Studies on the purification and characterization of malate dehydrogenase from Mycobacterium phlei.

Malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) was purified from Mycobacterium phlei using (NH4)2SO4 precipitation followed by chromatography on Sephadex G-200, DEAE-cellulose on DEAE Sephadex A-50. The purified preparation homogeneous on column chromatography, polyacrylamide gel electrophoresis and sodium dodecyl sulphate gel electrophoresis had a molecular weight of 86 860. The native enzyme was composed of four subunits of equal molecular weight (21 550) and was thermostable upto 50 degrees C for 15 min. Some kinetic constants of the enzyme was determined. Tyrosine and isoleucine were identified as the N- and C-terminal amino acids respectively. The effects of various activators and inhibitors on the activity of the purified enzyme were studied. The purified enzyme exhibited maximum excitation and emission at 278 and 345 nm respectively. Amino acid composition of the enzyme was determined. Treatment of the enzyme with acid and urea resulted in dissociation of the enzyme followed by loss of catalytic activity. The dissociated enzyme could however be reconstituted by bringing the pH back to neutrality or by removal of urea from the enzyme solution.