Regulation of hepatic l-serine dehydratase and l-serine-pyruvate aminotransferase in the developing neonatal rat

1. The activities of l-serine dehydratase and l-serine–pyruvate aminotransferase were determined in rat liver during foetal and neonatal development. 2. l-Serine–pyruvate aminotransferase activity begins to develop in late-foetal liver, increases rapidly at birth to a peak during suckling and then decreases at weaning to the adult value. 3. l-Serine dehydratase activity is very low prenatally, but increases rapidly after birth to a transient peak. After a second transient peak around the time weaning begins, activity gradually rises to the adult value. Both of these peaks have similar isoenzyme compositions. 4. In foetal liver both l-serine dehydratase and l-serine–pyruvate aminotransferase activities are increased after injection in utero of glucagon or dibutyryl cyclic AMP. Cycloheximide or actinomycin D inhibited the prenatal induction of both enzymes and actinomycin D blocked the natural increase of l-serine dehydratase immediately after birth. Glucose or insulin administration also blocked the perinatal increase of l-serine dehydratase. 5. After the first perinatal peak of l-serine dehydratase, activity is increased by cortisol and this is inhibited by actinomycin D. After the second postnatal peak, activity is increased by amino acids or cortisol and this is insensitive to actinomycin D inhibition. Glucose administration blocks the cortisol-stimulated increase in l-serine dehydratase and also partially lowers the second postnatal peak of activity. 6. The developmental patterns of the enzymes are discussed in relation to the pathways of gluconeogenesis from l-serine. The regulation of enzyme activity by hormonal and dietary factors is discussed with reference to the changes in stimuli that occur during neonatal development and to their possible mechanisms of action.     

Glutamate-glyoxylate aminotransferase in rat liver cytosol. Purification, properties and identity with alanine-2-oxoglutarate aminotransferase

After cortisone injection, virtually identical increases in rat liver cytosol alanine-2-oxoglutarate aminotransferase and glutamate-glyoxylate aminotransferase activities were observed. The two activities were co-purified to homogeneity from rat liver cytosol. The purified enzyme was specific for L-alanine with 2-oxoglutarate as amino acceptor. With glyoxylate, however, the enzyme utilized various L-amino acids as amino donors in the following order of activity: glutamate greater than alanine greater than glutamine greater than methionine. The ratio of alanine-2-oxoglutarate aminotransferase activity to glutamate-glyoxylate aminotransferase activity remained constant during purification and was unchanged by a variety of treatments of the purified enzyme. These results suggest that glutamate-glyoxylate aminotransferase is identical with alanine-2-oxoglutarate aminotransferase. Evidence was obtained that the two enzyme activities in the cytosol of dog, cat and human liver are also properties of the same protein.     

Crystallization and characterization of human liver kynurenine–glyoxylate aminotransferase. Identity with alanine–glyoxylate aminotransferase and serine–pyruvate aminotransferase

Kynurenine–glyoxylate aminotransferase, alanine–glyoxylate aminotransferase and serine–pyruvate aminotransferase were co-purified and crystallized as yellow cubes from human liver particulate fraction. The crystalline enzyme was homogeneous by the criteria of electrophoresis, isoelectric focusing, gel filtration, sucrose-density-gradient centrifugation and analytical ultracentrifugation. The molecular weight of the enzyme was calculated as approx. 90000, 89000 and 99000 by the use of gel filtration, analytical ultracentrifugation and sucrose-density-gradient centrifugation respectively, with two identical subunits. The enzyme has a s_20,w value of 5.23S, an isoelectric point of 8.3 and a pH optimum between 9.0 and 9.5. The enzyme solution showed absorption maxima at 280 and 420nm. The enzyme catalysed transamination between several l-amino acids and pyruvate or glyoxylate. The order of effectiveness of amino acids was alanine>serine>glutamine>glutamate>methionine>kynurenine = phenylalanine = asparagine>valine>histidine>lysine>leucine>isoleucine>arginine>tyrosine = threonine>aspartate, with glyoxylate as amino acceptor. The enzyme was active with glyoxylate, oxaloacetate, hydroxypyruvate, pyruvate, 4-methylthio-2-oxobutyrate and 2-oxobutyrate, but showed little activity with phenylpyruvate, 2-oxoglutarate and 2-oxoadipate, with kynurenine as amino donor. Kynurenine–glyoxylate aminotransferase activity was competitively inhibited by the addition of l-alanine or l-serine. From these results we conclude that kynurenine–glyoxylate aminotransferase, alanine–glyoxylate aminotransferase and serine–pyruvate aminotransferase activities of human liver are catalysed by a single protein. Kinetic parameters for the kynurenine–glyoxylate aminotransferase, alanine–glyoxylate aminotransferase, serine–pyruvate aminotransferase and alanine–hydroxypyruvate aminotransferase reactions of the enzyme are presented.     

Maturation in liver mitochondria of Ruthenium Red-sensitive calcium-ion-transport activity and the influence of glucagon administration in vivo and in utero

The maturation of Ca²⁺ transport in mitochondria isolated from rat liver was examined, from 5 days before birth. The mitochondria used were isolated from liver homogenates by centrifugation at 22000g-min. Ca²⁺ transport by mitochondria isolated from foetal liver is energy-dependent and Ruthenium Red-sensitive. The transmembrane pH gradient in these mitochondria is higher by about 7mV and the membrane potential lower by about 20mV than in adult mitochondria. The inclusion of 2mm-P_i in the incubation medium enhances the protonmotive force by approx. 30mV. The rate of Ca²⁺ influx in foetal mitochondria measured in buffered KCl plus succinate is low until about 2–3h after birth, when it increases to about 60% of adult values; approx. 24h later it has reached near-adult values. Higher rates of Ca²⁺ influx are observed in the presence of 2mm-P_i; 3–5 days before birth the rates are about one-third of adult values and decline slightly as birth approaches. By 2–3h post partum they have reached adult values. The inclusion of 12.5μm-MgATP with the P_i stimulates further the initial rate of Ca²⁺ influx in foetal mitochondria. The rates observed are constant over the prenatal period examined and are 50–60% of those observed in adult mitochondria. Mitochondria isolated from foetal livers 4–5 days before birth retain the accumulated Ca²⁺ for about 50min in the presence of 2mm-P_i. In the period 2 days before birth to birth, this ability is largely lost, but by 2–3h after birth Ca²⁺ retention is similar to that of adult mitochondria. The presence of 12.5μm-MgATP progressively enhances the Ca²⁺ retention time as development proceeds until 2–3h after birth, when it becomes less sensitive to added MgATP. Glucagon administration to older foetuses in utero enhances both the rate of mitochondrial Ca²⁺ influx assayed in the presence of 2mm-P_i and the time for which mitochondria retain accumulated Ca²⁺ in the presence of 12.5μm-MgATP and 2mm-P_i. Its administration to neonatal animals leads to an increase in mitochondrial Ca²⁺ retention similar to that seen in adult mitochondria. The data provide evidence that the Ruthenium Red-sensitive Ca²⁺ transporter is potentially as active in foetal mitochondria 5 days before birth as it is in adult mitochondria. They also show that foetal mitochondria have an ability to retain accumulated Ca²⁺ reminiscent of mitochondria from tumour cells and from hormone-challenged rat liver.     

The effects of salicylate on the activity of rat liver tyrosine–2-oxoglutarate aminotransferase in vitro and in vivo

1. Salicylate, in concentrations of 0.25mm and above, enhances the basal activity of tyrosine–2-oxoglutarate aminotransferase in homogenates of rat liver incubated in the absence of added pyridoxal 5′-phosphate (endogenous activity). The effect is decreased by increasing the concentration of the cofactor. 2. The intraperitoneal administration of sodium salicylate enhances the activity of rat liver tyrosine aminotransferase; the major effect during the first hour being on the enzyme in the absence of added pyridoxal phosphate. Actinomycin D prevents the induction of the enzyme by cortisol and tryptophan. Induction by pyridoxine or salicylate is 50% inhibited by actinomycin D. The effects of the injections of various combinations of cortisol, pyridoxine and salicylate were also studied in the absence or presence of actinomycin D. 3. It is suggested that salicylate induces rat liver tyrosine aminotransferase by displacing its protein-bound cofactor and that a cofactor-type induction of the hepatic enzyme occurs in pyridoxine-treated rats.     

The effect of disulphides on mitochondrial oxidations

1. Nicotinamide nucleotide-linked mitochondrial oxidations were inhibited by the disulphides NNN′N′-tetraethylcystamine, cystamine and cystine diethyl ester, whereas l-homocystine, oxidized mercaptoethanol, oxidized glutathione, NN′-diacetylcystamine and tetrathionate were only slightly inhibitory. Mitochondrial oxidations were not blocked by the thiol cysteamine. 2. NAD-independent oxidations were not inhibited by cystamine. The oxidation of choline was initially stimulated. 3. The inactivation of isocitrate, malate and β-hydroxybutyrate oxidation of intact mitochondria could be partially reversed by external NAD. For the reactivation of α-oxoglutarate oxidation a thiol was also required. 4. A leakage of nicotinamide nucleotides from the mitochondria is suggested as the main cause of the inhibition. In addition, a strong inhibition of α-oxoglutarate dehydrogenase by cystamine was observed. A mixed disulphide formation with CoA and possibly also lipoic acid and lipoyl dehydrogenase is suggested to explain this inhibition.     

Identity of alanine:glyoxylate aminotransferase with alanine:2-oxoglutarate aminotransferase in rat liver cytosol

Rat liver soluble fraction contained 3 forms of alanine: glyoxylate aminotransferase. One with a pI of 5.2 and an Mr of approx. 110,000 was found to be identical with cytosolic alanine:2-oxoglutarate aminotransferase. The pI 6.0 enzyme with an Mr of approx. 220,000 was suggested to be from broken mitochondrial alanine:glyoxylate aminotransferase 2 and the pI 8.0 enzyme with an Mr of approx. 80,000 enzyme from broken peroxisomal and mitochondrial alanine:glyoxylate aminotransferase 1. These results suggest that the cytosolic alanine: glyoxylate aminotransferase activity is due to cytosolic alanine: 2-oxoglutarate aminotransferase.     

Cytoplasmic binding of dexamethasone and induction of tyrosine aminotransferase in neonatal rat liver

Dexamethasone administration markedly increases the activity of tyrosine aminotransferase in postnatal rat liver. The glucocorticoid fails to induce the enzyme in foetal rats when administered in utero. Dexamethasone binding activity of rat liver cytoplasm is low or absent in foetal animals but increases to adult levels 1–2 days after birth. In vitro experiments with isolated nuclei indicate that foetal nuclei have the capacity to accumulate dexamethasone but only when presented with cytosol-bound glucocorticoid.     

The subcellular distribution of rat liver l-alanine–glyoxylate aminotransferase in relation to a pathway for glucose formation involving glyoxylate

1. The distribution of l-alanine-glyoxylate aminotransferase activity between subcellular fractions prepared from rat liver homogenates was investigated. The greater part of the homogenate activity (about 80%) was recovered in the ;total-particles' fraction sedimented by high-speed centrifugation and the remainder in the cytosol fraction. 2. Subfractionation of the particles by differential sedimentation and on sucrose density gradients revealed a specific association between the aminotransferase and the mitochondrial enzymes glutamate dehydrogenase and rhodanese. 3. The aminotransferase activities in the cytosol and the mitochondria are due to isoenzymes. The solubilized mitochondrial enzyme has a pH optimum of 8.6, an apparent K(m) of 0.24mm with respect to glyoxylate and is inhibited by glyoxylate at concentrations above 5mm. The cytosol aminotransferase shows no distinct pH optimum (over the range 7.0-9.0) and has an apparent K(m) of 1.11mm with respect to glyoxylate; there is no evidence of inhibition by glyoxylate. 4. The mitochondrial location of the bulk of the rat liver l-alanine-glyoxylate aminotransferase activity is discussed in relation to a pathway for gluconeogenesis involving glyoxylate.     

The synergistic decarboxylation of glyoxylate and 2-oxoglutarate by an enzyme system from pig-liver mitochondria

1. An enzyme system that catalyses a synergistic decarboxylation of glyoxylate and 2-oxoglutarate has been purified from pig-liver mitochondria. 2. The purified system is specific for glyoxylate and 2-oxoglutarate as substrates, although in earlier stages of purification glycine and l-glutamate are also active. 3. The reaction is inhibited strongly by EDTA and N-ethylmaleimide. Substrate analogues, present at concentrations equimolar with respect to the substrates, are not effective as inhibitors. 4. The reaction proceeds in the absence of added cofactors. Magnesium chloride, mercaptoethanol and sucrose stimulate the reaction, and stabilize the activity of the enzyme. 5. The pH optimum of the reaction is 7·0. The K_m values of glyoxylate and 2-oxoglutarate, at saturating concentration of the corresponding co-substrate, are 16mm and 3·6mm respectively. 6. Isotopic work with specifically labelled [¹⁴C]glyoxylate and 2-oxo[¹⁴C]-glutarate suggests that the enzyme system catalyses an initial condensation of glyoxylate and 2-oxoglutarate that results in, or leads to, release of C-1 of both substrates as carbon dioxide. C-2 of glyoxylate and C-5 of 2-oxoglutarate do not appear as carbon dioxide. 7. The stoicheiometry of the reaction is complex. During the initial stages of the reaction, more carbon dioxide is recovered from 2-oxoglutarate than from glyoxylate. Subsequently, there is a disproportionate increase with time of carbon dioxide evolution from the carboxyl group of glyoxylate. The excess of decarboxylation of glyoxylate over 2-oxogluturate is further increased by treatment of reaction products with acid.     

REGIONAL AND SUBCELLULAR DISTRIBUTION OF AMINOTRANSFERASES IN RAT BRAIN

Abstract— Aminotransferase activity was measured in various areas of the nervous system of the rat (cortical grey matter, midbrain, corpus callosum, spinal cord and sciatic nerve) and in subcellular fractions of rat brain (nuclei, mitochondria and cytosol). Activity was low or absent in the sciatic nerve relative to that in the other areas, with the exception of incubation of glutamate with oxaloacetate (25 per cent of the activity found in brain) and of asparagine with 2-oxoglutarate (65 per cent of the activity found in brain). The distribution of enzymic activity was not homogeneous; alanine-2-oxoglutarate aminotransferase was highest in cortical grey matter; leucine- and GABA-2-oxoglutarate aminotransferases were highest in midbrain. Incubation of phenylalanine or tyrosine with 2-oxoglutarate gave similar activities in grey matter and midbrain. Activity generally was higher in the grey matter than in corpus callosum or spinal cord. However, incubations of methionine with 2-oxoglutarate, or glutamine with glyoxylate, gave similar activities in the three areas studied from the brain, whereas incubations of glutamate with glyoxylate gave highest activity in the corpus callosum. Only incubations of asparagine with 2-oxoglutarate, and glutamate with glyoxylate, gave significant activity in the nuclear subcellular fraction. Aminotransferase activity of phenylalanine, tyrosine or GABA with 2-oxoglutarate, or ornithine or glutamine with glyoxylate, was localized to mitochondria. The remaining reactions studied (glutamate with oxaloacetate; leucine, alanine, methionine or asparagine with 2-oxoglutarate and glutamate with glyoxylate) demonstrated activity in both the mitochondrial fraction and the soluble supernatant fraction.     

The metabolism of galactarate, d-glucarate and various pentoses by species of Pseudomonas

1. When NAD⁺ was present, cell extracts of Pseudomonas (A) grown with d-glucarate or galactarate converted 1mol. of either substrate into 1mol. each of 2-oxoglutarate and carbon dioxide; 70–80% of the gas originated from C-1 of the hexarate. 2. The enzyme system that liberated carbon dioxide from galactarate was inactive in air and was stabilized by galactarate or Fe²⁺ ions; the system that acted on d-glucarate was more stable and was stimulated by Mg²⁺ ions. 3. When NAD⁺ was not added, 2-oxoglutarate semialdehyde accumulated from either substrate. This compound was isolated as its bis-2,4-dinitrophenylhydrazone, and several properties of the derivative were compared with those of the chemically synthesized material. Methods were developed for the determination of 2-oxoglutarate semialdehyde. 4. Synthetic 2-oxoglutarate semialdehyde was converted into 2-oxoglutarate by an enzyme that required NAD⁺; the reaction rate with NADP⁺ was about one-sixth of that with NAD⁺. 5. For extracts of Pseudomonas (A) grown with d-glucarate or galactarate, or for those of Pseudomonas fragi grown with l-arabinose or d-xylose, specific activities of 2-oxoglutarate semialdehyde–NAD oxidoreductase were much higher than for extracts of the organisms grown with (+)-tartrate and d-glucose respectively. 6. Extracts of Pseudomonas fragi grown with l-arabinose or d-xylose converted l-arabonate or d-xylonate into 2-oxoglutarate when NAD⁺ was added to reaction mixtures and into 2-oxoglutarate semialdehyde when NAD⁺ was omitted.     

Biosynthesis of Phytosiderophores : In Vitro Biosynthesis of 2'-Deoxymugineic Acid from l-Methionine and Nicotianamine

2′ -Deoxymugineic acid (DMA), one of mugineic acid-family phytosiderophores (MAs), was synthesized in vitro both from l-methionine and from nicotianamine (NA) with a cell-free system derived from root tips of iron-deficient barley (Hordeum vulgare L.). The reactions producing DMA from NA needed an amino group acceptor (i.e. 2-oxoglutarate, pyruvate, or oxalacetic acid) and a reductant (i.e. NADH or NADPH). The activity of the enzymes to produce NA from l-methionine was the highest at about pH 9. This biosynthetic activity was markedly induced by iron-deficiency stress. The synthesis of NA from S-adenosyl-l-methionine was more efficient than from l-methionine. From the results with the cell-free system reported here, we propose a revised biosynthetic pathway of MAs.     

Glycolate and glyoxylate metabolism by isolated peroxisomes or chloroplasts

Chloroplasts, mitochondria, and peroxisomes from leaves were separated by isopycnic sucrose density gradient centrifugation. The peroxisomes converted glycolate-¹⁴C or glyoxylate-¹⁴C to glycine, and contained a glutamate: glyoxylate aminotransferase as indicated by an investigation of substrate specificity. The pH optimum for the aminotransferase was between 7.0 and 7.5, and the Km for l-glutamate was 3.6 mm and for glyoxylate, 4.4 mm. The reaction of glutamate plus glyoxylate was not reversible. The isolated peroxisomes did not convert glycine to glyoxylate nor glycine to serine.     

Developmental profiles and properties of hepatic peroxisomal apo- and mitochondrial holoalanine:glyoxylate aminotransferase during chick embryogenesis

Alanine:glyoxylate aminotransferase was present as the apoenzyme in the peroxisomes and as the holoenzyme in the mitochondria in chick embryos. The peroxisomal enzyme predominated in the early stage and gradually decreased during embryonic development and disappeared after hatching. In contrast, the mitochondrial enzyme gradually increased and predominated in the later stage of chick embryos. Peroxisomal alanine:glyoxylate aminotransferase in chick embryos was a single peptide with a molecular weight of about 40,000. The enzyme differed from the mitochondrial enzyme in the embryos, and mammalian alanine:glyoxylate aminotransferases 1 (with a molecular weight of about 80,000 with two identical subunits) and 2 (with a molecular weight of about 200,000 with four identical subunits) in molecular weights and immunological properties. Mitochondrial alanine:glyoxylate aminotransferase in chick embryos had an identical molecular weight and immunologically cross-reacted with mammalian mitochondrial alanine:glyoxylate aminotransferase 2. Pyridoxal 5'-phosphate dissociated easily from the peroxisomal enzyme saturated with pyridoxal 5'-phosphate. Hepatic aspartate:2-oxoglutarate aminotransferase and alanine:2-oxoglutarate aminotransferase in chick embryos, and hepatic alanine:glyoxylate aminotransferases in different animal species were all present as the holoenzyme.     

Studies of inhibition of rat spermidine synthase and spermine synthase

1. S-Adenosyl-l-methionine, S-adenosyl-l-homocysteine, 5′-methylthioadenosine and a number of analogues having changes in the base, sugar or amino acid portions of the molecule were tested as potential inhibitors of spermidine synthase and spermine synthase from rat ventral prostate. 2. S-Adenosyl-l-methionine was inhibitory to these reactions, as were other nucleosides containing a sulphonium centre. The most active of these were S-adenosyl-l-ethionine, S-adenosyl-4-methylthiobutyric acid, S-adenosyl-d-methionine and S-tubercidinylmethionine, which were all comparable in activity with S-adenosylmethionine itself, producing 70–98% inhibition at 1mm concentrations. Spermine synthase was somewhat more sensitive than spermidine synthase. 3. 5′-Methylthioadenosine, 5′-ethylthioadenosine and 5′-methylthiotubercidin were all powerful inhibitors of both enzymes, giving 50% inhibition of spermine synthase at 10–15μm and 50% inhibition of spermidine synthase at 30–45μm. 4. S-Adenosyl-l-homocysteine was a weak inhibitor of spermine synthase and practically inactive against spermidine synthase. Analogues of S-adenosylhomocysteine lacking either the carboxy or the amino group of the amino acid portion were somewhat more active, as were derivatives in which the ribose ring had been opened by oxidation. The sulphoxide and sulphone derivatives of decarboxylated S-adenosyl-l-homocysteine and the sulphone of S-adenosyl-l-homocysteine were quite potent inhibitors and were particularly active against spermidine synthase (giving 50% inhibition at 380, 50 and 20μm respectively). 5. These results are discussed in terms of the possible regulation of polyamine synthesis by endogenous nucleosides and the possible value of some of the inhibitory substances in experimental manipulations of polyamine concentrations. It is suggested that 5′-methylthiotubercidin and the sulphone of S-adenosylhomocysteine or of S-adenosyl-3-thiopropylamine may be particularly valuable in this respect.     

The permeability of mitochondria to oxaloacetate and malate

1. A spectrophotometric assay of the rates of penetration of oxaloacetate and l-malate into mitochondria is described. The assay is based on the measurement of the oxidation of intramitochondrial NADH by oxaloacetate and of the reduction of intramitochondrial NAD⁺ by malate. 2. The rate of entry of both oxaloacetate and l-malate into mitochondria is restricted, as shown by the fact that disruption of the mitochondrial structure can increase the rate of interaction between the dicarboxylic acids and intramitochondrial NAD⁺ and NADH by between 100- and 1000-fold. 3. The rates of entry of oxaloacetate and malate into liver, kidney and heart mitochondria increased by up to 50-fold on addition of a source of energy, either ascorbate plus NNN′N′-tetramethyl-p-phenylenediamine aerobically, or ATP anaerobically. 4. In the absence of a source of energy the changes in the concentrations of intramitochondrial NAD⁺ and NADH brought about by the addition of l-malate or oxaloacetate were followed by parallel changes in the concentrations of NADP⁺ and NADPH, indicating the presence in the mitochondria of an energy-independent transhydrogenase system. 5. The results are discussed in relation to the hypothesis that malate acts as a carrier of reducing equivalents between mitochondria and cytoplasm.     

Oxaloacetate transport into plant mitochondria

The properties of oxaloacetate (OA) transport into mitochondria from potato (Solanum tuberosum) tuber and pea (Pisum sativum) leaves were studied by measuring the uptake of ¹⁴C-labeled OA into liposomes with incorporated mitochondrial membrane proteins preloaded with various dicarboxylates or citrate. OA was found to be transported in an obligatory counterexchange with malate, 2-oxoglutarate, succinate, citrate, or aspartate. Phtalonate inhibited all of these countertransports. OA-malate countertransport was inhibited by 4,4′-dithiocyanostilbene-2,2′-disulfonate and pyridoxal phosphate, and also by p-chloromercuribenzene sulfonate and mersalyl, indicating that a lysine and a cysteine residue of the translocator protein are involved in the transport. From these and other inhibition studies, we concluded that plant mitochondria contain an OA translocator that differs from all other known mitochondrial translocators. Major functions of this translocator are the export of reducing equivalents from the mitochondria via the malate-OA shuttle and the export of citrate via the citrate-OA shuttle. In the cytosol, citrate can then be converted either into 2-oxoglutarate for use as a carbon skeleton for nitrate assimilation or into acetyl-coenzyme A for use as a precursor for fatty acid elongation or isoprenoid biosynthesis.     

Transport of methionine and proline by rat liver slices and the effect of certain hormones

1. Transport characteristics of l-methionine and l-proline in rat liver slices in vitro were studied. 2. Intracellular concentration gradients for methionine were obtained. 3. Methionine uptake was inhibited by iodoacetate, dinitrophenol, Na⁺-free media and also by glycine, lysine, cysteine and dithiothreitol but not by α-aminoisobutyrate. 4. The rate of methionine metabolism in the slice was slow. 5. Puromycin inhibited methionine incorporation into protein, but not methionine uptake. 6. Methionine inhibited the transport of α-aminoisobutyrate but not of cystine. 7. Efflux and exchange diffusion of methionine was studied. 8. Amino acid transport in rat liver slices was not affected by thyroidectomy. 9. Addition of insulin, glucagon, adrenaline or cortisol did not affect the transport of methionine. 10. Addition of 6-N,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate increased methionine transport after a 120min incubation period in some experiments. 11. Studies of l-proline transport were invalidated because of the rapid evolution of CO₂ from the substrate.     

Biosynthesis of the β-Methylarginine Residue of Peptidyl Nucleoside Arginomycin in Streptomyces arginensis NRRL 15941

The peptidyl nucleoside arginomycin is active against Gram-positive bacteria and fungi but displays much lower toxicity to mice than its analog blasticidin S. It features a rare amino acid, β-methylarginine, which is attached to the deoxyhexose moiety via a 4′-aminoacyl bond. We here report cloning of the complete biosynthetic gene cluster for arginomycin from Streptomyces arginensis NRRL 15941. Among the 14 putative essential open reading frames, argM, encoding an aspartate aminotransferase (AAT), and adjacent argN, encoding an S-adenosyl methionine (SAM)-dependent methyltransferase, are coupled to catalyze arginine and yield β-methylarginine in Escherichia coli. Purified ArgM can transfer the α-amino group of l-arginine to α-ketoglutaric acid to give glutamate and thereby converts l-arginine to 5-guanidino-2-oxopentanoic acid, which is methylated at the C-3 position by ArgN to form 5-guanidino-3-methyl-2-oxopentanoic acid. Iteratively, ArgM specifically catalyzes transamination from the donor l-aspartate to the resulting 5-guanidino-3-methyl-2-oxopentanoic acid, generating β-methylarginine. The complete and concise biosynthetic pathway for the rare and bioactive amino acid revealed by this study may pave the way for the production of β-methylarginine either by enzymatic conversion or by engineered living cells.