Search PubMed⌕ Search

Biomedical subjects

J M Phang

Publications and source records attributed to J M Phang.

At least 73 records · Page 4Linked to original sources
PubMed1981

Regulation of proline biosynthesis: the inhibition of pyrroline-5-carboxylate synthase activity by ornithine.

Mammalian cells have the capacity for proline biosynthesis from ornithine or glutamic acid. Using a radioisotopic assay, we have studied the regulation by ornithine of delta 1-pyrroline-5-carboxylate synthase, the enzyme that catalyzes the first step of proline biosynthesis from glutamic acid. In homogenates from Chinese hamster ovary cells, ornithine was found to be a potent inhibitor of pyrroline-5-carboxylate synthase activity(50% inhibition at 0.37 mM). The effect was reversible and did not occur with amino acids other than ornithine. Preliminary findings suggest that the inhibition does not result from altered requirements for the cofactors NADPH and ATP. Significant inhibition was observed in four different Chinese hamster cell lines. Ornithine was also shown to inhibit the conversion of 3H-glutamic acid to 3H-proline in intact human skin fibroblasts. Cells from patients with a rare ocular disease, gyrate atrophy of the choroid and retina, were used for these studies since they lack interfering ornithine aminotransferase activity. We conclude that ornithine may be a physiologic regulator of the rate of proline formation from glutamic acid. This information allows us to construct an hypothetical model for the overall regulation of proline biosynthesis and also to suggest a pathophysiologic mechanism for the disease gyrate atrophy.

Animals↗
PubMed1981

Pyrroline-5-carboxylate reductase in human erythrocytes.

Pyrroline-5-carboxylate reductase, which converts pyrroline-5-carboxylate to proline, has been identified in human erythrocytes. The level of pyrroline-5-carboxylate reductase activity in these cells is comparable to the activity levels of major erythrocyte enzymes. The physiologic function of the enzyme in erythrocytes cannot be related to its function in other tissues, i.e., producing proline for protein synthesis. We examined the kinetic properties of erythrocyte pyrroline-5-carboxylate reductase and compared them to the properties of the enzyme from proliferating cultured human fibroblasts. We found that the kinetic properties and regulation of the erythrocyte enzyme are distinctly different from those for human fibroblast pyrroline-5-carboxylate reductase. These characteristics are consistent with the interpretation that the function of the enzyme in human erythrocytes may be to generate oxidizing potential in the form of NADP+.

Adult↗
PubMed1980

Pyrroline-5-carboxylate synthase activity in mammalian cells.

Although glutamic acid is known to be a precursor for proline biosynthesis, the enzymatic conversion of glutamic acid to pyrroline-5-carboxylic acid, the immediate precursor of proline, has not been demonstrated in cell-free systems. By providing appropriate concentrations of ATP and NADPH and blocking further metabolism of pyrroline-5-carboxylic acid, we have developed a method for measuring the formation of pyrroline-5-carboxylic acid from glutamic acid in homogenates of mammalian cells. We have designated this activity pyrroline-5-carboxylate synthase. To confirm that our assay is a valid measure of the initial step in proline biosynthesis from glutamic acid, we have compared two mutant lines of Chinese hamster ovary cell. Proline prototrophic cells, which can synthesize proline from glutamic acid, have easily measurable pyrroline-5-carboxylate synthase activity (5.97 nmol of pyrroline-5-carboxylic acid per hr per mg of homogenate protein). In contrast, proline auxotrophic cells, which are unable to synthesize proline from glutamic acid, have no detectable pyrroline-5-carboxylate synthase activity.

Adenosine Triphosphate↗
PubMed1979

Changes in proline synthetic and degradative enzymes during matrix-induced cartilage and bone formation.

Proline biosynthetic and degradative enzymes are unevenly distributed in differentiated mammalian tissues. Activities of the synthetic enzymes are relatively high in collagenous tissues, whereas activities of the degradative enzymes are high in noncollagenous tissues. In order to further characterize tissue-specific proline biosynthesis and degradation, we have determined proline enzyme activities during cartilage and bone formation induced by demineralized bone matrix. We can thus follow temporal changes in enzyme activity in a single tissue as different cell types develop. Ornithine aminotransferase and pyrroline-5-carboxylate reductase have peaks of activity which correlate with maximal type II collagen synthesis by chondrocytes. Both enzymes also are active during bone formation. In contrast, proline oxidase and pyrroline-5-carboxylate dehydrogenase are present at low levels and do not change as new cell types appear. Arginase activity peaks during the first 3 days and then rapidly decreases by the time cartilage and bone formation begin. These observations further substantiate the importance of proline biosynthesis in collagenous tissues. The close correlation between ornithine aminotransferase activity and type II collagen synthesis suggests that the pathway from ornithine to proline may be especially important during formation of type II collagen.

Animals↗
PubMed1979

The importance of ornithine as a precursor for proline in mammalian cells.

Ornithine aminotransferase catalyzes the reversible transamination of L-ornithine to delta1-pyrroline-5-carboxylate, the immediate precursor of proline. The direction and flux through this pathway in mammalian cells has not been established. Glutamate has generally been considered to be the most important precursor for proline biosynthesis, but recent studies in xiphoid cartilage indicate that a significant fraction of cellular proline is derived from ornithine. Using newly isolated mutant Chinese hamster ovary cells with defined defects in the proline biosynthetic pathways, we now have established that cells can grow at a maximal rate with ornithine as the sole source of proline. Furthermore, we have measured the rate of proline formation from ornithine (1.6 nmol/h/10(6) cells); Future studies with these mutant Chinese hamster ovary cells may offer insight into the regulatory mechanism which coordinates proline biosynthesis from ornithine and glutamate.

Animals↗
PubMed1979

Genetic evidence for a common enzyme catalyzing the second step in the degradation of proline and hydroxyproline.

The initial step in the degradation pathways of proline and hydroxyproline is catalyzed by proline oxidase and hydroxyproline oxidase, yielding delta 1-pyrroline-5-carboxylate and delta 1-pyrroline-3-hydroxy-5-carboxylate, respectively. The second step is the oxidation of delta 1-pyrroline-5-carboxylate to glutamate and delta 1-pyrroline-3-hydroxy-5-carboxylate to gamma-hydroxy-glutamate. To determine if this second step in the degradation of proline and hydroxyproline is catalyzed by a common or by separate enzyme(s), we developed a radioisotopic assay for delta 1-pyrroline-3-hydroxy-5-carboxylate dehydrogenase activity. We then compared delta1-pyrroline-3-hydroxy-5-carboxylate dehydrogenase activity with that of delta 1-pyrroline-5-carboxylate dehydrogenase in fibroblasts and leukocytes from type II hyperprolinemia patients, heterozygotes, and controls. We found that cells from type II hyperprolinemia patients were deficient in both dehydrogenase activities. Furthermore, these activities were highly correlated over the range found in the normals, heterozygotes, and patients. We conclude from these data that a common delta 1-pyrroline-5-carboxylate dehydrogenase catalyzes the oxidation of both delta 1-pyrroline-5-carboxylate and delta 1-pyrroline-3-hydroxy-5-carboxylate, and that this activity is deficient in type II hyperprolinemia.

1-Pyrroline-5-Carboxylate Dehydrogenase↗
PubMed1978

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.

Animals↗
PubMed1977

Proline oxidase in cultured mammalian cells.

We sought a cultured cell line with Proline Oxidase activity to study the regulation and physiologic role of the enzyme in mammalian tissues. Among the cell lines tested, only LLC-RK1 cells, derived from rabbit kidney, had significant Proline Oxidase activity; the Km for proline of the enzyme from these cells was similar to that for the liver enzyme. LLC cells, Proline Oxidase positive, were able to convert proline to CO2. In contrast, CHL cells, Proline Oxidase negative, did not have this capability. The presence of Proline Oxidase in LLC cells and the absence of the enzyme in fibroblasts suggest that Proline Oxidase may serve as a marker enzyme for distinguishing parenchymal kidney cells from fibroblasts in culture. Cells transformed by SV40 virus and cells transformed by methylcholanthrene had activities higher that the parent cell line, but this effect of transformation could not be generalized to all transformed cells. Finally, L-hydroxy proline at 100-fold greater concentration than substrate L-proline failed to decrease proline oxidation. This finding suggests distinct degradative enzymes for these two amino acids.

Animals↗
PubMed1977

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 three-fold 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.

Adrenalectomy↗
PubMed1977

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.

Animals↗
PubMed1976

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 delta 1-pyrroline-5-carboxylate (PC), undergoes 15- and eight-fold increases in liver and kidney, respectively, as rats mature from term-fetal 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. delta 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. delta 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.

Aging↗