Isolation and properties of glycerol-3-phosphate oxidoreductase from human placenta

Abstract

Glycerol‐3‐phosphate oxidoreductase (sn‐glycerol 3‐phosphate:NAD+ 2‐oxidoreductase, EC 1.1.1.8) from human placenta has been purified by chromatography on 2,4,6‐trinitrobenzenehexamethylenediamine‐Sepharose, DEAE‐Sephadex A‐50 and 5′‐AMP‐Sepharose 4B approximately 15800‐fold with an overall yield of about 19%. The final purified material displayed a specific activity of about 88 μmol NADH min−1 mg protein−1 and a single protein band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The native molecular mass, determined by Ultrogel AcA 44 filtration, was 62000 ± 2000 whereas the subunit molecular mass, established on polyacrylamide gel in the presence of 0.1% sodium dodecyl sulphate, was 38000 ± 500. The isoelectric point of the enzyme protein, determined by column isoelectric focusing, was found to be 5.29 ± 0.09. The pH optimum of the placental enzyme was in the range 7.4–8.1 for dihydroxyacetone phosphate reduction and 8.7–9.2 for sn‐glycerol 3‐phosphate oxidation. The apparent Michaelis constants (Km) for dihydroxyacetone phosphate, NADH, sn‐glycerol 3‐phosphate and NAD+ were 26 μM, 5 μM, 143 μM and 36 μM respectively. The activity ratio of cytoplasmic glycerol‐3‐phosphate oxidoreductase to mitochondrial glycerol‐3‐phosphate dehydrogenase in human placental tissue was 1:2. The consumption of oxygen by human placental mitochondria incubated with the purified glycerol‐3‐phosphate oxidoreductase, NADH and dihydroxyacetone phosphate was similar to that observed in the presence of sn‐glycerol 3‐phosphate. The possible physiological role of glycerol‐3‐phosphate oxidoreductase in placental metabolism is discussed.

Increased Activity of Glycerol 3-phosphate Dehydrogenase and Other Lipogenic Enzymes in Human Bladder Cancer

“…The supernatant was combined with that obtained after the first centrifugation step and used for enzyme assay. The activity of glycerol 3-phospate dehydrogenase (EC 1.1.1.8), fatty acid synthase (EC 2.3.1.85), ATP-citrate lyase (EC 4.1.3.8), glucose 6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.42) and citrate synthase (EC 4.1.3.7) were measured as described previously [1,3,16,17]. All assays were performed in duplicate at 37 8C using a Beckman DU 68 spectrophotometer (Beckman Instruments, Fullerton, CA).…”

GPD1L links redox state to cardiac excitability by PKC-dependent phosphorylation of the sodium channel SCN5A

“…The purified proteins were cleaved by factor Xa (Novagen). G3P oxidation activity of the GPD1L WT and mutants were measured spectrophotometrically at 25°C by following the disappearance of the oxidation of G3P and formation of NADH at 340 nm (28). The assay mixture contained 1 mM EDTA and 1 mM 2-mercaptoethanol, 5 mM NAD ϩ and 5 mM G3P, buffered by 50 mM Tris ⅐ HCl (for pH 6.5-7.4).…”