Succinylated bovine heart mitochondrial cytochrome c oxidase.
Cytochrome c oxidase was prepared by sequential extraction of bovine heart muscle submitochondrial particles with sodium deoxycholate, followed by fractional precipitation with ammonium sulfate and chromatography on Sephadex G-75. The resulting preparation had typical absorption spectra, an activity of 1.28 sec(-1) (mg protein)(-1) (3 ml)(-1) in deoxycholate or 4.13 sec(-1) (mg protein)(-1) (3 ml)(-1) in 0.5% Tween 80, and a minimum molecular weight of 120,000 daltons as calculated from the heme content and the total protein. Amino acid analyses of nine preparations yielded a molecular weight per heme of 86,500 daltons. The net charge was calculated to be +8.7 at pH 7.0. Succinylation of cytochrome c oxidase in the presence of 500 molar excess of succinic anhydride produced a soluble preparation having a negative charge at neutral pH. The modified enzyme was highly autoxidizable and had little or no activity toward ferrocytochrome c as a substrate. Its average s20,w was 5.8 and its apparent D was 4.0 x 10(-7) cm2 sec(-1), from which a molecular weight of 126,000 daltons was calculated. This size of enzyme is considered to be that of the monomer, because the value is practically the same as the minimum molecular weight reported herein, and since it is approximately one-half the value obtained in our laboratory (and in others) for the unmodified enzyme.
Hillman
K
K
Department of Biochemistry and the Bureau of Biological Research, Rutgers College, Rutgers University, The State University of New Jersey, New Brunswick, New Jersey 08903, USA.
Wainio
W W
WW
D000596:Amino Acids / Q000032:analysis*; D000818:Animals; D002417:Cattle; D002845:Chromatography; D003911:Dextrans; D003576:Electron Transport Complex IV / Q000378:metabolism*; D005260:Female; D006418:Heme / Q000737:chemistry*; D008928:Mitochondria / Q000201:enzymology*; D009206:Myocardium / Q000166:cytology* / Q000201:enzymology; D013387:Succinic Anhydrides / Q000378:metabolism*
D000596:Amino Acids; D003911:Dextrans; D013387:Succinic Anhydrides; D006418:Heme; C031801:succinic anhydride; C025614:sephadex; D003576:Electron Transport Complex IV