Analysis of the native forms of the 90 kDa heat shock protein (hsp90) in plant cytosolic extracts：（分析的原生形式90 kDa的热休克蛋白(一半)在植物胞质提取物）.pdfVIP

Plant Molecular Biology 33: 457–466, 1997. 457 c 1997 Kluwer Academic Publishers. Printed in Belgium. Analysis of the native forms of the 90 kDa heat shock protein (hsp90) in plant cytosolic extracts 1 1 1 2 Priti Krishna , Ramachandra K. Reddy , Melanie Sacco , J. Roger H. Frappier and Roderick F. Felsheim3 1Department of Plant Sciences, and 2Department of Zoology, The University of Western Ontario, London, Ontario, Canada N6A 5B7; 3Department of Biochemistry, University of Minnesota, St. Paul, MN 55108, USA  ( author for correspondence) Received 7 June 1996; accepted in revised form 10 October 1996 Key words: hsp90, native forms, R2 antibody Abstract A polyclonal antibody, R , was raised against a fusion protein consisting of a portion of plant hsp90 fused to the 2 trpE protein of Escherichia coli. This antibody was found to be specific towards plant hsp90, showing little or no cross-reactivity with mouse and human hsp90 proteins. The R2 antibody identified an 83 kDa protein as the hsp90 homologue in cytosolic extracts of several dicot and monocot plants. Two-dimensional gel electrophoresis indicated that at least two different isoforms of hsp90 are expressed in Brassica napus seedlings. An examination of the native state of hsp90 by non-denaturing gel electrophoresis showed that this protein exists as a monomer, dimer and as a high-molecular-mass complex of ca. 680 kDa in cell extracts of spinach cotyledons and leaves, B. napus seedlings and wheat germ. Native gel analysis and cross-linking studies of purified hsp90 showed that plant hsp90 exists predominantly as a monomer. When 35 S-labelled B. napus cytosolic extracts were immunoprecipitated with the R2