Copurification of small heat shock protein with alpha B crystallin from human skeletal muscle.
Immunoreactive alpha B crystallin and a 28-kDa protein in an extract of human pectoral muscle were precipitated by (NH4)2SO4 at 40% saturation, and coeluted during column chromatography on DEAE-Sepharose and on Bio-Gel A-5m. The two proteins were separated on a column of S-Sepharose HP in the presence of 7 M urea. Further chromatography of each of the two resultant fractions on a column of Superdex 75 pg and on a TSK-SP 5PW column in the presence of urea yielded preparations of alpha B crystallin and the 28-kDa protein each of which gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The final preparation of 28-kDa protein contained at least two subtypes, which were separable on the TSK-SP column. However, fragmentation patterns of the two major 28-kDa proteins after digestion with endoproteinase Asp-N were identical. Amino acid sequences of peptides formed by cleavage of the purified 28-kDa protein and alpha B crystallin were identical to those of particular regions of the deduced amino acid sequences of human small heat shock protein (HSP28) and lens alpha B crystallin, respectively. Using an immunoassay method, with antibodies raised in rabbits, we found that HSP28 was present in all human tissues tested and at high levels (greater than 1 micrograms/mg protein) in the heart and other tissues composed of striated and smooth muscles. HSP28, found with alpha B crystallin, in extracts of several human and bovine tissues was trapped on and coeluted with alpha B crystallin from an affinity column prepared with antibodies against alpha B crystallin. This result suggests that the two proteins are associated in cells.
Kato
K
K
Department of Biochemistry, Institute for Developmental Research, Aichi Prefectural Colony, Japan.
Shinohara
H
H
Goto
S
S
Inaguma
Y
Y
Morishita
R
R
Asano
T
T
D000595:Amino Acid Sequence; D000918:Antibody Specificity; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003459:Crystallins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006360:Heat-Shock Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009132:Muscles / Q000737:chemistry*; D016415:Sequence Alignment
D003459:Crystallins; D006360:Heat-Shock Proteins