The effect of temperature on protein metabolism and antioxidant activity in the spotted wolffish, Anarhichas minor

Lamarre, Simon G. (2009) The effect of temperature on protein metabolism and antioxidant activity in the spotted wolffish, Anarhichas minor. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Temperature has profound effects on the rate of physiological processes in ectothermic vertebrates. Protein metabolism is no exception and the effects of temperature have mostly been studied with respect to protein synthesis. Temperature generally has a parabolic effect on protein synthesis with a maximum rate being observed at optimal growth temperature. The effect of temperature on protein degradation is poorly understood. The 20S proteasome is mainly responsible for the degradation of short-lived and oxidatively modified proteins. It has been recently identified as a potentially good proxy for protein degradation in fish. -- In the first experiment, the relationships between the rate of protein synthesis, 20S proteasome activity, oxidative stress markers and antioxidant capacity in white muscle of juvenile spotted wolffish (Anarhichas minor) acclimated at three temperatures (4, 8 and 12 °C) were examined. The rate of protein synthesis was lower at 4 °C than at 8 °C while it was intermediate at 12 °C. Despite the decrease in protein synthesis at low temperature, 20S proteasome activity was maintained at a high level, reaching 130% of that of fish acclimated at 8 °C when measured at a common temperature. The oxidative stress markers TBARS and carbonyl-protein content did not change amongst temperature groups, but the concentration of reduced glutathione was higher in cold acclimated fish suggesting a higher antioxidant capacity in this group. The data suggest that lower growth rate at cold temperatures results from both high 20S proteasome activity and a reduced rate of protein synthesis. -- In a second experiment, the relationship between specific growth rate (SGR) and 20S proteasome activity in heart ventricle, liver and white muscle was assessed in fish acclimated at 4 and 12 °C in order to determine if protein degradation via the proteasome pathway could impose a limitation on somatic growth in fish of weight ranging from 150 to 1500 g in mass. The data show that white muscle 20S proteasome activity is negatively correlated to SGR (partial Pearson's r = -0.609) in white muscle at the cold acclimation temperature (4 °C) but not at 12 °C or heart and liver at either temperature. Contrary to the first experiment, the white muscle 20S proteasome activity was not higher in the group acclimated at 4 °C. This observation suggests that the effect of temperature on protein degradation may change during fish ontogeny. Nevertheless, the results from the first two experiments suggest that interindividual variation of 20S proteasome activity has an impact on SGR. -- The third part of this study documents the effects of acclimation to high and low temperature (4 and 12 °C) on mitochondrial and antioxidant capacities in white muscle, heart ventricle and liver of spotted wolffish. Following an acclimation period of 51 days, mitochondrial capacity was measured as the activities of the Complex I of the mitochondrial electron transport system (CPLXI) and citrate synthase (CS). Glutathione disulfide reductase (GR) and catalase (CAT) activities as well as glutathione concentration were also measured to estimate antioxidant capacities. Following acclimation to 4 °C, mitochondrial capacities were compensated in liver and heart ventricle but not in white muscle. GR activity was increased at cold temperature in the three tissues while CAT activity was increased at the higher acclimation temperature in heart and white muscle. The relationships between mitochondrial and antioxidant enzyme activities, when observed, were always positive. In white muscle only, the activity of 20S proteasome was positively related to the complex I (r² = 0.450) and to CS (r² = 0.411) activities. Also, only in white muscle, positive relationships were observed between 20S proteasome, CAT and GSH (only at 12 °C for the latter) activities. These results suggest a connection between mitochondrial capacity and protein degradation by the 20S proteasome but whether or not this link is mediated by the necessity to degrade protein oxidatively modified by mitochondrial ROS production remains an open question. -- A supplementary section presents a simple method devised to measure the fractional rate of protein synthesis in fish using the stable isotope labelled tracer (ring-D₅L-phenylalanine) instead of radioactive phenylalanine. The method takes advantage of the increasingly available technology of liquid chromatography with tandem mass spectrometry detection (LC-MSMS). The technique was validated by measuring the fractional rate of protein synthesis in the gills of goldfish (Carrassius auratus). The modified technique requires fewer steps compared to previously available procedures and allows studies on fish protein metabolism to be carried out in situations where the use of radioactivity is not possible, such as in free living animals.

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