Purification and characterization of cysteine protease from germinating cotyledons of horse gram
1 Center for Cellular and Molecular Biology, Uppal Road, Hyderabad - 500 007, India
2 Department of Biotechnology and Bioinformatics, Yogi Vemana University, Kadapa - 516 003. A.P. India
3 Department of Biochemistry, Sri Krishnadevaraya University, Anantapur - 515 003. Andhra Pradesh, India
BMC Biochemistry 2009, 10:28 doi:10.1186/1471-2091-10-28Published: 17 November 2009
Proteolytic enzymes play central role in the biochemical mechanism of germination and intricately involved in many aspects of plant physiology and development. To study the mechanism of protein mobilization, undertaken the task of purifying and characterizing proteases, which occur transiently in germinating seeds of horse gram.
Cysteine protease (CPRHG) was purified to homogeneity with 118 fold by four step procedure comprising Crude extract, (NH4)2SO4 fractionation, DEAE-Cellulose and CM-sephacel chromatography from the 2 day germinating cotyledons of horse gram (Macrotyloma uniflorum (Lam.) Verdc.). CPRHG is a monomer with molecular mass of 30 k Da, was determined by SDS-PAGE and gel filtration. The purified enzyme on IEF showed two isoforms having pI values of 5.85 and 6.1. CPRHG composed of high content of aspartic acid, glutamic acid and serine. The enzyme activity was completely inhibited by pCMB, iodoacetate and DEPC indicating cysteine and histidine residues at the active site. However, on addition of sulfhydryl reagents (cysteine, dithiothreitol, glutathione and beta-ME) reverse the strong inhibition by pCMB. The enzyme is fairly stable toward pH and temperature. Immunoblot analysis shows that the enzyme synthesized as zymogen (preproenzyme with 81 kDa) and processed to a 40 kDa proenzyme which was further degraded to give 30 kDa active enzyme.
It appears that the newly synthesized protease is inactive, and activation takes place during germination. CPRHG has a broad substrate specificity and stability in pH, temperature, etc. therefore, this protease may turn out to be an efficient choice for the pharmaceutical, medicinal, food, and biotechnology industry.