PROTON-ATPASE ACTIVITIES INVOLVED IN THE UPTAKE OF AN S-ADENOSYLMETHIONINE ANALOG

Characteristics of the transport of sinefungin (SF) were studied in Le ishmania donovani promastigotes grown in vitro in a semi-defined mediu m. The uptake is time and pH dependent, temperature sensitive, saturab le and independent of the growth phase. Metabolic inhibitors decrease the influx, indicating that sinefungin uptake is an energy requiring p rocess. The presence of Na+ is unnecessary for activity. The uptake is sensitive to valinomycin and nigericin and to the H+-ATPases inhibito rs such as N'N'-dicyclohexylcarbodiimide, bafilomycin A and oligomycin . Sulfhydryl group(s) are involved in carrier activity. Use of SF anal ogues shows, stereospecificity of the transporter, recognition of the 6'-amino group and to a lesser degree of the 9'-amino group of the lat eral chain, whereas the 9'-carboxyl group of the lateral chain is not implicated in the recognition. Adenosine and ornithine do not interfer e with the uptake. No significant amount of SF is tightly bound to mac romolecules. In a SF-resistant clone, though the uptake of SF is reduc ed (the apparent V-max is 276 pmoles mg protein(-1) 30 min(-1) compare d with 2061 pmoles mg protein(-1) 30 min(-1) for the wild-type clone), the apparent affinity for SF is similar to that of wild-type cells (K -m 0.7 and 0.6 mu M respectively). This lower uptake activity is not t he reflection of an increased efflux of the drug. In these resistant c ells, the susceptibility of SF uptake to variation of the external pH, as well as to azide, NaF, and valinomycin are decreased, that to nige ricin is lost. (C) 1998 Elsevier Science B.V. All rights reserved.