes and/or their association with the enzyme forming systems (polysomes) might be expected correspondingly to affect induction and repression. For example, when glycogen is deposited in the liver cell after feeding carbohydrate, it appears to become associated with the endoplasmic reticulum. Numerous workers have shown that this endoplasmic reticulum is morphologically quite different (tubular network, no ribosomes attached to membranes) from that not associated with glycogen (lamellar network, ribosomes attached to membranes) and it is possible that such morphological differences may be accompanied by differences in metabolic properties. In the further exploration of this hypothesis we have tested the effects of a variety of hormones on the induction and repression of certain amino acid catabolizing enzymes (serine dehydrase, ornithine transaminase, and tyrosine transaminase), the rationale being that hormones may exert their metabolic action through an effect on membrane structure. We observed that in protein depleted rats which have been pretreated with cortisone (5 mg/day for 3 days) glucose repression of enzyme induction is significantly less than that in rats not given cortisone. We also found, however, that induction by casein hydrolysate without glucose is substantially less in rats treated with cortisone as compared to those not receiving cortisone. The administration of cortisone alone does not produce significant enzyme induction under the conditions of these experiments. These results suggest that cortisone exerts a “protective” effect on the induction process, permitting induction to occur in the face of carbohydrate, but not itself causing induction. Similar effects are produced by triamcinolone, but deoxycorticosterone has no effect on the induction or repression phenomena. Experiments were also conducted to study the possibility that carbohydrate repression may in fact be a consequence of increased insulin production stimulated by the carbohydrate feeding. No suppression of induction occurs when rats receiving casein hydrolysate are injected with insulin at dosages just below lethality.

Glucose repression and induction of enzyme synthesis in rat liver

Summary Glucose has long been known as a key metabolite in intermediary metabolism. Its importance as a regulator of protein synthesis is emphasized by its ability to repress the induced synthesis of threonine dehydrase and ornithine transaminase in rat liver. Simultaneous with its repressive effect, glucose administration per os with casein induces glucose-6-phosphate dehydrogenase after a lag of 12 hr. Neither glucose alone nor casein alone were capable of inducing Zwischenferment. In contrast, glucokinase induction by glucose does not require the concomitant administration of protein. Glucokinase induction was inhibited by puromycin and actinomycin D. Insulin administration along with glucose resulted in some inhibition of induction while glucagon suppressed glucokinase induction more than 80 per cent. Fructose administration resulted in glucokinase induction to about two thirds that with glucose. Other carbohydrates tested gave little or no induction. A model to explain these results based on work with microorganisms is presented.

Effect of Gamma Radiation on Dietary and Hormonal Induction of Enzymes in Rat Liver

The dietary induction of serine dehydrase, produced by the oral intubation of hydrolyzed casein to protein-depleted rats, is markedly inhibited by doses of γ-radiation of 400 roentgens or higher provided the irradiation is given within an hour after the initial dose of casein. If the irradiation is delayed until 7 hours after the initial dose of casein, induction is not inhibited. In contrast, the cortisone induction of tyrosine α-ketoglutarate transaminase is not inhibited by doses of γ-radiation up to 3200 roentgens; in some instances hormonal induction of this enzyme appears to be enhanced by irradiation.

Concentrations of free amino acids in blood plasma of rats force-fed l-glutamic acid, l-glutamine or l-alanine☆

Abstract The concentrations of glutamic acid, glutamine, alanine, proline, threonine, glycine, valine, leucine-isoleucine, phenylalanine, and lysine were measured in the portal and systemic plasma of rats that had been forced-fed l -glutamic acid, l -glutamine, or l -alanine. The administration of glutamic acid resulted in a marked rise in the concentration of this amino acid in the portal plasma and a smaller one in the systemic plasma. It also caused the concentration of alanine to increase in the portal and to a lesser extent in the systemic plasma and the concentration of glutamine to increase substantially in both. Administration of glutamine (same molar quantity as for glutamic acid) resulted in greater increases in portal and systemic plasma concentrations of glutamine than were observed for glutamic acid after glutamic acid administration. It also caused the concentration of glutamic acid to increase in the portal and to a lesser extent in the systemic plasma, and the alanine concentration to increase markedly in both. Administration of alanine (1.6× the molar quantity used for glutamic acid or glutamine) resulted in a maximum increase in the portal plasma concentration of alanine which was 8× (on a molar basis) that observed for glutamic acid and 3× that observed for glutamine in the previous experiments. The systemic plasma alanine concentration also rose but much less than the portal concentration. The concentration of glutamic acid increased measurably in the portal plasma while the concentration of glutamine increased more in the systemic than in the portal plasma. The plasma concentrations of the other amino acids tested were not significantly altered in any of the experiments.

Template stability in liver and hepatoma

Abstract The recent advances in the comparative biochemistry of liver tumors of different growth rates were outlined. 1. 1. The four key gluconeogenic enzymes, glucose-6-phosphatase, fructose-1,6-diphosphatase, phosphoenolpyruvate carboxykinase and pyruvate carboxylase decreased with the increasing growth rate and were absent in the rapidly-growing tumors. It was suggested that the genes for these enzymes may be localized on the same Functional Genome Unit. 2. 2. The RNA amount showed no correlation with growth rate in a spectrum of hepatomas. 3. 3. The incorporation of formate into total hepatoma RNA progressively increased with the increasing growth rate. The incorporation of orotate into RNA was markedly decreased in all tumors examined. The incorporation of formate and orotate into RNA was increased in the regenerating liver. 4. 4. Triamcinolone treatment in control, normal liver caused an increase in total RNA; however, there was no change in the hepatomas. The RNA specific activity was increased in normal liver by steroid injections and it also showed some rise in a slow-growing hepatoma. There was no response in the rapidly-growing liver tumor. 5. 5. The concentration of free amino acid did not correlate with the growth rate of hepatomas. Glucocorticoid injection caused a marked elevation in hepatic amino acid level; however, there was a complete failure of response in all hepatomas examined. 6. 6. Triamcinolone injection caused a progressive increase in hepatic glycogen, nitrogen and free amino acid content and in the activities of glucose-6-phosphatase and fructose-1,6-diphosphatase. There was a failure of response in these biochemical parameters in the kidney cortex. 7. 7. The various biochemical parameters and metabolic responses were correlated with the growth rate of hepatomas.