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
References
Gerlach G, Turay L, Malik TA, Lida J, Scutt A, Goldspink G (1990) Mechanisms of temperature acclimation in the carp: a molecular approach. Am J Physiol 259:R237-R244
Hazel JR, Prosser CL (1974) Molecular mechanisms of temperature compensation in poikilotherms. Physiol Rev 54:620–677
Heap SP, Watt PW, Goldspink G (1985) Consequences of thermal change on the myofibrillar ATPase of five freshwater teleosts. J Fish Biol 26:733–738
Hwang GC, Watabe S, Hashimoto K (1990) Changes in carp myosin ATPase induced by temperature acclimation. J Comp Physiol B 160:233–239
Hwang GC, Ochiai Y, Watabe S, Hashimoto K (1991) Changes of carp myosin subfragment-1 induced by temperature acclimation. J Comp Physiol B 161:141–146
Inostroza J, Vera MI, Goicoechea O, Amthauer R, Krauskopf M (1990) Apolipoprotein A-I synthesis during the acclimatization of the carp (Cyprinus carpio). J Exp Zool 256:8–15
Johnston IA, Davison W, Goldspink G (1975) Adaptation in Mg2+-activated myofibrillar ATPase activity induced by temperature acclimation. FEBS Lett 50:293–295
Koban M, Graham G, Prosser CL (1987) Induction of heat-shock protein synthesis in teleost hepatocytes: effects of acclimation temperature. Physiol Zool 60:290–296
Kobayashi M, Hiura N, Matsuda K (1985) Isolation of enzymes from polyacrylamide disk gels by a centrifugal homogenization method. Anal Biochem 145:351–353
Ku CC, Chen SN (1991) Heat shock proteins in cultured gill cells of color carp Cyprinus carpio L. Bull Inst Zool Academia Sinica 30:319–330
Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227:680–985
Lindquist S (1986) The heat shock response. Annu Rev Biochem 55:1156–1191
Margulis BA, Antropiva Y, Kharazova AD (1989) 70 kDa heat shock proteins from mollusc and human cells have common structural and functional domains. Comp Biochem Physiol 94 B:621–623
Matsudaira P (1987) Sequence from picomole quantities of protein electroblotted onto polyvinylidene difluoride membranes. J Biol Chem 262:10035–10038
Nakagawa T, Watabe S, Hashimoto K (1988) Identification of three major components in fish sarcoplasmic proteins. Nippon Suisan Gakkaishi 54:999–1004
O'Farrell PH (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250:4007–4021
Oda S, Mitani O, Naruse K, Shima A (1991) Synthesis of heat shock protein in the isolated fin of the medaka, Oryzias latipes, acclimated to various temperatures. Comp Biochem Physiol 98 B:587–591
Pelham HRB (1986) Speculations on the functions of the major heat shock and glucose regulated proteins. Cell 46:959–961
Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of protein from polyacrylamide gel to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354
Watabe S, Hwang GC, Nakaya M, Guo XF, Okamoto Y (1992) Fast skeletal myosin isoforms in thermally acclimated carp. J Biochem 111:113–122
Watabe S, Kikuchi K, Aida K (1993) Cold- and warm-temperature acclimation induces specific cytosolic protein in goldfish and carp. Nippon Suisan Gakkaishi 59:151–156
Welch WJ, Feramisco JR (1985) Rapid purification of mammalian 70,000-dalton stress proteins: affinity of the proteins for nucleotides. Mol Cell Biol 5:1229–1237
Zafarullah M, Wisniewsky J, Shworak NW, Schieman S, Misra S, Gedamu L (1992) Molecular cloning and characterization of a constitutively expressed heat-shock-cognate hsc71 gene from rainbow trout. Eur J Biochem 204:893–900
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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