Proline Oxidase Research Articles

Glucocorticoid induction of proline oxidase in LLC-RK1 cells.

Dexamethasone induced proline oxidase in cultured LLC-RK1 cells, an epithelial cell line derived from rabbit kidney. The dexamethasone-mediated increase in enzyme activity was concentration and time dependent. Although the effect could be dissociated from cell growth and cell density, it was dependent on protein and RNA synthesis. A comparison of the enzyme isolated from control and dexamethasone-treated cells showed that the increased activity was not due to an alteration in the affinity of the enzyme for proline. These findings suggest that glucocorticoids induce the synthesis of proline oxidase in mammalian cells.

A single mutation affects L-serine deaminase, L-leucyl-, L-phenylalanyl-tRNA protein transferase, and proline oxidase activity in Escherichia coli K-12

A mutation at a single locus, wyb, results in several phenotypic changes in Escherichia coli K-12. The Wyb- phenotype includes: (i) an increase in L-serine deaminase activity, together with a loss of inducibility by L-leucine; (ii) an absence of L-leucyl-, L-phenylalanyl-tRNA protein transferase activity; (iii) inducibility of proline oxidase by proline; and (iv) a loss of ability to use maltose as a carbon and energy source.

Proline metabolism in cartilage: The importance of proline biosynthesis

Mammalian cells have the capacity to synthesize proline from ornithine or glutamic acid, although preformed proline usually is available from extracellular sources. We have measured the rate of proline formation from ornithine in the presence of physiologic concentrations of proline. In rat xiphoid cartilage organ cultures with ornithine and proline present at equimolar concentrations, we have found that at least 20% of the proline incorporated is derived from ornithine. In addition, we have studied the proline biosynthetic and degradative enzymes in several different tissues. The rate of proline incorporation is threefold greater in xiphoid cartilage than in liver or kidney; and in each of these tissues, the levels of the proline biosynthetic and degradative enzymes correlate with the relative demand for proline incorporation. The biosynthetic enzymes pyrroline-5-carboxylate reductase and ornithine aminotransferase are present at high levels in xiphoid cartilage, whereas the degradative enzymes proline oxidase and pyrroline-5-carboxylate dehydrogenase are absent and at very low levels, respectively. In contrast, in liver and kidney, pyrroline-5-carboxylate reductase is at a much lower level and proline oxidase and pyrroline-5-carboxylate dehydrogenase are present at high levels. It appears that proline biosynthesis is an important process, especially in collagenforming tissues, and that proline oxidation is limited to a few specialized mammalian tissues.

Nitrogen regulation of amino acid catabolism in Neurospora crassa.

Neurospora crassa can utilize numerous compounds including certain amino acids as a sole nitrogen source. Mutants of the nit-2 locus, a regulatory gene which is postulated to mediate nitrogen catabolite repression, are deficient in the ability to utilize several amino acids as well as other nitrogen sources used by wild type. Various enzymes involved in amino acid catabolism were found to be regulated in distinct ways. Arginase, ornithine transaminase, and pyrroline-5-carboxylate dehydrogenase are all inducible enzymes but are not subject to nitrogen catabolite repression. By contrast, proline oxidase and the amino acid transport system(s) are controlled by nitrogen repression and their synthesis is increased markedly when nitrogen source is limiting. Unlike wild type, the nit-2 mutant cannot derepress amino acid transport, although proline oxidase is regulated in a normal fashion.

Regulation of the major proline permease gene of Salmonella typhimurium

The structural gene for the major proline permease is located in a tight cluster with genes coding for the proline degradative enzymes, proline oxidase and pyrroline-5-carboxylic acid dehydrogenase. Expression of the permease is regulated in parallel with the two degradative enzymes, and all three functions are subject to catabolite repression. Regulatory mutants (putC) have constitutively high levels of all three activities, suggesting that all are regulated by a single mechanism.

Cluster of genes controlling proline degradation in Salmonella typhimurium

A cluster of genes essential for degradation of proline to glutamate (put) is located between the pyrC and pyrD loci at min 22 of the Salmonella chromosome. A series of 25 deletion mutants of this region have been isolated and used to construct a fine-structure map of the put genes. The map includes mutations affecting the proline degradative activities, proline oxidase and pyrroline-5-carboxylic dehydrogenase. Also included are mutations affecting the major proline permease and a regulatory mutation that affects both enzyme and permease production. The two enzymatic activities appear to be encoded by a single gene (putA). The regulatory mutation maps between the putA gene and the proline permease gene (putP).

Reduced expression of a distal gene of the prn gene cluster in deletion mutants of Aspergillus nidulans: Genetic evidence for a dicistronic messenger in an eukaryote

The prn gene cluster involved in L-proline catabolism in Aspergillus nidulans, has the gene order prnA-prnD-regulatory region-prnB-prnC. prnB, prnD, and prnC specify proline permease, proline oxidase, and delta1-pyrroline-5-carboxylate (P5C) dehydrogenase, respectively. prnA is probably a positive regulatory gene whose product is necessary for expression of the prn activities. Proline induces proline permease and P5C dehydrogenase in prnD- mutants which lack proline oxidase, showing that proline does not have to be converted to P5C to act as inducer. Deletion mutations extending from within prnD to within prnB result in considerably reduced expression of prnC, whereas a prnD- prnB- double mutant shows normal prnC expression. This strongly suggests that the deletion mutations eliminate a promotor/initiator site for transcription of a dicistronic messenger for prnB and prnC. The fact that the deletions do not eliminate prnC expression altogether indicates that at least one other species of prnC transcript (monocistronic, tricistronic, or tetracistronic) can be made.

GltB gene and regulation of nitrogen metabolism by glutamine synthetase in Escherichia coli.

A mutant (gltB) of Escherichia coli lacking glutamate synthase (GOGAT) was unable to utilize a wide variety of compounds as sole nitrogen source (e.g., arginine, proline, gamma-aminobutyrate, and glycine). Among revertants of these Asm- strains selected on one of these compounds (e.g., arginine, proline, or gamma-aminobutyrate) were those that produce glutamine synthetase (GS) constitutively (GlnC phenotype). These revertants had a pleiotropically restored ability to grow on compounds that are metabolized to glutamate. This suggested that the expression of the genes responsible for the metabolism of these nitrogen sources was regulated by GS. An examination of the regulation of proline oxidase confirmed this hypothesis. The differential sensitivities of GlnC and wild-type strains to low concentrations (0.1 mM) of the glutamine analog L-methionine-DL-sulfoximine supported the conclusion that the synthesis of a glutamine permease was also positively controlled by GS. During the course of this study we found that the reported position of the locus (gltB) for glutamate synthase is incorrect. We have relocated this gene to be 44% linked to the argG locus by P1 transduction. Further mapping has shown that the locus previously called aspB is in reality the gltB locus and that the "suppressor" of the aspB mutation (A. M. Reiner, J. Bacteriol. 97:1431-1436, 1969) is the locus for glutamate dehydrogenase (gdhA).

Regulation of proline oxidase activity by lactate.

We found that proline oxidase, the first enzyme of the proline degradative pathway, is inhibited by lactate. The Km of the enzyme for proline increases with increasing concentrations of lactate. Since proline can be a source for gluconeogenesis, regulation of proline degradation by lactate may serve as a mechanism for allocation of metabolic fuel sources. The marked inhibition of proline oxidase at levels of lactate that commonly occur in both genetic and acquired lactic acidosis may cause the previously unexplained hyperprolinemia seen in these metabolic disorders.

Metabolic effects of hydrocortisone in mouse brain

The effect of intramuscular administration of hydrocortisone (10 mg/day per animal) for 5 days has been studied on the content of the amino acids belonging to the glutamate family, in the different regions of the mouse brain, along with the activities of glutamine synthetase, glutamate dehydrogenase, and aspartate, alanine, tyrosine, and ornithine aminotransferases. Further, since proline too is related to glutamate metabolism, the activity of proline oxidase was also studied in these regions. As hydrocortisone is known to influence the ionic fluxes in different tissues and the nitrogen metabolism, the activities of Na(+),K(+)-ATPase together with the content of RNA and protein have also been estimated. A fall in the amino acids of the glutamate family in all three regions was observed with an increase in glutamate dehydrogenase activity in cerebral cortex. A significant fall in the protein content was also observed, mainly in the brain stem. A universal increase in Na(+),K(+)-ATPase activity was observed in all three regions, with the highest in the cerebral cortex. The results indicate that hydrocortisone triggers increased utilization of glutamate in brain as an alternative to glucose, thereby shifting the nitrogen metabolism toward catabolism. The increased activity of Na(+),K(+)-ATPase under these conditions would further aggravate the same and may lead to membrane stabilization.

Glucocorticoid control of hepatic proline oxidase

Since adrenal corticosteroids are known to affect amino acid metabolism and gluconeogenesis, we examined the relationship of these hormones to hepatic proline oxidase, the mitochondrial enzyme degrading L-proline. In adrenalectomized rats hepatic proline oxidase activity decreased to about 50% of control levels within 5–6 days. This depressed activity can be restored to normal by 4 days of corticosteroid repletion. Treatment of intact rats with supraphysiologic doses of corticosteroid further elevated proline oxidase activity. The level in treated intact rats was threefold that of adrenalectomized rats. Kinetic analysis revealed that corticosteroid increased the amount of enzyme without altering enzyme affinity for proline. Administration of a single dose of corticosteroid to either adrenalectomized or intact animals increased enzyme activities, with a lag time of less than 2 hr. The maximum effect occurred 5–6 hr following injection. Since proline degraded by this pathway can contribute carbon skeletons directly to carbohydrate, corticosteroid induction of proline oxidase may play an important role in hepatic gluconeogenesis occurring with corticosteroid action.

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.

Regulation of enzyme synthesis by the glutamine synthetase of Salmonella typhimurium: a factor in addition to glutamine synthetase is required for activation of enzyme formation.

In Klebsiella aerogenes but not in Salmonella typhimurium glutamine synthetase can function during nitrogen-limited growth to increase the rate of synthesis of histidase from the hut genes of S. typhimurium 15-59 (hutS. 15-59). Formation of proline oxidase is also not increased in nitrogen-limited cultures of S. typhimurium. However, in hybrid strains of Escherichia coli or K. aerogenes, the glutamine synthetase of S. typhimurium activates synthesis of histidase from the hutS. 15-59 genes. Apparently, glutamine synthetase is necessary but not sufficient for activation of transcription of the hut genes; another factor must also be present. This factor is active in both K. aerogenes and E. coli but is missing or altered in S. typhimurium.

Lack of a regulatory function for glutamine synthetase protein in the synthesis of glutamate dehydrogenase and nitrite reductase in Escherichia coli K12.

Synthesis of glutamine synthetase (GS) in anaerobic batch cultures of Escherichia coli was repressed when excess NH4+ was available, but derepressed during growth with a poor nitrogen source. In wild-type bacteria there was only a weak inverse correlation between the activities of GS and glutamate dehydrogenase (GDH) during growth in various media. No positive correlations were found between the activities of GS and nitrite reductase, or between GS and cytochrome c552: both of these proteins were synthesized normally by mutants that contained no active GS. Although activities of GS and GDH were low in two mutants that are unable to synthesize cytochrome c552 or reduce nitrite because of defects in the nirA gene, the nirA defect was separated from the GS and GDH defects by transduction with bacteriophage P1. Attempts to show that catabolite repression of proline oxidase synthesis could be relieved during NH4+ starvation also failed. It is, therefore, unlikely that nitrite reduction or proline oxidation by E. coli are under positive control by GS protein. The regulation of the synthesis of enzymes for the utilization of secondary nitrogen sources in E. coli, therefore, different from that in Klebsiella aerogenes, but is similar to that in Salmonella typhimurium.

Metabolism of proline and its physiological significance in a blowfly, Aldrichina grahami

1. (1) In the larval stage of Aldrichina grahami the high proline diet (semi-synthetic diet consisting mainly of milk casein) resulted in a much higher concentration of free proline in the haemolymph than did the low proline diet (semi-synthetic diet consisting mainly of egg albumin). Therefore, proline accumulation in 4-day-old larvae reared on the high proline diet, is mainly due to the proline derived from the diet, and its accumulation seems to have no physiological significance. 2. (2) The synthesis of proline from glutamic acid in A. grahami was first observed in the adult stage. Proline was synthesized much more from glutamic acid than from arginine in the adult stage. 3. (3) Unnecessary interconversion between Δ 1-pyrroline-5-carboxylic acid (P5A) and proline seems to be prevented by separate compartmentation of P5A reductase and proline oxidase in the cell.

Partial purification and some properties of delta1-pyrroline-5-carboxylate reductase from Escherichia coli.

delta1-Pyrroline-5-carboxylate (PCA) reductase [L-proline:NAD(P)+5-oxidoreductase, EC 1.5.1.2] has been purified over 200-fold from Escherichia coli K-12. It has a molecular weight of approximately 320,000. PCA reductase mediates the pyridine nucleotide-linked reduction of PCA to proline but not the reverse reaction (even at high substrate concentrations). The partially purified preparation is free of competing pyridine nucleotide oxidase, PCA dehydrogenase, and proline oxidase activities. The Michaelis constant (Km) values for the substrate, PCA, with reduced nicotinamide adenine dinucleotide phosphate (NADPH) or NADH as cofactor are 0.15 and 0.14 mM, respectively. The Km values determined for NADPH and NADH are 0.03 and 0.23 mM, respectively. Although either NADPH or NADH can function as cofactor, the activity observed with NADPH is severalfold greater. PCA reductase is not repressed by growth in the presence of proline, but it is inhibited by the reaction end products, proline and NADP.

Biosynthesis of prodigiosin by non-proliferating wild-type Serratia marcescens and mutants deficient in catabolism of alanine, histidine, and proline

Mutants of Serratia marcescens Nima, designated as Aut, Hut, or Put, did not utilize L-alanine, L-histidine, or L-proline, respectively, as a sole carbon source but did utilize other amino acids or glycerol as carbon sources. The bacteria were permeable to alanine, histidine, and proline but lacked the enzymes responsible for degradation of these amino acids. The Aut mutant contained no L-alanine dehydrogenase activity, whereas the Hut and Put mutants contained only 7 and 4% of the histidase and proline oxidase activities, respectively, found in the wild-type strain. Rates of oxygen uptake and protein synthesis were significantly lower when the mutants were incubated in the presence of amino acids they could not degrade. Studies of L-[14C]alanine, L-[14C]histidine, and L-[14C]proline incorporation into prodigiosin synthesized by these mutants and the wild-type strain revealed that proline was incorporated intact, whereas all of alanine except the carboxyl group was incorporated into the pigment molecule. Histidine did not enter prodigiosin directly. These data suggested that the presence of unique biosynthetic pathways, independent of primary metabolism, leads to formation of prodigiosin from specific amino acids.

Alterations in proline metabolic enzymes with mammalian development

Through the use of specific radioisotopic assays, the activities of enzymes degrading and synthesizing proline were examined in rat liver and kidney as a function of development. Proline oxidase (PO), the enzyme converting proline to Δ 1-pyrroline-5-carboxylate (PC), undergoes 15- and eight-fold increases in liver and kidney, respectively, as rats mature from termfetal to adult life (6–12 wk). The differences are not due to enzyme inhibitors or activators, and kinetic analysis reveals the change to be one of greater tissue content of the same enzyme. Δ 1-Pyrroline-5-carboxylate dehydrogenase, which converts PC to glutamate, shows a two- to three-fold increase in both tissues, paralleling the changes in PO with development. Δ 1-Pyrroline-5-carboxylate reductase (PCR), the enzyme which catalyzes the committed step in endogenous proline formation, undergoes oppositely directed changes, such that adult levels are only 20%–25% of fetal levels in liver and kidney. PO/PCR ratios are 25- to 50-fold greater in adult central tissues than they are in fetal tissues. Thus, the central tissues of adult rats appear to function as proline utilizers, whereas those of young rats are chiefly proline formers. This difference may relate to different rates of utilization of proline for protein synthesis in young and adult rats.

Role of L-proline in the biosynthesis of prodigiosin.

Nonproliferating cells of Serratia marcescens, wild-type strain Nima, synthesized the pigment, prodigiosin, when saline suspensions were incubated with aeration at 27 degrees C in the presence of proline or alanine. Mutants PutS1 and PutS2 derived from strain Nima formed prodigiosin from alanine, but not from proline, unless alanine also was added. Strain Nima utilized proline as a sole source of carbon and of nitrogen for growth, whereas Put mutants did not. Investigation of enzymes degrading proline showed that the wild-type strain contained proline oxidase, which was absent in Put mutants. The wild type, as well as the mutants, utilized alanine as the sole source of carbon and nitrogen for growth. Although nonproliferating cells of Put mutants failed to synthesize prodigiosin from proline, addition of L-[U-14C]proline to suspensions metabolizing and synthesizing the pigment because of addition of alanine resulted in the incorporation of radioactive label into prodigiosin, as well as into cellular protein. Since Put mutants could not catabolize proline, the incorporation of [14C]proline into the prodigiosin molecule indicated that proline was incorporated directly into the pigment.

Enzymes of orithine metabolism in adult developing rat intestine

The levels of 11 enzymes, most of them involved in the metabolism of orithine, were measured in whole upper intestine, or in duodenum, small intestine and colon of adult rats. The developmental formations in small intestine of arginase, orithine aminotransferase, and orithine transcarbamylase were compared with those in liver. Changes with age (late gestation to adult) of the intestinal activities of pyrroline-5-carboxylate reductase, proline oxidase and glutamyl transpeptidase are also described. The results suggests 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 aminotraferase 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 orithine 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.