Identification of type-2 phosphatidic acid phosphohydrolase (PAPH-2) in neutrophil plasma membranes.
Plasma membrane phosphatidic acid phosphohydrolase (PAPH) plays an important role in signal transduction by converting phosphatidic acid to diacylglycerol. PAPH-2, a Mg(2+)-independent, detergent-dependent enzyme involved in cellular signal transduction, is reportedly absent from the plasma membranes of neutrophilic leukocytes, a cell that responds to metabolic stimulation with abundant phospholipase D-dependent diacylglycerol generation. The present study was designed to resolve this discrepancy, focusing on the influence of cellular disruption techniques, detergent availability and cation sensitivity on the apparent distribution of PAPH in neutrophil subcellular fractions. The results clearly indicate the presence of two distinct types of PAPH within the particulate and cytosolic fractions of disrupted cells. Unlike the cytosolic enzyme, the particulate enzymes was not potentiated by magnesium and was strongly detergent-dependent. The soluble and particulate enzymes displayed dissimilar pH profiles. Separation of neutrophil particulate material into fractions rich in plasma membranes, specific granules and azurophilic granules by high speed discontinuous density gradient centrifugation revealed that the majority of the particulate activity was confined to plasma membranes. This activity was not inhibited by pretreatment with n-ethyl-maleimide in concentrations as high as 25 mM. PAPH activity recovered in the cytosolic fraction of disrupted neutrophils was almost completely inhibited by 5.0 mM n-ethylmaleimide. We conclude that resting neutrophils possess n-ethylmaleimide-resistant PAPH (type 2) within their plasma membranes. This enzyme may markedly influence the kinetics of cell activation by metabolizing second messengers generated as a result of activation of plasma membrane phospholipase D.
Boder
E
E
Bone Marrow Transplant Laboratory, Methodist Hospital of Indiana, Indianapolis, USA.
Taylor
G
G
Akard
L
L
Jansen
J
J
English
D
D
D002462:Cell Membrane / Q000201:enzymology*; D005033:Ethylmaleimide / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D009504:Neutrophils / Q000201:enzymology*; D010711:Phosphatidate Phosphatase / Q000097:blood* / Q000187:drug effects / Q000502:physiology; D015398:Signal Transduction
D010711:Phosphatidate Phosphatase; D008274:Magnesium; D005033:Ethylmaleimide