Functional reassembly of lymphocyte lentil lectin receptor glycoproteins into lipid bilayer vesicles.
Plasma membrane vesicles purified from pig mesenteric lymph nodes were solubilized using the mild, readily-dialyzable detergent dodecyltrimethylammonium bromide, and lentil lectin receptor glycoproteins were isolated by affinity chromatography. The receptor fraction showed 12 major bands on SDS-polyacrylamide gel electrophoresis representing 8-9% of the membrane protein. 5'-Nucleotidase (EC 3.1.3.5) was very effectively solubilized by the detergent and was recovered in high yield in the receptor fraction. Receptor glycoproteins were reassembled into large unilamellar vesicles of phosphatidylcholine/phosphatidylserine (mean diameter 0.235 microm) using a detergent dialysis technique. Sixty to seventy percent of the glycoprotein and most of the 5'-nucleotidase activity is associated with the phospholipid vesicles. 5'-Nucleotidase is reassembled in a symmetrical fashion and is inhibited by binding of concanavalin A, lentil lectin and pea lectin but not by succinyl-concanavalin A. Measured values for Ki and maximal inhibition are similar to those observed with intact plasma membrane vesicles. Hemagglutination inhibition studies showed that the reassembled receptors effectively bind lentil lectin. Thus lymphocyte membrane glycoproteins reassembled into phospholipid vesicles seem to retain at least part of their function in that enzyme activities such as 5'-nucleotidase remain intact and the receptors effectively bind lentil lectin.
D000818:Animals; D003208:Concanavalin A / Q000031:analogs & derivatives / Q000494:pharmacology; D006023:Glycoproteins / Q000378:metabolism*; D006385:Hemagglutination Inhibition Tests; D008051:Lipid Bilayers / Q000378:metabolism*; D008214:Lymphocytes / Q000378:metabolism*; D008854:Microscopy, Electron; D000644:Quaternary Ammonium Compounds; D011975:Receptors, Mitogen / Q000302:isolation & purification / Q000378:metabolism*; D013552:Swine
D006023:Glycoproteins; D008051:Lipid Bilayers; D000644:Quaternary Ammonium Compounds; D011975:Receptors, Mitogen; D003208:Concanavalin A; C019558:succinylconcanavalin A; C022806:dodecyltrimethylammonium