Probing the active site of the reconstituted carnitine carrier from rat liver mitochondria with sulfhydryl reagents. A cysteine residue is localized in or near the substrate binding site.
The interaction of sulfhydryl reagents with the carnitine carrier of rat liver mitochondria was studied in detail in proteoliposomes. The addition of N-ethylmaleimide, mercurials at low concentrations, Cu(2+)-phenanthroline and diamide modified a single sulfhydryl group (the class II group) that is involved in transport function. The treatment of the inhibited protein with 1,4-dithioerythritol led to full recovery of carnitine exchange except for N-ethylmaleimide. Evidence is provided that the addition of carnitine to the carrier blocks the interaction of the sulfhydryl reagents with the protein. This result strongly suggests that the critical cysteine residue is localized in, or near, the substrate binding site. Interaction of other cysteine residues in the carrier protein with high concentrations of mercurials modified another class of sulfhydryl groups (the class I group) that are not directly involved in carnitine transport. The oxidized and reduced forms of the carnitine carrier show slightly different molecular masses on SDS/PAGE. Disulfide bridge(s) induced by Cu(2+)-phenanthroline and diamide are present in a single polypeptide part of the protein and induced no disulfide bridges between two polypeptide chains.
Indiveri
C
C
Department of Pharmaco-Biology, University of Bari, Italy.
Tonazzi
A
A
Giangregorio
N
N
Palmieri
F
F
D000818:Animals; D001665:Binding Sites; D002331:Carnitine / Q000378:metabolism* / Q000494:pharmacology; D002352:Carrier Proteins / Q000737:chemistry*; D003545:Cysteine; D004220:Disulfides; D008930:Mitochondria, Liver / Q000737:chemistry*; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010618:Phenanthrolines / Q000494:pharmacology; D051381:Rats; D013439:Sulfhydryl Reagents / Q000494:pharmacology*
D002352:Carrier Proteins; D004220:Disulfides; D010618:Phenanthrolines; D013439:Sulfhydryl Reagents; D003545:Cysteine; D002331:Carnitine; C025205:1,10-phenanthroline