58] dl-Lactate dehydrogenases (NAD+-independent) from Lactobacillus arabinosus

Abstract

Publisher Summary The NAD+-independent lactate dehydrogenases are assayed by following the rate of reduction of 2,6-dichlorophenolindophenol at 600 mμ with either D- or L-lactate as a substrate. The combined lactate dehydrogenase activity may be measured using lithium DL-lactate as substrate. One unit of enzyme activity is defined as that amount catalyzing the oxidation of 1 micromole of lactate per minute and specific activity as units per milligram protein, determined by the biuret method. The amount of lactate oxidized is determined from the amount of indophenol reduced. This assay procedure may be applied to crude bacterial extracts, as it has been found that 10-3 M oxalate, a competitive inhibitor of both the D- and L- nicotinamide adenine dinucleotide+ (NAD+)-independent lactate dehydrogenases, completely abolishes the activity in crude extracts while the NAD+-dependent enzymes are not affected. The D-lactate dehydrogenase is considerably less stable than the L-lactate dehydrogenase during the fractionation procedure. The final D-lactate dehydrogenase preparation retains less than 10% of its original activity on storage at–15 ° for 2 weeks whereas the L-lactate dehydrogenase retains 30% of its original activity after 2 months' storage under similar conditions. Highly purified L-lactate dehydrogenase contains flavin mononucleotide (FMN) in the approximate ratio of 1 molecule of FMN per molecule of enzyme.