PROTEIN EXPRESSIONS IN MITOCHONDRIA ISOLATE FROM KALANCHOË DAIGREEMONTIANA
Abstract
In this study, we isolated and purified mitochondria from leaves of Kalanchoë daigreemontiana by using Percoll gradients. The purified mitochondrial were used to isolate protein and mitochondrial protein were determined. Mitochondrial purified protein was extracted in lysis buffer containing protease inhibitor mix, followed by precipitation with ice-cold acetone. Two-dimensional electrophoresis (2DE) was used to investigate and identify mitochondrial protein expression of K. daigreemontiana. This investigation focused on proteins in the isoelectric point range of 3-10 and molecular weight range from 14.4 to 97.4 kDa. Staining of 2 DE gel with Sypro Ruby revealed more than 150 major protein spots from mitochondria. The 2DE maps of K. daigreemontiana mitochondrial soluble proteins showed less vertical streaking indicating that well proteins were transferred from strips into the gel.
References
Bardel, J., Louwagie, M., Jaquinod, M., Luche, S., Rabilloud, T., Macherel, D., Garin, J. and Bourguignon, J. A survey of the plant mitochondrial proteome in relation to development. Proteomics, 2, (2002), 880-898.
Berkelman, T., Stenstedt, T. and Bjellqvist, B. Sample preparation. In: Berkelman, T., Stenstedt, T., Bjellqvist, B. Laird, N. McDowell, M. Olsson, I. and Westermeier, R., eds. 2-D electrophoresis using immobilized pH gradients. Principles and methods, Handbooks from Amersham Biosciences, (1998), 1-104.
Bradford, M.M. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analysis Biochemistry, 72, (1976), 248-254.
Heazlewood, J.L., Howell, K.A., Whelan, J. and Millar, A.H. Towards an analysis of the rice mitochondrial proteome. Plant Physiology, 132, (2003), 230-242.
Hoa, L.T.P., Nomura, M., Kajiwara, H., Day, D.A. and Tajima, S. Proteomic analysis on symbiotic differentiation of mitochondria in soybean nodules. Plant Cell Physiology, 45, (2004), 300-308.
Hong HTK, Nose A, Agarie S. Oxidation of substrate in Percoll-purified mitochondria isolated from Kalanchoë daigremontiana. Bulletin of the Agriculture, Saga University 89, (2004a), 121–129.
Hong, H.T.K., Nose, A., and Agarie, S. Respiratory properties and malate metabolism in Percoll-purified mitochondria isolated from pineapple, Ananas comosus (L.) Merr. cv. smooth cayenne. Journal Experimental Botany, 55, (2004b) 2201-2211.
Kruft, V., Eubel, H., Jansch, L., Werhahn, W. and Braun, H.P. Proteomic approach to identify novel mitochondrial proteins in Arabidopsis. Plant Physiology, 127, (2001), 1694–1710.
Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, (1970), 680-685.
Millar, A.H., Sweetlove, L.J., Giege, P. and Leaver, C.J. Analysis of the Arabidopsis mitochondrial proteome. Plant Physiology, 127, (2001) 1711–1727.
Patricia, M.L., Xiaosi, Z., Vasant, G. H., Drena, L.D, Aigen, F., and Steve, R.R. A proteomic analysis of maize chloroplast biogenesis. Plant Physiology. 134 (2004), 560-574.
Stasyk, T., Hellman, U. and Souchelnytskyi, S. Optimizing sample preparation for 2-D electrophoresis, Amersham Bioscience, Innovations forum, Life Science News, 9, (2001), 1-4.
