Target size analysis of the peptide/H(+)-symporter in kidney brush-border membranes.
The apparent functional molecular mass of the kidney peptide/H(+)-symporter was determined by radiation inactivation in brush-border membrane vesicles (BBMV) of rat kidney cortex. Purified BBMV were irradiated at low temperatures with high energy electrons generated by a 10-MeV linear accelerator at doses from 0 to 30 megarads. Uptake studies were performed with [3H]cefadroxil, a beta-lactam antibiotic which serves as a substrate for the kidney peptide/H(+)-symporter. Inhibition of influx of [3H]cefadroxil into BBMV was used to determine the functional molecular mass of the transporter. Additionally, direct photoaffinity labeling of the transport- and/or binding proteins for [3H]cefadroxil in control and irradiated BBMV was performed to determine the molecular mass of the putative transporter by SDS-polyacrylamide gel electrophoresis. Initial rates of pH-gradient dependent uptake of [3H]cefadroxil decreased progressively as a function of radiation dose. The apparent radiation inactivation size (RIS) of the transport function was found to be 414 +/- 16 kDa. Direct photoaffinity labeling yielded labeled membrane proteins with apparent molecular masses of 130 kDa and 105 kDa, respectively. The proteins displayed different labeling characteristics with respect to incubation time, specificity and the response to irradiation. It appears that only a 105 kDa protein is directly involved in transport function since (a) only it showed a specific pH gradient dependent labeling pattern and (b) the covalent incorporation of [3H]cefadroxil into this protein decreased parallel to the loss of transport function in irradiated BBMV. The peptide/H(+)-symporter in kidney brush-border membranes therefore appears to have a monomer mass of 105 kDa and may function in an oligomeric arrangement.
Boll
M
M
Institute of Nutritional Sciences, Justus-Liebig-University Giessen, Germany.
Daniel
H
H
D000345:Affinity Labels; D000818:Animals; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000378:metabolism / Q000528:radiation effects*; D002434:Cefadroxil / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007672:Kidney Cortex / Q000648:ultrastructure*; D008297:Male; D008871:Microvilli; D008970:Molecular Weight; D010315:Particle Accelerators; D000074011:Peptide Transporter 1; D010777:Photochemistry; D051381:Rats; D017208:Rats, Wistar; D027981:Symporters
D000345:Affinity Labels; D002352:Carrier Proteins; D000074011:Peptide Transporter 1; C489940:Slc15a1 protein, rat; D027981:Symporters; D002434:Cefadroxil