Modification of system A amino acid carrier by diethyl pyrocarbonate.
Sodium-dependent alanine transport in plasma membrane vesicles from rat liver was inactivated in a time- and concentration-dependent fashion by prior treatment of membranes with the acylating reagent diethyl pyrocarbonate (DEPC). Both components of Na+/alanine cotransport (systems A and ASC) were inhibited. Exposure of vesicles to p-bromophenacyl bromide and methyl p-nitrobenzenesulfonate, which share with DEPC reactivity against histidine residues, also led to inhibition of alanine transport through systems A and ASC. The presence of Na+ (100 mM NaCl) and L-alanine (10 mM) during exposure to vesicles to DEPC protected against inactivation of system A (but not system ASC) transport activity. This protective effect was specific and required the presence of L-alanine since the presence of L-phenylalanine alone (10 mM) or L-phenylalanine plus Na+ (100 mM NaCl) did not cause any detectable protection. This overall pattern of protection is opposite to that previously found against specific sulfhydryl reagents (i.e. N-ethylmaleimide), where protection of system ASC was nearly maximal. The pH profile for DEPC-dependent inhibition of system A transport activity suggests modification of amino acid residue(s) with a pKr of approximately 7, most likely histidine(s), in close parallel with the pH dependence of system A transport activity. Our results suggest the presence of critical histidine residues on the system A carrier that may be responsible for the pH dependence of system A transport activity.
Bertran
J
J
Departament de Bioquimica i Fisiologia, Facultat de Biologia, Universitat de Barcelona, Spain.
Roca
A
A
Pola
E
E
Testar
X
X
Zorzano
A
A
Palacín
M
M
D000409:Alanine / Q000097:blood*; D000818:Animals; D001692:Biological Transport; D004047:Diethyl Pyrocarbonate; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D008099:Liver / Q000187:drug effects; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D012964:Sodium / Q000378:metabolism
D007202:Indicators and Reagents; D012964:Sodium; D004047:Diethyl Pyrocarbonate; D000409:Alanine