Isolation of plasma membrane from human neutrophils and determination of cytochrome b and quinone content.
Analyses of plasma membrane and other subcellular fractions indicate that the primary location of cytochrome b in human neutrophils is not the plasma membrane. The procedure developed for the purification of plasma membrane from fresh human neutrophils yielded a 14-fold enrichment in the marker enzyme 5'-nucleotidase and a 10-fold enrichment in ouabain-sensitive ATPase. On sucrose density gradients, the peak density of 5'-nucleotidase activity was 1.12 g/ml, and was shifted after digitonin addition to 1.15 g/ml. Protein in the plasma membrane equalled approximately 8 percent of the whole cell protein. A b-type cytochrome was found to be present in the plasma membrane fraction at a concentration of 205 pmol/mg of protein, which is three times greater than that in the neutrophil overall. Although this cytochrome has been reported previously in the neutrophil, this is the first determination for purified plasma membrane and may indicate that b-type cytochrome has a dual localization in the human neutrophil. Differential centrifugation results suggest that the primary location is in the granules, probably specific granules. Quinone content in the plasma membrane was found to be 740 pmol/mg of protein, a concentration two times greater than in the whole cell. Such a small enhancement of quinone indicates that quinone also is not primarily located in the plasma membrane.
D002462:Cell Membrane / Q000032:analysis; D003573:Cytochrome b Group; D003580:Cytochromes / Q000032:analysis*; D006596:Hexosaminidases / Q000032:analysis; D006801:Humans; D009504:Neutrophils / Q000032:analysis* / Q000276:immunology; D009708:Nucleotidases / Q000032:analysis; D011809:Quinones / Q000032:analysis*; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis
D003573:Cytochrome b Group; D003580:Cytochromes; D011809:Quinones; D009708:Nucleotidases; C018775:adenosine monophosphatase; D006596:Hexosaminidases; D000254:Sodium-Potassium-Exchanging ATPase