The tricarboxylate (or citrate) carrier was purified from eel liver mitochondria and functionally reconstituted into liposomes. Incubation of the proteoliposomes with various sulfhydryl reagents led to inhibition of the reconstituted citrate transport activity. Preincubation of the proteoliposomes with reversible SH reagents, such as mercurials and methanethiosulfonates, protected the eel liver tricarboxylate carrier against inactivation by the irreversible reagent N-(1-pyrenyI)maleimide (PNI). Citrate and L-malate, two substrates of, the tricarboxylate carrier, protected the protein against inactivation by sulfhydryl reagents and decreased the fluorescent I'M bound to the purified protein. These results suggest that the eel liver tricarboxylate carrier requires a single population of free cysteine(s) in order to manifest catalytic activity. The reactive cysteine(s) is most probably located at or near the substrate binding site of the carrier protein.
The mitochondrial tricarboxylate carrier of silver eel: chemical modification by sulfhydryl reagents
CAPOBIANCO, Loredana;FERRAMOSCA, Alessandra;ZARA, Vincenzo
2004-01-01
Abstract
The tricarboxylate (or citrate) carrier was purified from eel liver mitochondria and functionally reconstituted into liposomes. Incubation of the proteoliposomes with various sulfhydryl reagents led to inhibition of the reconstituted citrate transport activity. Preincubation of the proteoliposomes with reversible SH reagents, such as mercurials and methanethiosulfonates, protected the eel liver tricarboxylate carrier against inactivation by the irreversible reagent N-(1-pyrenyI)maleimide (PNI). Citrate and L-malate, two substrates of, the tricarboxylate carrier, protected the protein against inactivation by sulfhydryl reagents and decreased the fluorescent I'M bound to the purified protein. These results suggest that the eel liver tricarboxylate carrier requires a single population of free cysteine(s) in order to manifest catalytic activity. The reactive cysteine(s) is most probably located at or near the substrate binding site of the carrier protein.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
