Purification and characterization of a 20-kDa protein that is highly homologous to alpha B crystallin.
A 20-kDa protein (p20) that had internal amino acid sequences highly similar to those of alpha B crystallin was purified from rat and human skeletal muscle. p20 co-eluted with alpha B crystallin and HSP27/28 during column chromatography on DEAE-Sepharose and on Bio-Gel A-5m. p20 was separated from alpha B crystallin and HSP27/28 and was resolved into two fractions, a minor first peak and a major second peak, by column chromatography on S-Sepharose in the presence of 7 M urea. During chromatography on a column of Superdex 75pg, even in the presence of 7 M urea, p20 in the second peak was eluted as aggregates near the exclusion volume of the column, whereas p20 in the first peak was eluted in fractions that corresponded to a lower molecular mass. Further chromatography on a TSK-SP-5PW column yielded pure preparations of each of the two forms of rat and human p20. The fragmentation patterns of the two forms of the respective p20 proteins generated by digestion with endoproteinase Asp-N were identical. The primary structures of rat and human p20, determined with an NH2-terminal sequenator, were highly homologous to those of alpha B crystallin and HSP27/28. p20 was present in all rat tissues examined and at high levels (> 1 micrograms/mg protein) in the soleus muscle, heart, and diaphragm, as are alpha B crystallin and HSP27. Centrifugation on sucrose density gradients allowed detection of the aggregated form and the small form of p20, as well as of HSP27, in extracts of rat muscle tissues. During heating at 45 degrees C of tissue in vitro, p20 in rat diaphragm was redistributed from the cytoplasm to the insoluble fraction, and dissociation of the aggregated p20 to the small form was enhanced. These results suggest that p20 is related to stress proteins.
Kato
K
K
Department of Biochemistry, Institute for Developmental Research, Aichi Prefectural Colony, Japan.
Goto
S
S
Inaguma
Y
Y
Hasegawa
K
K
Morishita
R
R
Asano
T
T
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003459:Crystallins / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D050886:HSP20 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000235:genetics / Q000302:isolation & purification*; D009132:Muscles / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution
D003459:Crystallins; D050886:HSP20 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D009124:Muscle Proteins; C087458:Hspb6 protein, rat