Purification of a novel type of SDS-dependent protease in maize [Zea mays] using a monoclonal antibody

• Published in: Plant and cell physiology Vol. 39; no. 1; pp. 106 - 114
• Main Authors: Yamada, T. (Tokyo Inst. of Technology (Japan)), Ohta, H, Masuda, T, Ikeda, M, Tomita, N, Ozawa, A, Shioi, Y, Takamiya, K
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 01.01.1998
• Subjects: 5-bisphos-phate carboxylase/oxygenase (EC 4.1.1.39); Activation by SDS; adenosine triphosphate; Agronomy. Soil science and plant productions; Animals; Antibodies, Monoclonal; ANTICORPS MONOCLONAL; ANTICUERPOS MONOCLONALES; Biological and medical sciences; Conformational change of protease; DENATURATION; DESNATURALIZACION; Detergents - pharmacology; e-64; Economic plant physiology; ECOTIPOS; ECOTYPE; ECOTYPES; EDTA (chelating agent); Endopeptidases - drug effects; Endopeptidases - immunology; Endopeptidases - isolation & purification; Endopeptidases - metabolism; enzyme activators; enzyme activity; enzyme inhibitors; Enzyme Inhibitors - pharmacology; Enzymes; fatty acids; Female; Fundamental and applied biological sciences. Psychology; Hydrogen-Ion Concentration; kinetics; leaves; leupeptin; lysine; Maize (Zea mays); Mercaptoethanol; Metabolism; Metals; Mice; Mice, Inbred BALB C; molecular conformation; MONOCLONAL ANTIBODIES; Nutrition. Photosynthesis. Respiration. Metabolism; physicochemical properties; Plant Leaves - enzymology; Plant physiology and development; PROTEASAS; PROTEASE; PROTEASES; protein composition; proteinases; proteolysis; PURIFICACION; PURIFICATION; ribulose-bisphosphate carboxylase; Ribulose-l; SH-protease; sodium dodecyl sulfate; Sodium Dodecyl Sulfate - pharmacology; Substrate Specificity; substrates; sulfates; ZEA MAYS; Zea mays - enzymology; Monocotyledones; Purification; Zea mays; Enzyme; Monoclonal antibody; Cereal crop; Lauryl sulfate; Sodium Compounds; Characterization; Peptidases; Gramineae; Activator; Angiospermae; Hydrolases; Spermatophyta; Catalyst activity
• ISSN: 0032-0781; 1471-9053
• DOI: 10.1093/oxfordjournals.pcp.a029281

A protease which was activated by SDS was purified to homogeneity from maize laves. 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