A novel endoprotease responsible for the specific cleavage of transducin gamma subunit.
Isoprenylated/methylated heterotrimeric G proteins play important roles in a large number of signal transduction processes. While the enzymology of isoprenylated/methylated protein biosynthesis is well understood, nothing is known about how these proteins are degraded. In this article, a novel endoproteolytic activity has been identified from bovine retina and is shown specifically to remove the glycylfarnesylcysteine moiety from the carboxyl terminus of T gamma. When tested in a GTP binding assay, freshly prepared proteolyzed T beta gamma was unable to catalyze the binding of guanosine 5'-(gamma-thio)triphosphate (GTP-gamma-S) to T alpha in the presence of detergent solubilized rhodopsin. The optimum pH for this proteolytic activity is approximately 6, and the pH profile corresponds to an enzyme having pKa's of 4.4 +/- 0.1 and 7.7 +/- 0.1 for its active site residues. After analyzing a series of protease inhibitors, we found E-64, a specific thiol protease inhibitor, to be the most effective irreversible inhibitor of this enzyme, suggesting that the endoprotease might be a thiol protease. Affinity labeling studies using biotinylated affinity labeling probes have identified a 35 kDa protein as a candidate for the endoprotease.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
H
H
Cheng
C A
CA
Parish
B A
BA
Gilbert
R R
RR
Rando
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D010450:Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008745:Methylation; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D011487:Protein Conformation; D017368:Protein Prenylation; D012160:Retina / Q000201:enzymology; D013379:Substrate Specificity; D015293:Transducin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D010446:Peptide Fragments; D011480:Protease Inhibitors; D006160:Guanosine Triphosphate; D010450:Endopeptidases; D015293:Transducin