Characterization of a halophilic glyceraldehyde-3-phosphate dehydrogenase from the archaebacterium Haloarcula vallismortis.

Abstract

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purified to electrophoretic homogeneity from the halophilic archaebacterium Haloarcula (Halobacterium) vallismortis. The purification was achieved by (NH4)2SO4-mediated chromatography on Sepharose 4B and DEAE- cellulose, and hydrophobic and hydroxylapatite chromatography. In contrast to nonhalophilic archaebacteria, only a single NAD+-specific GAPDH was found in the halobacterium. However, it shares the property of insensitivity to the antibiotic pentalenolactone with the enzymes from other archaebacterial sources. Like all other GAPDHs the enzyme from H. vallismortis is a homomeric tetramer with catalytic properties comparable to the NAD+-specific enzymes characterized so far. The molecular mass of the subunit, deduced under denaturing conditions in the presence of a cationic detergent, was 40±2kDa. The halobacterial GAPDH is a halophilic enzyme requiring high concentrations of salt for activation and tability and is an acidic protein. Immunological tests between the halobacterial enzyme and its counterparts from eubacterial, mammalian and nonhalophilic archaebacterial sources were negative.