Purification and characterization of a Z-Leu-Leu-Leu-MCA degrading protease expected to regulate neurite formation: a novel catalytic activity in proteasome.
A tripeptide aldehyde protease inhibitor, benzyloxycarbonyl(Z)-Leu-Leu-leucinal (ZLLLa1), initiates neurite outgrowth in PC12 cells at an optimal concentration of 30nM. This result suggests the existence of a protease which regulates neurite formation in PC12 cells. We report here an attempt to identify this target protease in bovine brain using Z-Leu-Leu-Leu-4-methylcoumaryl-7-amide (ZLLL-MCA), in which the aldehyde moiety of ZLLLa1 was changed to 4-methylcoumaryl-7-amide to serve as a substrate for the protease. As a result, we have purified a proteasome with a molecular weight of about 660 kDa as a ZLLL-MCA degrading protease. The activity of the proteasome was inhibited efficiently by ZLLLa1, and was different from known catalytic activities of proteasome in some aspects, suggesting it to be a novel one. Thus, the proteasome may be involved in the regulation of neurite formation in PC12 cells.
Tsubuki
S
S
Department of Molecular Biology, Tokyo Metropolitan Institute of Medical Science, Japan.
Kawasaki
H
H
Saito
Y
Y
Miyashita
N
N
Inomata
M
M
Kawashima
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D002852:Chromatography, Ion Exchange; D003546:Cysteine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D007976:Leupeptins / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016501:Neurites / Q000187:drug effects / Q000502:physiology*; D009842:Oligopeptides / Q000378:metabolism* / Q000494:pharmacology; D016716:PC12 Cells; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D007976:Leupeptins; D009842:Oligopeptides; C084513:benzyloxycarbonylleucyl-leucyl-leucyl-4-methyl-coumaryl-7-amide; D003546:Cysteine Endopeptidases; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde