Abstract
(LDH) obtained from larvae of Drosophila melanogaster was purified to homogeneity by affinity chromatography on oxamate-Sepharose. The purification procedure is simple to operate and gives a homogeneous preparation in a good yield (34.86%) after only two steps. Utilizing the homogeneous LDH preparation, an attempt was made to characterize the LDH molecule. Thus, it was found that the N-terminal amino acid is isoleucine, and the enzyme is tetrameric and composed of four identical subunits of apparent molecular weight 38,000, suggesting that it is controlled by a single gene. Homogeneous LDH preparations exhibit one band on neutral acrylamide gels when the substrate is either DL-lactic acid or L-(+)-lactate. The optimum temperature is 45 degrees C for the purified enzyme and 40 degrees C for the crude homogenate. The Km values for pyruvate and NADH are 0.154 and 0.027 mM, respectively, while the Km values for lactate and NAD are 29.4 and 1.33 mM, respectively. A discontinuity in the Ea slope was observed at a transition temperature of 30 degrees C. The Ea value between 20 and 30 degrees C was calculated as 12.06 kcal/mol, while between 30 and 45 degrees C the Ea value was 4.01 kcal/mol. This evidence, together with other observations reported in the literature, suggests that the LDHs of invertebrates and vertebrates have arisen by divergent evolution from a common ancestral gene.
