Purification, Properties and Immunological Relationship of l(+)‐Lactate Dehydrogenase from Lactobacillus casei

Abstract

The fructose‐1,6‐bisphosphate‐activated l‐lactate dehydrogenase (EC 1.1.1.27) from Lactobacillus casei ATCC 393 has been purified to homogenity by including affinity chromatography (cibacronblue‐Sephadex‐G‐200) and preparative polyacrylamide gel electrophoresis into the purification procedures. The enzyme has an Mr of 132000–135000 with a subunit Mr of 34000. The pH optimum was found to be 5.4 in sodium acetate buffer. Tris/maleate and citrate/phosphate buffers inhibited enzyme activity at this pH. The enzyme was completely inactivated by a temperature increase from 60 °C to 70 °C. Pyruvate saturation curves were sigmoidal in the absence of fructose 1,6‐bisphosphate. In the presence of 20μM fructose 1,6‐bisphosphate a Km of 1.0 in M for pyruvate was obtained, whereas fructose 1,6‐bisphosphate had no effect on the Km of 0.01 mM for NADH. The use of pyruvate analogues revealed two types of pyruvate binding sites, a catalytic and an effector site. The enzyme from L. casei appears to be subject to strict metabolic control, since ADP, ATP, dihydroxyacetone phosphate and 6‐phosphogluconate are strong inhibitors. Immunodiffusion experiments with a rabbit antiserum to L. casei lactate dehydrogenase revealed that L. easel ATCC 393 l(+)‐lactate dehydrogenase is probably not immunologically related to group D and group N streptococci. Of 24 lactic acid bacterial strains tested only 5 strains did cross‐react: L. casei ATCC 393 = L. casei var. rhamnosus ATCC 7469 = L. casei var. alactosus NCDO 680 > L. casei UQM 95 > L. plantarum ATCC 14917. Copyright © 1976, Wiley Blackwell. All rights reserved