Oxidized nicotinamide-adenine dinucleotide-independent lactate dehydrogenases of Lactobacillus arabinosus 17.5

Abstract

1. 1. The object of this work was to investigate the nature and properties of an l- and a d-lactate dehydro g enase of Lactobacillus arabinosus 1 and to establish the relationship of these two enzymes to the two lactate dehydrogenases purified from the same organism by Dennis and Kaplan 2. 2. 2. The d- and l-lactate dejydrogenases were partially purified from cell-free extracts by ammonium sulphate and protamine sulphate treatments followed by chromatography on DEAE-cellulose columns. Finally the two enzymes were completely separated on CM-cellulose columns. 3. 3. The l-lactate dehydrogenase was further purified (a total of 1200-fold) by chromatography on TEAE- and Ecteola-cellulose columns. This enzymes contained 1 molecule of FMN per molecule of enzyme and approx. 20% of the flavin was reduced by the addition of sodium l-lactate. 4. 4. Both enzymes were NAD +-independent and only reduced oxidation-reduction dyes. The K m and optimum pH values were quite different from those reported for the d- and l-lactate dehydrogenases isolated by Dennis and Kaplan, also the former enzymes readily separated by electrophoresis in contrast to the latter enzymes. 5. 5. Both the NAD +-independent d- and l-lactate dehydrogenases were separated from the NAD +-dependent enzymes on a TEAE-cellulose column. Thus, Lactobacillus arabinosus appears to contain two pairs of lactate dehydrogenases, two of which are NAD +-linked and another two which are NAD +-independent. Of these latter two enzymes the l-lactate dehydrogenase is a flavoprotein while the d-lactate dehydrogenase is probably a flavoprotein also.