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At least 19 recordsLinked to original sources
PubMed2005

High activity of mitochondrial glycerophosphate dehydrogenase and glycerophosphate-dependent ROS production in prostate cancer cell lines.

Most malignant cells are highly glycolytic and produce high levels of reactive oxygen species (ROS) compared to normal cells. Mitochondrial glycerophosphate dehydrogenase (mGPDH) participates in the reoxidation of cytosolic NADH by delivering reducing equivalents from this molecule into the electron transport chain, thus sustaining glycolysis. Here, we investigate the role of mGPDH in maintaining an increased rate of glycolysis and evaluate glycerophosphate-dependent ROS production in prostate cancer cell lines (LNCaP, DU145, PC3, and CL1). Immunoblot, polarographic, and spectrophotometric analyses revealed that mGPDH abundance and activity was significantly elevated in prostate cancer cell lines when compared to the normal prostate epithelial cell line PNT1A. Furthermore, both the glycolytic capacity and glycerophosphate-dependent ROS production was increased 1.68- to 4.44-fold and 5- to 7-fold, respectively, in prostate cancer cell lines when compared to PNT1A cells. Overall, these data demonstrate that mGPDH is involved in maintaining a high rate of glycolysis and is an important site of electron leakage leading to ROS production in prostate cancer cells.

Cell Line, Tumor↗
PubMed1997

Nucleotide sequence of cDNA fragments coding for the FAD-,glycerophosphate- and calcium-binding domains of human islet mitochondrial glycerophosphate dehydrogenase.

The cDNA fragments coding for the FAD-, glycerophosphate- and calcium-binding domains of mitochondrial glycerophosphate dehydrogenase (mGDH) were synthetized using RNA extracted from freshly isolated pancreatic islets of a normal subject and two-non-insulin-dependent diabetic patients. Single strand conformation polymorphism analysis of the PCR products yielded the same mobility as control cDNA probes. Likewise, the nucleotide sequence and corresponding amino acid sequence were identical to the normal gene bank sequence. These findings argue against the presence, in pancreatic islets, of an mGDH isoform distinct from that previously characterized in extrapancreatic organs.

Aged↗
PubMed1980

The reconstitution of L-3-glycerophosphate-cytochrome c oxidoreductase from L-3-glycerophosphate dehydrogenase, ubiquinone-10 and ubiquinol-cytochrome c oxidoreductase.

Purified L-3-glycerophosphate dehydrogenase from pig brain mitochondria interacts with ubiquinone-10 and ubiquinol-cytochrome c oxidoreductase (Complex III) from bovine heart mitochondria to reconstitute antimycin-sensitive L-3-glycerophosphate- cytochrome c oxidoreductase. This activity is completely dependent on the two enzymes and largely dependent on ubiquinone-10. Reconstitution requires that the two enzymes should be simultaneously present in the same membranous aggregate produced by removal of detergent from the enzymes. Reconstitution by removing detergent by dialysis or dilution is inefficient because of self-aggregation of the dehydrogenase. Highly efficient reconstitution can be achieved if the enzymes are co-precipitated by addition of ethanol. The rate with reconstituted enzyme approaches that expected from the turnover of the dehydrogenase with ubiquinone-1 as acceptor. The behaviour of the reconstituted system shows some of the characteristics expected for a stoicheiometric association of one molecule of dehydrogenase with one molecule of Complex III. On raising the phospholipid/protein ratio, the dehydrogenase and Complex III appear to operate as independent enzymes acting in sequence. These effects are very similar to those observed for the interaction of NADH dehydrogenase and Complex III and are explained in terms of the model proposed by Heron, Ragan & Trumpower [(1978) biochem. J. 174, 791-800].

Animals↗
PubMed1977

Origin of alpha-glycerophosphate dehydrogenase isozymes in Drosophila melanogaster and their functional relationship in the alpha-glycerophosphate cycle.

The basis for the differentiation of L-glycerol-3-phosphate dehydrogenase (alpha-GPDH) into larval and adult isozymes in Drosophila melanogaster was investigated by the correlation of a lack of appearance of each isozyme during development within Drosophila bearing alpha-GPDH "null" alleles and by the study of a putative conversion factor. Conversion studies indicate the presence of a heat-labile RNase-resistant conversion factor present in crude larval extracts with the ability to convert GPDH-1 to GPDH-2 and GPDH-3 but not vice versa. In addition, "null" mutations at the Gpdh locus obliterate all isozymatic species of alpha-GPDH in all developmental stages. These observations suggest that all alpha-GPDH isozymes are the product of a single structural gene and that the multiple forms of this enzyme arise during successive developmental stages through an epigenetic modification of the primary Gpdh+ polypeptide. Finally, observations are reported which bear on the functional divergence of the alpha-glycerophosphate cycle in the adult and larval stage of development.

Alleles↗
PubMed1976

Regulation of alpha-glycerophosphate dehydrogenase activity in human term placental mitochondria.

1. alpha-Glycerophosphate dehydrogenase (sn-glycerol-3-phosphate:(acceptor) oxidoreductase, EC 1.1.99.5) activity in mitochondria isolated from human term placenta was found to be inhibited by ethyleneglycolbis (beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA). Addition of an excess of calcium ions to the incubation medium completely restored the original activity. The concentration of free calcium ion required to activate the alpha-glycerophosphate dehydrogenase was found to vary between 10 and 100 nM. 2. The pH optimum for alpha-glycerophosphate dehydrogenase activity varied with substrate concentration. The pH optima were 7.4 and 8.0 in the presence of 2 or 8 mM alpha-glycerophosphate, respectively. The apparent Km for alpha-glycerophosphate also varied with pH; the values being 0.4 mM at pH 7.05, 1.5 mM at pH 7.8, and 3.5 mM at pH 8.5. 3. alpha-Glycerophosphate dehydrogenase activity was inhibited by palmitoyl-CoA in a competitive manner with an apparent Ki value of about 10 muM. This inhibition was less pronounced in the presence of calcium or magnesium ions. 4. The activity of alpha-glycerophosphate dehydrogenase was inhibited by phosphoenolpyruvate, D- and DL-glyceraldehyde 3-phosphate and 3-phosphoglyceric acid, in a competitive manner, the apparent Ki values being 0.5, 0.95, 0.12 and 1.5 mM, respectively. 5. alpha-Glycerophosphate dehydrogenase activity in human placental mitochondria was found to be more sensitive to phosphoenolpyruvate, than the activity of the same enzyme in rat skeletal muscle mitochondria. alpha-Glycerophosphate dehydrogenase activity in rat brown adipose tissue mitochondria was only slightly affected by phosphenolpyruvate under the same conditions. 6. The data obtained suggest that the activity of alpha-glycerophosphate dehydrogenase in human placental mitochondria may be controlled by changes of the cytosolic level of palmitoyl-CoA, some glycolytic intermediates, and pH.

Adipose Tissue, Brown↗
PubMed1966

The components of an alpha-glycerophosphate cycle and their relation to oxidative metabolism in the lens.

1. The concentration of ATP in a lens brei is maintained when the brei is incubated in oxygen with alpha-glycerophosphate. Lack of alpha-glycerophosphate or incubation in nitrogen causes the concentration to decrease. alpha-Glycerophosphate has some effect under anaerobic conditions but this is not sufficient to account for the maintenance in oxygen. 2. Manometric experiments show that alpha-glycerophosphate enhances the respiration of lens preparations. This respiration can be further increased by the addition of ADP and is abolished by cyanide and antimycin. The inference from these experiments is that a mitochondrial system able to oxidize alpha-glycerophosphate is present, i.e. the particulate half of the alpha-glycerophosphate cycle. 3. More than the calculated proportion of NADH is used when limiting amounts of dihydroxyacetone phosphate are added to lens tissue in spectrophotometric experiments. Dihydroxyacetone phosphate is therefore regenerated and an alpha-glycerophosphate cycle is operative. 4. A preparation of a particulate alpha-glycerophosphate dehydrogenase that takes up oxygen with methylene blue as electron acceptor is described. 5. Methods for obtaining mitochondria from lens are compared, and a useful extraction medium is defined. 6. Mitochondria with activities of the same order of magnitude as those obtained from liver, with alpha-glycerophosphate and glutamate as substrates, are prepared from epithelium detached from the capsule; some respiratory control is observed.

Adenine Nucleotides↗
PubMed1965

Growth stasis by accumulated L-alpha-glycerophosphate in Escherichia coli.

Cozzarelli, N. R. (Harvard Medical School, Boston, Mass.), J. P. Koch, S. Hayashi, and E. C. C. Lin. Growth stasis by accumulated l-alpha-glycerophosphate in Escherichia coli. J. Bacteriol. 90:1325-1329.1965.-Cells of Escherichia coli K-12 can grow on either glycerol or l-alpha-glycerophosphate as the sole source of carbon and energy. The first step in the dissimilation of glycerol requires a kinase, and the initial process of utilization of l-alpha-glycerophosphate involves an active transport system. In either case, intracellular l-alpha-glycerophosphate is an intermediate whose further metabolism depends upon a dehydrogenase. When this enzyme is lost by mutation, the cells not only fail to grow on glycerol or l-alpha-glycerophosphate, but are subject to growth inhibition in the presence of either compound. Resistance to inhibition by glycerol can be achieved by the loss of glycerol kinase. Such cells are still susceptible to growth inhibition by l-alpha-glycerophosphate. Similarly, in dehydrogenase-deficient cells, immunity to exogenous l-alpha-glycerophosphate can be achieved by genetic blocking of the active transport system. Such cells are still sensitive to free glycerol in the growth medium. Reversal of inhibition by glycerol or l-alpha-glycerophosphate in cells lacking the dehydrogenase can also be brought about by the addition of glucose. Glucose achieves this effect without recourse to catabolite repression. Our results suggest that growth stasis associated with the over-accumulation of l-alpha-glycerophosphate is due to interference with other cellular processes by competition with physiological substrates rather than to depletion of cellular stores of adenosine triphosphate or inorganic phosphate.

Escherichia coli↗
PubMed1993

Formation of 1,3-cyclic glycerophosphate by the action of phospholipase C on phosphatidylglycerol.

The action of phospholipase C (PLC) from Bacillus cereus on phosphatidylglycerol (PG), derived from egg yolk phosphatidylcholine (PC), was examined in an ether-water mixture. The PLC cleavage of PG and PC followed a Michaelis-Menten kinetics with apparent Vmax values per 1 microgram enzyme of 0.26 and 0.91 mumol.min-1 and Km values of 10 and 12 mM, respectively. When the same enzymic reaction was carried out in minimally buffered aqueous solution of 1% Triton X-100, the decrease in pH with respect to phospholipid cleavage was as expected with PC but much less pronounced with PG. This could be accounted for by the formation of a cyclic glycerophosphate, rather than alpha-glycerophosphate, in the PLC hydrolysis of PG. Examination of the chemical nature of the water-soluble product of PG by phosphorus nuclear magnetic resonance (31P NMR) revealed a single band at 2.31 ppm, while the bands of alpha-glycerophosphate and beta-glycerophosphate appeared at 5.12 and 4.57 ppm, respectively. Basic hydrolysis of the phospholipase cleavage product of PG (0.1 M NaOH for 1 min at 80 degrees C) followed by neutralization shifted its 31P NMR band to 5.18 ppm, which practically coincided with that of alpha-glycerophosphate. Analogous experiments were carried out with PG labeled with 3H at the carbon 2 of the glycerol headgroup ([3H]PG). Autoradiography of thin layer chromatography (TLC) of the [3H]PG enzymic hydrolyzate displayed a single 3H-labeled compound, which could be converted to alpha-glycerophosphate by basic hydrolysis. These results strongly suggest that the phosphate headgroup of PG is cleaved off by PLC as 1,3-cyclic glycerophosphate. A series of PLC experiments with phosphatidyl dihydroxyacetone and phosphatidyl 1,3-propanediol as model substrates supported this assignment. Two-dimensional homonuclear 1H NMR correlated spectra as well as infrared spectra carried out on the isolated sodium salt of this product could further confirm such a structure. The unique structure and chemical nature of 1,3-cyclic glycerophosphate may bear a distinct physiological function.

Bacillus cereus↗
PubMed1970

Specificity and locale of the L-3-glycerophosphate-flavoprotein oxidoreductase of mitochondria isolated from the flight muscle of Sarcophaga barbata thoms.

1. The oxidation of l-3-glycerophosphate by flight-muscle mitochondria isolated from the flesh fly Sarcophaga barbata has been studied. Use of substrate analogues indicates that the catalytic and effector l-3-glycerophosphate binding sites on the allosteric l-3-glycerophosphate-flavoprotein oxidoreductase differ markedly in specificity. 2. The l-3-glycerophosphate-cyanoferrate oxidoreductase system in these mitochondria is antimycin-insensitive whereas the corresponding NADH-cyanoferrate oxidoreductase is extremely sensitive to this respiratory-chain inhibitor. Also no swelling is observed when these mitochondria are suspended in iso-osmotic solutions of ammonium glycerophosphate in contrast with the extensive swelling seen in similar solutions of ammonium pyruvate. These observations indicate that l-3-glycerophosphate does not penetrate the mitochondrial matrix whereas pyruvate does. 3. Submitochondrial particles catalyse the ATP-driven reduction of NAD(+) by l-3-glycerophosphate but at a far lower rate than that seen when succinate is the electron donor. These particles do not have an energy-linked pyridine nucleotide transhydrogenase activity. 4. We conclude that the l-3-glycerophosphate-flavoprotein oxidoreductase is located on the outer surface of the inner membrane of the flight-muscle mitochondria.

Adenosine Triphosphate↗
PubMed1979

Purification and properties of L-alpha-glycerophosphate oxidase from Streptococcus faecium ATCC 12755.

A procedure was developed to purify the Streptococcus faecium ATCC 12755 L-alpha-glycerophosphate oxidase. The molecular weight of the purified enzyme was 131,000 and the subunit molecular weight was 72,000. Two moles of FAD were bound/mol of enzyme. Apo-L-alpha-glycerophosphate oxidase displayed physical properties similar to the holoenzyme as judged by electrophoresis in 10% buffer gels at pH 8.5 and by centrifugation in a 5 to 20% linear sucrose gradient. The apoenzyme was completely reactivated by incubation with FAD. L-alpha-Glycerophosphate oxidase was specific for L-alpha-glycerophosphate when compared with several other pohsphorylated glycerol and sugar derivatives. Oxygen was the preferred electron acceptor. At 10 mM DL-alpha-glycerophosphate (below the Km of 26 mM for L-alpha-glycerophosphate), activity was increased from 2.6- to 10-fold by increasing the buffer concentration from 0.01 to 0.1 m. This buffer effect was observed with potassium phosphate and other anionic buffers. In 0.001 m potassium phosphate buffer, pH 7.0, activity was increased by several divalent metal ions, including 10 mM CaCl2 (7.7-fold activation) and 10 mM MgCl, (6.8-fold activation). Fructose 6-phosphate and fructose1-phosphate were inhibitors of the L-alpha-glycerophosphate oxidase.

Cations, Divalent↗