Enzymes of ornithine metabolism in adult and developing rat intestine.

The levels of 11 enzymes, most of them involved in the metabolism of ornithine, were measured in whole upper intestine, or in duodenum, small intestine and colon of adult rats. The developmental formations in small intestine of arginase, ornithine aminotransferase, and ornithine transcarbamylase were compared with those in liver. Changes with age (late gestation of adult) of the intestinal activities of pyrroline-5-carboxylate reductase, proline oxidase and glutamyl transpeptidase are also described. The results suggest that the proximal part of the intestine is well endowed with enzymes involved in the conversion of ornithine to proline as well as to citrulline. Fetal intestine is rich in proline oxidase and pyrroline-5-carboxylate reductase. The peak levels of ornithine aminotransferase found in intestine in the first 3 postnatal weeks were higher than seen in any other rat tissue. Some of the properties of arginase, ornithine aminotransferase and pyrroline-5-carboxylate reductase in small intestine were compared with those in liver. Isozymes of arginase in small intestine differed from those in liver; the kinetic properties of ornithine aminotransferase were similar in the two tissues. In intestine of 14-day-old rats, the ornithine aminotransferase reaction was reversible, forming ornithine from pyrroline-5-carboxylate. The intestinal pyrroline-5-carboxylate reductase was cold-labile as was the hepatic enzyme in rat.     

Amino acid metabolizing enzymes in rat submaxillary gland, normal or neoplastic, and in pancreas.

The activities of 12 enzymes, many related to ornithine metabolism, were measured in rat submaxillary gland, submaxillary gland tumors and pancreas. In submaxillary gland, the activities of arginase, ornithine aminotransferase, pyrroline-5-carboxylate reductase and glutamine synthetase were high, but no ornithine transcarbamylase or proline oxidase could be detected. In the fetal submaxillary gland, arginase was at almost adult levels while ornithine aminotransferase reached 50% of its adult value postnatally. Submaxillary tumors deviated from their cognate tissue by lower levels of amino acid metabolizing enzymes and by high concentrations of thymidine kinase. In pancreas, none of the pyrroline-5-carboxylate metabolizing enzymes were as high as in either liver or submaxillary gland. The outstanding activities were those of gamma-glutamyl transpeptidase and glutamate dehydrogenase. Although arginase activities in submaxillary gland and pancreas were quantitatively similar, they differed qualitatively: submaxillary gland contained the same variant as liver while the pancreatic isozymes resembled those of other nonhepatic tissues.     

Metabolism of arginine in lactating rat mammary gland.

Significant activities of the four enzymes needed to convert arginine into proline and glutamate (arginase, ornithine aminotransferase, pyrroline-5-carboxylate reductase and pyrroline-5-carboxylate dehydrogenase) develop co-ordinately in lactating rat mammary glands in proportion to the increased production of milk. No enzymes were detected to carry out the reactions of proline oxidation or reduction of glutamate to pyrroline-5-carboxylate. Minces of the gland converted ornithine into proline and into glutamate plus glutamine. These conversions increased during the cycle of lactation in proportion to the increased milk production and to the content of the necessary enzymes. The minced gland did not convert labelled ornithine into citrulline, confirming the absence from the gland of a functioning urea cycle, and did not convert labelled proline or glutamate into ornithine. A metabolic flow of labelled arginine to proline and glutamate in mammary gland was confirmed in intact animals with experiments during which the specific radioactivity of proline in plasma remained below that of the proline being formed from labelled arginine within the gland. It was concluded that arginase in this tissue had a metabolic role in the biosynthesis of extra proline and glutamate needed for synthesis of milk proteins.     

Enzymes metabolizing delta1-pyrroline-5-carboxylate in rat tissues.

The direction and capacity for the metabolism of delta1-pyrroline-5-carboxylate in a number of rat tissues ere investigated by measuring the activities of delta1-pyrroline-5-carboxylate reductase, delta1-pyrroline-5-carboxylate dehydrogenase and proline oxidase. Each of these enzymes catalyzed unidirectional reactions in which delta1-pyrroline-5-carboxylate was either the substrate or product. Delta1-Pyrroline-5-carboxylate reductase activities that were much higher than any previously reported were obtained by avoiding its inactivation in the cold. delta1-Pyrroline-5-carboxylate dehydrogenase, previously said to act on both D- and L-isomers of delta1-pyrroline-5-carboxylate, acted only on the L-isomer. Proline oxidase could not be measured in two adult tissues, in which an inhibitor appeared after birth. The activity of delta1-pyrroline-5-carboxylate reductase significantly paralleled that of ornithine aminotransferase in 23 tissues, showing a widespread potential for proline synthesis from ornithine. An independently distributed potential in fewer tissues for proline degradation to alpha-oxoglutarate was shown by the significantly similar tissue distributions of proline oxidase. Delta1-pyrroline-5-carboxylate dehydrogenase and glutamate dehydrogenase. Reverse metabolism of glutamate or proline to ornithine would be atypical in rat tissues with these distributions of unidirectional enzyme reactions.     

Alterations in the activities of the enzymes of proline metabolism in Ragi (Eleusine coracana) leaves during water stress

Free proline content in Ragi (Eleusine coracana) leaves increased markedly (6 to 85 fold) as the degree of water stress, created by polyethylene gylcol treatment, was prolonged There was also a marginal increase in soluble proteins in the stressed leaves as compared to that in the controls. Water stress stimulated the activities of ornithine aminotransferase and pyrroline-5-carboxylate reductase, the enzymes of proline biosynthesis and markedly inhibited the enzymes involved in proline degradation viz., proline oxidase and pyrroline-5-carboxylate dehydrogenase. These results suggest that increase in free proline content of Ragi leaves could be due to enhanced activities of the enzymes synthesizing proline but more importantly due to severe inhibition of the enzymes degrading proline. These observations establish for the first time, the pathway of proline metabolism in plants by way of detection of the activities of all the enzymes involved and also highlight the role of these enzymes in proline accumulation during water stress.     

Assay and subcellular localization of pyrroline-5-carboxylate dehydrogenase in rat liver

Delta1-pyrroline-5-carboxylate dehydrogenase (P5CDh) catalyzes the conversion of Delta1-pyrroline-5-carboxylate to glutamate in a reaction requiring NADP+ as a cofactor. Delta1-pyrroline-5-carboxylate is formed in liver from proline by proline oxidase (EC number not assigned) or from ornithine via ornithine aminotransferase. A spectrophotometric assay for P5CDh was shown to be valid if rotenone was included in the assay to prevent reoxidation of NADH. Using this new assay, liver was fractionated using differential centrifugation and the distribution of P5CDh was compared to that of appropriate marker enzymes. P5CDh is enriched only in the mitochondrial fractions, as are the mitochondrial enzymes, succinate cytochrome c reductase, proline oxidase, glutaminase, and ornithine aminotransferase. Thus, it can be concluded that P5CDh occurs only in mitochondria, not in both mitochondria and cytoplasm, as had previously been reported.     

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.     

Arginine metabolism in insects. Role of arginase in proline formation during silkmoth development

1. Ornithine delta-transaminase (l-ornithine-2-oxo acid aminotransferase, EC 2.6.1.13) and Delta(1)-pyrroline-5-carboxylate reductase [l-proline-NAD(P) 5-oxidoreductase, EC 1.5.1.2] were demonstrated in fat-body and flight-muscle tissues of the silkmoth Hyalophora gloveri. Arginase (l-arginine ureohydrolase, EC 3.5.3.1) is also present in these tissues. 2. Arginase, ornithine transaminase and pyrroline-carboxylate reductase are generally considered to make up the catabolic pathway for the conversion of arginine into proline. The conversion of l-[U-(14)C]arginine into [(14)C]proline by intact fat-body tissue was used to show that the enzymes in insect fat body also function in this capacity. 3. Of the three enzymes of the catabolic pathway, only arginase increased during adult development and the increase coincided with the emergence of the winged adult moth. Since proline appears to be a major substrate utilized in insect flight metabolism, the increase in arginase activity at this stage suggests a major role for arginase in proline formation.     

On the possible significance of the transamidination reaction in evolution

The origin of the various muscle phosphagens during evolution is considered in the context of the need to conserve ornithine for the synthesis of proline for connective tissue necessary for structural strength and flexibility and/or a complicated musculature. In each phosphagen, arginine is known to have contributed its amidine moiety thus maintaining the function of the phosphagen and setting free the proline precursor ornithine. Tissues from an earthworm, a starfish and a sea-squirt have been found to contain the enzymes arginase, ornithine aminotransferase and pyrroline-5-carboxylate reductase which are necessary to convert arginine to proline. For each of the animals studied analysis for the relevant free amino acids and for the characteristic amino acids (Pro, Oh-Pro, Oh-Lys, Gly) of collagen are presented. The amino acid composition of the diet of the sea-squirt Pyura stolonifera and of the starfish Coscinasterias calamaria is presented along with the level of the phosphagen kinases of the animals studied. The significance of the experimental results is discussed in connection with the importance of the transamidination reaction.     

Proline dehydrogenase and pyrroline-5-carboxylate reductase from pumpkin cotyledons

Activity of proline dehydrogenase and pyrroline-5-carboxylate reductase was greatest after 5 and 7 days germination in green and etiolated cotyledons respectively of pumpkin (Cucurbita moschata Poir. cv. Dickinson Field). The ratio of pyrroline-5-carboxylate reductase to proline dehydrogenase activity was constant throughout germination. Both enzymes were purified 30-fold but the ratio pyrroline-5-carboxylate reductase—proline dehydrogenase activity was constant throughout purification. However, this ratio decreased with storage, especially in purified preparations. Both enzymes were stable at high temperature and the ratio pyrroline-5-carboxylate reductase—proline dehydrogenase remained unchanged on heating. Proline dehydrogenase and pyrroline-5-carboxylate reductase were inhibited by sodium bisulfite and cysteine. ATP, ADP and NADP caused inhibition of both enzymes. Proline dehydrogenase utilized NAD but not NADP. Pyrroline-5-carboxylate reductase had a 2.5-fold greater activity with NADH than NADPH. Most of the data presented suggest that proline dehydrogenase and pyrroline-5-carboxylate reductase activities occur on the same protein molecule.     

The stimulation of purine nucleotide production by pyrroline-5-carboxylic acid in human erythrocytes

We previously reported that pyrroline-5-carboxylate (PC), the intermediate in the interconversions of proline, ornithine and glutamate markedly stimulates hexosemonophosphate-pentose pathway activity in human erythrocytes. The stimulation is mediated by pyrroline-5-carboxylate reductase which generates NADP⁺ accompanying the conversion of pyrroline-5-carboxylate to proline. We now report that the previously demonstrated effect of pyrroline-5-carboxylate on glucose oxidation through the hexose-monophosphate-pentose pathway is accompanied by increased phosphoribosyl-pyrophosphate production and increased formation of nucleotides via the salvage pathway. The demonstrated effect of pyrroline-5-carboxylate on purine processing may provide a regulatory link between amino acid and nucleotide metabolism.     

Changes of glucose metabolism and skin-collagen neogenesis in vitamin B6 deficiency

The mechanism of pellagrous changes in skin caused by a deficiency of vitamin B6 was studied in respect to neogenesis of proline in skin collagen and glucose metabolism. In vitamin B6 deficiency the insulin/glucagon coefficient in serum decreased significantly from 3.02 to 2.32, indicating a metabolic change towards gluconeogenesis. A deficiency of vitamin B6 caused a decrease in the levels of vitamin B6-dependent enzymes, such as ornithine aminotransferase, alanine aminotransferase, and aspartate aminotransferase, which also contribute to gluconeogenesis. Because the conversion of ornithine to proline via pyrroline-5-carboxylate was suppressed due to the decrease in ornithine aminotransferase activity, the amount of proline in the skin collagen fraction also decreased significantly in vitamin B6-deficient rats compared with the pair-fed control. These results suggest that the pellagrous lesions in vitamin B6-deficiency are caused by an impaired synthesis of proline from ornithine, which results in the suppression of collagen neogenesis in the skin.     

Does altered nitrogen metabolism and H2O2 accumulation explain the vitrified status of the fully habituated callus of Beta vulgaris (L.)?

The habituated callus is a vitrified tissue which has two main biochemical characteristics both leading to production of toxic forms of oxygen: first the blockage of the porphyrin pathway and a lack of H₂O₂ detoxifying enzymes (catalase and peroxidases); secondly a deviation of the nitrogen metabolism induced by NH₃ accumulation. Ammonia detoxification is ensured by increased glutamate dehydrogenase activity and accumulation of both proline and polyamines. A putative linkage between proline synthesis and the HMP pathway, as proposed for animal proliferating tissues (Phang 1985), might explain a high purine biosynthesis and cytokinin autonomy.Abbreviations FFA free fatty acids - 6PG-DH 6-phosphogluconate dehydrogenase - G6P-DH glucose-6-phosphate dehydrogenase - GLU glutamate - GDH glutamate dehydrogenase - GR glutathion reductase - H habituated callus - HMP hexoses-monophosphate - IAA indolyl-acetic acid - LOX lipoxygenase - MDA malondialdehyde - N normal callus - OAT ornithine aminotransferase - ORN ornithine - PAs polyamines - P₅C pyrroline-5-carboxylate - P₅CR pyrroline-5-carboxylate reductase - PP-ribose-P phosphoribosyl pyrophosphate - SOD superoxide dismutase     

Water stress-induced alterations in the proline metabolism of drought-susceptible and -tolerant cassava (Manihot esculenta) cultivars

The free proline levels and activities of ornithine aminotransferase (EC 2.6.1.13) and proline oxidase (EC 1.5.2.2), two of the enzymes involved in proline metabolism were studied during the induction of water stress in a drought susceptible (M-4) and a drought tolerant (S-1315) cultivar of cassava ( Manihot esculenta Crantz). Water stress induced by polyethylene glycol (MW 6000, osmotic potential — 1.65 MPa) caused a ca 25-fold increase in proline in young excised leaves of the susceptible cultivar (M-4) while the increase was about 9-fold in the tolerant cultivar (S-1315). The activity of ornithine aminotransferase (OAT), a key enzyme involved in the biosynthesis of proline, was found to increase 3-fold in water stressed leaves of M-4 and about 2-fold in those of S-1315. The activity of proline oxidase, which is involved in the degradation of proline to pyrroline-5-carboxylate, was reduced by 50% in M-4 and nearly 25% in S-1315 on water stress. Comparison of the kinetic properties of OAT showed that the enzyme from water-stressed leaves is more stable to heat inactivation compared to that of control. These results indicate that during water stress there are alterations in the metabolism of proline in cassava, and the extent of alteration varies between drought-susceptible and -tolerant cultivars.     

Stimulation of the hexose-monophosphate pentose pathway by delta 1-pyrroline-5-carboxylic acid in human fibroblasts.

L-pyrroline-5-carboxylic acid, an intermediate in the interconversions of glutamic acid, ornithine and proline, is a potent stimulator of the hexose-monophosphate pentose pathway in cultured human fibroblasts. These studies suggest that pyrroline-5-carboxylate reductase, which catalyzes the conversion of pyrroline-5-carboxylate to proline coupled with the oxidation of NADPH, provides the NADP for the observed activation of the hexose-monophosphate pentose pathway.     

Linkage of the HMP pathway to ATP generation by the proline cycle

The reactions catalyzed by proline oxidase and pyrroline-5-carboxylate reductase form a catalytic cycle linking the hexose-monophosphate pentose (HMP) pathway to mitochondrial ATP generation. The cycling of proline and pyrroline-5-carboxylate couples glucose oxidation to ATP generation by a mechanism independent of the Embden-Meyerhof pathway and the tricarboxylic acid cycle.     

Proline and Proline Metabolising Enzymes in in-vitro Selected NaCl-tolerant Brassica juncea L. under Salt Stress

The effect of salt stress was studied on proline accumulationand the activities of proline metabolic pathway enzymes in seedlingand leaf tissue of two genetically stable lines (SR2P1-2 andSR3P6-2) of in vitro selected NaCl-tolerant plants and parentcultivar Prakash of Brassica juncea L. Salt stress caused differentialenhancement in proline level in both seedlings and leaf tissueof plants at different developmental stages. The magnitude ofincrease in proline content was higher in SR3P6-2 line in seedlings(34 fold at 140 meq^-1 NaCl) as well as leaves (16 fold at 40d after sowing at 100 meq^-1 NaCl) compared to the parent cv.Prakash (29 fold in seedlings and five fold in leaves) and SR2P1-2(21 fold in seedlings and five fold in leaves) at similar stresslevels. Salt stress also resulted in changes in the activitiesof enzymes of proline metabolism. The activities of prolinebiosynthetic enzymes, pyrroline-5-carboxylate reductase andornithine aminotransferase, increased under salt stress bothin the seedlings and leaves. The range of increase in the activitiesof the two enzymes was relatively higher in SR3P6-2 (3·3-3·9fold) compared to the SR2P1-2 (1·8-2·8 fold) andparent cv. Prakash (1·5-2·8 fold). The activityof proline degrading enzyme, proline oxidase, decreased undersalt stress in both the tissues of all the lines; the reductionin activity was relatively greater in SR3P6-2 compared to SR2P1-2or cv. Prakash. The trend of changes in the enzyme activitieswas in tune with the increase in proline level, the magnitudeof change did not match the extent of increase in proline level.Copyright1995, 1999 Academic Press Brassica juncea L., NaCl-tolerant somaclones, proline content, ornithine aminotransferase, proline oxidase, pyrroline 5-carboxylate reductase     

The oxidation of proline by mitochondrial preparations

The oxidation by mitochondria of various rat tissues of proline, pyrroline-5-carboxylate (P5C) and a number of aldehydes has been studied and ADP/O ratios determined for liver mitochondria. High oxidative activity for proline and P5C was found only in the liver and kidney. During the oxidation by liver and kidney mitochondria of proline and P5C; glutamate, ammonia, aspartate and some ornithine accumulated, thus suggesting that proline may normally be converted to ornithine by mitochondria. The oxidation of P5C (glutamic acid semialdehyde) by a mitochondrial dehydrogenase may be the same enzyme that oxidizes succinic acid semi-aldehyde but different from that oxidizing acetaldehyde.     

Development of pyrroline-5-carboxylate synthase and N-acetylglutamate synthase and their changes in lactation and aging

Using newly developed assay procedures, we studied the development of pyrroline-5-carboxylate synthase (PCS) and N-acetylglutamate synthase (AGAS) activity in rat tissues. PCS in the small intestine of fetuses was 1/5 that of adults and reached an adult level as early as postnatal Day 1. The highest peak was observed at Day 14, and then activity decreased to the adult level. However, PCS in the brain was highest at birth and quickly inactivated in a few days. AGAS in the fetus small intestine was 1/3 that of adults and became higher than the adult level by 40% at Day 1 but was reduced to 1/2 that of adults at Day 3. Subsequently activity increased gradually to the adult level at Day 24. On the contrary, AGAS in the fetus liver was only 1/20 that of adults, and activity increased slowly up to 10 weeks and more. Pregnancy and lactation reduced liver AGAS markedly up to Day 8 and intestinal PCS considerably up to Day 14 after parturition. PCS in the small intestine of senescent rats was almost halved compared to young controls on a whole tissue basis. AGAS in the small intestine was also halved on a gram wet weight basis. Nonetheless the liver AGAS of 430-day-old rats was higher than that of the controls, although that of 630-day rats was lower. The results indicate that the arginine synthesizing enzymes in the small intestine are highly activated in suckling and weaning, and raise a question whether arginine remains fully dispensable in pregnancy, lactation, and senescence.     

Arginine utilization by Chlorella autotrophica and Chlorella saccharophila

Chlorella saccharophila can utilize the amino acids arginine, glutamate. ornithine and proline as sole sources of nitrogen for growth. By comparison C. autotrophica utilized only arginine and ornithine. Following osmotic shock of Chlorella autotrophica from 50 to 150% artificial seawater rapid synthesis of proline (the main osmoregulatory solute in this alga) occurred in cells grown on arginine or citrulline. However, little proline synthesis occurred in ornithine-grown cells. Distribution of radiolabelled carbon from [¹⁴C]-arginine assimilation following osmotic shock of C. autotrophica agrees with the following pathway of arginine utilization: arginine→citrulline→ornithine→glutamate semialdehyde→pyrroline-5-carboxylate→proline. These 4 steps are catalysed by arginine deiminase (EC 3.5.3.6), citrullinase (EC 3.5.1.20), ornithine transaminase (EC 2.6.1.13) and pyrroline-5-carboxylate reductase (EC 1.5.1.2), respectively. Of these 4 enzymes, only arginine deiminase and pyrroline-5-carboxylate reductase were detected in the crude extract of the 2 Chlorella species. Arginine deiminase did not require specific cations for optimal activity. The deimi-nase showed maximal activity at pH 8.0 and followed Michaelis-Menten kinetics with an apparent K_m for L-arginine of 0.085 m M for the C. autotrophica enzyme and 0.097 m M for that of C. saccharophila. The activity of arginine deiminase was not influen-ced by growing C. saccharophila on arginine. Ornithine competitively inhibited arginine deiminase with an apparent K, of 2.4 m M for the C. autotrophica enzyme, and 3.8 m M for that of C. saccharophila . Arginine utilization by Chlorella is discussed in relation to that of other organisms.