Abstract
Goldfish Carassius auratus were acclimated to either 10 or 30°C for a minimum of 5 weeks. A 65-kDa protein specific to warm-temperature-acclimated fish was extracted from the gel with 70% formic acid after two-dimensional electrophoresis of the muscle cytoplasmic protein fraction. The 65-kDa protein thus prepared to homogeneity was used to raise specific antibodies in rabbit by conventional methods. The antibody produced exhibited specific reaction with a protein having the same molecular weight from brain and liver tissue, suggesting that the 65-kDa protein is a ubiquitous cytosolic component in warm-acclimated goldfish. When water temperature was increased from 20 to 30°C over a 20-h period, a prominent amount of the 65-kDa protein was observed in muscle tissue extracts within 5 days of additional rearing; this was demonstrated by immunoblotting with the specific antibody. The N-terminal amino acid sequence of the 65-kDa protein was determined as Asp-Glu-Pro-Gln-Gly-His-Gln-His (or Asp)-Glu-Leu, differing from that of a family of known heat-shock proteins having about 70 kDa in molecular mass (hsp 70). No interaction between ATP and the 65-kDa protein revealed by ATP-agarose affinity chromatography further confirmed the different properties of the 65-kDa protein from those of hsp 70.
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Abbreviations
- ATP:
-
adenosine 5′-triphosphate
- hsp:
-
heat-shock protein(s)
- IgG:
-
immunoglobulin G
- mRNA:
-
messenger ribonucleic acid
- PMSF:
-
phenylmethylsulphonyl fluoride
- PVDF:
-
polyvinylidene difluoride
- SDS:
-
sodium dodecyl sulphate
- SDS-PAGE:
-
SDS-polyacrylamide gel electrophoresis
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Kikuchi, K., Watabe, S., Suzuki, Y. et al. The 65-kDa cytosolic protein associated with warm temperature acclimation in goldfish, Carassius auratus . J Comp Physiol B 163, 349–354 (1993). https://doi.org/10.1007/BF00265637
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DOI: https://doi.org/10.1007/BF00265637