Sensitivity of system A and ASC transport activities to thiol-group-modifying reagents in rat liver plasma-membrane vesicles. Evidence for a direct binding of N-ethylmaleimide and iodoacetamide on A and ASC carriers.
1. In the present study we have examined the sensitivity of A and ASC amino-acid-carrier activities in rat liver plasma-membrane vesicles to the thiol-group modifying reagents N-ethylmaleimide (NEM) and iodoacetamide (IA). To this end, the different Na(+)-dependent entities involved in alanine transport were assessed. 2. NEM inactivated Na(+)-dependent alanine transport as a result of the inhibition of both system A and ASC transport activities. The functional sensitivity of system A to NEM was greater than that of system ASC. 3. The presence of L-alanine (10 mM) during the exposure of vesicles to NEM afforded partial protection to system A, but not to the ASC, carrier. This effect was specific, since the presence of L-phenylalanine (10 mM) did not cause any protection. 4. Na+ did not protect A or ASC carriers against NEM inactivation; however, the presence of Na+ (100 mM-NaCl) and L-alanine (10 mM) during the exposure of the vesicles to NEM protected against inactivation of system A and ASC transport activities. The extent of protection was greater in the case of the system ASC transport activity than in the case of the A carrier. 5. IA also diminished Na(+)-dependent alanine transport by inhibition of A and ASC transport activities. Sodium and L-alanine afforded protection to both A and ASC transport activities from the inhibitory action of IA. The extent of protection induced by substrates was similar for both carriers. 6. It is concluded that there is one, or several, free thiol groups in A and ASC carriers, the integrity of which is essential for transport activity. Sensitivity to thiol-group-specific reagents and the pattern of protection with substrates against inactivation is different in A and ASC carriers. That suggests the existence of topological dissimilarities regarding the thiol-group containing site(s) in A and ASC amino acid carriers.
Pola
E
E
Department de Bioquimica i Fisiologia, Facultat de Biologia, Universitat de Barcelona, Spain.
Bertran
J
J
Roca
A
A
Palacín
M
M
Zorzano
A
A
Testar
X
X
D000409:Alanine / Q000378:metabolism* / Q000494:pharmacology; D026905:Amino Acid Transport Systems; D000621:Aminoisobutyric Acids / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D005033:Ethylmaleimide / Q000494:pharmacology*; D007460:Iodoacetamide / Q000494:pharmacology*; D008099:Liver / Q000378:metabolism*; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D012964:Sodium / Q000494:pharmacology; D013861:Thiocyanates / Q000494:pharmacology
D026905:Amino Acid Transport Systems; D000621:Aminoisobutyric Acids; D002352:Carrier Proteins; D013861:Thiocyanates; C017911:2-(methylamino)isobutyric acid; C024553:sodium thiocyanate; D012964:Sodium; D005033:Ethylmaleimide; D000409:Alanine; C009941:potassium thiocyanate; D007460:Iodoacetamide