Cloning of a cDNA coding for an amino acid carrier from Ricinus communis (RcAAP1) by functional complementation in yeast: kinetic analysis, inhibitor sensitivity and substrate specificity

Marvier, A.C., Neelam, A, Bick, J.A. and Williams, LE , et al. (1998) Cloning of a cDNA coding for an amino acid carrier from Ricinus communis (RcAAP1) by functional complementation in yeast: kinetic analysis, inhibitor sensitivity and substrate specificity. Biochimica et biophysica acta. General Subjects, 1373 (2), 321-331. (doi:10.1016/S0005-2736(98)00117-5).

Abstract

A cDNA for the amino acid permease gene RcAAP1 has been isolated from Ricinus communis by yeast complementationand subjected to a detailed kinetic analysis. RcAAP1 cDNA is 1.5 kb with an open reading frame that codes for a proteinwith 486 amino acids and a calculated molecular mass of 53.1 kDa. RcAAPl-mediated histidine uptake was pH dependentwith highest transport rates at acidic pH; it was sensitive to protonophores and uncouplers and the Km for histidine uptakewas 96 gM. The substrate specificity was investigated by measuring the levels of inhibition of histidine uptake by a range ofamino acids. The basic amino acids (histidine, lysine and arginine) showed strongest inhibition of uptake whereas acidicamino acids competed less effectively. Alanine was the most efficient competitor of the neutral amino acids. Glutamine,serine, asparagine, methionine and cysteine showed moderate inhibition whereas threonine, isoleucine, leucine, phenylalanine, tyrosine and tryptophan showed only low levels of inhibition. Glycine, proline and citrulline caused slightstimulation. More detailed competition kinetics indicated that both lysine and arginine showed simple competitive inhibitionof histidine uptake. When direct uptake measurements were carried out, both lysine and arginine were found to be effectivesubstrates for RcAAP1

This record has no associated files available for download.

Contributors

Download statistics