Purification and characterization of cysteine protease from germinating cotyledons of horse gram.

BMC Biochemistry
Rajeswari JinkaRamakrishna P Rao

Abstract

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 a...Continue Reading

References

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Citations

Dec 12, 2012·Preparative Biochemistry & Biotechnology·S Ishwarya, R Sangeetha
Jun 28, 2011·Journal of the Science of Food and Agriculture·Runqiang YangCuijuan Li
Sep 1, 2017·Preparative Biochemistry & Biotechnology·Ngangoum Eric SergeMookambeswaran Vijayalakshmi
Apr 24, 2018·Chemické Zvesti·Rafał I RawskiKlaudia Kijowska
Nov 18, 2020·International Journal of Biological Macromolecules·Jihen ElleuchSlim Abdelkafi

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Methods Mentioned

BETA
electrophoresis
gel filtration

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