The effect of native bull sperm lipids on liposome and proteoliposome structure and function

Na+ K+ - ATPase is an integral membrane protein active in many cell types. In sperm cells, Na+ K + - ATPase is important for capacitation and fertility. This study uses liposomes created with lipids from bovine sperm heads as model membrane systems to evaluate factors affecting ATPase function. The objective is to develop methods for the study of Na+ K+ - ATPase so that a lipid environment that optimizes its function during cryopreservation can be determined.;CHAPS fast and filtration methods of liposome production gave the most stable size distribution and showed the most likelihood of being consistently unilamellar, so these two methods were selected for evaluation of ATPase activity. Fluorescence resonance energy transfer confirmed that the enzyme was incorporated into the proteoliposome bilayer. Filtration incorporated more functional Na + K+ - ATPase into proteoliposomes (measuring function as release of phosphate from exogenous ATP) and so was selected as the method of choice. Na+ K+ - ATPase in proteoliposomes made by filtration is primarily right-side out, although much is not incorporated. Altering the lipid composition significantly changed this orientation.;The measure of Na+ K+ - ATPase function, incorporation and orientation in a native lipid environment as well as the establishment of methodology and evaluation techniques developed in this study can now act as a base for ongoing research in sperm membrane function.;First, it was essential to prepare liposomes in a simple, repeatable fashion that would support enzyme activity. Liposomes were made with two detergents (CHAPS and C12E8), two detergent removal methods (fast and slow) and one non-detergent method (high pressure filtration). All five methods were compared by dynamic light scattering and scanning electron microscopy for liposome size, shape, and consistency. Na+ K+ - ATPase was then inserted into liposomes and evaluated for function, incorporation, and orientation.