Analysis of native forms and isoform compositions of the mouse 90-kDa heat shock protein, HSP90.
The 90-kDa heat shock protein, HSP90, of the mouse has two isoforms, alpha and beta, which are electrophoretically separable. We have investigated the native forms of HSP90 molecules under physiological conditions and determined their isoform compositions. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that HSP90 purified from mouse lymphoma L5178Y cells consists of approximately 40% alpha and 60% beta isoforms. Analysis by nondenaturing polyacrylamide gel electrophoresis showed that the purified HSP90 exists predominantly as a dimer, but a considerable amount of monomer was also detected. Western blotting using polyclonal anti-mouse HSP90 antibodies revealed that the native forms of HSP90 in the crude L5178Y cell lysates are also dimer and monomer. The nondenaturing polyacrylamide gel electrophoresis resolved the dimeric forms into two separate bands that were identified as alpha/alpha and beta/beta homodimers by two methods: sodium dodecyl sulfate-polyacrylamide gel electrophoresis and peptide mapping. In addition, the results showed that the monomeric form consists mainly of the beta isoform. Both the alpha and beta isoforms were shown to bind equally to actin filaments.
Minami
Y
Y
Department of Cell Biology, Tokyo Metropolitan Institute of Medical Science, Japan.
Kawasaki
H
H
Miyata
Y
Y
Suzuki
K
K
Yahara
I
I
D000199:Actins / Q000378:metabolism; D000818:Animals; D003488:Cyanogen Bromide; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006360:Heat-Shock Proteins / Q000378:metabolism*; D007940:Leukemia L5178 / Q000378:metabolism / Q000473:pathology; D051379:Mice; D014407:Tumor Cells, Cultured
D000199:Actins; D006360:Heat-Shock Proteins; D003488:Cyanogen Bromide