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 gamma-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 at 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 beta-alanine and L-lysine were much less affected by radiation. The radiation-inactivation size of the Na(+)-dependent beta-alanine carrier was 127 kDa and that of the L-lysine carrier was 90 kDa.
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Département de Chimie, Université du Québec à Montréal, Quebec, Canada.
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D000409:Alanine / Q000378:metabolism; D026905:Amino Acid Transport Systems; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001565:Benzoates / Q000494:pharmacology; D019817:Benzoic Acid; D001692:Biological Transport / Q000528:radiation effects; D002352:Carrier Proteins / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000528:radiation effects; D004307:Dose-Response Relationship, Radiation; D005609:Free Radicals; D005720:Gamma Rays; D005971:Glutamates / Q000378:metabolism; D018698:Glutamic Acid; D007672:Kidney Cortex / Q000378:metabolism / Q000528:radiation effects* / Q000648:ultrastructure; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D008871:Microvilli / Q000378:metabolism / Q000528:radiation effects; D011392:Proline / Q000378:metabolism; D051381:Rats; D012964:Sodium / Q000494:pharmacology
D026905:Amino Acid Transport Systems; D000596:Amino Acids; D001565:Benzoates; D002352:Carrier Proteins; D005609:Free Radicals; D005971:Glutamates; D018698:Glutamic Acid; D019817:Benzoic Acid; D011392:Proline; D012964:Sodium; D007930:Leucine; D000409:Alanine