Group-selective reagent modification of the sodium- and chloride-coupled glycine transporter under native and reconstituted conditions.
Glycine transporter from rat brain stem and spinal cord is inactivated by specific sulfhydryl reagents. Modification of lysine residues also promotes a decrease of the transporter activity but in a lesser extent than that promoted by thiol group reagents. Mercurials showed a more marked inhibitory effect than maleimide derivatives. SH groups display a similar reactivity for p-chloromercuribenzenesulfonate (pCMBS) and mersalyl in synaptosomal membrane vesicles and proteoliposomes reconstituted with the solubilized transporter. However, different reactivity is observed with N-ethylmaleimide (MalNEt), the greatest effect being attained in membrane vesicles. The rate of inactivation by pCMBS and MalNEt is pseudo-first-order showing time- and concentration-dependence. pCMBS and MalNEt decrease the Vmax for glycine transport and to a lesser extent act on the apparent Km. Treatment with dithiothreitol (DTT) of the transporter modified by pCMBS results in a complete restoration of transporter activity indicating that the effect exercised by the reagent is specific for cysteine residues on the protein. It is concluded that SH groups are involved in the glycine transporter function and that these critical residues are mostly located in a relatively hydrophilic environment of the protein.
Alcántara
R
R
Departamento de Biología Molecular, Centro de Biología Molecular, Facultad de Ciencias, Universidad Autónoma, Madrid, Spain.
López-Corcuera
B
B
Aragón
C
C
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D026921:Amino Acid Transport Systems, Neutral; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D001933:Brain Stem / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D005998:Glycine / Q000378:metabolism*; D050487:Glycine Plasma Membrane Transport Proteins; D007202:Indicators and Reagents; D007700:Kinetics; D008297:Male; D008566:Membranes / Q000378:metabolism; D008634:Mersalyl / Q000494:pharmacology; D011510:Proteolipids / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D012964:Sodium / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000378:metabolism; D013574:Synaptosomes / Q000378:metabolism*; D014827:Vocal Cords / Q000378:metabolism
D026921:Amino Acid Transport Systems, Neutral; D002352:Carrier Proteins; D002712:Chlorides; D050487:Glycine Plasma Membrane Transport Proteins; D007202:Indicators and Reagents; D011510:Proteolipids; D013438:Sulfhydryl Compounds; C020485:proteoliposomes; D008634:Mersalyl; D002731:4-Chloromercuribenzenesulfonate; D012964:Sodium; D005033:Ethylmaleimide; D005998:Glycine