Magnesium-dependent adenosine triphosphatase as a marker enzyme for the plasma membrane of human polymorphonuclear leukocytes.
The adenosine triphosphatase (ATPase) activities of human polymorphonuclear leukocytes (PMNL) were studied with an assay that monitored the release of 32P-labeled inorganic pyrophosphate (32P1) from gamma-[32P]adenosine 5'-triphosphate (ATP). In cell homogenates, (Na+ + K+)-sensitive, ouabain-inhibitable ATPase comprised an insignificant fraction of the total ATPase activity. Additions of p-nitrophenyl phosphate and beta-glycerophosphate (substrates for nonspecific acid and alkaline phosphatases) and of tartrate (inhibitor of acid phosphatase) gave no indication of inhibition. This suggested that the assay was relatively specific for ATP hydrolysis. The activity was found to have a pH optimum of 8.7 and a Km for ATP of 0.6 mM. There was an absolute requirement for Mg2+, with other divalent cations substituting less efficiently. When the Mg2+-dependent ATPase activity of intact cells was compared with that in homogenized cells, no significant difference was observed. The activity in intact cells was linear with respect to incubation time up to at least l0 min. Trypan blue staining and lactate dehydrogenase assays revealed that greater than 92% of the PMNL remained intact and viable during the assay. No soluble ATPase was released from the cells under assay conditions. In following the distribution of gamma[32P]ATP and 32P2 counts became cell associated. Since the experimental evidence supports the observation that PMNL remain intact and viable and that ATP does not penetrate the cell under assay conditions, it is proposed that greater than 90% of the Mg2+-dependent ATPase of the human PMNL is associated with a plasma membrnae enzyme. This would qualify the enzyme for the role of a plasma membrane marker for future fractionation and isolation attempts.
D000251:Adenosine Triphosphatases / Q000032:analysis* / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D005994:Glycerophosphates / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009504:Neutrophils / Q000201:enzymology* / Q000648:ultrastructure; D010710:Phosphates / Q000494:pharmacology; D013644:Tartrates / Q000494:pharmacology
D005994:Glycerophosphates; D010710:Phosphates; D013644:Tartrates; D000251:Adenosine Triphosphatases; D008274:Magnesium; D002118:Calcium