Isolation and localization of plasma membrane-bound invertase in yeast (Saccharomyces cerevisiae).
Latex spheres of 60 nm diameter (synthesized by aqueous emulsion copolymerization of methacrylate derivatives according to [22]) were coated with bovine serum albumin (BSA) and concanavalin A. By virtue of their size and their high density (1.32-1.35 g/ml) they are well suited as scanning electron microscopy markers and as affinity density perturbation reagents. Yeast protoplasts could be labeled with these spheres and the amount of binding depended upon incubation time and temperature. Isolated and solubilized yeast plasma membranes were incubated with these spheres and by density gradient centrifugation the membrane glycoproteins could be separated from the other proteins by the method of affinity density perturbation. Since the yeast plasma membrane glycoproteins exhibit invertase activity [1, 19] the activity of the different fractions was either detected on gels by staining for invertase activity or measured in vitro and quantified; a 6 to 7fold purification of the enzyme was achieved. Protoplasts labeled with antibodies directed against these glycoproteins exhibited a distribution of ferritin marker molecules that was very similar to that of the intramembranous particles. Antibodies against extracellular invertase cross reacted with the plasma membrane of glycoproteins and showed the same distribution of markers as the antibodies against the glycoproteins. It can therefore be concluded that the yeast plasma membrane glycoproteins exhibit invertase activity and that they are associated with the intramembranous particles.
D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000201:enzymology; D006023:Glycoproteins / Q000302:isolation & purification; D007840:Latex; D008854:Microscopy, Electron; D008863:Microspheres; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000648:ultrastructure; D013393:Sucrase / Q000302:isolation & purification*
D006023:Glycoproteins; D007840:Latex; D013393:Sucrase