A Phospholipid-requiring Enzyme, Malate-Vitamin K Reductase: PURIFICATION AND CHARACTERIZATION

Abstract

Abstract Malate-vitamin K reductase has been purified to near homogeneity from Mycobacterium phlei. The purified enzyme is dependent upon an added phospholipid for activity and requires FAD as a cofactor. The enzymatic activity was found to be affected by the degree of enzyme aggregation. At high salt concentrations the enzyme existed in a monomeric form which was more active than the aggregated form. The enzyme was reversibly aggregated into a less active form by either dilution or dialysis against a buffer of low salt concentration. An enzyme-phospholipid complex was isolated by glycerol gradient centrifugation. It is suggested that a phospholipid binding site (or sites) seems to be involved in the aggregation-disaggregation process. The molecular weight of the monomeric form was determined to be 53,000 by Sephadex G-200 chromatography and 51,000 by sodium dodecyl sulfate gel electrophoresis, whereas the aggregated form had a molecular weight of approximately 164,000, as estimated by Sephadex G-200.