Purification and properties of succinyl-coenzyme A-3-oxo acid coenzyme A-transferase from sheep kidney

Abstract

CoA-transferase (succinyl-CoA-3-oxo acid CoA-transferase, EC 2.8.3.5) isolated from sheep kidney was purified to homogeneity. The purified enzyme has a specific activity of approx. 200 units/mg. A mol.wt. of 110000 was obtained by gel filtration on Sephadex G-200, and a lower mol.wt. of 102000 was determined by analytical ultracentrifugation. A sedimentation coefficient of 5.6S was also determined. A subunit mol.wt. of 56000 was obtained by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Isoelectric focusing of sheep kidney extracts indicated the presence of a single band of CoA-transferase activity with pI9.0. However, isoelectric focusing of purified CoA-transferase showed the presence of two peaks of CoA-transferase activity with pI values of 8.7 and 8.4, suggesting the presence of proteolytic activity during purification. Evidence for sheep kidney CoA-transferase being a dimer of two identical subunits has been obtained from sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the amino acid composition, peptide 'mapping' and N-terminal analysis.

Purification and Properties of Succinyl‐CoA:3‐Oxo‐Acid CoA‐Transferase from Rat Brain

“…of 161 pmoVmin/mg (seven separate preparations), with approximately 300-fold purification. This specific activity is comparable with homogenous preparations from pig heart (Hersh and Jencks, 1967) and sheep kidney (Sharp and Edwards, 1978).…”

“…3-0x0-acid CoA-transferase from rat brain binds to Blue Sepharose (Fig. 2) as has been shown with this enzyme from other tissues (White and Jencks, 1976~;Sharp and Edwards, 1978). In our initial studies, the enzyme was eluted from the Blue Sepharose column with a stepwise gradient of 10, 100, and 250 mM potassium phosphate buffer (Fig.…”

“…The partially purified preparation of 3-0x0-acid CoA-transferase (reportedly 300-fold purification) from brains of weanling rats (Tildon and Sevdalian, 1972) had extremely low activity (0.9 pmoYmidmg of protein in the reverse direction) as compared to the specific activity of approximately 200 pmol/ midmg of protein found in both pig heart (White and Jencks, 1976c) and sheep kidney (Sharp and Edwards, 1978). Also, the ratio of the activities in the reverse to forward directions was sixfold lower in rat brain preparation (Tildon and Sevdalian, 1972) compared to a highly purified enzyme preparation (White and Jencks, 1 9 7 6~) and partially purified preparations from several mammalian tissues (Blair, 1969; Fenselau and Wallis,197&,6).…”

Cloning and Characterization of Helicobacter pyloriSuccinyl CoA:Acetoacetate CoA-transferase, a Novel Prokaryotic Member of the CoA-transferase Family

“…They have been identified in many prokaryotes (5)(6)(7)(8)(9)(10)(11)(12) and in mammalian tissues (13)(14)(15)(16)(17)(18). Although the CoA-transferases appear to be mechanistically and functionally very similar (5,6,10), their substrate ranges and activities differ.…”

Characterization of the genes encoding beta-ketoadipate: succinyl-coenzyme A transferase in Pseudomonas putida

“…and mammalian tissues (3,44,48,59). Although the substrate ranges and subunit structures may differ, CoA transferases appear to be mechanistically and functionally very similar (2,24,51,57,62).…”