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J M Phang

Publications and source records attributed to J M Phang.

At least 19 recordsLinked to original sources
PubMed1992

A new functional role for P-glycoprotein: efflux pump for benzo(alpha)pyrene in human breast cancer MCF-7 cells.

We propose that the cellular burden of certain carcinogens may be mitigated by P-glycoprotein (P-gp), the putative drug efflux pump. In a series of multidrug resistant human breast cancer MCF-7 cells with increasing P-gp expression we examined this hypothesis using benzo(alpha)pyrene, a widely distributed environmental and dietary carcinogen. We found that multidrug resistant cells were cross-resistant to benzo(alpha)pyrene and the rates of efflux for benzo(alpha)pyrene were higher in multidrug resistant cells than in wild type cells. Evidence supporting the involvement of P-gp included the inhibition of azidopine binding to P-gp benzo(alpha)pyrene and the inhibition of benzo(alpha)pyrene efflux by Adriamycin and verapamil. Our findings suggest that P-gp may play a role in the cellular defense to carcinogens. The expression of P-gp and the modulation of its function may affect the susceptibility of normal tissues to transformation by carcinogens.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

Structural analogues of pyrroline 5-carboxylate specifically inhibit its uptake into cells.

Pyrroline 5-carboxylate, a naturally occurring intermediate, is a potent activator of redox-dependent metabolic pathways. The effect of pyrroline 5-carboxylate is due, at least in part, to the special mechanism mediating its entry into cells. Using Chinese hamster ovary cells we recently characterized the cellular uptake of pyrroline 5-carboxylate as a process transferring oxidizing potential pari passu with cell entry, a process consistent with group translocation. We sought to identify specific inhibitors to probe this unique uptake mechanism, to blockade the metabolic effects of pyrroline 5-carboxylate, and to provide strategies to identify the putative carrier protein. Because pyrroline 5-carboxylate, a ring structure with a tertiary nitrogen, is in spontaneous equilibrium with glutamic-gamma-semialdehyde, an open-chain structure, we tested analogues of both. Most open-chain aldehydes at 10 mM had little effect on the uptake of pyrroline 5-carboxylate. Although succinic semialdehyde did inhibit, its effect was nonspecific in that the uptake of alpha(methylamino) isobutyric acid was inhibited as much as the uptake of pyrroline 5-carboxylate. In contrast, pyrroline 2-carboxylate and other cyclic compounds with tertiary nitrogens, e.g., pyridines, were specific inhibitors of pyrroline 5-carboxylate uptake. Respective potencies of pyridine derivatives depended on the nature and location of constituent groups. Kinetics studies showed that these inhibitors were competitive with pyrroline 5-carboxylate and the most potent inhibitor, 2,6-pyridinedicarboxaldehyde, exhibited a K12 of 0.27 +/- 0.05 mM.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Accumulation of pyrroline 5-carboxylic acid in conditioned medium of cultured fibroblast: stimulatory effects of serum, insulin, and IGF-1.

Pyrroline 5-carboxylate, an intermediate of amino acid metabolism, is released into medium by cultured normal human fibroblasts. With cells made quiescent by serum starvation, the addition of 10% fetal bovine serum augmented the release of pyrroline 5-carboxylate into medium by 2.5-fold. Although platelet-derived growth factor was without effect, both insulin and insulinlike growth factor-1 nearly reproduced the serum effect. The dose-dependence of insulin and insulinlike growth factor 1 effects suggested their mediation by their own respective receptors. Although the mechanism for the stimulatory effect remains unknown, these effects of insulin and insulinlike growth factor 1 on pyrroline 5-carboxylate suggest hormonal regulation of pyrroline 5-carboxylate release.

Blood↗
PubMed1989

Purified human erythrocyte pyrroline-5-carboxylate reductase. Preferential oxidation of NADPH.

Pyrroline-5-carboxylate reductase catalyzes the final step in proline synthesis by NAD(P)H-dependent reduction of pyrroline-5-carboxylate. We have purified and characterized this enzyme from human erythrocytes. Purification to homogeneity (approximately 600,000-fold) was accomplished by sonication, ultracentrifugation, 2',5'-ADP-Sepharose affinity chromatography, and DEAE-Sephacel ion exchange chromatography. The enzyme runs as a single band of 30,000 Mr on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sizing chromatography under nondenaturating conditions demonstrates activity in the 300,000-350,000 Mr range, suggesting that the native enzyme exists as a 10- to 12-mer. The purified enzyme exhibits kinetic characteristics similar to those previously described for whole red cell homogenates. The Vmax is 10-fold higher and the Km for pyrroline-5-carboxylate is 7-fold higher with NADH versus NADPH as cofactor. The affinity for NADPH is 15-fold higher than that for NADH. Erythrocyte pyrroline-5-carboxylate reductase is competitively inhibited by NADP+. Unlike the enzyme from some other sources, erythrocyte pyrroline-5-carboxylate reductase is not inhibited by proline or ATP. Double label studies using [14C]pyrroline-5-carboxylate and [3H]exNADPH in the presence of both NADH and NADPH were performed to determine the preferred source of reducing equivalents. In the presence of physiologic concentrations of pyrroline-5-carboxylate and both pyridine nucleotides, all of the reducing equivalents came from NADPH. We suggest that, in some cell types including human erythrocytes, a physiologic function of pyrroline-5-carboxylate reductase is the generation of NADP+.

1-Pyrroline-5-Carboxylate Dehydrogenase↗
PubMed1989

Type II hyperprolinaemia in a pedigree of Irish travellers (nomads).

We describe a study of 312 subjects in 71 families near related to a proband with type II hyperprolinaemia. The subjects were Irish travellers (nomads) among whom consanguineous marriage and high fertility are common. Thirteen additional cases of type II hyperprolinaemia were discovered; all were offspring of consanguineous unions. A further 50 subjects were found to have mild hyperprolinaemia. We found a strong association between type II hyperprolinaemia and seizures during childhood but no significant association with mental handicap. Most adults with type II hyperprolinaemia enjoyed normal health and there was no evidence that maternal hyperprolinaemia compromised fetal development. The documented association between type II hyperprolinaemia and seizures may be related to the neuromodulatory or reducing-oxidising effects of proline and pyrroline-5-carboxylate, respectively, that has been shown in vitro. Alternatively, another genetic defect closely linked to the type II hyperprolinaemia allele could be the explanation.

1-Pyrroline-5-Carboxylate Dehydrogenase↗
PubMed1989

Fluctuations in plasma pyrroline-5-carboxylate concentrations during feeding and fasting.

Pyrroline-5-carboxylate (P5C) is the oxidized metabolite of proline. In cultured cells redox-sensitive metabolic pathways are influenced by these two amino acids. To determine whether plasma P5C levels fluctuate enough physiologically to regulate such pathways in vivo, we measured venous plasma P5C concentrations hourly for 48 h in five normal subjects while eating and in four subjects while fasting. Two fed subjects had one or more plasma P5C excursions per day exceeding 10 times the baseline level; the other fed subjects had lesser variations. These patterns suggest a relationship of plasma P5C peaks to meals. This relationship is supported by the finding of no significant P5C elevations above baseline levels in four subjects during fasting for 24 h. Despite the changes in plasma P5C concentrations, those of proline and other metabolically related amino acids were constant. The variation among subjects in the degree of plasma P5C fluctuation could not be explained by differences in total protein and caloric intake or body weight. Among plasma constituents, the large peak to basal plasma P5C excursions in some of the normal subjects we studied are matched only by those of peptide hormones.

Adult↗
PubMed1988

Stimulation of phosphoribosyl pyrophosphate and purine nucleotide production by pyrroline 5-carboxylate in human erythrocytes.

Recent studies have shown that pyrroline 5-carboxylate, the intermediate in the interconversions of proline, ornithine, and glutamate, can regulate the metabolism of erythrocytes. We now report that the formation of 5-phosphoribosyl 1-pyrophosphate (PP-Rib-P) was markedly stimulated by pyrroline 5-carboxylate in intact red cells. The production of PP-Rib-P is an important point of regulation in nucleotide metabolism. We found that pyrroline 5-carboxylate increased glucose metabolism through the oxidative arm of the pentose shunt, ribose 5-phosphate formation, and PP-Rib-P production and subsequently augmented purine nucleotide production through the salvage pathway in erythrocytes. We now report that pyrroline 5-carboxylate markedly stimulated the net synthesis of inosine monophosphate from hypoxanthine in intact human red cells so that the pool of inosine monophosphate became 20-30% of the total pool of purine nucleotides. Inosine monophosphate has been considered to be a "mobile pool" of purines, i.e. a reservoir from which peripheral tissues can be supplied; the effect of pyrroline 5-carboxylate on the inosine monophosphate pool may be a mechanism for regulating the function of erythrocytes in purine delivery.

Adenine Nucleotides↗
PubMed1988

The uptake of pyrroline 5-carboxylate. Group translocation mediating the transfer of reducing-oxidizing potential.

The cellular uptake of pyrroline 5-carboxylate (P5C) is of interest because this nutritionally responsive constituent of human plasma can mediate the transfer of oxidizing potential into cells and stimulate the production of phosphoribosyl pyrophosphate. Using a cloned line of Chinese hamster ovary cells, we found that the uptake of P5C was saturable, temperature-dependent, and sensitive to metabolic inhibitors. Furthermore, this uptake of P5C exhibited unusual features. It was independent of sodium ion and had a pH optimum of 6.4. The kinetics characteristics of P5C uptake included an apparent Km of 0.46 +/- 0.04 mM and a Vmax of 19.6 +/- 1.8 nmol/min/mg. Although the Vmax for P5C was comparable to those for certain other amino acids, e.g. leucine, it was significantly higher than that for alpha-methylaminoisobutyric acid in these cells. Importantly, there was no interaction between these amino acids and the uptake mechanism for P5C. Twenty naturally occurring amino acids, each at a concentration of 5 mM, were without effect on the uptake of P5C. Interestingly, the uptake mechanism for P5C is unusual in that it is linked to the transfer of reducing-oxidizing potential. Over wide ranges of P5C concentration and duration of incubation, P5C entry is coupled to its conversion to proline and the concomitant oxidation of reduced pyridine nucleotide with stimulation of the pentose phosphate shunt. In fact, no free P5C derived from the medium could be detected in cells. Our interpretation of these findings is that P5C uptake occurs by its own unique mechanism, a group translocation that mediates the transfer of reducing-oxidizing potential.

Amino Acids↗
PubMed1987

Regulation of the pentose phosphate pathway in human astrocytes and gliomas.

Several aspects of the regulation of the pentose phosphate pathway were examined in cultured normal human cortical astrocytes and gliomas of pathological grades I-IV. The generation of radiolabeled CO2 from [1-14C]glucose by the oxidative arm of the pentose phosphate pathway is a saturable process and has a maximum flux rate of 8-9 nmol/hr/mg cell protein. The flux can be blocked by the glycolytic inhibitor iodoacetamide but is unaffected by agents which inhibit oxidative phosphorylation. The magnitude of the pentose phosphate flux is directly related to the glioma grade. Grade IV gliomas (glioblastoma) show a pentose phosphate flux rate of approximately 4% of the total glucose flux. The flux rate can be increased by pharmacological agents which decrease the NADPH/NADP+ ratio. Both the activity and the regulation of glioma glucose-6-phosphate dehydrogenase (G6PDH) are altered in high-grade gliomas. While the affinity constants for cofactors in whole homogenates were not significantly different in glioma or normal astrocyte homogenates, normal astrocytes have a lower Km for glucose-6-phosphate and a G6PDH activity which is 10-fold greater than that of gliomas. NADPH is a powerful regulator of G6PDH activity in the normal astrocytes and in gliomas. At a NADPH/NADP+ ratio of 7:1 the normal astrocyte G6PDH is entirely inhibited, while the glioma enzyme is only 70% inhibited even at a ratio of 20:1. Increased metabolic flux through the oxidative arm of the pentose phosphate pathway is apparently due to an altered form of G6PDH.

Astrocytes↗
PubMed1986

Catalytic transfer of hydride ions from NADPH to oxygen by the interconversions of proline and delta 1-pyrroline-5-carboxylate.

Studies in reconstituted systems indicate that the interconversions of proline and delta 1-pyrroline-5-carboxylate can constitute a shuttle which transfers reducing equivalents into mitochondria as proline and oxidizing potential out as delta 1-pyrroline-5-carboxylate. The studies reported here determine if the transfer of reducing equivalents can be stoichiometrically greater than the utilization of shuttle intermediates. First, 3HOH production from [5-3H]proline was used to quantitate proline oxidation under conditions where delta 1-pyrroline-5-carboxylate could recycle back to proline in a system containing mitochondrial particles and pyrroline-5-carboxylate reductase. In parallel incubations the metabolic fate of [U-14C]proline was determined. 3HOH production continued to increase while no net change occurred in 14C-labeled proline and delta 1-pyrroline-5-carboxylate indicating catalytic activity in this system. In another system that contained intact mitochondria and pyrroline-5-carboxylate reductase, proline oxidation was quantitated by both [U-14C]proline recoveries and 3HOH production from [5-3H]proline. Proline oxidation was the same by both methods in incubations lacking NADPH and pyrroline-5-carboxylate reductase. However, it was significantly underestimated by [U-14C]proline recoveries as compared to 3HOH production in incubations containing all shuttle components. These results indicate catalytic functioning of the proposed shuttle in a reconstituted system containing intact mitochondria. This shuttle may function at specific times to catalytically generate cytosolic NADP+ and in turn regulate enzymes limited by [NADP+]. We suggest that the proposed shuttle may function to increase ribose-5-phosphate synthesis by the oxidative limb of the pentose phosphate pathway and inturn increase PP-ribose-P and purine synthesis during the initiation of cell growth.

Adenosine Triphosphate↗
PubMed1986

The aqueous humor of rabbit contains high concentrations of pyrroline-5-carboxylate.

Accumulating evidence suggests that the interconversion of proline and pyrroline-5-carboxylate (P5C) transfers oxidizing or reducing potential within or between cells. We report here that rabbit aqueous humor contains P5C at a concentration at least 20 times that found in rabbit plasma or in biologic fluids from other animals, and, in isolated lens, P5C stimulates the activity of the pentose phosphate shunt. However, aqueous humor in other species contains P5C in the same range of concentrations as their respective plasma. The high P5C concentration in rabbit aqueous humor may be due to comparatively low levels of P5C reductase activity in ocular tissues, especially the cornea. The levels in lens epithelium, though lower in the rabbit, nevertheless could mediate the transfer of oxidizing potential from P5C and stimulate the pentose-phosphate shunt. These results suggest that the eye may serve as a convenient model for the study of proline and P5C-related transfer of reducing or oxidizing potential between tissues.

Animals↗
PubMed1985

Deficiency of pyrroline-5-carboxylate synthase in the intestinal mucosa of the cat.

Pyrroline-5-carboxylate synthase activity of the intestinal mucosa of cats was compared to that of rats and found to be only 18% as high per gram of mucosa and only 5% as high per kilogram body weight. This severe limitation in the first step in the de novo synthesis of ornithine may be the metabolic basis for the severe hyperammonemia found in cats fed an arginine-deficient diet. This lack of ornithine synthesis makes the cat completely dependent on dietary arginine for the ornithine required for the removal of ammonia via urea synthesis in the liver.

Animals↗
PubMed1984

The effect of pyrroline-5-carboxylic acid on nucleotide metabolism in erythrocytes from normal and glucose-6-phosphate dehydrogenase-deficient subjects.

Pyrroline-5-carboxylate, the intermediate in the interconversion of proline, ornithine, and glutamate, increases 5-phosphoribosyl 1-pyrophosphate (PP-ribose-P) and purine nucleotide formation in intact human erythrocytes. We proposed that: 1) pyrroline-5-carboxylate is converted to proline by pyrroline-5-carboxylate reductase with concomitant oxidation of NADPH, 2) NADP+ augments glucose-6-phosphate dehydrogenase activity, and 3) production of ribose-5-phosphate via the pentose shunt is increased. Since glucose-6-phosphate dehydrogenase plays a central role in this proposed mechanism, we examined the responsiveness of glucose-6-phosphate dehydrogenase-deficient erythrocytes to pyrroline-5-carboxylate. We compared erythrocytes from four Sardinian glucose-6-phosphate dehydrogenase-deficient subjects and four Sardinian normal controls. Without pyrroline-5-carboxylate treatment, the levels of pentose shunt activity, PP-ribose-P, and inosine monophosphate were comparable in the two populations. However, the response to pyrroline-5-carboxylate in erythrocytes from normal and glucose-6-phosphate dehydrogenase-deficient subjects was markedly different. In normal erythrocytes, pyrroline-5-carboxylate treatment increased pentose shunt activity 600%, PP-ribose-P formation 250%, and the incorporation of hypoxanthine into inosine monophosphate 260%. In contrast, pyrroline-5-carboxylate had no effect on glucose-6-phosphate dehydrogenase-deficient erythrocytes. These findings strongly support our proposed mechanism for the pyrroline-5-carboxylate effect on nucleotides. Furthermore, the markedly different capacities for nucleotide synthesis in the two populations with pyrroline-5-carboxylate treatment suggest a role for pyrroline-5-carboxylate-mediated modulation of nucleotide metabolism in normal cells.

Erythrocytes↗
PubMed1984

Pyrroline-5-carboxylate in human plasma.

Pyrroline-5-carboxylate is the intermediate in the interconversions of proline, ornithine, and glutamate. Recent studies have suggested that pyrroline-5-carboxylate has regulatory properties in a wide range of tissues. Physiologic studies in humans have been limited by the unavailability of a sufficiently sensitive assay for this compound. Until now pyrroline-5-carboxylate has not been detectable in plasma of normal humans or even in plasma of patients with type II hyperprolinemia. We now report a method for measuring pyrroline-5-carboxylate made possible by a preparation of purified Escherichia coli pyrroline-5-carboxylate reductase. This method is more sensitive than currently available methods by two or three logs and is applicable for all biologic fluids. We have quantitated pyrroline-5-carboxylate in normal plasma, urine, and saliva. In addition, we have found pyrroline-5-carboxylate levels 10x to 20x normal in two patients with type II hyperprolinemia. The ability to measure physiologic concentrations of pyrroline-5-carboxylate now enables further characterization of its role as a regulatory molecule.

Chromatography, Ion Exchange↗
PubMed1983

Pyrroline-5-carboxylate stimulates the conversion of purine antimetabolites to their nucleotide forms by a redox-dependent mechanism.

The activation of purine antimetabolites to their respective nucleotides is a step critical to their effectiveness as chemotherapeutic agents. Erythrocytes, with their relatively simple purine metabolism, are useful as a model for identifying mechanisms which enhance this 5-phosphoribosyl 1-pyrophosphate (P-Rib-PP)-dependent activation. We previously showed that pyrroline-5-carboxylate, a physiologic intermediate in the interconversions of proline, ornithine, and glutamate, markedly stimulated the pentose phosphate pathway, increased the formation of P-Rib-PP, and increased purine incorporation into nucleotides. We now report that the events initiated by pyrroline-5-carboxylate markedly increased the activation of 6-thiohypoxanthine, 6-thioguanine, and azathioprine to their respective nucleotides in intact human erythrocytes. The mechanism of this effect was directly demonstrated in studies using the conversion of hypoxanthine to inosine monophosphate as a model for pyrroline-5-carboxylate-mediated stimulation of P-Rib-PP-dependent nucleotide formation. Since the P-Rib-PP-dependent activation of these chemotherapeutic agents may be important to their clinical effectiveness, the events initiated by pyrroline-5-carboxylate may provide new insight into the nature of tumor sensitivity and resistance to these agents.

Azathioprine↗
PubMed1983

Transfer of reducing equivalents into mitochondria by the interconversions of proline and delta 1-pyrroline-5-carboxylate.

Direct evidence is presented for a proline cycle using a cell-free experimental system which sequentially transfers 3H from [1-3H]glucose to NADP+ to delta 1-pyrroline-5-carboxylate and yields [3H]proline. The formation of [3H]proline depends on the presence of NADP, delta 1-pyrroline-5-carboxylate, and the enzymes glucose-6-phosphate dehydrogenase and delta 1-pyrroline-5-carboxylate reductase. The production of [3H]proline from unlabeled proline in the presence of mitochondria provides direct evidence for one complete turn of a proline cycle which transfers reducing equivalents produced by glucose oxidation in the pentose pathway into mitochondria. In this cycle, proline is oxidized to delta 1-pyrroline-5-carboxylate by mitochondrial proline oxidase. delta 1-pyrroline-5-carboxylate is released from mitochondria and is recycled back to proline by delta 1-pyrroline-5-carboxylate reductase with concomitant oxidation of NADPH. At the maximal rate observed, 60% of delta 1-pyrroline-5-carboxylate produced is recycled back to proline. This cycle provides a mechanism for transferring reducing equivalents from NADPH into mitochondria and is linked to glucose oxidation in the pentose pathway by NADPH turnover.

Animals↗