α-Glycerophosphate dehydrogenase

Uses

α-Glycerophosphate Dehydrogenase from rabbit muscle has been used:

• in the reaction mixture to measure the glycerol kinase activity
• to demonstrate compartmentalized enzymatic reactions where NADH is involved
• in the reaction mixture assay for L-fuculose-1-phosphate aldolase

General Description

α-Glycerophosphate dehydrogenase is a membrane protein and a flavin-linked primary dehydrogenase.

Biochem/physiol Actions

α-Glycerophosphate dehydrogenase catalyzes the oxidation of L-α-glycerophosphate (Glp) to form dihydroxyacetone phosphate (DHAP) and hydrogen peroxide (H2O2). This enzyme is associated with respiratory electron transport chain, glycolysis, and phospholipid metabolism.

Purification Methods

The dehydrogenase is recrystallised by adding (NH4)2SO4 to 0.45 saturation at pH 5.5 at 4oC, and the small amount of precipitate is removed, then a saturated solution of (NH4)2SO4 is added dropwise from time to time over several days in the cold room. The crystals are collected and recrystallised until they have maximum activity. The enzyme is stable in half-saturated (NH4)2SO4 for several weeks at 4o. The equilibrium [dihydroxyacetone][NADH][H+]/[G-3-P][NAD] is 1.0 x 10 -12M in Tris buffer at 25o. It uses NAD ten times more efficiently than NADP. The Km for G-3-P is 1.1 x 10 -4M, for NAD it is 3.8 x 10-4M and for dihydroxyacetone it is 4.6 x 10-4M in phosphate buffer pH 7.0 and at 23.3o. Dihydroxyacetone phosphate and fructose-1,6-diphosphate are inhibitors. [Branowski J Biol Chem 180 515 1949, The Enzymes 7 85 1963, Young & Pace Arch Biochem Biophys 75 125 1958, Walsh & Sallach Biochemistry 4 1076 1965.]