Preparation of right-side-out plasma membrane vesicles from Penicillium cyclopium: a critical assessment of markers.
A plasma membrane fraction was obtained by the combined use of differential centrifugation and aqueous polymer two-phase partitioning techniques. Vanadate-inhibited ATPase and glucan synthase activities were highly enriched in this fraction, although the presence of ATPase activity which was not inhibited by vanadate, nitrate, molybdate, anyimycin A or azide was also detected. Other intracellular membrane marker activities were present at very low or undetectable levels. A further separation step using Percoll density gradient centrifugation resulted in the separation of a fraction which exclusively contained vanadate-inhibited ATPase activity, and was enriched with silicotungstic-acid-staining membrane material. Latency tests performed on the plasma membrane markers showed that the membrane vesicles were in the right-side-out orientation.
Ugalde
U O
UO
Dept. of Applied Chemistry, University of the Basque Country, San Sebastian, Spain.
Hernandez
A
A
Galindo
I
I
Pitt
D
D
Barnes
J C
JC
Wakley
G
G
D015720:5'-Nucleotidase / Q000032:analysis; D000251:Adenosine Triphosphatases / Q000032:analysis / Q000187:drug effects; D015415:Biomarkers; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000737:chemistry* / Q000201:enzymology / Q000648:ultrastructure; D016764:Cell Polarity; D002500:Centrifugation, Isopycnic; D005964:Glucosyltransferases / Q000032:analysis; D009245:NADH Dehydrogenase / Q000032:analysis; D010407:Penicillium / Q000737:chemistry* / Q000201:enzymology; D012995:Solubility; D014638:Vanadates / Q000494:pharmacology
D015415:Biomarkers; D014638:Vanadates; D009245:NADH Dehydrogenase; D005964:Glucosyltransferases; C040232:glucan synthase; D015720:5'-Nucleotidase; D000251:Adenosine Triphosphatases