Reconstitution of brush border membrane proteins in phosphatidylcholine vesicles. Biochemical and functional characterization.
Horse kidney brush border membrane proteins were incorporated into phosphatidylcholine vesicles. Structural analysis of proteoliposomes prepared with various lipid:protein ratios showed that: (a) only a few of the proteins present in the crude brush border extract are integrated, (b) all known membrane hydrolases are integrated, and (c) these proteoliposomes are homogeneous vesicles. Papain solubilization of brush border membrane hydrolases, i.e. aminopeptidase M, neutral alpha-glucosidase, gamma-glutamyltransferase and alkaline phosphatase, performed in parallel on native membrane vesicles and proteoliposomes, revealed similar kinetics. Analysis of membrane vesicles and proteoliposomes on sucrose density gradients either without any treatment, or after papain treatment showed that: (a) in proteoliposomes, neutral alpha-glucosidase is associated with radiolabelled phosphatidylcholine, and (b) papain-treated vesicles and proteoliposomes released enzyme activity in the same way. These results suggest that the integration mechanism of brush border membrane proteins may be similar in proteoliposomes and native membrane vesicles. Transport experiments under equilibrium exchange conditions showed that the uptake properties of proteoliposomes are similar to those of brush border membrane vesicles.
D000409:Alanine / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D006736:Horses; D007672:Kidney Cortex / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008871:Microvilli / Q000378:metabolism; D010713:Phosphatidylcholines; D011510:Proteolipids / Q000378:metabolism
D008565:Membrane Proteins; D010713:Phosphatidylcholines; D011510:Proteolipids; C020485:proteoliposomes; D000409:Alanine