Studies on the oligomeric state of isolated cytochrome oxidase using cross-linking reagents.
(1) Sucrose gradient centrifugation of cytochrome oxidase in the presence of Triton X-100 gave one slowly sedimenting green band. After cross-linking with dithiobis(succinimidylpropionate) (DSP), two green bands were observed, one sedimenting like the control and the other one more rapidly. Only the slowly sedimenting band was observed if the cross-linker was cleaved by dithiothreitol before centrifugation. (2) The rapidly sedimenting band in the Triton-containing sucrose gradient is probably the internally cross-linked dimer of cytochrome oxidase; the one sedimenting slowly is the monomeric enzyme. (3) Cross-linking with DSP after monomerization yields a small fraction of internally cross-linked dimers in addition to the internally cross-linked monomers. Under similar conditions, but using the shorter cross-linker disuccinimidyl tartarate (DST), no dimers are detected. (4) Both DSP and DST cross-link the dimeric enzyme so that it could no longer be monomerized by centrifugation in Triton, unless the cross-link is cleaved. (5) Polypeptide analysis using two-dimensional gel electrophoresis of cross-linked dimers and monomers suggest that subunit VIb is involved in intermonomeric cross-linking of dimeric enzyme by DSP.
Finel
M
M
Department of Medical Chemistry, University of Helsinki, Finland.
D000818:Animals; D002417:Cattle; D002499:Centrifugation, Density Gradient; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D003576:Electron Transport Complex IV; D004591:Electrophoresis, Polyacrylamide Gel; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D011108:Polymers; D013388:Succinimides / Q000494:pharmacology
D003432:Cross-Linking Reagents; D011108:Polymers; D013388:Succinimides; D011092:Polyethylene Glycols; D017830:Octoxynol; D003576:Electron Transport Complex IV; C011240:dithiobis(succinimidylpropionate)