The mitochondrial carnitine carrier: characterization of SH-groups relevant for its transport function.
The transport function of the purified and reconstituted carnitine carrier from rat liver mitochondria was correlated to modification of its SH-groups by various reagents. The exchange activity and the unidirectional transport, both catalyzed by the carnitine carrier, were effectively inhibited by N-ethylmaleimide and submicromolar concentrations of mercurial reagents, e.g., mersalyl and p-(chloromercuri)benzenesulfonate. When 1 microM HgCl2 or higher concentrations of the above mentioned mercurials were added, another transport mode of the carrier was induced. After this treatment, the reconstituted carnitine carrier catalyzed unidirectional substrate-efflux and -influx with significantly reduced substrate specificity. Control experiments in liposomes without carrier or with inactivated carrier protein proved the dependence of this transport activity on the presence of active carnitine carrier. The mercurial-induced uniport correlated with inhibition of the 'physiological' functions of the carrier, i.e., exchange and substrate specific unidirectional transport. The effect of consecutive additions of various reagents including N-ethylmaleimide, mercurials, Cu(2+)-phenanthroline and diamide on the transport function revealed the presence of at least two different classes of SH-groups. N-Ethylmaleimide blocked the carrier activity by binding to SH-groups of one of these classes. At least one of these SH-groups could be oxidized by the reagents forming S-S bridges. Besides binding to the class of SH-groups to which N-ethylmaleimide binds, mercurials also reacted with SH-groups of the other class. Modification of the latter led to the induction of the efflux-type of carrier activity characterized by loss of substrate specificity.
Indiveri
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C
Department of Pharmaco-Biology, University of Bari, Italy.
Tonazzi
A
A
Dierks
T
T
Krämer
R
R
Palmieri
F
F
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002333:Carnitine Acyltransferases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D008634:Mersalyl / Q000494:pharmacology; D008930:Mitochondria, Liver / Q000378:metabolism*; D011510:Proteolipids; D051381:Rats; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D013997:Time Factors
D011510:Proteolipids; D013438:Sulfhydryl Compounds; C020485:proteoliposomes; D008634:Mersalyl; D002333:Carnitine Acyltransferases; D005033:Ethylmaleimide