UBC Theses and Dissertations
Temperature and enzyme activity in poikilotherms : liver soluble NADP+-linked isocitrate dehydrogenase from trout Moon, Thomas William
The effect of temperature on the oxidative-decarboxylation of isocitrate by the soluble NADP+-linked isocitrate dehydrogenase (NADP-IDH, EC 22.214.171.124) from rainbow trout (Salmo gairdnerii) liver has been investigated. A particular interest was given those properties of the enzyme which might help to explain the temperature-independent function of the Krebs cycle and the large increase in fatty acid synthesis known to occur during low temperature acclimation. Within the thermal range experienced by rainbow trout, control of catalysis by this enzyme is temperature-independent. Acclimation to an altered thermal regime is accompanied by an increase in the relative proportion of the slowest migrating isozyme of liver NADP-IDH on starch-gel electrophoresis. These cold- and warm-isozyme variants display different and adaptive Km-temperature relationships, and allow for temperature-independent modulation of enzyme activity through the entire thermal range this species is likely to encounter in nature. Other trout species, including the brook (Salvelinus frontinalis), lake (Salmo namaycush) and their hybrid, the splake trout, were investigated for similar responses. The elaboration of enzyme variants in brook and splake trout are complexly regulated by temperature changes, but the lake trout genome contains a single gene coding for liver NADP-IDH which is not affected by temperature. Catalysis by the trout liver enzyme is modulated not only by temperature, but also ADP and ɣ-KGA. Both of these metabolites alter the Km of DL-isocitrate; at physiological ADP concentrations, the Km is reduced as it is with ɣ-KGA below 0.05 mM, but at higher ɣ-KGA concentrations it is markedly increased. These two controls suggest this enzyme may be important for the Krebs cycle oxidation of isocitrate. The availability of a purified NADP-IDH from pig heart allowed a study of the kinetic properties of homologous enzymes from both a poikilotherm and a homeotherm. Even though the molecular weights, Ea values, substrate, cofactor and inhibitor specificities are similar, subtle changes in enzyme structure and/or conformation as identified by electrophoresis, may result in the observed differences in temperature characteristics. These apparent adaptive enzyme responses are of importance to the rainbow trout which lives in a fluctuating thermal regime, but not the pig which does not experience these changes. In vivo, the response of enzymes to temperature fluctuations may be quite different to those seen in vitro. The locus(i) coding for rainbow trout liver NADP-IDH was found to contain a large amount of heterogeneity; in fact, seven distinct phenotypes were found to coexist in one hatchery population. The kinetics of three of these phenotypes were investigated and it was found that by increasing the number of slow moving isozymes, an increase in Km(DL-isocit) at high assay temperatures occurs. This suggests that irrespective of changes in the cellular milieu, isozymal content can determine the Km-temperature response. The data from this study suggest that changes in enzyme-substrate affinity with temperature as a result of either the temperature directed production of enzyme variants and/or their genetic expression, are important in controlling the catalytic activity of NADP-IDH from the eurythermal rainbow trout. Also, unlike the mammalian enzyme, the trout liver enzyme may be important in both fatty acid synthesis and the Krebs cycle oxidation of isocitrate.
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