Digitized by Kansas Correctional Industries

Thesis (Ph. D.)--University of Washington, 1993. / Vita. Includes bibliographical references (leaves [115]-120).

Diss. Nr. 6049 Naturwiss. ETH Zürich. (KA).

The regulatory properties of fructose 1,6-diphosphatase (FDPase) from liver of rainbow trout (Salmo gairdneri), South American lungfish (Lepidosiren paradoxa) and migrating pink salmon (Oncorhynchus gorbuscha) were examined over the physiological temperature ranges of the organisms. In rainbow trout saturation curves for substrate (fructose 1,6-diphosphate), and a cofactor (Mg²⁺ ) are sigmoidal, and the evidence suggests cooperative interaction between the binding sites for these ligands. The affinity of the trout enzyme is approximately 50-to 100-fold higher for Mn²⁺ than for Mg²⁺ and the Mn²⁺ saturation curve is hyperbolic. The enzyme is inhibited by Ca²⁺ and Zn²⁺ and this inhibition appears to be competitive with respect to cofactor. The trout FDPase has an alkaline pH optimum and high pH values enhance FDPase affinity for cofactor. Low concentrations of 5'AMP inhibit the rainbow trout FDPase and the enzyme-AMP interaction is sensitive to temperature; thus, Ki for AMP at 25° is approximately 30-fold higher than at 0°. Furthermore, increasing concentrations of the cofactors tend to reduce and/or reverse the AMP inhibition and it is suggested that these ions may play a role in regulating the efficiency of AMP as an inhibitor of FDPase activity. Similar to the trout FDPase, substrate and cofactor saturation curves for the lungifsh FDPase are sigmoid and Hill plots of the data suggest homotropic interaction between their respective binding sites. Affinity of the lungfish enzyme for substrate increases markedly at low temperature, with the result that at physiological concentrations of substrate, the velocity of the reaction is essentially independent of temperature. In addition, increasing pH values stimulate enzyme activity and increase affinity of the lungfish FDPase for cofactor. The lungfish enzyme is inhibited by AMP, as is the trout FDPase, but affinity for the allosteric inhibitor is reduced and this FDPase-AMP interaction is independent of temperature. It is suggested that the decreased sensitivity to AMP is adaptive in view of the aestivating habit of the South American lungfish, under which condition the animal would be starved for long periods of time and a low energy charge (high concentrations of AMP) would probably exist in the liver cell. The regulatory properties of FDPase from migrating salmon were investigated. The calculated energy charge of the liver cell from these fish is very low (0.464) in keeping with the extended starvation and high rates of muscular and biosynthetic activity in these organisms. As in the trout and lungfish enzymes, affinity of salmon FDPase for substrate increases with a decrease in temperature. As a result at physiological concentrations of substrate enzyme activity is independent of temperature. Arrhenius plots of the saturation kinetics are complex and suggest an inter-conversion of one or more forms of the enzyme. Subsequent examination of the enzyme by sucrose density gradient centrifugation at different temperatures indicates that such thermally-dependent changes in conformation of the protein do occur, a finding which also offers a basis for the observed temperature-dependent changes in Km for substrate. The affinity of salmon FDPase for its allosteric inhibitor, AMP, is lower than in other FDPases and this enzyme-AMP interaction is largely insensitive to temperature. The functional significance of this reduced sensitivity to AMP is that it allows normal, indeed high, FDPase activity during conditions of low energy charge. These findings suggest mechanisms for the maintenance of high rates of gluconeogenesis in salmon during spawning migration. / Science, Faculty of / Zoology, Department of / Graduate

The thermogenic effects in hypothermia of four substrates--alanine, glycine, ethano, and pyruvate - were studied in seventeen experiments. Albino rats were decapitated, and their livers were removed. The livers were homogenized with phosphate buffer at -5° C. After equilibration in a refrigerated Warburg apparatus at 20° C, the substrates were added and tissue respiration was recorded over three hours. Heat production was calculated from O2 uptake and CO2 production. Results showed that alanine, glycine, and pyrvate yielded 93.19, 89.86, and 89.89 x 10^6 kg-cal compared to a control value of 86.11 x 10^-6 kg-cal. Ethanol provided 110.31 x 10^-6 kg-cal, a value significantly greater than for the other substrates. The substrates studied, especially ethanol, did, therefore increase heat production in an artificially hypothermic environment in homogenized rat livers.

Digitized by Kansas Correctional Industries

Digitized by Kansas Correctional Industries

Endothermy refers to the ability of an individual to produce heat from internal sources, and allows animals to maintain a body temperature that is higher than their external environment. Although much is known about the benefits of endothermy, its origin is highly debated. Nonetheless, due to environmental variation, endotherms have to regulate their heat production (thermogenesis) in order to remain normothermic. An endotherms regulatory response seems to be body size dependent. Keeping warm during cold periods is energetically expensive, and for small mammals this is exacerbated by their high rate of heat loss due to high surface area to volume ratios. To compensate for the heat lost, small non-hibernating mammals must increase their level of thermogenesis. Much of our current understanding of thermogenic responses of small mammals is derived from laboratory acclimated animals, and studies on naturally acclimatized animals are uncommon. In addition, most studies on thermogenesis tend to focus on one level of animal organisation, such as subcellular, tissue or in-vivo, but seldom integrate these data. The aim of this study was to measure year-round variation in thermogenesis across all levels of organisation, using naturally acclimatized Rhabdomys pumilio individuals from the Nelson Mandela Metropolitan University, Port Elizabeth. It was predicted that the level of thermogenesis would be significantly higher during winter relative to other seasons in order to cope with the low ambient temperatures (Tas) experienced during this season. Open flow respirometry was used to measure the animal's oxygen consumption, as a proxy for metabolism; the by product of which is heat production. The animal's basal metabolic rate (BMR), nonshivering thermogenesis (NST) capacity and summit metabolic rate (MSUM) were measured. A Western blot analysis was used to determine the expression of uncoupling protein 1 (UCP 1) in the animals' brown adipose tissue (BAT), as well as determine its relative concentration. The cytochrome c oxidase (COX) activity of the animals' visceral organs and BAT was measured, as an indicator of the tissues' metabolic activity. COX activity was determined as the difference in the tissues' oxygen consumption before and after the addition of horse cytochrome c.

No description available.

A group of red-eared turtles (Pseudemys scripta elegans) was acclimated to 18-20˚C and artificial photo-period for 21 days. The animals were then individually placed in a thermal gradient and their selected temperatures were continuously recorded for 24 hours. A second group of turtles was acclimatized to 7.5-11.0˚C and natural photoperiods for 28 days, and subsequently tested in the same manner as the first group. Lastly, the turtles in the first group, after being acclimated to 3.0˚C and the previous artificial photoperiod for 14 days, were retested in the gradient.