Phospholipase D activity in phagocytic leucocytes is synergistically regulated by G-protein- and tyrosine kinase-based mechanisms.

摘要: The regulation of phospholipase D (PLD)-type effector enzymes by G-proteins and protein kinases/phosphatases was characterized in the U937 human promonocytic leucocyte line. PLD activity assayed measuring (in presence 1% ethanol) accumulation phosphatidylethanol cells permeabilized with beta-escin, a saponin-like detergent. Basal very low when were incubated cytosol-like medium containing micromolar [Ca2+]. When this supplemented exogenous MgATP or guanosine 5'-[gamma-thio]triphosphate (GTP[S]), increased 9- 14-fold respectively. Cells absence exogenously added MgATP, but 1 microM vanadate/100 H2O2, also exhibited modest 12-fold increase activity. However, simultaneous either GTP[S] plus vanadate/H2O2 (and endogenous MgATP) induced similar 60-75-fold increases rate extent accumulation. These latter effects strongly correlated rapid multiple tyrosine-phosphorylated proteins. Other studies utilized which then washed before assay PLD. retained approximately 60% MgATP-regulatable (EC50 = to 100 observed freshly non-washed cells. In pre-treatment, minimal Genistein, tyrosine kinase inhibitor, significantly attenuated ability stimulate proteins GTP[S]-treated data suggest that myeloid leucocytes involves co-ordinate both G-protein(s) phosphorylation.