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THE USE OF HYBRIDIZATION BY COSONICATION FOR INVESTIGATING PROTEIN-PROTEIN INTERACTIONS IN PIGEON ERYTHROCYTE PLASMA MEMBRANE
Abstract
Hybrid vesicles formed from exogenous neutral lipids and membrane, which are so small and dilute that they contain either one or no protein molecules or molecular assemblages, should be separable on the basis of the proteins or molecular assemblages they contain. Vesicles were formed by sonicating a mixture of neutral lipids (phosphatidylethanolamine, phosphatidylcholine and cholesterol) and membrane. These vesicles retained functions of the original membrane including vesicle sealing, anion transport and ATP dependent calcium transport. These vesicles had properties consistent with the formation of hybrids. Very dilute hybrids were then made with a membrane:exogenous lipid ratio such that the calculated ratios of vesicles with no proteins to those with one protein or molecular assemblage was 6.7. Freeze fracture electron microscopy showed 200-400 Angstrom vesicles with at most one intramembrane particle per vesicle. Density gradient centrifugation and density gradient stabilized isoelectric focusing were used to fractionate hybrids. These techniques produced distinct band patterns in the gradients. Examination of individual bands by isoelectric focusing, SDS gel electrophoresis or 2-D gel electrophoresis showed very few protein differences between these fractions. Despite numerous changes in the fractionation procedures it was not possible to show that individual fractions contained a minority of the total components of the membrane, the result necessary for a useable procedure. To do the experiments outlined above, procedures were developed for labeling membrane proteins by low-level reductive methylation. Membrane proteins were labeled with 83 nmoles formaldehyde and 28 nmoles tritiated sodium borohydride/mg protein in borate or phosphate buffer, pH 8.3. This gave a labeling level of 28 uCi/mg protein. The isoelectric focusing patterns of labeled and unlabeled membrane proteins were indistinguishable. This, plus the correspondence between the radioautograph and stain patterns obtained from two dimensional electrophoretograms indicates that the labeled proteins are good tracers for unlabeled ones. Membrane labeled in phosphate buffer (but not borate buffer) retained full capacities for vesicle sealing, ATP dependent calcium transport and sodium dependent glycine transport. A procedure was also developed for isoelectric focusing at 0.154 u ionic strength.
Subject Area
Biochemistry
Recommended Citation
JONES, STEVEN WAYNE, "THE USE OF HYBRIDIZATION BY COSONICATION FOR INVESTIGATING PROTEIN-PROTEIN INTERACTIONS IN PIGEON ERYTHROCYTE PLASMA MEMBRANE" (1983). ETD collection for University of Nebraska-Lincoln. AAI8318660.
https://digitalcommons.unl.edu/dissertations/AAI8318660