Purification and characterization of an enzymically active cleavage product of pig kidney aldehyde reductase.
During the purification of pig kidney aldehyde reductase by an established procedure [Flynn, Cromlish & Davidson (1982) Methods Enzymol. 89, 501-506] a second enzyme with aldehyde reductase activity may be purified. When the procedure was performed in the presence of 5 mM-EDTA, only traces of the second reductase, pig kidney aldehyde reductase (minor form), were present. By the criterion of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, pig kidney aldehyde reductase (minor form) had Mr 35 000, in comparison with Mr 40 200 found for pig kidney aldehyde reductase. Amino acid analysis of both enzymes and tryptic-peptide-map comparisons indicated differences in primary structure. The N-terminus of pig kidney aldehyde reductase (minor form) had the sequence Lys-Val-Leu, in contrast with the blocked (acetylated) N-terminus of pig kidney aldehyde reductase. The C-terminal sequence of both enzymes was the same. Both reductases were immunologically identical by double immunodiffusion and rocket immunoelectrophoresis. Pig kidney aldehyde reductase (minor form) had 50% of the specific activity of pig kidney aldehyde reductase when tested with a variety of aldehyde substrates. Michaelis constants of both enzymes for these substrates and for NADPH were similar, but values for kcat. and kcat./Km indicated that catalytically pig kidney aldehyde reductase was the more efficient enzyme. Typical aldehyde reductase inhibitors, such as phenobarbital and sodium valproate, had the same effect on both enzymes. It was concluded that pig kidney aldehyde reductase (minor form) is an enzymically active cleavage product of pig kidney aldehyde reductase which is formed when the latter is purified in the absence of the metalloproteinase inhibitor EDTA.
D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002384:Catalysis; D002850:Chromatography, Gel; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D005779:Immunodiffusion; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D013552:Swine
D000596:Amino Acids; D007527:Isoenzymes; D010446:Peptide Fragments; D000429:Alcohol Oxidoreductases; C019642:alcohol dehydrogenase (NADP+)