IMMONUGOLD ELECTRON-MICROSCOPIC DETECTION OF HEAT-SHOCK PROTEIN-60 (HSP60) IN MITOCHONDRIA OF RAT HEPATOCYTES AND MYOCARDIOCYTES

We characterize the specificity of a polyclonal antibody against heat shock protein 60 (hsp60) and present an application for ultrastructura l localization studies of this protein. The antibody was obtained from an IgG fraction (AB 121) originally raised against the calcium bindin g protein calsequestrin by immunoabsorption on isolated rat liver hsp6 0. As shown by partial N-terminal amino acid sequence analysis of immu noprecipitated proteins AB 121 contained reactivities against hsp60, c alsequestrin and the glycoprotein fetuin. In rat heart AB 121 recogniz ed calsequestrin and hsp60. In human and rat liver the only reacting p rotein was hsp60. In rat erythrocytes the antibody bound to 61 kDa and 58 kDa isoforms of fetuin. According to published data no amino acid sequence homologies nor common motifs are found between calsequestrin, hsp60 and fetuin. As the first application the anti-hsp60 antibody wa s used for immune-gold electron microscopical localization of hsp60: i n myocardiocytes and hepatocytes of the rat strong labelling was obtai ned exclusevely in mitochondria. No extramitochondrial structures were labelled. The specificity of the antibody and its ability to be visua lized by immune-gold electron microscopy offers the possibility to stu dy the expression of this protein in the liver and in other organs. Po ssible clinical applications of these studies are discussed, since hsp 60 could be a target antigen of autoantibodies in diseases such as aut oimmune hepatitis, primary sclerosing cholangitis or primary biliary c irrhosis.