Partial Purification and Properties of Respiratory Chain-Linked L-Glycerol 3-Phosphate Dehydrogenase from a Marine Bacterium, Vibrio alginolyticus

Abstract

Respiratory chain-linked L-glycerol 3-phosphate (G3P) dehydrogenase [EC 1.1.99.5] of marine bacterium, Vibrio alginolyticus, was extracted from the membrane fraction by treatment with Tween 20, and fractionation on DEAE-Sephacel and QAE-Sephadex in the presence of 0.05% Liponox DCH(a1kyl polyoxyethylene ether) yielded a preparation having a specific activity of 22.1 units/mg protein when assayed by phenazine methosulfate (PMS)-coupled reduction of thiazolyl blue tetrazolium (MTT). The purified enzyme had an apparent molecular weight of 300,000 as determined by chromatography on Sephacryl S-300 in 0.05% Liponox DCH, and had noncovalently bound FAD as its coenzyme. The enzyme had a pH optimum of 8.5–9.0, and required 200 mM NaCl or KC1 and an appropriate detergent (such as Tween, Brij or Liponox DCH) for maximum activation. The activating effect of NaCl was due to a decrease in K_m for G3P and that of Tween 20 was due to both a decrease in K_m and an increase in V_m. Triton X-100 could not activate the enzyme and was inhibitory in the presence of phospholipids. The reaction followed a ping-pong mechanism. In addition to PMS, 2,6-dichlorophenol indophenol and duroquinone, the enzyme could reduce ubiquinone-5 (Q-5) in the presence of Liponox DCH at a rate of 46% of the PMS reductase activity. The enzyme was strongly inhibited by heavy metal ions and by p-chloromercuribenzoate. The activity for Q-5, but not for PMS, was inhibited by o-phenanthroline and bathophenanthroline, suggesting the participation of nonheme iron protein in the Q-5 reduction.