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. Acad. Sci. U.S.A. 89, 6275-6279] membrane-associated isoprenylated protein endoprotease is important in the processing of isoprenylated proteins terminating with CAAX. The enzyme is of substantial interest because specific inhibitors of it block the processing and functioning of ras in vivo. The enzyme appears to be an integral membrane protein, as it can only be removed from microsomal membranes with detergent. The enzyme is effectively solubilized by the detergent CHAPSO and can be partially purified (approximately 10-fold) by anion ion exchange and size exclusion chromatography. Attempts to further purify the enzyme by other column means, including affinity chromatography, were unsuccessful. The partially purified enzyme is very sensitive to thiol reagents but insensitive to other kinds of protease inhibitors, suggesting that the enzyme is a thiol protease. Potent and specific chloroketone containing affinity labeling agents have been developed. These novel inactivators owe their potency to an S-farnesylcysteine moiety which is recognized by the enzyme. Specific inhibitors of this type should allow for the identification and cloning of this protease, which is important for signal transduction.
Chen
Y
Y
Department of Biological Chemistry and Molecular, Harvard Medical School, Boston, Masssachutes 02115, USA.
Ma
Y T
YT
Rando
R R
RR
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000595:Amino Acid Sequence; D002852:Chromatography, Ion Exchange; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D004795:Enzyme Stability; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D008861:Microsomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000494:pharmacology; D017368:Protein Prenylation
D000590:Amino Acid Chloromethyl Ketones; D008565:Membrane Proteins; D011480:Protease Inhibitors; D010450:Endopeptidases; C095298:prenylated peptide-specific endoprotease