Summary
Basal-lateral membranous vesicles prepared from rabbit renal cortex exhibited Mg2+-stimulated, probenecid-inhibitable transport ofp-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 poypeptides (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+-dependentl-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. Furthermore, modification of the vesicles with phenylglyoxal led to a 38% loss of PAH uptake activity. This suggests that arginine residues may play an important role in the transport system.
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References
Berner, W., Kinne, R. 1976. Transport ofp-aminohippuric acid by plasma membrane vesicles isolated from rat kidney cortex.Pfluegers Arch. 361, 269–277
Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 72:248–254
Churchill, L., Hokin, L.E. 1976. The susceptibility of the glycoprotein from the purified (Na+,K+)-activated adenosine triphosphatase to tryptic and chymotryptic degradation with and without Na+ and K+.Biochim. Biophys. Acta 434:258–264
Cuatrecasas, P. 1974. Membrane receptors.Annu. Rev. Biochem. 43:169–214
Dixon, H.B.F., Perham, R.N. 1968. Reversible blocking of amino groups with citraconic anhydride.Biochem. J. 109:312–314
Ebel, H., Aulbert, E., Merker, H.J. 1976. Isolation of the basal and lateral plasma membranes of rat kidney tubules cells.Biochim. Biophys. Acta 433:531–546
Fields, R. 1972. The rapid determination of amino groups with TNBS.Methods Enzymol. 25:464–468
Glossmann, H., Neville, D.M., Jr. 1972. Phlorizin receptors in isolated kidney brush border membranes.J. Biol. Chem. 247:7779–7789
Heidrich, H., Kinne, R., Kinne-Saffran, E., Hannig, K. 1972. The polarity of the proximal tubule cell in rat kidney. Different surface charges for the brush-border microvilli and plasma membranes from the basal infoldings.J. Cell Biol. 54:232–245
Hittelman, K., Mamelok, R.D., Prusiner, S.B. 1978. Preservation by freezing of glucose and alanine transport into kidney membrane vesicles.Anal. Biochem. 89:324–331
Holohan, P.D., Pessah, N.I., Pessah, I.N., Ross, C.R. 1979. Reconstitution of N′-methylnicotinamide andp-aminohippuric acid transport in phospholipid vesicles with a protein fraction isolated from dog kidney membranes.Mol. Pharmacol. 16:343–356
Hsu, B.Y., Corcoran, S.M., Marshall, C.M., Segal, S. 1982. The effect on amino acid transport of trypsin treatment of rat renal brush border membranes.Biochim. Biophys. Acta 689:181–193
Inui, K., Okano, T., Takano, M., Kitazawa, S., Hori, R. 1981. A simple method for the isolation of basolateral plasma membrane vesicles from rat kidney cortex. Enzyme activities and some properties of glucose transport.Biochim. Biophys. Acta 647:150–154
Jain, M.K. 1973. Enzymic hydrolysis of various components in biomembranes and related systems.In: Current Topics in Membranes and Transport. F. Bronner and A. Kleinzeller, editors. Vol. 4, pp. 175–254. Academic, New York
Jorgensen, P.L. 1975. Purification and characterization of (Na+, K+)-ATPase. V. Conformational changes in the enzyme. Transitions between the Na-form and the K-form studied with tryptic digestion as a tool.Biochim. Biophys. Acta 401:399–415
Kinsella, J.L., Holohan, P.D., Pessah, N.I., Ross, C.R. 1979a. Isolation of luminal and antiluminal membranes from dog kidney cortex.Biochim. Biophys. Acta 552:468–477
Kinsella, J.L., Holohan, P.D., Pessah, N.I., Ross, C.R. 1979b. Transport of organic ions in renal cortical luminal and antiluminal membrane vesicles.J. Pharmacol. Exp. Ther. 209:443–450
Knauf, P.A. 1979. Erythrocyte anion exchange and the band 3 protein: Transport kinetics and molecular structure.In: Current Topics in Membranes and Transport. F. Bronner and A. Kleinzeller, editors. Vol. 12, pp. 231–365. Academic, New York
Kotaki, A., Harada, M., Yagi, K. 1964. Reaction between sulfhydryl compounds and 2,4,6-trinitrobenzene-1-sulfonic acid.J. Biochem. 55:553–561
Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature (London) 227:680–683
Liang, C.T., Sacktor, B. 1977. Preparation of renal cortex basallateral and brush border membranes. Localization of adenylate cyclase and guanylate cyclase activities.Biochim. Biophys. Acta466:474–487
Lineweaver, H., Murray, C.W. 1947. Identification of the trypsin inhibitor of egg white with ovomucoid.J. Biol. Chem. 171:565–581
Mamelok, R.D., Tse, S., Bildstein, C., Liu, D. 1981. Purification and characterization of rabbit kidney basal-lateral membranes prepared in a zonal rotor.J. Cell Biol. 91:102a
Mamelok, R.D., Tse, S.S., Newcomb, K., Bildstein, C.L., Liu, D. 1982. Basal-lateral membranes from rabbit renal cortex prepared on a large scale in a zonal rotor.Biochim. Biophys. Acta 692:115–125
Mitchell, W.M., Harrington, W.F. 1971. Clostripain.In: The Enzymes. P.D. Boyer, editor. Vol. III, pp. 699–719. Academic, New York
Noonan, K.D. 1978. Proteolytic modification of cell surface macromolecules: Mode of action in stimulating cell growth.In: Current Topics in Membranes and Transport. R.L. Juliano and A. Rothstein, editors. Vol. 11, pp. 397–461. Academic, New York
Okuyama, T., Satake, T. 1960. On the preparation and properties of 2,4,6-trinitrophenyl-amino acids and-peptides.J. Biochem. (Tokyo) 47:454–466
Plapp, B.V., Moore, S., Stein, W.H. 1971. Activity of bovine pancreatic deoxyribonuclease A with modified amino groups.J. Biol. Chem. 246:939–945
Sacktor, B., Rosenbloom, I.L., Liang, C.T., Cheng, L. 1981. Sodium gradient- and sodium plus potassium gradient-dependentl-glutamate uptake in renal basolateral membrane vesicles.J. Membrane Biol. 60:63–71
Satake, K., Okuyama, T., Ohashi, M., Shinoda, T. 1960. The spectrophotometric determination of amine, amino acid and peptide with 2,4,6-trinitrobenzene-1-sulfonic acid.J. Biochem. (Tokyo) 47:654–660
Scalera, V., Huang, Y.K., Hildmann, B., Murer, H. 1981. A simple isolation method for basal-lateral plasma membranes from rat kidney cortex.Membr. Biochem. 4:49–61
Seaman, G.V.F., Uhlenbruck, G. 1962. The action of proteolytic enzymes on the red cells of some animal species.Biochim. Biophys. Acta 64:570–572
Sheikh, M.I., Møller, J.V. 1982. Na+-gradient-dependent stimulation of renal transport ofp-aminohippurate.Biochem. J. 208:243–246
Takahashi, K. 1968. The reaction of phenylglyoxal with arginine residues in proteins.J. Biol. Chem. 243:6171–6179
Tse, S., Bildstein, C., Liu, D., Mamelok, R.D. 1982. Inactivation ofp-aminohippurate uptake in basal-lateral membrane vesicles by trypsin.Clin. Res. 30:465A
Tse, S.S., Bildstein, C.L., Liu, D., Mamelok, R.D. 1983. Effects of divalent cations and sulfhydryl reagents on the PAH transporter of renal basal-lateral membranes.J. Pharmacol. Exp. Ther. 226:19–26
Tse, S.S., Bildstein, C.L., Liu, D., Mamelok, R.D. 1984. Effects of analogues of salicylate onp-aminohippurate uptake into basal-lateral membraneous vesicles.J. Pharmacol. Exp. Ther. (in press)
Umezawa, H., Aoyagi, T. 1977. Activities of proteinase inhibitors of microbial origin.In: Proteinases in Mammalian Cells and Tissues. A.J. Barrett, editor. pp. 637–662. Elsevier/North Holland, Amsterdam
Wang, Y-J., Gurd, J.W., Mahler, H.R. 1975. Topography of synaptosomal high affinity uptake systems.Life Sci. 17:725–734
Weber K., Osborn, M. 1969. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis.J. Biol. Chem. 244:4406–4412
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Tse, S.S., Liu, D., Bildstein, C.L. et al. Effects of trypsin and protein modification on the renal transporter ofp-aminohippurate. J. Membrain Biol. 82, 249–257 (1984). https://doi.org/10.1007/BF01871634
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DOI: https://doi.org/10.1007/BF01871634