Orientation of the cytoplasmically made subunits of beef heart cytochrome c oxidase determined by protease digestion and antibody binding experiments.
The topology of several of the cytoplasmically made subunits of beef heart cytochrome c oxidase has been determined by protease digestion of oriented membrane preparations, using subunit-specific antibodies to identify cleavage products. Reconstituted vesicles of cytochrome c oxidase and asolectin were used as a vesicle preparation with the C domain of the enzyme available for protease digestion. Submitochondrial particles were used as vesicles with the M domain outermost. Trypsin and/or proteinase K cleaved polypeptides CIV, ASA, AED, STA, and IHQ. Cleavage of CIV, STA, and IHQ was from the M domains only and involved the removal of a fragment from the N-terminus in each case. Polypeptide AED was cleaved from the C side in the N-terminal part, while ASA was cleaved from both the C and M domains. Polypeptide fragments were electroblotted from polyacrylamide gels onto derivatized glass paper and sites of proteolytic cleavage determined by N-terminal sequence analysis.
Zhang
Y Z
YZ
Institute of Molecular Biology, University of Oregon, Eugene 97403.
Lindorfer
M A
MA
Capaldi
R A
RA
D000595:Amino Acid Sequence; D000818:Animals; D000936:Antigen-Antibody Complex; D002417:Cattle; D003593:Cytoplasm / Q000201:enzymology; D003576:Electron Transport Complex IV / Q000096:biosynthesis*; D007700:Kinetics; D008081:Liposomes; D046911:Macromolecular Substances; D008929:Mitochondria, Heart / Q000201:enzymology*; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D010446:Peptide Fragments / Q000032:analysis; D010447:Peptide Hydrolases / Q000378:metabolism; D010713:Phosphatidylcholines; D010743:Phospholipids; D011487:Protein Conformation; D013367:Submitochondrial Particles / Q000201:enzymology*
D000936:Antigen-Antibody Complex; D008081:Liposomes; D046911:Macromolecular Substances; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D010743:Phospholipids; C024990:asolectin; D003576:Electron Transport Complex IV; D010447:Peptide Hydrolases