Intracellular localization of N-formyl chemotactic receptor and Mg2+ dependent ATPase in human granulocytes.
Human granulocytes were disrupted by nitrogen cavitation and the lysates fractionated by sucrose density gradient centrifugation at 83000 X g for 20 min (rate zonal) or 3.5 h (isopycnic). The distribution of marker enzymes allowed the identification of the following subcellular components: plasma membrane, Golgi, endoplasmic reticulum, azurophil granules, specific granules, mitochondria and cytosol. Examination of the gradient fractions by electron microscopy confirmed the biochemical marker analysis. The protocol permitted isolation of vesicles highly enriched in either plasma membrane or Golgi (galactosyl transferase) activities. Absolute plasma membrane yields of 40-60% were achieved with a 20-70-fold increase in specific activity of surface marker over the cells. Plasma membrane sedimented to an average density of 1.14 g X cm-3. Galactosyl transferase activity was bimodal in distribution. The denser peak cosedimented with specific granules (p = 1.19). The lighter peak sedimented to unique position at an average density of 1.11, was enriched 18-fold over the low speed supernatant, and contained structures resembling Golgi. N-Formyl-Met-Leu-Phe binding and Mg2+-ATPase activities cosedimented with the plasma membrane as well as specific granule and/or high density galactosyl transferase fractions. These findings suggest that Mg2+-ATPase and N-formyl chemotactic peptide receptor activities may be localized in an internal pool of membranes as well as in the plasma membrane and that Golgi may have been a contaminant of previous granulocyte plasma membrane or specific granule preparations.
D000251:Adenosine Triphosphatases / Q000097:blood*; D017301:Ca(2+) Mg(2+)-ATPase; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D002630:Chemotactic Factors / Q000378:metabolism; D006098:Granulocytes / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D009239:N-Formylmethionine / Q000031:analogs & derivatives / Q000378:metabolism; D009240:N-Formylmethionine Leucyl-Phenylalanine; D009842:Oligopeptides / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D044042:Receptors, Formyl Peptide
D002630:Chemotactic Factors; D009842:Oligopeptides; D011956:Receptors, Cell Surface; D044042:Receptors, Formyl Peptide; D009239:N-Formylmethionine; D009240:N-Formylmethionine Leucyl-Phenylalanine; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase