MOLECULAR SIZES OF AMINO-ACID TRANSPORTERS IN THE LUMINAL MEMBRANE FROM THE KIDNEY CORTEX, ESTIMATED BY THE RADIATION-INACTIVATION METHOD

Renal brush-border membrane vesicles from rat kidney cortex were irradiated in frozen state with a γ-radiation source. Initial rates of influx into these vesicles were estimated for substrates such as L-glutamic acid, L-alanine, L-proline and L-leucine to establish the molecular sizes of their carriers. Transport was measured in initial-rate conditions to avoid artifacts arising from a decrease in the driving force caused by a modification of membrane permeability. Initial rates of Na+-independent uptakes for those four substrates appeared unaffected in the dose range used (0-6 Mrad), indicating that the passive permeability of the membrane towards these substrates was unaffected. However, at higher doses of irradiation the Na+ influx and the intravesicular volume evaluated by the uptake of glucose equilibrium were altered by radiation. Thus Na+-dependent influx values were corrected for volume changes, and the corrected values were used to compute radiation-inactivation sizes of the transport systems. Their respective values for L-glutamic acid, L-proline, L-leucine and L-alanine carriers were 250, 224, 293 and 274 kDa. The presence of the free-radicals scavenger benzoic acid in the frozen samples during irradiation did not affect the uptake of glucose, phosphate and alkaline phosphatase activity. These results indicate that freezing samples in a cryoprotective medium was enough to prevent secondary inactivation of transporters by free radicals. Uptakes of β-alanine and L-lysine were much less affected by radiation. The radiation-inactivation size of the Na+-dependent β-alanine carrier was 127 kDa and that of the L-lysine carrier was 90 kDa.