Metabolic and secretory responses of parotid cells to cationic amino acids. Oxidation of the amino acids and interference with the oxidation of D-glucose or endogenous nutrients.

Cationic amino acids were recently found to stimulate amylase release from rat parotid cells. The possible relevance of their oxidative catabolism to such a secretory stimulation was investigated. D-Glucose, which was efficiently metabolized in parotid cells and which augmented O2 uptake above basal value, failed to affect basal or stimulated amylase release. L-Arginine, L-lysine and L-histidine failed to stimulate the oxidation of either exogenous D-[6-14C]glucose or endogenous nutrients in cells pre-labelled with [U-14C]palmitate or L-[U-14C]glutamine. The oxidation of L-[U-14C]arginine, L-[U-14C]ornithine, L-[U-14C]lysine and L-[U-14C]histidine, all tested at a 10 mM concentration, was much lower than that of D-[U-14C]glucose (5.6 mM). These findings argue against the view that the stimulation of amylase release by cationic amino acids would be related to their role as a source of energy in the parotid cells.     

Participation of ornithine aminotransferase in the synthesis and catabolism of ornithine in mice. Studies using gabaculine and arginine deprivation.

Gabaculine, a potent suicide inhibitor of ornithine aminotransferase (OAT), at a dose of 50 mg/kg inhibited this enzyme in mouse tissues and dramatically increased tissue ornithine concentrations, whether or not arginine was present in the diet. Thus even under arginine deprivation there is catabolism of ornithine which involves OAT. This was confirmed by administration of [14C]ornithine to arginine-deprived mice. Gabaculine (3-amino-2,3-dihydrobenzoic acid) drastically decreased the release of 14CO2 and increased the radioactivity in the basic amino acids in the tissues. When [1-14C]glutamate was injected into mice deprived of arginine, a significant amount of radioactivity was recovered in tissue ornithine and arginine, and gabaculine decreased this labelling by about two-thirds, indicating that ornithine was synthesized in vivo from glutamate via OAT. In addition, we failed to detect in liver and small intestine alpha-N-acetylornithine, N-acetylglutamate kinase or N-acetylornithine aminotransferase, which are obligatory components of a potential route of ornithine synthesis from N-acetylglutamate. Our results indicate that at least 45 mumol of ornithine was synthesized and catabolized daily via OAT in the mouse deprived of arginine.     

Utilization of ornithine and arginine as specific precursors of clavulanic acid.

Ornithine and arginine (5 to 20 mM), but not glutamic acid or proline, exerted a concentration-dependent stimulatory effect on the biosynthesis of clavulanic acid in both resting-cell cultures and long-term fermentations of Streptomyces clavuligerus. Ornithine strongly inhibited cephamycin biosynthesis in the same strain. [1-14C]-, [5-14C]-, or [U-14 C] ornithine was efficiently incorporated into clavulanic acid, whereas the incorporation of uniformly labeled glutamic acid was very poor. [U-14C] citrulline were not incorporated at all. Mutant nca-1, a strain that is blocked in clavulanic acid biosynthesis, did not incorporate arginine into clavulanic acid. S. clavuligerus showed arginase activity, converting arginine into ornithine, but not amidinotransferase activity. Both arginase activity and clavulanic acid formation were enhanced simultaneously by supplementing the production medium with 10 mM arginine.     

Utilization of ornithine and arginine as specific precursors of clavulanic acid

Ornithine and arginine (5 to 20 mM), but not glutamic acid or proline, exerted a concentration-dependent stimulatory effect on the biosynthesis of clavulanic acid in both resting-cell cultures and long-term fermentations of Streptomyces clavuligerus. Ornithine strongly inhibited cephamycin biosynthesis in the same strain. [1-14C]-, [5-14C]-, or [U-14 C] ornithine was efficiently incorporated into clavulanic acid, whereas the incorporation of uniformly labeled glutamic acid was very poor. [U-14C] citrulline were not incorporated at all. Mutant nca-1, a strain that is blocked in clavulanic acid biosynthesis, did not incorporate arginine into clavulanic acid. S. clavuligerus showed arginase activity, converting arginine into ornithine, but not amidinotransferase activity. Both arginase activity and clavulanic acid formation were enhanced simultaneously by supplementing the production medium with 10 mM arginine.     

The role of mitochondrially bound arginase in the regulation of urea synthesis: studies with [U-15N4]arginine, isolated mitochondria, and perfused rat liver

The main goal of the current study was to elucidate the role of mitochondrial arginine metabolism in the regulation of N-acetylglutamate and urea synthesis. We hypothesized that arginine catabolism via mitochondrially bound arginase augments ureagenesis by supplying ornithine for net synthesis of citrulline, glutamate, N-acetylglutamate, and aspartate. [U-(15)N(4)]arginine was used as precursor and isolated mitochondria or liver perfusion as a model system to monitor arginine catabolism and the incorporation of (15)N into various intermediate metabolites of the urea cycle. The results indicate that approximately 8% of total mitochondrial arginase activity is located in the matrix, and 90% is located in the outer membrane. Experiments with isolated mitochondria showed that approximately 60-70% of external [U-(15)N(4)]arginine catabolism was recovered as (15)N-labeled ornithine, glutamate, N-acetylglutamate, citrulline, and aspartate. The production of (15)N-labeled metabolites was time- and dose-dependent. During liver perfusion, urea containing one (U(m+1)) or two (U(m+2)) (15)N was generated from perfusate [U-(15)N(4)]arginine. The output of U(m+2) was between 3 and 8% of total urea, consistent with the percentage of activity of matrix arginase. U(m+1) was formed following mitochondrial production of [(15)N]glutamate from [alpha,delta-(15)N(2)]ornithine and transamination of [(15)N]glutamate to [(15)N]aspartate. The latter is transported to cytosol and incorporated into argininosuccinate. Approximately 70, 75, 7, and 5% of hepatic ornithine, citrulline, N-acetylglutamate, and aspartate, respectively, were derived from perfusate [U-(15)N(4)]arginine. The results substantiate the hypothesis that intramitochondrial arginase, presumably the arginase-II isozyme, may play an important role in the regulation of hepatic ureagenesis by furnishing ornithine for net synthesis of N-acetylglutamate, citrulline, and aspartate.     

Metabolism of [14C]citrulline in the perfused sheep and goat udder

1. A lactating-sheep mammary gland was perfused for 12h in the presence of l-[2-(14)C]-citrulline and received adequate quantities of glucose, acetate and amino acids. Two lactating-goat udders were similarly perfused in the presence of either l-[carbamoyl-(14)C,-2-(14)C]citrulline or l-[carbamoyl-(14)C,1-(14)C]citrulline and l-[4-(3)H]arginine. 2. In these experiments, [(14)C]citrulline was substantially oxidized to CO(2) and converted into arginine and proline of casein. 3. The specific radioactivities of arginine, ornithine and proline of the plasma increased after passage through the udders, demonstrating that [(14)C]citrulline is metabolized by the mammary gland. 4. The presence of two unknown radioactive metabolites of [(14)C]citrulline was detected in the perfusate. These substances were not found after incubation in vitro of oxygenated blood in the presence of the radioactive precursor. 5. From these experiments, it is concluded that citrulline is metabolized in mammary tissue by way of arginine to urea, ornithine and proline.     

Enhanced intestinal synthesis of polyamines from proline in cortisol-treated piglets

This study was conducted to determine a role for cortisol in regulating intestinal ornithine decarboxylase (ODC) activity and to identify the metabolic sources of ornithine for intestinal polyamine synthesis in suckling pigs. Thirty-two 21-day-old suckling pigs were randomly assigned to one of four groups with eight animals each and received daily intramuscular injections of vehicle solution (sesame oil; control), hydrocortisone 21-acetate (HYD; 25 mg/kg body wt), RU-486 (10 mg/kg body wt, a potent blocker of glucocorticoid receptors), or HYD plus RU-486 for two consecutive days. At 29 days of age, pigs were killed for preparation of jejunal enterocytes. The cytosolic fraction was prepared for determining ODC activity. For metabolic studies, enterocytes were incubated for 45 min at 37 degrees C in 2 ml of Krebs-bicarbonate buffer (pH 7.4) containing 1 mM [U-(14)C]arginine, 1 mM [U-(14)C]ornithine, 1 mM [U-(14)C]glutamine, or 1 mM [U-(14)C]proline plus 1 mM glutamine. Cortisol administration increased intestinal ODC activity by 230%, polyamine (putrescine, spermidine, and spermine) synthesis from ornithine and proline by 75-180%, and intracellular polyamine concentrations by 45-83%. Polyamine synthesis from arginine was not detected in enterocytes of control pigs but was induced in cells of cortisol-treated pigs. There was no detectable synthesis of polyamines from glutamine in enterocytes of all groups of pigs. The stimulating effects of cortisol on intestinal ODC activity and polyamine synthesis were abolished by coadministration of RU-486. Our data indicate that an increase in plasma cortisol concentrations stimulates intestinal polyamine synthesis via a glucocorticoid receptor-mediated mechanism and that proline (an abundant amino acid in milk) is a major source of ornithine for intestinal polyamine synthesis in suckling neonates.     

Arginine catabolism in the cyanobacterium Synechocystis sp. Strain PCC 6803 involves the urea cycle and arginase pathway

Cells of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 supplemented with micromolar concentrations of L-[(14)C]arginine took up, concentrated, and catabolized this amino acid. Metabolism of L-[(14)C]arginine generated a set of labeled amino acids that included argininosuccinate, citrulline, glutamate, glutamine, ornithine, and proline. Production of [(14)C]ornithine preceded that of [(14)C]citrulline, and the patterns of labeled amino acids were similar in cells incubated with L-[(14)C]ornithine, suggesting that the reaction of arginase, rendering ornithine and urea, is the main initial step in arginine catabolism. Ornithine followed two metabolic pathways: (i) conversion into citrulline, catalyzed by ornithine carbamoyltransferase, and then, with incorporation of aspartate, conversion into argininosuccinate, in a sort of urea cycle, and (ii) a sort of arginase pathway rendering glutamate (and glutamine) via Delta(1)pyrroline-5-carboxylate and proline. Consistently with the proposed metabolic scheme (i) an argF (ornithine carbamoyltransferase) insertional mutant was impaired in the production of [(14)C]citrulline from [(14)C]arginine; (ii) a proC (Delta(1)pyrroline-5-carboxylate reductase) insertional mutant was impaired in the production of [(14)C]proline, [(14)C]glutamate, and [(14)C]glutamine from [(14)C]arginine or [(14)C]ornithine; and (iii) a putA (proline oxidase) insertional mutant did not produce [(14)C]glutamate from L-[(14)C]arginine, L-[(14)C]ornithine, or L-[(14)C]proline. Mutation of two open reading frames (sll0228 and sll1077) putatively encoding proteins homologous to arginase indicated, however, that none of these proteins was responsible for the arginase activity detected in this cyanobacterium, and mutation of argD (N-acetylornithine aminotransferase) suggested that this transaminase is not important in the production of Delta(1)pyrroline-5-carboxylate from ornithine. The metabolic pathways proposed to explain [(14)C]arginine catabolism also provide a rationale for understanding how nitrogen is made available to the cell after mobilization of cyanophycin [multi-L-arginyl-poly(L-aspartic acid)], a reserve material unique to cyanobacteria.     

Incorporation of dl-[2-14C]ornithine and dl-[5-14C]arginine in milk constituents by the isolated lactating sheep udder

1. Lactating mammary glands of sheep were perfused for several hours in the presence of dl-[2-(14)C]ornithine or dl-[5-(14)C]arginine and received adequate quantities of acetate, glucose and amino acids. 2. In the [(14)C]ornithine experiment 1.4% of the casein and 1% of the expired carbon dioxide came from added ornithine; 96% of the total radioactivity in casein was recovered in proline; 13% of the proline of casein originated from plasma ornithine. 3. In this experiment the results of chemical degradation of proline of casein as well as relative specific activities in the isolated products are consistent with the view that ornithine is metabolized, by way of glutamic gamma-semialdehyde, to proline or glutamic acid. 4. In the [(14)C]arginine experiments 3% of the casein and 1% of the expired carbon dioxide came from arginine; 84% of the arginine and 9% of the proline of casein originated from plasma arginine. 5. In these experiments the relative specific activities of arginine, ornithine and proline in plasma are in agreement with the view that arginine is metabolized by way of ornithine to proline. The conversion of arginine into ornithine is probably catalysed by arginase, so that arginase in mammary tissue may be involved in the process of milk synthesis.     

Hepatic pyruvate kinase. Regulation by glucagon, cyclic adenosine 3'-5'-monophosphate, and insulin in the perfused rat liver.

A reversible interconversion of two kinetically distinct forms of hepatic pyruvate kinase regulated by glucagon and insulin is demonstrated in the perfused rat liver. The regulation does not involve the total enzyme content of the liver, but rather results in a modulation of the substrate dependence. The forms of pyruvate kinase in liver homogenates are distinguished by measurements of the ratio of the enzyme activity at a subsaturating concentration of P-enolpyruvate (1.3 mM) to the activity at a saturating concentration of this substrate (6.6 mM). A low ratio form of pyruvate kinase (ratio between 0.1 and 0.2) is obtained from livers perfused with 10(-7) M glucagon or 0.1 mM adenosine 3':5'-monophosphate (cyclic AMP). A high ratio form of the enzyme is obtained from livers perfused with no hormone (ratio = 0.35 to 0.45). The regulation of pyruvate kinase by glucagon and cyclic AMP occurs within 2 min following the hormone addition to the liver. Insulin (22 milliunits/ml) counteracts the inhibition of pyruvate kinase caused by 5 X 10(-11) M glucagon, but has only a slight influence on the enzyme properties in the absence of the hyperglycemic hormone. The low ratio form of pyruvate kinase obtained from livers perfused with glucagon or cyclic AMP is unstable in liver extracts and will revert to a high ratio form within 10 min at 37 degrees or within a few hours at 0 degrees. Pyruvate kinase is quantitatively precipitated from liver supernatants with 2.5 M ammonium sulfate. This precipitation stabilizes the enzyme and preserves the kinetically distinguishable forms. The kinetic properties of the two forms of rat hepatic pyruvate kinase are examined using ammonium sulfate precipitates from the perfused rat liver. At pH 7.5 the high ratio form of the enzyme has [S]0.5 = 1.6 +/- 0.2 mM P-enolpyruvate (n = 8). The low ratio form of enzyme from livers perfused with glucagon or cyclic AMP has [S]0.5 = 2.5 +/- 0.4 mM P-enolpyruvate (n = 8). The modification of pyruvate kinase induced by glucagon does not alter the dependence of the enzyme activity on ADP (Km is approximately 0.5 mM ADP for both forms of the enzyme). Both forms are allosterically modulated by fructose 1,6-bisphosphate, L-alanine, and ATP. The changes in the kinetic properties of hepatic pyruvate kinase which follow treating the perfused rat liver with glucagon or cyclic AMP are consistent with the changes observed in the enzyme properties upon phosphorylation in vitro by a clyclic AMP-stimulated protein kinase (Ljungstr?m, O., Hjelmquist, G. and Engstr?m, L. (1974) Biochim. Biophys. Acta 358, 289--298). However, other factors also influence the enzyme activity in a similar manner and it remains to be demonstrated that the regulation of hepatic pyruvate kinase by glucagon and cyclic AMP in vivo involes a phosphorylation.     

A cortisol surge mediates the enhanced polyamine synthesis in porcine enterocytes during weaning

This study was conducted to determine whether a cortisol surge mediates the enhanced expression of intestinal ornithine decarboxylase (ODC) in weanling pigs. Piglets were nursed by sows until 21 days of age, when 40 pigs were randomly assigned into one of four groups (10 animals/group). Group 1 continued to be fed by sows, whereas groups 2-4 were weaned to a corn and soybean meal-based diet. Weanling pigs received intramuscular injections of vehicle solvent (sesame oil), RU-486 (a potent blocker of glucocorticoid receptors; 10 mg/kg body wt), and metyrapone (an inhibitor of adrenal cortisol synthesis; 5 mg/kg body wt), respectively, 5 min before weaning and 24 and 72 h later. At 29 days of age, pigs were used to prepare jejunal enterocytes for ODC assay and metabolic studies. To determine polyamine (putrescine, spermidine, and spermine) synthesis, enterocytes were incubated for 45 min at 37 degrees C in 2 ml Krebs-bicarbonate buffer containing 1 mM [U-(14)C]arginine, 1 mM [U-(14)C]ornithine, 1 mM [U-(14)C]glutamine, or 1 mM [U-(14)C]proline plus 1 mM glutamine. Weaning increased intestinal ODC activity by 230% and polyamine synthesis from ornithine, arginine, and proline by 72-157%. Arginine was a quantitatively more important substrate than proline for intestinal polyamine synthesis in weaned pigs. Administration of RU-486 or metyrapone to weanling pigs prevented the increases in intestinal ODC activity and polyamine synthesis, reduced intracellular polyamine concentrations, and decreased villus heights and intestinal growth. Our results demonstrate an essential role for a cortisol surge in enhancing intestinal polyamine synthesis during weaning, which may be of physiological importance for intestinal adaptation and remodeling.     

Regulation of the glycine cleavage system in the isolated perfused rat liver

The catabolism of glycine in the isolated perfused rat liver was investigated by measuring the production of 14CO2 from [1-14C]- and [2-14C]glycine. Production of 14CO2 from [1-14C]glycine was maximal as the perfusate glycine concentration approached 10 mM and exhibited a maximal activity of 125 nmol of 14CO2 X g-1 X min-1 and an apparent Km of approximately 2 mM. Production of 14CO2 from [2-14C]glycine was much lower, approaching a maximal activity of approximately 40 nmol of 14CO2 X g-1 X min-1 at a perfusate glycine concentration of 10 mM, with an apparent Km of approximately 2.5 mM. Washout kinetic experiments with [1-14C]glycine exhibited a single half-time of 14CO2 disappearance, indicating one metabolic pool from which the observed 14CO2 production is derived. These results indicate that the glycine cleavage system is the predominant catabolic fate of glycine in the perfused rat liver and that production of 14CO2 from [1-14C]glycine is an effective monitor of metabolic flux through this system. Metabolic flux through the glycine cleavage system in the perfused rat liver was inhibited by processes which lead to reduction of the mitochondrial NAD(H) redox couple. Infusion of beta-hydroxybutyrate or octanoate inhibited 14CO2 production from [1-14C]glycine by 33 and 50%, respectively. Alternatively, infusion of acetoacetate stimulated glycine decarboxylation slightly and completely reversed the inhibition of 14CO2 production by octanoate. Metabolic conditions which are known to cause a large consumption of mitochondrial NADPH (e.g. ureogenesis from ammonia) stimulated glycine decarboxylation by the perfused rat liver. Infusion of pyruvate and ammonium chloride stimulated production of 14CO2 from [1-14C]glycine more than 2-fold. Lactate plus ammonium chloride was equally as effective in stimulating glycine decarboxylation by the perfused rat liver, while alanine plus ammonium chloride was ineffective in stimulating 14CO2 production.     

Metabolism of valine and 3-methyl-2-oxobutanoate by the isolated perfused rat kidney.

Metabolism of branched-chain amino and 2-oxo acids was studied in the isolated perfused kidney. Significant amounts of 2-oxo acids were released by perfused kidney with all concentrations of amino acids tested (0.1-1.0 mM each), despite the high activity of branched-chain 2-oxo acid dehydrogenase in kidney. As perfusate valine concentration was increased from 0.2 to 1.0 mM, [1-14C]valine transamination (2-oxo acid oxidized + released) increased roughly linearly; [1-14C]valine oxidation, however, increased exponentially. Increasing perfusate concentration of 3-methyl-2-oxo[1-14C]butanoate from 0 to 1.0 mM resulted in a linear increase in the rate of its oxidation and a rise in perfusate valine concentration; at the same time significant decreases occurred in perfusate isoleucine and leucine concentrations, with corresponding increases in rates of release of their respective 2-oxo acids. Comparison of rates of oxidation of [1-14C]valine and 3-methyl-2-oxo[1-14C]butanoate suggests that 2-oxo acid arising from [1-14C]valine transamination has freer access to the 2-oxo acid dehydrogenase than has the 2-oxo acid from the perfusate. The observations indicate that, when branched-chain amino and 2-oxo acids are present in perfusate at near-physiological concentrations, rates of transamination of the amino and 2-oxo acids by isolated perfused kidney are greater than rates of oxidation.     

Effect of phenylephrine on glutamate and glutamine metabolism in isolated perfused rat liver.

Addition of phenylephrine to isolated perfused rat liver is followed by an increased 14CO2 production from [1-14C]glutamate, [1-14C]glutamine, [U-14C]proline and [3-14C]pyruvate, but by a decreased 14CO2 production from [1-14C]pyruvate. Simultaneously, there is a considerable decrease in tissue content of 2-oxoglutarate, glutamate and citrate. Stimulation of 14CO2 production from [1-14C]glutamate is also observed in the presence of amino-oxyacetate, suggesting a stimulation of glutamate dehydrogenase and 2-oxoglutarate dehydrogenase fluxes by phenylephrine. Inhibition of pyruvate dehydrogenase flux by phenylephrine is due to an increased 2-oxoglutarate dehydroxygenase flux. Phenylephrine stimulates glutaminase flux and inhibits glutamine synthetase flux to a similar extent, resulting in an increased hepatic glutamine uptake. Whereas the effects of NH4+ ions and phenylephrine on glutaminase flux were additive, activation of glutaminase by glucagon was considerably diminished in the presence of phenylephrine. The reported effects are largely overcome by prazosin, indicating the involvement of alpha-adrenergic receptors in the action of phenylephrine. It is concluded that stimulation of gluconeogenesis from various amino acids by phenylephrine is due to an increased flux through glutamate dehydrogenase and the citric acid cycle.     

Acute Effect of Ammonia on Branched-Chain Amino Acid Oxidation and Incorporation into Proteins in Astrocytes and in Neurons in Primary Cultures

14CO2 production and incorporation of label into proteins from the labeled branched-chain amino acids, leucine, valine, and isoleucine, were determined in primary cultures of neurons and of undifferentiated and differentiated astrocytes from mouse cerebral cortex in the absence and presence of 3 mM ammonium chloride. Production of 14CO2 from [1-14C]leucine and [1-14C]valine was larger than 14CO2 production from [U-14C]leucine and [U-14C]valine in both astrocytes and neurons. In most cases more 14CO2 was produced in astrocytes than in neurons. Incorporation of labeled branched-chain amino acids into proteins varied with the cell type and with the amino acid. Addition of 3 mM ammonium chloride greatly suppressed 14CO2 production from [1-14C]-labeled branched chain amino acids but had little effect on 14CO2 production from [U-14C]-labeled branched-chain amino acids in astrocytes. Ammonium ion, at this concentration, suppressed the incorporation of label from all three branched-chain amino acids into proteins of astrocytes. In contrast, ammonium ion had very little effect on the metabolism (oxidation and incorporation into proteins) of these amino acids in neurons. The possible implications of these findings are discussed, especially regarding whether they signify variations in metabolic fluxes and/or in magnitudes of precursor pools.     

Nonparticipation of extracellular glutathione in renal transport of dibasic amino acids

Microinjections of L-[14C]arginine (2.9 mM) and L-[14C]ornithine (3.4 mM) were made into renal proximal tubules of rats in the presence of methionine sulfoximine (MSO) (10, 20 mM), ATP (10 mM), and MgCl2 (20 mM) together. Absorption of both labelled amino acids dropped, respectively, by 31.1 and 49.1% compared with control microinjections. The MSO alone or ATP plus MgCl2 had no effect. These data suggest that the inhibition by MSO plus ATP plus MgCl2 is not due to direct competition between MSO and dibasic amino acids but rather to suppression of the renewal of intracellular glutathione. Such an effect is discussed in comparison with cycloleucine inhibition of dibasic amino acid transport. Addition of exogenous glutathione to microinjectates die not reverse either type of inhibition. This study shows that while intracellular glutathione may affect amino acid transport, extracellular glutathione has no effect.     

Stimulation by glucagon of the incorporation of U-14C-labeled substrates into glucose by isolated hepatocytes from fed rats.

The effect of glucagon on the incorporation of U-14C-labeled lactate, pyruvate or alanine into glucose has been studied using isolated hepatocytes from livers of fed rats. Rates of incorporation into glucose were about the same as observed in perfused liver preparations provided precautions were taken to avoid depletion of certain metabolities by the preparative procedures. With each substrate, stimulation of the incorporation into glucose by a maximally effective concentration of glucagon (10 nM) was associated with about a 75% reduction in the substrate concentration required for a half-maximal rate and with about a 30% increase in maximum rate. Consequently, the hormone caused a substantial (2--4-fold) stimulation when any one of the above substrates was present at a near physiological concentration, but brought about only a relatively small stimulation (1.4-fold) when very high substrate concentrations were used. Provision of cytoplasmic reducing equivalents (by ethanol addition), or of precursor for acetyl-coenzyme A formation (by acetate addition)-stimulated incorporation of labeled alanine into glucose and their effects were additive with that of glucagon. This suggested that provision of either of these intermediates was not a means by which the hormone increased the incorporation of labeled substrate into glucose. NH4+ stimulated the incorporation of 20 mM [U-14C] lactate into glucose 2-fold, probably by promoting glutamate synthesis and thus enhancing the transamination of oxaloacetate to aspartate. Evidence was obtained to support the view that glucagon also increases glutamate production (presumably from endogenous protein). However, the stimulation of incorporation into glucose from 20 mM [U-14C] lactate by NH4+ plus glucagon was synergistic. This suggested that glucagon also stimulated the incorporation of labeled substrate into glucose by additional means. Stimulation of the incorporation of [U-14C] alanine into glucose by beta-hydroxybutyrate plus glucagon was also synergistic. This suggested that another action of glucagon may be to provide more intramitochondrial reducing potential.     

Metabolism of L-(U-14C)valine, L-(U-14C)leucine, L-(U-14C)histidine and L-(U-14C) phenylalanine by the isolated perfused lactating guinea-pig mammary gland.

1. The incorporation of L-[U-14C]leucine, L[U-14C]histidine and L-[U-14C]phenylalanine into casein secreted during perfusion of isolated guinea-pig mammary glands was demonstrated. 2. The extent of incorporation of label into casein residues was consistent with their being derived from free amino acids of the perfusate plasma. 3. The mean transit time of the amino acids from perfusate into secreted casein was approx. 100 min. 4. Whereas radioactive histidine and phenylalanine were incorporated solely into milk protein, radioactivity from [U-14C]valine was also transferred to CO2 and to an unidentified plasma component, and from [U-14C]leucine to plasma glutamic acid. 5. Evidence from experiments with [U-14C]phenylalanine suggests that, as in rats, but in contrast with ruminant species, guinea-pig mammary tissue does not possess phenyl alanine hydroxylase activity. 6. The results are discussed in relation to the possible role of essential amino acid catabolism in the control of milk-protein synthesis.     

Metabolism of Proline, Glutamate, and Ornithine in Proline Mutant Root Tips of Zea mays (L.)

In excised pro_1-1 mutant and corresponding normal type roots of Zea mays L. the uptake and interconversion of [¹⁴C]proline, [¹⁴C]glutamic acid, [¹⁴C]glutamine, and [¹⁴C]ornithine and their utilization for protein synthesis was measured with the intention of finding an explanation for the proline requirement of the mutant. Uptake of these four amino acids, with the exception of proline, was the same in mutant and normal roots, but utilization differed. Higher than normal utilization rates for proline and glutamic acid were noted in mutant roots leading to increased CO₂ production, free amino acid interconversion, and protein synthesis. Proline was synthesized from either glutamic acid (or glutamine) or ornithine in both mutant and normal roots; it did not accumulate but rather was used for protein synthesis. Ornithine was not a good precursor for proline in either system, but was preferentially converted to arginine and glutamine, particularly in mutant roots. The pro_1-1 mutant was thus not deficient in its ability to make proline. Based on these findings, and on the fact that ornithine, arginine, glutamic acid and aspartic acid are elevated as free amino acids in mutant roots, it is suggested that in the pro_1-1 mutant proline catabolism prevails over proline synthesis.     

Ornithine transport and exchange in Streptococcus lactis.

Resting cells of Streptococcus lactis 133 appeared to accumulate [14C]ornithine to a high concentration in the absence of an exogenous energy source. However, analysis of intracellular amino acid pool constituents and results of transport experiments revealed that the accumulation of ornithine represented a homoexchange between extracellular [14C]ornithine and unlabeled ornithine in the cell. The energy-independent exchange of ornithine was not inhibited by proton-conducting uncouplers or by metabolic inhibitors. Intracellular [14C]ornithine was retained by resting cells after suspension in a buffered medium. However, addition of unlabeled ornithine to the suspension elicited rapid exit of labeled amino acid. The initial rate of exit of [14C]ornithine was dependent on the concentration of unlabeled ornithine in the medium, but this accelerative exchange diffusion process caused no net loss of amino acid. By contrast, the presence of a fermentable energy source caused a rapid expulsion of and net decrease in the concentration of intracellular ornithine. Kinetic analyses of amino acid transport demonstrated competitive inhibition between lysine and ornithine, and data obtained by two-dimensional thin-layer chromatography established the heteroexchange of these basic amino acids. The effects of amino acids and of ornithine analogs on both entry and exit of [14C]ornithine have been examined. The data suggest that a common carrier mediates the entry and exchange of lysine, arginine, and ornithine in cells of S. lactis.