title: Constitutive heat shock protein 70 (HSC70) expression in rainbow trout hepatocytes: effect of heat shock and heavy metal exposure.
abstract: The 70-kDa family of heat shock proteins plays an important role as molecular chaperones in unstressed and stressed cells. The constitutive member of the 70 family (hsc70) is crucial for the chaperoning function of unstressed cells, whereas the inducible form (hsp70) is important for allowing cells to cope with acute stressor insult, especially those affecting the protein machinery. In fish, the role of hsc70 in the cellular stress response process is less clear primarily because of the lack of a fish-specific antibody for hsc70 detection. In this study, we purified hsc70 to homogeneity from trout liver using a three-step purification protocol with differential centrifugation, ATP-agarose affinity chromatography and electroelution. Polyclonal antibodies to trout hsc70 generated in rabbits cross-reacted strongly with both purified trout hsc70 protein and also purified recombinant bovine hsc70. Two-dimensional electrophoresis followed by Western blotting confirmed that the isoelectric point of rainbow trout hsc70 was more acidic than hsp70. Using this antibody, we detected hsc70 content in the liver, heart, gill and skeletal muscle of unstressed rainbow trout. Primary cultures of trout hepatocytes subjected to a heat shock (+15 degrees C for 1 h) or exposed to either CuSO(4) (200 microM for 24 h), CdCl(2) (10 microM for 24 h) or NaAsO(2) (50 microM for 1 h) resulted in higher hsp70 accumulation over a 24-h period. However, hsc70 content showed no change with either heat shock or heavy metal exposure suggesting that hsc70 is not modulated by sublethal acute stressors in trout hepatocytes. Taken together, we have for the first time generated polyclonal antibodies specific to rainbow trout hsc70 and this antibody will allow for the characterization of the role of hsc70 in the cellular stress response process in fish.
authors.affiliation: Department of Biology, University of Waterloo, Waterloo, Ont., Canada N2L 3G1.
authors.forename: Adrienne N
authors.initials: AN
authors.lastname: Boone
authors.forename: Mathilakath M
authors.initials: MM
authors.lastname: Vijayan
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D018053:Arsenites / Q000633:toxicity; D015153:Blotting, Western; D002104:Cadmium / Q000633:toxicity*; D002623:Chemistry Techniques, Analytical / Q000379:methods; D003300:Copper / Q000633:toxicity*; D005260:Female; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D018869:Heat-Shock Response / Q000502:physiology*; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism*; D017686:Oncorhynchus mykiss / Q000378:metabolism*; D009928:Organ Specificity; D011817:Rabbits
chemical_list: D000906:Antibodies; D018053:Arsenites; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D002104:Cadmium; D003300:Copper; C015001:arsenite