The purine nucleotide cycle and ammonia formation from glutamine by rat kidney slices.

To test the significance of the purine nucleotide cycle in renal ammoniagenesis, studies were conducted with rat kidney cortical slices using glutamate or glutamine labelled in the alpha-amino group with 15N. Glucose production by normal kidney slices with 2 mM-glutamine was equal to that with 3 mM-glutamate. With L-[15N]glutamate as sole substrate, one-third of the total ammonia produced by kidney slices was labelled, indicating significant deamination of glutamate or other amino acids from the cellular pool. Ammonia produced from the amino group of L-[alpha-15N]glutamine was 4-fold higher than from glutamate at similar glucose production rates. Glucose and ammonia formation from glutamine by kidney slices obtained from rats with chronic metabolic acidosis was found to be 70% higher than by normal kidney slices. The contribution of the amino group of glutamine to total ammonia production was similar in both types of kidneys. No 15N was found in the amino group of adenine nucleotides after incubation of kidney slices from normal or chronically acidotic rats with labelled glutamine. Addition of Pi, a strong inhibitor of AMP deaminase, had no effect on ammonia formation from glutamine. Likewise, fructose, which may induce a decrease in endogenous Pi, had no effect on ammonia formation. The data obtained suggest that the contribution of the purine nucleotide cycle to ammonia formation from glutamine in rat renal tissue is insignificant.

Significance of CoA-transferase in the reaction of maleate with amino acids and proteins.

Maleate reacts, in the presence of acetoacetyl-CoA and CoA-transferase with amino acids containing no sulphydryl groups. The physico-chemical properties of the reaction products are the same as those of the derivatives obtained in the reaction of amino acids with maleic anhydride, which indicates that the amino groups became acylated. In the presence of the transferase system, maleate also reacts with such proteins as histone IIA, cytochrome c and albumin. The reaction also occurs after blocking SH groups, thus it could involve amino groups as well. The reacting agent is not maleate itself but a product of the reaction of maleate with acetoacetyl-CoA, catalysed by the transferase, i.e. maleyl-CoA.

Effects of maleate on the content of CoA and its derivatives in rat kidney mitochondria.

Effects of maleate on the content of CoA derivatives in isolated mitochondria and in the tissues of maleate-intoxicated rats have been studied. The addition of maleate to kidney mitochondria incubated with 2-oxo-glutarate decreased CoA-SH and acid-soluble acyl-CoA concentrations while acid insoluble acyl-CoA content remained unchanged. As a result, a substantial loss (depletion) of the total CoA occurred. Similar changes in CoA content were found in vivo in the kidneys of maleate-treated rats. Neither in the isolated liver mitochondria nor in the liver of intoxicated animals have such changes been observed before. Acetoacetate, the substrate for CoA transferase, added to kidney mitochondria before maleate, abolished its inhibitory effect on oxidation of 2-oxoglutarate and prevented the decrease of CoA content. The data are in accord with the previous findings indicating that maleate can bind and sequester CoA in the form of a stable and metabolically inert compound.

Studies on chemical and enzymatic synthesis of maleyl-CoA.

In the course of the enzymatic reaction of acetoacetyl-CoA with maleate, catalyzed by CoA transferase, a transient appearance of free CoA-SH occurred. Subsequently, both free CoA and acyl-CoA decreased with time, indicating the formation of an unusual CoA derivative resistant to alkaline hydrolysis. During the chemical reaction of CoA-SH with maleic anhydride, the SH groups of CoA disappeared quickly, but not more than 30% could be accounted for as thioester. The product is unstable at neutrality and is hydrolyzed by nitroprusside reagent. Another product having an acyl bond of low reactivity, which reacts with hydroxylamine and does not undergo ammonolysis, but is susceptible to alkaline hydrolysis, slowly accumulated, accounting for about 25% of the CoA that disappeared. The main product appears to be formed by the addition of CoA-SH to the double bond of maleic anhydride. Column chromatography of the products of the chemical and the enzymatic reaction revealed two products, designated at X1 and X2, showing absorbance of the adenine moiety of CoA, containing 14C-labeled maleate and no free SH groups. Alkaline hydrolysis of X1 resulted in the recovery of CoA and in an increase of X2. The results are interpreted as indicating that maleyl-CoA readily hydrolyzes and reacts spontaneously with the SH group of free CoA to form an addition compound. The thioester of this product slowly hydrolyzes to give rise to the final product, which appears to be the thioether, a stable and metabolically inert compound.

Metabolism of glutamine in rat kidney mitochondria in the presence of aminooxyacetate.

1. Aminooxyacetate has no effect on respiration of rat kidney cortex mitochondria in the presence of glutamine but it inhibits respiration in the presence of glutamate to the values of endogenous respiration. 2. Rat kidney mitochondria in state 3 produce aspartate from glutamine in the amount corresponding to almost 50% of the glutamate formed intramitochondrially. 3. Aminooxyacetate affects neither formation of ammonia nor of aspartate from glutamine but it inhibits aspartate synthesis in the mitochondria treated with Triton X-100.

Effects of maleate on CoA metabolism in rat kidney.

CoA transfer from acetoacetyl-CoA to maleate catalyzed by purified CoA transferase was studied. The thioester product of the reaction undergoes further chemical transformations and the final product appeared to be stable CoA derivative showing neither free SH group nor hydrolyzable thio-acyl bond. Similar product was obtained as the result of the reaction of addition of CoA to the double bond of maleic anhydride. This was confirmed by DEAE-chromatography. The decrease of free CoA and acid soluble acyl-CoA in kidney mitochondria respiring in the presence of maleate or in the kidney of maleate-treated rats occured without appreciable changes in acid insoluble acyl-CoA derivatives. As the result substantial loss of total content of CoA was found. The data indicate that maleate binds and removes CoA in the form of stable and metabolically inert compound.

Substrate specificity of succinyl-CoA transferase from rat kidney mitochondria.

1. Succinyl-CoA: 3-oxoacid transferase (EC 2.8.3.5) from rat kidney mitochondria, purified about 200-fold, catalyses the CoA transfer from acetoacetyl-CoA to succinate, acetoacetate, maleate, glutarate and malonate; maleate proved to be a true substrate of the enzyme. 2. Double-reciprocal plots of the initial reaction rates against substrates concentrations arb best fitted by parallel lines. Inhibition by each acid product of the reaction is competitive with respect to the acid acceptor of CoA. 3. CoA-transferase from rat kidney shows similar kinetics as, but different substrate specificity than, the enzyme from other sources.

Effect of extracellular pH and inhibitors on the gluconeogenesis and ammoniagenesis relationship in rat kidney cortex slices.

1. Gluconeogenesis from glutamine, fumarate, pyruvate, glutamine plus fumarate, and glutamine plus pyruvate, was generally higher at pH 7.1 than at pH 7.4 and 7.7, whereas ammoniagenesis did not depend on the pH of the medium. 2. The intermediates of the Krebs cycle decreased ammonia formation from glutamine, raising at the same time gluconeogenesis. 3. Arsenite, malonate, maleate, hydrazine and 2,4-dinitrophenol inhibited gluconeogenesis, and enhanced simultaneously ammonia formation irrespective of the pH of the medium.

Effects of maleate on carbohydrate metabolism in rats.

Intraperitoneal administration of maleate produced an increase in blood alpha-ketoacid, acetoacetate, and free fatty acids. The effect of this treatment on blood glucose levels depended on whether the rats were fed or fasted. In fed rats it was accompanied by slight, transient hyperglycemia connected with depletion of liver glycogen stores. In fasted animals moderate hypoglycemia was observed. The in vivo conversion of various precursors into blood glucose was not inhibited, suggesting that maleate does not affect hepatic gluconeogenesis. Neither was a direct effect on liver glycogenolysis observed. On the other hand, maleate inhibited renal gluconeogenesis from various substrates and stimulated anerobic glycolysis in kidney cortical alices. The data are interpreted in terms of increased utilization and decreased production of glucose by the kidney followed by secondary changes in liver carbohydrate metabolism.

Glutamate metabolism in rat kidney mitochondria.

1. Kidney cortex mitochondria did not swell in ammonium or potassium salts of glutamate even in the presence of valinomycin or 2,4-dinitrophenol. Aminooxyacetate diminished the reduction of nicotinamide nucleotides in intact mitochondria in the presence of glutamate. 2. Transamination with oxaloacetate appeared to be the main pathway of glutamate metabolism in isolated rat kidney cortex mitochondria under various metabolic conditions. Ammonia formation was negligible. The gamma-aminobutyrate pathway was found to be of almost no importance.

Transfer of cholesterol and lecithin between erythrocytes and serum in vitro.

1. In human blood incubated in vitro, the transfer of free cholesterol and lecithin from erythrocytes to serum was not related to glycolytic activity in erythrocytes or esterification of cholesterol in serum. 2. The stability of free cholesterol concentration in serum was dependent on the activity of lecithin:cholesterol acyltransferase (EC 2.3.1.43), and the stability of lecithin concentration in erythrocytes, on glycolytic activity.