Purification and characterization of bovine liver 3-cis-2-trans-enoyl-CoA isomerase.
The purification to homogeneity of 3-cis-2-trans-enoyl-CoA isomerase from bovine liver is described. This procedure has also been successfully applied to the isolation of the enzyme from rat liver mitochondria. Its molecular mass was determined to be 30 kDa by several methods. Km of the isomerase for 3-cis-dodecanoyl-CoA, the physiological intermediate in the beta-oxidation of oleic acid and of the 3-trans-isomer was determined to be 3.2 x 10(-5) M. The velocity of isomerization of the 3-trans-substrate is reduced 10-15 times. Inhibition experiments and pH-dependency of the reaction kinetics suggest the participation of a histidine residue in the isomerization. Protein analytical studies revealed that the N-terminus is blocked (acetylated). Cyanogen bromide cleavage of the purified enzyme resulted in five fragments. The N-terminus of one fragment is blocked. Partial amino-terminal sequences of two of the fragments were obtained by Edman degradation, a prerequisite for further studies on the structure of this isomerase on the DNA level.
Euler-Bertram
S
S
Institut für Biochemie, Medizinische Fakultät, Universität zu Köln.
Stoffel
W
W
D000595:Amino Acid Sequence; D000818:Animals; D019748:Carbon-Carbon Double Bond Isomerases; D002417:Cattle; D003488:Cyanogen Bromide; D064006:Dodecenoyl-CoA Isomerase; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007535:Isomerases / Q000302:isolation & purification*; D007536:Isomerism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D013552:Swine
D007535:Isomerases; D019748:Carbon-Carbon Double Bond Isomerases; D064006:Dodecenoyl-CoA Isomerase; D003488:Cyanogen Bromide