A rapid isolation procedure of plasma membranes from human neutrophils using self-generating Percoll gradients. Importance of pH in avoiding contamination by intracellular membranes.
In this study we report an overall procedure for the isolation of both human polymorphonuclear neutrophils and their plasma membrane, by means of self-generating Percoll gradients. After efficient purification (40% yield), neutrophils were lysed by nitrogen cavitation and cellular structures quickly isolated in a one-step procedure. Plasma membrane recovery was monitored by [3H]concanavalin A and 5'-nucleotidase (EC 3.1.3.5) activity. We showed the latter activity is indeed present in human neutrophils. The procedure resulted in a good yield of plasma membrane, since 45% and 55% of total 5'-nucleotidase and [3H]concanavalin A activity, respectively, were recovered within two gradient fractions. Depending on the final pH of the Percoll gradient medium, endoplasmic reticulum markers contaminated either the plasma membrane or the granule fractions. At pH 9.05, NADH-ferricyanide reductase activity clearly separated from plasma membrane markers and displayed the same profile as CDPcholine:diacylglycerolcholine phosphotransferase (EC 2.7.8.2), a typical enzyme of endoplasmic reticulum. These results emphasize the need for strict monitoring of the pH of the gradient medium in subcellular fractionation of neutrophils.
D015720:5'-Nucleotidase; D002458:Cell Fractionation / Q000379:methods*; D002462:Cell Membrane / Q000201:enzymology; D002469:Cell Separation; D002499:Centrifugation, Density Gradient / Q000379:methods; D005966:Glucuronidase / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes; D008854:Microscopy, Electron; D009113:Muramidase / Q000378:metabolism; D009245:NADH Dehydrogenase / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D009504:Neutrophils / Q000648:ultrastructure*; D009708:Nucleotidases / Q000378:metabolism; D011205:Povidone; D012822:Silicon Dioxide
C016039:Percoll; D012822:Silicon Dioxide; D009247:NADH, NADPH Oxidoreductases; C014227:ferricyanide reductase; D009245:NADH Dehydrogenase; D009708:Nucleotidases; D015720:5'-Nucleotidase; D009113:Muramidase; D005966:Glucuronidase; D011205:Povidone