Abstract
The NADPH-oxidase of human neutrophils can be activated in a cell-free system comprised of plasma membrane, cytosol, and an anionic amphiphile such as arachidonate or sodium dodecyl sulfate (SDS). Recently, we showed that diacylglycerol acts synergistically with SDS in the cell-free system to stimulate superoxide generation, with concurrent phosphorylation of a 47-kDa cytosolic protein which is thought to be a component of the oxidase (Burnham, D. N., Uhlinger, D. J., and Lambeth, J. D. (1990) J. Biol. Chem. 265, 17550-17559). We report herein that when undialyzed cytosol is used along with either SDS alone or SDS plus diacylglycerol as activators, adenosine 5'-(gamma-thio)triphosphate (ATP gamma S) and guanosine 5'-(gamma-thio)triphosphate (GTP gamma S) both stimulated superoxide generation several fold, yielding about the same maximal velocity. ATP and GTP showed lower levels of stimulation. Stimulation by ATP gamma S and GTP gamma S was nonadditive, and showed a 5-7-fold greater specificity for GTP gamma S. ATP gamma S stimulation was inhibited by the nucleoside diphosphate (NDP) kinase inhibitor UDP. In contrast, when extensively dialyzed cytosol was used, most of the stimulation by ATP gamma S was lost, while most of that by GTP gamma S was retained. Addition of GDP restored the ability of ATP gamma S to stimulate, consistent with NDP kinase-catalyzed formation of GTP gamma S from ATP gamma S plus GDP. This activity was demonstrated directly in both cytosol and plasma membrane. Using undialyzed cytosol, phosphorylation of p47 showed a similar nonspecificity for nucleoside triphosphates, due to NDP kinase activity, but revealed the expected ATP specificity when dialyzed cytosol was used. Neither ATP gamma S nor GTP gamma S were good substrates for protein phosphorylation. Under a variety of conditions, phosphorylation of p47 or other neutrophil proteins failed to correlate with oxidase activation. The present studies indicate that SDS and diacylglycerol stimulation of superoxide generation in the cell-free system is independent of protein kinase C or other protein kinase activity, and suggest a novel role for diacylglycerol in cell regulation.
Highlights
The NADPH-oxidase of human neutrophils can be organisms
The oxidase exists in a dormant state in unstimactivated in a cell-free system comprised of plasma ulated neutrophils, but canbe activated by a variety of stimuli, membrane, cytosol, and ananionic amphiphile such as including the chemoattractant formyl-methionyl-leucylarachidonate or sodium dodecyl sulfate (SDS)
Re- phenylalanine, protein kinase C activators such as phorbol cently, we showed that diacylglycerol acts synergisti- 12-myristate 13-acetate, and particulates such as opsonized cally with SDS in the cell-free system to stimulate bacteriaor zymosan
Summary
Effect of ~ ~ leoside ~ ripho sopn~Stuepseroxide Generation inthe Cell-freeSystem-Results using plasma membrane plus cytosol, activated with SDS, are shown in the two left panels of Fig. 1. ATP (100pM) augmented the activity slightly less than 2-fold (Fig 1, upper left panel, filled circles),and therewas no further stimulationby concentrations up to 400 p~ (not shown). A larger (2.5-fold) augmentation which occurred at lower concentrations (optimum at 20 p ~wa) s seen with ATPyS (Fig. 1,upper leftpanel, open circles). DiC8 alone augmented the basal rate by more than 2-fold to 544nmol/min/mg plasma membrane protein. ATPyS resulted in a larger (2.6-fold) enhancement of oxidase activity to more than 1400 nmol/ min/mg plasma membrane protein (upper right, open circles). There was a modest increase with GTP alone (1.5-fold at 100 p ~ bu) t a larger (3.3-fold)effect was seen with GTPyS (10pM), which resulted in a rate of more than 1500 nmoi/min/mg plasma membrane protein. The basal rates in nmol cytochrome c reduced/min/mg plasma membrane (PM) protein were as follows: in the native (undialyzed system) the rate with SDS alone with 245, and 544 with SDS plus diC8, and with dialyzed cytosol the rate with SDS was 56, and that with SDS Dlus diC8 was 155
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
