Purification of a novel type of SDS-dependent protease in maize using a monoclonal antibody.
A protease which was activated by SDS was purified to homogeneity from maize leaves. On the basis of its proteolytic activity towards ribulose-1,5-bisphosphate carboxylase/ oxygenase (Rubisco) or a synthesized peptide, the purification was carried out using immunoaffinity chromatography with a monoclonal antibody raised against a partially purified enzyme by native gradient PAGE. The purified protease showed three bands at 40, 15, and 13 kDa on SDS-PAGE, indicating that it was composed of heterogeneous subunits. The protease was specifically activated by SDS (optimum = 0.4% for Rubisco proteolysis), but not by poly-L-lysine, fatty acids, or ATP. The protease had a pH optimum around 4.9. beta-Mercaptoethanol stimulated the activity only in the presence of SDS. The proteolytic activity was sensitive to E-64 and leupeptin but was resistant to EDTA, suggesting that the enzyme was an SH-protease. Thus, this enzyme is a novel type of SDS-dependent protease which differs from proteasome, matrix metalloproteinase, and other proteases reported in many organisms.
Yamada
T
T
Department of Biological Sciences, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan.
Ohta
H
H
Masuda
T
T
Ikeda
M
M
Tomita
N
N
Ozawa
A
A
Shioi
Y
Y
Takamiya
K
K
D000818:Animals; D000911:Antibodies, Monoclonal; D003902:Detergents / Q000494:pharmacology*; D010450:Endopeptidases / Q000187:drug effects / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006863:Hydrogen-Ion Concentration; D008623:Mercaptoethanol; D008670:Metals; D051379:Mice; D008807:Mice, Inbred BALB C; D018515:Plant Leaves / Q000201:enzymology; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology*; D013379:Substrate Specificity; D003313:Zea mays / Q000201:enzymology*
D000911:Antibodies, Monoclonal; D003902:Detergents; D004791:Enzyme Inhibitors; D008670:Metals; D012967:Sodium Dodecyl Sulfate; D008623:Mercaptoethanol; D010450:Endopeptidases