Ketoacid Analogues Research Articles

Effect of valine on the control of fatty acid synthesis in white adipose tissue of the rat.

In adipocytes from fed rats, the rate of fatty acid synthesis in the presence of glucose and insulin was inhibited 40% by valine (5 mm). tthis inhibition was largely abolished by the addition to the incubation medium of the transaminase inhibitor aminooxy acetate, and of pyruvate and agents which raise the intracellular pyruvate levels such as N,N,N1,N1-tetramethyl-p-phenylenediamine. Pyruvate output into the incubation medium from fat pads obtained from fed rats and incubated with glucose and insulin was decreased significantly by the addition of valine. When adipocytes were incubated under similar conditions, the final concentration of pyruvate in the incubation medium was 42 +/- 1.6 muM under control conditions and approximately one third of this value in the presence of 2.5 mM valine. Valine had no significant effect on pyruvate dehydrogenase (lipoate) (EC 1.2.4.1) activity when assayed in homogenates prepared from adipose tissue previously incubated for 60 min with the amino acid. Although the ketoacid analogue of valine alpha-ketoisovaleric acid, is a competitive inhibitor of pyruvate dehydrogenase (lipoate) (K1 = 1.4 mM), this cannot solely account for the valine-induced reduced rate of lipogenesis. Rather, the mechanism involves a reduction in pyruvate concentration and thereby a diminished flow through pyruvate dehydrogenase (lipoate). Details of the possible mechanism are discussed.

UTILISATION OF KETOACID ANALOGUES OF VALINE AND PHENYLALANINE IN HEALTH AND URÆMIA

15N-labelled ammonium chloride was given to four healthy individuals and to two uræmic patients during studies of the ability of α-ketoisovaleric and β-phenylpyruvic acid to substitute for valine and phenylalanine. 15N enrichment of valine, phenylalanine, and other essential and non-essential aminoacids is reported, In three of the four healthy individuals the incorporation of 15N into the appropriate essential aminoacid increased. No significant uptake of 15N was found in the fourth individual. One uræmic patient showed a much greater enrichment of phenylalanine and valine than did healthy individuals. Her uptake of 15N was also much greater than the uptake by the other and more uræmic patient who had failed to maintain nitrogen balance with the ketoacids. It is concluded from these isotopic studies that a number of healthy and uræmic men can synthesise phenylalanine and valine from their ketoacid analogues.

The Mechanism of Transamination: FUNCTION OF THE HISTIDYL RESIDUE AT THE ACTIVE SITE OF SUPERNATANT ASPARTATE TRANSAMINASE

Controlled photooxidation inactivates supernatant glutamate aspartate transaminase through the destruction of histidyl residues only. Since previous studies have failed to show alterations in the over-all enzyme structure, the mechanism of transamination has been examined before and after photooxidation, taking advantage of the spectral changes associated with the enzyme prosthetic group, pyridoxal phosphate, after substrate or inhibitor binding. By spectrophotometric titration of the active site, photooxidized enzyme retains its ability to bind the dicarboxylic acids, glutarate, oxaloacetate, and α-ketoglutarate, to form non-covalent enzyme-substrate complexes. Binding of αmethyl aspartate was also measured spectrophotometrically. Because the interaction of the enzyme with radioactive substrate can be trapped by reduction of the enzyme-substrate complex with sodium borohydride, it is shown that aspartate in the forward reaction and α-ketoglutarate in the back reaction form the initial aldimine and ketimine complexes, respectively. The affinity for each substrate or substrate analogue is greater in the case of photooxidized enzyme under normal buffer conditions. Native enzyme exhibits relatively large changes in the dissociation constant with changes in the buffer anion concentration, while photooxidized enzyme fails to show these competitive anion effects. Photooxidized enzyme forms all of the visible enzyme substrate intermediates characteristic of the half-transamination reaction between an amino acid and its keto acid analogue, except for the semiquinoid intermediate which absorbs at 490 mµ. This inability correlates with the failure to catalyze the exchange of the α-hydrogen of the amino acid with tritium from tritiated water. From these data and from studies on the rate of the forward and back reaction with cysteine sulfinate and α-ketoglutarate, respectively, it is concluded that the primary defect after photooxidation of the active site histidine of supernatant glutamate aspartate transaminase occurs in the removal of the α-hydrogen from the aldimine Schiff's base in the forward reaction. A role of proton acceptor is proposed for the histidine residue at the active site.

UTILISATION OF AMMONIA NITROGEN FOR PROTEIN SYNTHESIS IN MAN, AND THE EFFECT OF PROTEIN RESTRICTION AND URÆMIA

The effect of dietary protein restriction on the incorporation of 15N-ammonia into body protein has been studied in 8 experiments on four healthy individuals and two uræmic patients. In healthy individuals, the more severe the protein restriction the greater was the incorporation of the isotope into the plasma-albumin pool, and the smaller the recovery of 15N in urine and fæces. The two stable uraemic patients who had been protein restricted for 9 months incorporated between three and five times as much ammonia nitrogen into the plasma-albumin as did two healthy individuals after 3 weeks on a 20 g. protein intake, with a total calculated incorporation of 700 and 975 mg. of ammonia nitrogen in 24 hours, equivalent to about 4.5 and 6.0 g. of protein, respectively. Evidence is cited that ammonia is the form in which man reincorporates urea nitrogen into protein, and that this ammonia is derived from urea by bacterial breakdown in the intestine. We suggest that the ability of uræmic patients to re-utilise their urea nitrogen in protein synthesis could be exploited by providing them with an artificial diet containing the ketoacid analogues of the essential aminoacids, but no other nitrogen except for lysine and threonine, which may not be formed in sufficient amount in the body from their ketoacid analogues.

BIOSYNTHESIS OF MUSTARD OIL GLUCOSIDES. VI. BIOSYNTHESIS OF GLUCOBARBARIN IN RESEDA LUTEOLA L.

A number of C14-labelled compounds were fed to Reseda luteola L.; after a 24-hour period of metabolism, the thioglucoside aglycone (5-phenyl-2-oxazolidinethione) was isolated and its specific activity determined. In some instances the aglycone was degraded to determine the distribution of C14.DL-γ-Phenylbutyrine (2-amino-4-phenylbutyric acid) was the most efficient precursor of the aglycone, followed by phenylalanine and acetate; the carboxyl carbon of these compounds was not incorporated into the thioglucoside aglycone. Little or no randomization of C14 in the aglycone resulted from feeding DL-γ-phenylbutyrine-2- and -3-C14, DL-phenylalanine-2- and -3-C14, and acetate-2-C14. The conversion of C14 from 10 additional compounds into the aglycone was less than that from D-glucose-G-C14. Isotope competition experiments suggest that β-benzylmalic acid also may be a precursor. It appears that the C6–C3 aglycone is formed from phenylalanine and acetate via C6–C5 and C6–C4 intermediates (including γ-phenylbutyrine or its keto acid analogue) in a manner analogous to the formation of gluconasturtiin in watercress. The carbon-14 and nitrogen-15 of L-phenylalanine-G-C14-N15 and of DL-γ-phenylbutyrine-2-C14-N15 were not incorporated as a unit into the aglycone of glucobarbarin.

Amino acid and alpha-keto acid-induced hyperinsulinism in the leucine-sensitive type of infantile and childhood hypoglycemia

Summary 1.Studies on 2 patients with leucine-induced “idiopathic” hypoglycemia showed that l-isoleucine also caused a striking fall in the concentration of blood glucose. d-leucine, l-valine, l-alanine, dl-threonine, and l-glycine did not have a significant effect. 2.The α-keto acid analogue of leucine, α-ketoisocaproic acid, induced hypoglycemia when injected intravenously but not its metabolite isovaleric acid. Its action was less effective orally. Pyruvic acid and α-ketoisovaleric acid failed to elicit a similar response. 3.No evidence of a defect in amino acid or α-keto acid metabolism was found, nor of a direct effect of leucine on glycolysis in whole blood. 4.It was shown that leucine caused increased peripheral utilization of glucose, suggesting an insulin-like action. Direct determinations of plasma insulin indicated a sharp rise in circulating insulin following the administration of leucine. 5.It is concluded that l-leucine, l-isoleucine, and their keto acid analogues induce an increase in the concentration of plasma insulin irrespective of the blood glucose level, an effect which may be attributed to their direct action on the mechanism of insulin secretion. Summary 1.Studies on 2 patients with leucine-induced “idiopathic” hypoglycemia showed that l-isoleucine also caused a striking fall in the concentration of blood glucose. d-leucine, l-valine, l-alanine, dl-threonine, and l-glycine did not have a significant effect. 2.The α-keto acid analogue of leucine, α-ketoisocaproic acid, induced hypoglycemia when injected intravenously but not its metabolite isovaleric acid. Its action was less effective orally. Pyruvic acid and α-ketoisovaleric acid failed to elicit a similar response. 3.No evidence of a defect in amino acid or α-keto acid metabolism was found, nor of a direct effect of leucine on glycolysis in whole blood. 4.It was shown that leucine caused increased peripheral utilization of glucose, suggesting an insulin-like action. Direct determinations of plasma insulin indicated a sharp rise in circulating insulin following the administration of leucine. 5.It is concluded that l-leucine, l-isoleucine, and their keto acid analogues induce an increase in the concentration of plasma insulin irrespective of the blood glucose level, an effect which may be attributed to their direct action on the mechanism of insulin secretion.

C14O2 ASSIMILATION IN LILIUM REGALE WITH REFERENCE TO γ-METHYLENEGLUTAMIC ACID

It was found that γ-methyleneglutamic acid, γ-methylglutamic acid, and their keto-acid analogues are natural constituents of Lilium regale. These compounds were not present in the seed but appeared soon after germination. It was shown that, in seedlings, these compounds are not formed from atmospheric carbon dioxide either in the light or in the dark. This was shown also for excised leaves under a variety of conditions. In entire young plants a synthesis of γ-methyleneglutamic acid was demonstrated in the bulbs and roots. The keto-acid analogue was synthesized in all parts of the plant including the leaves. The results suggest that the accumulation of γ-methyleneglutamic acid in leaves is through transport from the subterranean organs of the plant where it is synthesized.

C14O2 ASSIMILATION IN LILIUM REGALE WITH REFERENCE TO γ-METHYLENEGLUTAMIC ACID

It was found that γ-methyleneglutamic acid, γ-methylglutamic acid, and their keto-acid analogues are natural constituents of Lilium regale. These compounds were not present in the seed but appeared soon after germination. It was shown that, in seedlings, these compounds are not formed from atmospheric carbon dioxide either in the light or in the dark. This was shown also for excised leaves under a variety of conditions. In entire young plants a synthesis of γ-methyleneglutamic acid was demonstrated in the bulbs and roots. The keto-acid analogue was synthesized in all parts of the plant including the leaves. The results suggest that the accumulation of γ-methyleneglutamic acid in leaves is through transport from the subterranean organs of the plant where it is synthesized.