Modification of system A amino acid carrier by diethyl pyrocarbonate

• Published in: The Journal of biological chemistry 1991-01, Vol.266 (2), p.798-802
• Main Authors: Bertran, J, Roca, A, Pola, E, Testar, X, Zorzano, A, Palacín, M
• Format: Article
• Language: English
• Subjects: alanine; Alanine - blood; Animals; Biological and medical sciences; Biological Transport; Cell physiology; Diethyl Pyrocarbonate; diethylpyrocarbonate; Fundamental and applied biological sciences. Psychology; Hydrogen-Ion Concentration; Indicators and Reagents; Liver - drug effects; Male; Membrane and intracellular transports; Molecular and cellular biology; Rats; Rats, Inbred Strains; sodium; Sodium - metabolism; vesicles
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)35243-2

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.