Purification procedure and N-terminal amino acid sequence of yeast malate dehydrogenase isoenzymes.
A method has been devised for the rapid isolation of malate dehydrogenase isoenzymes. First, anionic proteins were precipitated with polyethyleneimine, whilst hydrophobic malate dehydrogenase remained in the supernatant fluid. Secondly, the supernatant was 30% saturated with ammonium sulfate and the two isoenzymes were separated by hydrophobic phenyl-Sepharose CL-4B chromatography. For further purification the enzymes were chromatofocused, and polybuffer was removed by hydrophobic chromatography. Affinity chromatography with blue Sepharose CL-6B [1] was used as final purification step. The purified isoenzymes were homogeneous as shown by isoelectric focusing and could be used for N-terminal sequencing. 34 amino acid residues could be identified for the cytoplasmic isoenzyme and 56 amino acid residues for the mitochondrial isoenzyme. Although there are regions of strong homology between both isoenzymes, the sequence differences clearly showed support that both isoenzymes are coded by different genes. Sequence comparison clearly indicated that the N-terminus of the cytoplasmic enzyme extended that of the mitochondrial enzyme by 12 amino acid residues. The amino acid sequence of the extending sequence resembled that of leading sequences known for enzymes which are transported into the mitochondria. The assumed leading sequence is discussed with respect to its possible role in glucose inactivation.
D000595:Amino Acid Sequence; D011232:Chemical Precipitation; D002845:Chromatography; D003593:Cytoplasm / Q000201:enzymology; D005947:Glucose / Q000494:pharmacology; D007525:Isoelectric Focusing; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D008291:Malate Dehydrogenase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D008928:Mitochondria / Q000201:enzymology; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D007527:Isoenzymes; D008291:Malate Dehydrogenase; D005947:Glucose