Purification and characterization of an endogenous inhibitor specific to the Z-Leu-Leu-Leu-MCA degrading activity in proteasome and its identification as heat-shock protein 90.
We previously identified a benzyloxycarbonyl(Z)-Leu-Leu-Leu-4-methylcoumaryl-7-amide (ZLLL-MCA) degrading activity in proteasome as a candidate for the regulator of neurite outgrowth. As its counterpart, we purified a protein from bovine brain that specifically inhibits the ZLLL-MCA degrading activity in proteasome. This protein is heat stable and has no effect on the other catalytic activities in proteasome, or on the activities of trypsin, chymotrypsin, or m- and mu-calpains either. The molar ratio of inhibitor-to-proteasome that inhibits 50% of the ZLLL-MCA degrading activity of proteasome is 1:1. The inhibitory mechanism of the inhibitor against proteasome is non-competitive. Finally, the inhibitor was identified as heat-shock protein 90 (HSP90) by partial amino acid sequencing and immunodetection. The results suggest that HSP90 initiates neurite outgrowth through the inhibition of the ZLLL-MCA degrading activity in proteasome.
Tsubuki
S
S
Department of Molecular Biology, Tokyo Metropolitan Institute of Medical Science, Japan.
Saito
Y
Y
Kawashima
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism*; D005456:Fluorescent Dyes; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000378:metabolism*; D011480:Protease Inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000494:pharmacology; D017421:Sequence Analysis
D005456:Fluorescent Dyes; D006360:Heat-Shock Proteins; D009842:Oligopeptides; D011480:Protease Inhibitors; C084513:benzyloxycarbonylleucyl-leucyl-leucyl-4-methyl-coumaryl-7-amide; D010450:Endopeptidases