Formula omitted] photoaffinity labeling of rat liver plasma membrane-associated proteins

Abstract

A method for the synthesis of the glutathione conjugate S-(4- azidophenacyl)[ 35 S]glutathione is desribed. The compound was used for photoaffinity labeling of proteins present in canalicular membrane vesicles (CMV), sinusoidal membrane vesicles (SMV), mitochondria and microsomes from rat liver. Most of the radioactivity introduced by photoaffinity labeling of CMV appeared in the 25–29 kDa range. Further labeled proteins were observed in bands at 37, 105 and about 120 kDa. 79% of the 25–29 kDa associated radioactivity was recovered in the supernatant after extensive revesiculation (washing) of the vesicles, together with the 37 kDa protein. CMV and SMV contained glutathione S-transferase (GST) activity which in CMV was decreased by 75% by washing. Photolabeling of a mixture of purified basic GST subunits from rat liver resulted in a band pattern at 25–29 kDa similar to that in the membrane preparations. Isoelectric focusing of the CMV indicated the presence of basic soluble GST subunits. S-Hexylglutathione-Sepharose affinity chromatography showed reversible binding of photolabeled proteins at 25–29 kDa. Difference photoaffinity bound by GST, but not that introduced into the 105 kDa protein band present in CMV. It is concluded that membrane-associated basic GST isoenzymes are present in standard membrane vesicle preparations. In the cell, the function may be transport of GST-bound compounds across the membrane and protection of the membranes against electrophiles.