Isolation and properties of oxaloacetate keto-enol-tautomerases from bovine heart mitochondria

Identification of the high‐molecular‐mass mitochondrial oxaloacetate keto‐enol tautomerase as inactive aconitase

“…Both mitochondrial enzymes were found to be quite different from that previously obtained from porcine kidney extracts [5,8]. We have pointed out that the proteins described may either be the unique mitochondrial oxaloacetate keto-enol tautomerases, or their tautomerase activity is the partial reaction of certain enzymes capable of oxaloacetate binding [7]. In this report it will be shown that the larger 80 kDa protein is identical to the mitochondrial inactive aconitase.…”

“…The molecular masses of OAT-2 (80 kDa as revealed by SDS gel electrophoresis and 89 kDa as revealed by Sephacryl gel filtration [7]) and that of the mitochondrial aconitase (83 kDa [14]) are very close. Concentrated solutions of OAT-2 have an amber color and aconitase is known as an iron-sulfur protein [15].…”

“…Electrophoretically homogeneous 80 kDa oxaloacetate ketoenol tautomerase (OAT-2) was prepared from the mitochondrial matrix as described [7]. The tautomerase activity (enol ---, ketone direction) was measured at 25°C using a coupled malate dehydrogenase (EC 1.1.1.37) assay [7] in a mixture containing 2 mM Tris-Cl-, 0.2 mM NAD.…”

“…The tautomerase activity (enol ---, ketone direction) was measured at 25°C using a coupled malate dehydrogenase (EC 1.1.1.37) assay [7] in a mixture containing 2 mM Tris-Cl-, 0.2 mM NAD. H, proper amounts of malate dehydrogenase and 35/~M enol-oxaloacetate added from a freshly prepared solution of solid acid in dry acetone (the pH of the assay mixture after addition of oxaloacetic acid was 9.0).…”

“…Recently we have described the malate dehydrogenase activity of succinate dehydrogenase (EC 1.3.99.1), and the enolisomer of oxaloacetate has been identified as the reaction product [6]. Subsequently, two highly purified proteins, with apparent molecular masses of 37 and 80 kDa, capable of the oxaloacetate ketoenol tautomerase activity were isolated from bovine heart mitochondrial matrix [7]. Both mitochondrial enzymes were found to be quite different from that previously obtained from porcine kidney extracts [5,8].…”