Cross-linking of mitochondrial matrix proteins in situ.
Different cross-linkers (10 mM) of varying specificity and arm length were found to cross-link mitochondria matrix proteins in situ in 2 min at pH 7.4. As seen by SDS-polyacrylamide electrophoresis, the disappearance of individual protein bands was accompanied by concomitant appearance of polymeric aggregates that failed to enter the 4% spacer gel. The disorganization of the mitochondrial matrix infrastructure either by swelling or sonication of the mitochondria resulted in a decrease in the rate of cross-linking. Leakage of citrate synthase, malate dehydrogenase and fumarase was found to be reduced when cross-linked mitochondria were made permeable with toluene. On lysing the cross-linked mitochondria, a major part of the matrix protein (75%) was found to sediment with the membrane fraction. The activities of citrate synthase malate dehydrogenase and fumarase in rat liver mitochondria were also found to increase in the percipitates with concomitant decrease in their activities in the soluble matrix fraction. These results indicate that the cross-linkers enters the mitochondria and cross-links matrix proteins including Krebs cycle enzymes either to the mitochondrial membranes, or to themselves resulting in very large molecular weight complexes. These results are interpreted to mean that in liver mitochondria, the Krebs cycle enzyme are preferentially located near the membrane.
D000818:Animals; D002950:Citrate (si)-Synthase / Q000378:metabolism; D002952:Citric Acid Cycle / Q000187:drug effects; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D005649:Fumarate Hydratase / Q000378:metabolism; D005976:Glutaral / Q000494:pharmacology; D007096:Imidoesters / Q000494:pharmacology; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000187:drug effects / Q000378:metabolism*; D008930:Mitochondria, Liver / Q000187:drug effects / Q000378:metabolism*; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains; D013329:Structure-Activity Relationship
D003432:Cross-Linking Reagents; D007096:Imidoesters; D008565:Membrane Proteins; D008291:Malate Dehydrogenase; D002950:Citrate (si)-Synthase; D005649:Fumarate Hydratase; D005976:Glutaral