Isolation of the protease component of maize cysteine protease-cystatin complex: release of cystatin is not crucial for the activation of the cysteine protease.
The maize cysteine protease complex, which required SDS for its activation in vitro, is a 179 kDa trimeric complex (P-I)3 of a cysteine protease (P) [EC 3.4.22] and a cysteine protease inhibitor (I), cystatin [Yamada et al. (1998) Plant Cell Physiol. 39: 106, Yamada et al. (2000) Plant Cell Physiol. 41: 185]. Here, we show the mechanism of the SDS-dependent activation of the trimeric (P-I) complex and stabilization of the activated protease by its specific substrates. The cystatin-free cysteine protease isolated by preparative SDS-PAGE was still specifically activated by SDS, and its profile of SDS-dependency was exactly the same as that of the trimeric (P-I) complex. It is, therefore, evident that an SDS-dependent conformational change of the protease itself, rather than the release of cystatin from the complex, is crucial for the activation. Pre-treatment analysis with SDS revealed that SDS was required for the initiation of the activation of the trimeric (P-I) complex. Furthermore, we found that once the protease was activated, if there was no substrate, it was rapidly inactivated under optimum conditions of proteolysis, and showed that such inactivation was not due to autolysis of the protease. In contrast, addition of specific substrates prevented the inactivation, and thus we presumed that the activity of the cysteine protease is regulated by both activation by conformational change and rapid inactivation after consumption of substrates.
Yamada
T
T
Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, 226-8501, Japan. tyamada@bio.titech.ac.jp
Kondo
A
A
Ohta
H
H
Masuda
T
T
Shimada
H
H
Takamiya
K
K
D015891:Cystatins / Q000302:isolation & purification / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D015853:Cysteine Proteinase Inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D018744:DNA, Plant; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D012333:RNA, Messenger; D003313:Zea mays / Q000201:enzymology*
D015891:Cystatins; D015853:Cysteine Proteinase Inhibitors; D018744:DNA, Plant; D012333:RNA, Messenger; D003546:Cysteine Endopeptidases