Incorporation of the human erythrocyte sialoglycoprotein into recombined membranes containing cholesterol
Glycophorin, the major sialoglycoprotein from the human erythrocyte membrane, has been isolated and recombined with phosphatidylcholine and cholesterol. Sucrose density gradient analysis of the recombinants shows that it is possible not only to recombine this protein with phospholipid, but also with phospholipid-cholesterol mixtures. Surprisingly, by the same analysis, it was possible to make a recombinant with cholesterol and glycophorin, only, in the absence of added phospholipid. The accessibility of the protein to trypsin was tested in each of these recombinants. In all the recombinants which contained either phospholipid, or phospholipid and cholesterol, the protein was protected from extensive hydrolysis. This is consistent with closed vesicles and incorporation of the protein into the recombinant membrane. Extensive hydrolysis of the protein occurred in the cholesterol-glycophorin recombinant indicating some differences in structure. Freeze-fracture electron microscopy of the phospholipid and the phospholipid-cholesterol recombinants showed mostly unilamellar vesicles, 1000 to 5000 Å in diameter. Intramembranous particles were observed on both fracture faces, and the fracture planes were those expected for phospholipid bilayers. The glycophorin-cholesterol recombinants also showed fracture planes consistent with bilayers, and revealed intramembranous particles. Pieces of membrane-like structures as well as apparent vesicular structures were observed. Finally in the recombinants of glycophorin with phospholipid and cholesterol, cholesterol is shown to reduce the population of the motionally restricted phospholipid headgroup environment, in proportion to the mole percent cholesterol content.
Key Wordscholesterol glycophorin membrane protein erythrocyte membrane lipid-protein interactions membrane structure
nuclear magnetic resonance
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