The inhibition by L-α-glycerophosphate of the reduction of dihydroxyacetone phosphate by crystalline rabbit muscle NAD+-linked L-α-glycerophosphate dehydrogenase has been examined. As a result of the measurement of the absorbance at 340 mμ in a photometric test system at 26° containing 0.08–2.0 mM dihydroxyacetone phosphate, 0.14 mM NADH, and 1–1.5 μg crystalline enzyme in 1.5 ml 10 mM EDTA −0.1 M phosphate buffer at pH 7-0, the apparent Michaelis constant (Km) for dihydroxyacetone phosphate was found to be 0.363 mM (± 0.025 S.E.). L-α-Giycerophosphate, but not D-α-glycerophosphate, acted as a competitive inhibitor in this system with an apparent inhibition constant (Ki) of 0.575 mM (± 0.030). Substitution of 50 mM triethanolarnine buffer for the 0.1 M phosphate buffer lowered the Kmto 0.088 mM (± 0.019) and the Kito 0.240 mM (± 0.013). To study the enzyme at lower NADH concentrations, a fluorometric system containing 20–75 μM NADH, 5–370 μM DHAP, and 0.5–2.0 μg enzyme in 1 ml 2 mM EDTA −50 mM triethanolarnine buffer, pH 7.0 at 23°, was used. The apparent Kmfor dihydroxyacetone phosphate and Kifor L-α-glycerophosphate were 0.075 μM (± 0.020) and 0.186 mM (± 0.006) respectively, at a NADH concentration of 75 μM. Lowering the NADH concentration to 20 μM further decreased the apparent Kmand Kivalues to 0.039 mM (± 0.008) and 0.056 mM (± 0.007) respectively.A consideration of the concentrations of dihydroxyacetone phosphate and L-α-glycerophosphate in muscle during contraction suggests that the competitive inhibition of cytoplasmic L-α-glycerophosphate dehydrogenase by its product, L-α-glycerophosphate, may influence the pathway of triose phosphate utilization and also the coupling, by way of the L-α-glycerophosphate cycle, of cytoplasmic NADH-generating reactions to the mitochondrial respiratory chain.
Mechanism of Action of Rabbit Muscle Phosphoglucomutase
