The protein component(s) of the isolated phosphate-transport system of mitochondria.
The ethylmaleimide-sensitive phosphate-transport system of heart mitochondria was isolated and the activity reconstituted in liposomes. The identification of the phosphate-carrier protein was complicated by several factors. 1. The phosphate-carrier fraction, isolated through different procedures in different laboratories, contains 4-5 protein components of very similar apparent molecular weight, as shown by gradient dodecylsulfate gel electrophoresis. 2. The amino acid composition of protein 1, 2 and 3(apparent Mr = 34 500, 34 000 and 33 000, respectively) is quite similar although not identical. 3. Including several protease inhibitors during isolation of the phosphate-carrier fraction does not influence the protein pattern. 4. Labeled N-ethylmaleimide binds only protein 1 and 3, labeled N,N'-dicyclohexylcarbodiimide bonds only to protein 1 and 2. 5. N,N'-Dicyclohexylcarbodiimide had no effect on the reconstituted phosphate-exchange activity after incubation with intact mitochondria or proteoliposomes. 6. In the presence of protecting mersalyl concentrations N-ethylmaleimide inhibits the phosphate transport in intact mitochondria. Under these conditions no label ethylmaleimide was bound to the phosphate-carrier fraction. 7. Comparison of the reconstituted transport activity with the protein pattern of fractions, isolated with or without cardiolipin, shows correlation of transport with the amount of protein 2 but not with that of protein 1, which binds ethylmaleimide. A model of the mitochondrial phosphate carrier is presented which suggests a proteolytic degradation of the phosphate-carrier protein during its isolation. The model explains the contradictory results of this investigation.
D000244:Adenosine Diphosphate / Q000378:metabolism; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000302:isolation & purification*; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D008928:Mitochondria / Q000378:metabolism*; D008929:Mitochondria, Heart / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D013045:Species Specificity; D013552:Swine
D000596:Amino Acids; D002352:Carrier Proteins; D010710:Phosphates; D011480:Protease Inhibitors; D004024:Dicyclohexylcarbodiimide; D000244:Adenosine Diphosphate; D005033:Ethylmaleimide