Modification of yeast plasma membrane density by concanavalin A attachment. Application to study of chitin synthetase distribution.
Yeast protoplasts were coated with different amounts of concanavalin A. Upon subsequent lysis and centrifugation in isopycnic density gradients, it was found that the buoyant density of plasma membranes was progressively increased from 1.125 to 1.21, according to the amount of bound concanavalin A. Enzymes that are attached to the plasma membrane showed the same density modifications and could thus be distinguished from constituents of intracellular membranes and organelles. With this methodology, it was confirmed that about two-thirds of yeast chitin synthetase is associated with the plasma membrane. The remainder of the enzyme was found in a peak at a lower density. Vanadate-sensitive ATPase showed a similar pattern, whereas GDP-mannose dolichyl-phosphate mannosyltransferase, an enzyme attached to the endoplasmic reticulum, remained in the same position in the gradients, irrespective of the amount of concanavalin A associated with the plasma membrane. Potential applications of this technique to the determination of plasma membrane markers and to the separation of subcellular organelles are discussed.
M S
MS
Kang
J
J
Au-Young
E
E
Cabib
D000251:Adenosine Triphosphatases / Q000378:metabolism; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology* / Q000378:metabolism / Q000648:ultrastructure; D002500:Centrifugation, Isopycnic; D002687:Chitin Synthase / Q000378:metabolism*; D003208:Concanavalin A / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005964:Glucosyltransferases / Q000378:metabolism*; D006897:Hydroxyindoleacetic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D008364:Mannosyltransferases / Q000378:metabolism; D011523:Protoplasts / Q000201:enzymology / Q000378:metabolism / Q000648:ultrastructure; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D003208:Concanavalin A; D006897:Hydroxyindoleacetic Acid; C046777:7-hydroxy-2-oxoindole-3-acetic acid 7'-O-glucopyranoside; D005964:Glucosyltransferases; D008364:Mannosyltransferases; C035777:chitobiosyldiphosphodolichol beta-mannosyltransferase; D002687:Chitin Synthase; D000251:Adenosine Triphosphatases