THE PARTIAL PURIFICATION OF A CARRIER-LIKE PROTEIN FOR ORGANIC BASES FROM THE KIDNEY

¹ Department of Pharmacology, State University of New York, Upstate Medical Center, Syracuse, New York

Dibenamine-C¹⁴, an irreversible inhibitor of the renal transport system of organic bases, was used in an attempt to specifically label the transport carrier molecule. The labeling of nonspecific sites was minimized by first allowing the tissue to react with unlabeled Dibenamine under conditions in which the presumed carrier was protected by the presence of substrates for transport (i.e., N-methylnicotinamide, tetraethyl-, tetramethyl- or tetrabutylammonium). Subsequent treatment of the tissue with Dibenamine-C¹⁴ alone resulted in relatively specific labeling. The extent of protection by the substrate bases was determined as the difference in C¹⁴ content of the "protected" and "unprotected" slices. This difference in labeling was observed only in the protein fraction. After shocking the tissue in a hypoosmotic medium, the differential labeling appeared in the medium and not in the residual slices. "Shock fluid" from untreated slices contained a macromolecular material which could react irreversibly with Dibenamine-C¹⁴, and this reaction was inhibited in the presence of any of the substrate bases. The material in shock fluid could be divided into pellet and supernatant fractions by centrifugation at 100,000 x g. Both fractions behaved similarly when tested as above. A preliminary description of the behavior of this material on diethylaminoethyl-cellulose columns is given.