Effects of trypsin and protein modification on the renal transporter of p-aminohippurate.
Basal-lateral membranous vesicles prepared from rabbit renal cortex exhibited Mg2+-stimulated, probenecid-inhibitable transport of p-aminohippurate (PAH). This uptake could be completely eliminated by incubating the membranes with trypsin at a weight ratio of 1:700 (trypsin/membrane protein). The loss of PAH uptake activity occurred in two stages. Over the first ten minutes of the vesicles' exposure to trypsin, there was a nearly linear loss, with respect to time, of about 80% of the PAH uptake activity. The remaining 20% of activity was resistant to further trypsin digestion for the next ten minutes, but by twenty-five minutes a total inactivation of the uptake activity occurred. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of normal and trypsin-treated vesicles showed very little degradation of proteins. However, two minor polypeptides (Mr - 410,000 and 388,000) were degraded during the first ten minutes of the membranes' exposure to trypsin. After twenty minutes of exposure, two other polypeptides (Mr = 94,500 and 87,500) were degraded. Chymotrypsin and clostripain also caused a loss of PAH transport activity. However, compared to the effects of trypsin, the effects of these two proteases were less complete, slower in onset, and for clostripain, a much higher concentration of enzyme was required. Other functions or properties of the vesicles including morphological appearance, degree of vesiculation, glucose space or Na+-dependent L-glutamate transport and Na+,K+-ATPase activity were not altered by the concentration of trypsin which abolished 80% of the transport of PAH. Thus, it is possible that one or more of the degraded polypeptides detected by polyacrylamide gel electrophoresis comprises the PAH transporter.(ABSTRACT TRUNCATED AT 250 WORDS)
D026905:Amino Acid Transport Systems; D000618:Aminohippuric Acids / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D007672:Kidney Cortex / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism*; D008970:Molecular Weight; D011339:Probenecid / Q000494:pharmacology; D011817:Rabbits; D014357:Trypsin; D010130:p-Aminohippuric Acid / Q000378:metabolism*
D026905:Amino Acid Transport Systems; D000618:Aminohippuric Acids; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D014357:Trypsin; D005947:Glucose; D011339:Probenecid; D010130:p-Aminohippuric Acid