Malate dehydrogenase from Chlorobium vibrioforme, Chlorobium tepidum, and Heliobacterium gestii: purification, characterization, and investigation of dinucleotide binding by dehydrogenases by use of empirical methods of protein sequence analysis.
Malate dehydrogenase (MDH; EC 1.1.1.37) from strain NCIB 8327 of the green sulfur bacterium Chlorobium vibrioforme was purified to homogeneity by triazine dye affinity chromatography followed by gel filtration. Purification of MDH gave an approximately 1,000-fold increase in specific activity and recoveries of typically 15 to 20%. The criteria of purity were single bands on sodium dodecyl sulfate (SDS) and nondenaturing polyacrylamide electrophoresis (PAGE) and the detection of a single N terminus in an Edman degradation analysis. MDH activity was detected in purified preparations by activity staining of gels in the direction of malate oxidation. PAGE and gel filtration (Sephadex G-100) analyses showed the native enzyme to be a dimer composed of identical subunits both at room temperature and at 4 degrees C. The molecular weight of the native enzyme as estimated by gel filtration was 77,000 and by gradient PAGE was 74,000. The subunit molecular weight as estimated by SDS-gradient PAGE was 37,500. N-terminal sequences of MDHs from C. vibrioforme, Chlorobium tepidum, and Heliobacterium gestii are presented. There are obvious key sequence similarities in MDHs from the phototrophic green bacteria. The sequences presented probably possess a stretch of amino acids involved in dinucleotide binding which is similar to that of Chloroflexus aurantiacus MDH and other classes of dehydrogenase enzymes but unique among MDHs.
Charnock
C
C
Department of Biology, University of Oslo, Norway.
Refseth
U H
UH
Sirevåg
R
R
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D046911:Macromolecular Substances; D008291:Malate Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D011487:Protein Conformation; D013696:Temperature
D046911:Macromolecular Substances; D009243:NAD; D008291:Malate Dehydrogenase