Isolation of human neutrophil plasma membranes employing neutrophil cytoplasts and changes in the cell-surface proteins upon cell activation.
A plasma membrane fraction, highly enriched in 5'-nucleotidase activity, was prepared from human neutrophils by disruption of previously formed neutrophil cytoplasts (enucleated neutrophils), which were devoid of intracellular organelles. This plasma membrane fraction shows an extremely low contamination by specific and azurophilic granule markers as compared to previous reported preparations. Nevertheless, a novel tertiary granule (Mollinedo, F. and Schneider, D.L. (1984) J. Biol. Chem. 259, 7143-7150), unlike specific and azurophilic granules, fuses partially with the cell surface under the experimental conditions used for cytoplast preparation. Comparison between the external cell-surface proteins in resting neutrophils and neutrophil cytoplasts by lactoperoxidase-catalyzed iodination showed some differences both in deletion and in addition of proteins. In resting cells, iodine was incorporated into at least 13 proteins ranging in size from over 200 to 30 kDa. A 140 kDa polypeptide, representing the major labeled surface component in resting neutrophils, was absent from cytoplasts. Furthermore, high-molecular-weight proteins (110 and over 160 kDa were more exposed to iodination after cytoplast preparation. Activation of human neutrophils by N-formylmethionylleucylphenylalanine induced some alterations in the pattern of labeled cell-surface proteins, which correlated to a certain degree with those observed during cytoplast preparation.
D015720:5'-Nucleotidase; D000954:Antigens, Surface / Q000032:analysis*; D002458:Cell Fractionation; D002462:Cell Membrane / Q000648:ultrastructure*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007455:Iodine / Q000378:metabolism; D007784:Lactoperoxidase / Q000378:metabolism; D008213:Lymphocyte Activation; D008565:Membrane Proteins / Q000032:analysis; D008970:Molecular Weight; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000166:cytology*; D009708:Nucleotidases / Q000097:blood
D000954:Antigens, Surface; D008565:Membrane Proteins; D009240:N-Formylmethionine Leucyl-Phenylalanine; D007455:Iodine; D007784:Lactoperoxidase; D009708:Nucleotidases; D015720:5'-Nucleotidase