Inducible Membrane-bound L-Lactate Dehydrogenase from Escherichia coZi

Membrane-bound L-lactate dehydrogenase was solubilized from the membranes ofEscherichia coli and purified to homogeneity using conventional procedures. The enzyme had a pH optimum of 8 to 9 and was specific for L-lactate. Its apparent K, for L-lactate and maximal velocity were 1.2 x lo-’ M and 31 pmol of tetrazolium dye reducedlminlmg of protein, respectively. It had a polypeptide molecular weight of 43,000 determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The average molecular weight of the purified enzyme after removal of detergent was estimated to be 480,000 by sucrose gradient centrifugation, suggesting that the enzyme was an oligomer in detergent-free solution. The purified enzyme was also an oligomer in the presence of Tween SO or cholate. It contained about 1 mol of flavin mononucleotidelmol of polypeptide with molecular weight of 43,000. The fluorescence of the flavin in the purified enzyme was 5.3% quantum yield of that of FMN. Specific antibody for this enzyme inhibited the enzyme activity and L-lactate-dependent uptake of oxygen in inverted membrane vesicles, suggesting that the purified enzyme was a primary dehydrogenase of the respiratory chain and was localizing on the inner surface of the cytoplasmic membranes. This enzyme was induced by growing the cells on DL-lactate, in confirmation of an earlier report (Kline, E. S., and Mahler, H. R. (1965)Ann. N. Y. Acud. Sci. 119, 905919). Cells grown aerobically on glycerol or anaerobically on glucose showed no appreciable activity of this enzyme or cross-reacting materials.