Studies on the succinate–neotetrazolium reductase system. Activation by vitamin K3

Abstract

The enzyme system linking succinate oxidation with neotetrazolium reduction has been investigated in rat-liver homogenates. In unsupplemented homogenates the production of formazan did not increase proportionately with increasing amounts of tissue. This non-linear response with succinate as substrate was unaffected by freezing and thawing the suspension; high-speed blending; homogenizing in hyper-osmotic sucrose; homogenizing in the presence of nicotinamide; addition of ethylenediaminetetraacetate, ox-plasma albumin, lipoic acid or riboflavin-5''-phosphate to the incubation medium. The variations produced in formazan production on varying the volume of the incubation medium and on adding boiled homogenate to the incubation medium suggested that a water-soluble cofactor was involved. Formazan production was stimulated by the addition of vitamin K3 to the incubation medium; this stimulation was, however, small with low final concentrations of tissue. Ascorbic acid or cysteine synergistically increased formazan production in the presence of vitamin K3 especially with low final concentrations of tissue. It is suggested that for optimum formazan production a heat-stable cofactor and reduced sulphydryl groups are required. The heat-stable cofactor appears to resemble vitamin K3 but not vitamin K1 in its properties; ascorbic acid and cysteine appear to function in the system by maintaining sulphydryl groups in the reduced form. Diphosphopyridine nucleotide does not appear to be directly involved in the succinate-neotetrazolium reductase system.