Interactions among mitochondrial aspartate aminotransferase, malate dehydrogenase, and the inner mitochondrial membrane from heart, hepatoma, and liver.
The inner mitochondrial membranes from bovine heart, rat liver, and Morris hepatoma 7777 all bound the mitochondrial isozymes of aspartate aminotransferase and malate dehydrogenase with comparable affinities and binding ratios (mg of enzyme bound per mg of membrane protein). A low molecular weight fraction separated from a detergent extract of the heart membrane by chromatography on Sephacryl S-300 contained most of the binding activity of the extract for the aminotransferase and had a dissociation constant for the aminotransferase of 0.2 microM. The protein component of the membrane binding sites for the aminotransferase was apparently present in this fraction because binding activity was largely eliminated by proteolysis with trypsin. When this fraction was chromatographed on an aminotransferase affinity column, only the portion that was bound and eluted by 0.25 M KCl associated with added aminotransferase. Unlike the membrane, which was markedly inhibited by the non-ionic detergent Genapol but was inhibited only 20% by trypsin, the binding activity of this subfraction was completely inhibited by trypsin but not by Genapol. This suggests, on the membrane, that the aminotransferase binds to the binding protein and is then transferred to lipids specifically associated with the binding protein. These putative lipids are presumably removed on the affinity column. Although the yield of the binding protein was low, there is probably ample binding protein in mitochondria to accommodate the aminotransferase. In every case, binding of the aminotransferase to the membrane inactivated the malate dehydrogenase binding site whereas malate dehydrogenase had little effect on the binding of the aminotransferase and only associated with the higher molecular weight fractions from the Sephacryl column that contained Complex I activity. Inactivation of the malate dehydrogenase site by the aminotransferase, but not vice versa, could result from aminotransferase associating with the binding protein and malate dehydrogenase with Complex I followed by association of the enzymes with lipids located in the same region of the membrane. However, since aminotransferase is more cationic, it is not displaced readily from the lipids by malate dehydrogenase. The relevance of these interactions to the organization of the enzymes is discussed.
Teller
J K
JK
Department of Pharmacology, University of Wisconsin Medical School, Madison 53706.
Fahien
L A
LA
Valdivia
E
E
D000818:Animals; D001219:Aspartate Aminotransferases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D001665:Binding Sites; D002417:Cattle; D002458:Cell Fractionation; D003902:Detergents / Q000494:pharmacology; D007425:Intracellular Membranes / Q000201:enzymology*; D007527:Isoenzymes / Q000378:metabolism; D008114:Liver Neoplasms, Experimental / Q000201:enzymology*; D008291:Malate Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D008929:Mitochondria, Heart / Q000201:enzymology* / Q000648:ultrastructure; D008930:Mitochondria, Liver / Q000201:enzymology* / Q000648:ultrastructure; D008970:Molecular Weight; D011092:Polyethylene Glycols / Q000494:pharmacology; D011485:Protein Binding; D051381:Rats; D011919:Rats, Inbred Strains; D014357:Trypsin / Q000494:pharmacology
D003902:Detergents; D007527:Isoenzymes; D008565:Membrane Proteins; D011092:Polyethylene Glycols; D008291:Malate Dehydrogenase; D001219:Aspartate Aminotransferases; D014357:Trypsin