Activities for nicotinamide adenine dinucleotide (NAD)-dependent and NAD-independent forms of lactate dehydrogenase (LDH) were measured in cell-free extracts of Staphylococcus aureus strain PS 6 for the d and l isomers of lactate. Data obtained for the NAD-dependent lactate dehydrogenases indicate that oxidation of both isomers of lactate is due to both an l -lactate-specific LDH and a lactate racemase. After acrylamide gel electrophoresis, two bands exhibiting LDH activity were detected in crude or in partially purified cell-free extracts. The fast band exhibited LDH activity that was not NAD-dependent for both isomers of lactate, whereas, the slow band had very high NAD-dependent LDH activity for the l isomer but just detectable activity or the d isomer. Both bands appeared when d -lactate was used as the substrate, but only the slow band was formed when l -lactate was the substrate. NAD-dependent LDH, in apparent association with a nonspecific tetrazolium-reducing protein, is responsible for the production of the slow band.
Lactate dehydrogenase (LDH) was studied in phage-propagating strains 29, 3A, 6, 81, and 42D of Staphylococcus aureus selected from the five groups in the International-Blair series. Cells were cultivated in Brain Heart Infusion (Difco) under nearly anaerobic conditions and were harvested near the end of the log phase. LDH activity-was maximal at the end of the exponential growth period and was measured spectrophotometrically by reduction of p-nitro-blue tetrazolium, with phenazine methosulfate as a coupling agent. Crude enzyme extracts were prepared both by an acetone extraction technique and by sonic treatment. LDH activity for these enzyme preparations was determined by the colorimetric method mentioned and also by measuring the rate of nicotinamide adenine dinucleotide reduction at 340 m,. The order of activity observed, by use of both assay methods, was 29 > 81 > 6 > 3A > 42D. LDH forms (possibly isoenzymes) for each of 15 strains, which represent the five phage-propagating groups of the International-Blair series, were separated by acrylamide gel electrophoresis. Five forms were distinguished and arbitrarily numbered on the basis of their rate of migration, no. 5 being the slowest component. No one strain had more than four, nor fewer than two, LDH forms. Form 3 appeared in 13 of the 15 strains and was followed in frequency by no. 2, 1, 4, and 5.
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