Assessment of enzyme inhibition or stimulation in plasma membrane preparations.
The purity of isolated plasma membranes is routinely judged by the activity of enzymes present both in this membrane and other locations in the cell. However, since enzyme inhibition and/or stimulation often occurs following disruption of the cell, the question as to which enzyme(s) provides a reliable marker of membrane purity should be considered. We have devised a simple method with which to address this problem. Inhibition or stimulation of plasma membrane marker enzymes can be rapidly assessed in cell homogenates and subfractions by mixing both samples, with known enzyme activity, and observing any deviation from the expected combined activity. Should the activity remain constant that enzyme can be used to gauge the purity of the plasma membrane preparation. Of the four putative plasma membrane marker enzymes examined only one, gamma-glutamyltranspeptidase appeared to give a reliable purity measurement in the cell system studied.
Mackinnon
W B
WB
Ludwig Institute for Cancer Research, University of Sydney, New South Wales, Australia.
Mountford
C E
CE
D015720:5'-Nucleotidase; D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D002458:Cell Fractionation; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure*; D009708:Nucleotidases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005723:gamma-Glutamyltransferase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D005723:gamma-Glutamyltransferase; D009708:Nucleotidases; D000469:Alkaline Phosphatase; D015720:5'-Nucleotidase; D000254:Sodium-Potassium-Exchanging ATPase