Doctor of Philosophy
Department of Biomedical Science
Wu, Ben Jing, Role of membranes in determining the molecular activity of the sodium pump, Doctor of Philosophy thesis, Department of Biomedical Science, University of Wollongong, 2000. http://ro.uow.edu.au/theses/1094
Metabolic rates of animals vary considerably. Major variations in standard or basal metabolic rate occur between vertebrate groups, as a result of changes in body size and during development. Recently, studies of membrane linked energy consuming processes have shown that membrane lipid composition may be correlated with the activity of these processes and therefore metabolism. Generally, high metabolic rate being associated with increased polyunsaturation of membranes. This implies that membranes may play a role in determining the rate or pace of metabolism.
In this thesis, the role of the membrane in determining the molecular activity of sodium pump (Na++K+-ATPase) will be assessed. The sodium pump is a membrane linked enzyme involved in energy turnover accounting for around 25 % of total resting energy consumption of organisms. The energy consumption of the sodium pump also appears to be involved in determining changes to metabolism that have arisen during the evolution of endothermy in mammals and occur during mammalian development.
The aims of this study are firstly to investigate the molecular activities of sodium pumps in endotherms and ectotherms, as well as to see if any similar changes occur for the sodium pump during mammalian development. The study will involve determining if the molecular activity differences persist in microsomal fraction preparations versus those previously found in tissue biopsies/homogenates; and secondly to determine the effect of the general membrane environment in determining these differences. These experiments will be performed using a simple membrane reconstitution method specifically developed within this study to allow for natural membrane cross-over experiments. The cross-over experiments involve those between endotherms and ectotherms and between young and adult mammals. A third aim of this study is to determine if any common factors are associated with sodium pump molecular activity, and membrane composition by a complete analysis of membrane composition. The final aim is to determine if simple physical properties of extracted lipids from the natural membranes, compared to those of purified lipids, are involved or correlated with molecular activity.
In comparisons of rat versus toad kidney and brain, of cow versus crocodile kidney, and of adult rat versus neonate rat brain, grossly different sodium pump molecular activities were shown to be present. These differences in sodium pump molecular activities matched the level of metabolism expressed by each animal. Animals with high metabolism had sodium pumps with high molecular activity, and animals with low metabolism had sodium pumps with low molecular activity.
It was further shown that these molecular activities could be altered. Shifts in molecular activity could be achieved by 'corrupting' the influence of the native membrane with the presence of a second membrane introduced under the influence of detergent in what was termed membrane cross-over experiments. The results of all three comparative membrane cross-over studies (rat versus toad kidney and brain, adult versus neonate rat brain and cow versus crocodile kidney) support the notion of membrane involvement in determining the molecular activity of sodium pumps. If the reconstituting membrane was formerly associated with sodium pumps possessing high molecular activity then when used as the reconstituting membrane, detergent-treated sodium pumps either regained their prior high activity or exhibited significantly higher activity if their molecular activity was formerly low. If the reconstituting membrane was formerly associated with sodium pumps possessing low molecular activity, then the reconstituted sodium pumps either returned to their prior low activity or exhibited significantly lower activity if their previous molecular activity was high.
Analysis of membrane composition shows that higher molecular activities were associated with lower cholesterohphospholipid (Chol:PL) and phosphatidylethanolamine: phosphatidylcholine (PE:PC) ratio's: Higher molecular activities were also associated with higher unsaturation index and therefore higher levels of long chain polyunsaturated fatty acids and lower n-6:n-3 fatty acid ratios. These change have all featured in previous work aimed at determining the optimal lipid requirements in relation to activity of membrane bound proteins.
Finally, a study of lipid monolayer behaviour offers a simple means of examining molecular packing and comparing differences between the phospholipids naturally present in the membranes of endotherms with those of ectotherms or between high and low molecular activity models. The results show that the phospholipid monolayer films extracted from rat tissues were more expanded than those from toad phospholipid monolayer films in both extracted total lipid (including total cholesterol content) and phospholipid. This different molecular packing is due to differences between the natural membrane phospholipid compositions. Evaluation of the relationship between extracted membrane lipid (including total cholesterol) and phospholipid molecular packing and sodium pump molecular activity showed higher sodium pump molecular activity associated with expanded phospholipid molecular packing, this latter variable may account for some 70-80% of the variance in the former. This highly significant positive relation between membrane phospholipid molecular packing and sodium pump activity would appear to indicate that sodium pump activity is strongly related to the overall physical state of the membrane, which is itself the result of the complex mix of membrane phospholipid amount, phospholipid fatty acid composition, cholesterol, etc. Therefore membrane physical state may partially be one reason why membranes with sodium pumps with higher molecular activities have relatively higher levels of polyunsaturated and lower levels of cholesterol.