THE IRREVERSIBLE INHIBITION OF THE P-AMIN OHIPPURATE TRANSPORT IN BRUSH-BORDER MEMBRANE-VESICLES ISOLATED FROM THE RAT-KIDNEY CORTEX - THE DETERMINATION OF THE KINETIC-PARAMETERS OF THE TRANSPORT

The inhibition properties of the bromoacetyl-p-aminohippuric acid as t he affinity probe of the organic anion transport system were studied. Bromoacetylated p-aminohippurate was shown to be able to inhibit irrev ersibly the p-aminohippurate (PAH) uptake in brush-border membrane ves icles. The inhibition depended on both the treatment time and the affi nity probe concentration. The treatment of brush-border membrane with 1 mM bromoacetyl-p-aminohippurate for 1.5 hour was resulted in 100% ir reversible inhibition of PAH transport but no changes were observed in the activity of membrane enzymes: alkaline phosphatase, gamma-glutamy l transpeptidase and maltase. After the treatment of brush border memb rane vesicles with bromoacetyl-p-aminohippurate the main part of coval ently bound affinity probe (85%) was associated with proteins and only 15% with lipids. The kinetic parameters of the PAH transport were mea sured by means of the affinity probe as the irreversible inhibitor of organic transport system. The apparent Michaelis constant was shown to be equal K(m) = 8 +/- 2 mM and the maximal rate V(max) = 20 +/- 3 mmo l/min per 1 mg protein. It was shown that the use of bromoacetyl-p-ami nohippurate enabled one to determine directly the contribution of the membrane surface substrate binding value in the total uptake of PAH by vesicles.