Ubiquitin in the stress response of a temperate and an Antarctic Fish.

Abstract

In this thesis results are presented from a study of the biochemical stress responses from the temperate marine reef teleost blue mao mao (Scorpis violaceus), and the cryopelagic antarctic teleost Pagothenia borchgrevinki. Ubiquitin is a polypeptide responsible for ATP-dependent degradation of abnormal proteins which increase in cells after stress. Using immunochemical methods originally developed for use in mammals, levels of the stress protein ubiquitin and ubiquitin protein conjugates were determined in the brain, gills, white muscle and erythrocytes. Confinement of blue mao mao resulted in a significant elevation of ubiquitin conjugates in erythrocytes after 30 min followed by a full return to baseline levels after 1 h. Tissue-specific changes of ubiquitin pools were also apparent in the other tissues examined. Elevations in plasma cortisol and plasma lactate coincided with modulations of tissue ubiquitin pools. Significant deviations in plasma electrolyte levels (Na+, Cl-) also occurred over the period of confinement. Confinement of P.borchgrevinki was associated with less marked alterations in primary and secondary stress parameters. Plasma cortisol levels were elevated after handling and transfer to the aquarium, but had returned to baseline levels after 12 h. Furthermore, plasma lactate levels and plasma electrolyte concentrations (Na+, K+, Cl-) were not significantly changed. The elevation of ubiquitin conjugates in erythrocytes was comparable to that seen in blue mao mao. In contrast, levels of ubiquitin conjugates fell in other tissues analysed. These reductions were significant versus control in the brain and gills. The conjugated fraction of total ubiquitin in the fish studied, as in comparable published data from other animal species, showed an inverse correlation with body temperature. With respect to free ubiquitin, higher levels were present in species with a wider range of temperature tolerance. Thus, the temperate reef fish blue mao mao had up to 10 times the amount of free ubiquitin compared with either the stenothermal P.borchgrevinki, or rats and rabbits. Activation of the ubiquitin system by confinement and heat shock was further demonstrated by investigation of polyubiquitin gene expression in the muscle of P.borchgrevinki. Elevated levels of ubiquitin mRNA transcript indicated that gene expression was increased after exposure to both stressors. Comparison of ubiquitin pools with levels of 3-methylhistidine in muscle from the epaxial myotome suggested that stress-induced protein degradation in P.borchgrevinki was mediated by the ubiquitin proteolytic pathway. Heat shock in P.borchgrevinki not only activated the polyubiquitin gene, but also induced high levels of a 72 kD heat shock protein. This is indicative of a heat shock response similar to that documented in other organisms. However, both confinement and nonthermal stress also elicited a similar response, confirming the role of ubquitin as a general stress protein.