Deactivation of neutrophil NADPH oxidase by actin-depolymerizing agents in a cell-free system.
The cell-free activation of human neutrophil NADPH oxidase (O(2)(-)-generating enzyme) is enhanced by actin [Morimatsu, Kawagoshi, Yoshida and Tamura (1997) Biochem. Biophys. Res. Commun. 230, 206--210]. In an attempt to elucidate the mechanism, we examined the effect of actin-depolymerizing agents on the duration of NADPH oxidase in a cell-free system. The addition of DNase I, an F-actin-depolymerizing protein, caused an accelerated deactivation of the oxidase. The deactivation was also facilitated by latrunculin A, a sponge toxin that depolymerizes F-actin. Exogenously added actin prevented the deactivation by DNase I or latrunculin A, whereas EDTA accelerated a dilution-induced deactivation of the oxidase and Mg(2+) ions retarded it. The stability in dilution was found to correlate well with free Mg(2+) concentration. Estimation of F-actin in the system showed that F-actin increased during the oxidase activation and that DNase I or EDTA decreased F-actin content in parallel with the activity. Treatment of the cell-free mixture with a chemical cross-linker prevented the deactivation and F-actin decrease by EDTA. Taken together, these results suggest that actin filaments which grow during the activation of NADPH oxidase prolong the lifetime of the oxidase.
Tamura
M
M
Department of Applied Biochemistry, Faculty of Engineering, Ehime University, Matsuyama, Ehime 790-8577, Japan. miketamu@en3.ehime-u.ac.jp
Kanno
M
M
Endo
Y
Y
D000199:Actins / Q000378:metabolism*; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002474:Cell-Free System; D003850:Deoxyribonuclease I / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004492:Edetic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D008274:Magnesium / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology* / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013347:Subcellular Fractions / Q000378:metabolism; D013844:Thiazoles / Q000494:pharmacology; D053778:Thiazolidines; D013997:Time Factors
D000199:Actins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D013844:Thiazoles; D053778:Thiazolidines; D012967:Sodium Dodecyl Sulfate; D006160:Guanosine Triphosphate; D004492:Edetic Acid; D019255:NADPH Oxidases; D003850:Deoxyribonuclease I; D008274:Magnesium; D005022:Ethyldimethylaminopropyl Carbodiimide; D010100:Oxygen; C037067:latrunculin A