SOLUBILIZATION, PARTIAL-PURIFICATION, AND AFFINITY LABELING OF THE MEMBRANE-BOUND ISOPRENYLATED PROTEIN ENDOPROTEASE

A previously described [Ma, Y.-T., & Rando, R. R. (1992) Proc. Natl. A cad. Sci. U.S.A. 89, 6275-6279] membrane-associated isoprenylated prot ein endoprotease is important in the processing of isoprenylated prote ins terminating with CAAX. The enzyme is of substantial interest becau se specific inhibitors of it block the processing and functioning of m s in vivo. The enzyme appears to be an integral membrane protein, as i t can only be removed from microsomal membranes with detergent. The en zyme is effectively solubilized by the detergent CHAPSO and can be par tially purified (similar to 10-fold) by anion ion exchange and size ex clusion chromatography. Attempts to further purify the enzyme by other column means, including affinity chromatography, were unsuccessful. T he partially purified enzyme is very sensitive to thiol reagents but i nsensitive to other kinds of protease inhibitors, suggesting that the enzyme is a thiol protease. Potent and specific chloroketone containin g affinity labeling agents have been developed. These novel inactivato rs owe their potency to an S-farnesylcysteine moiety which is recogniz ed by the enzyme. Specific inhibitors of this type should allow for th e identification and cloning of this protease, which is important for signal transduction.