Malate dehydrogenase isolated from extremely halophilic bacteria of the Dead Sea. 1. Purification and molecular characterization

Abstract

The complete purification of malate dehydrogenase (EC 1.1.1.37) from extremely halophilic bacteria of the Dead Sea is described. The purification procedure includes precipitation by (NH4)2SO4, fractionation on Sepharose 4B using a decreasing concentration gradient of (NH4)2SO4, gel permeation chromatography on Sephadex G-100, chromatography on hydroxylapatite and affinity chromatography on 8-(6-aminohexyl)amino-NAD+-Sepharose at 4.26 M NaCl. The absorption and fluorescence spectra of the native and denatured enzyme were measured, and the extinction coefficient at 280 nm in 4.26 M NaCl was 0.803 cm2mg-1. The amino acid composition analysis showed an excess of 10.4 mol % of acidic amino acids. The apparent specific volume .vphi.'' of the active enzyme at 4.26 M NaCl was 0.680 .+-. 0.015 ml/g. The MW of the native enzyme was 84,000 .+-. 4000 determined in 4.26 M NaCl from equilibrium sedimentation data. The MW of the subunits is 39,000 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Thus, the native enzyme is composed of 2 subunits.