Activation of the superoxide forming NADPH oxidase in a cell-free system by sodium dodecyl sulfate. Absolute lipid dependence of the solubilized enzyme.
The superoxide (O2-)-forming NADPH oxidase of resting macrophages can be activated in a cell-free system by certain anionic amphiphiles, such as sodium dodecyl sulfate (SDS). O2- production requires the cooperation of membrane-associated and cytosolic components. The membrane component can be solubilized by octyl glucoside yielding a highly active oxidase preparation. High performance gel filtration of the solubilized oxidase on Superose 12 in the presence of 40 mM octyl glucoside leads to the total loss of enzymatic activity. This can be restored in previously inactive eluate fractions by "reconstitution" with N-ethylmaleimide or heat (60 degrees C)-inactivated total solubilized membrane. Oxidase activity, that becomes evident upon reconstitution, is eluted from Superose 12 with molecules in the Mr range of 300,000-71,000. The material with reconstitutive capacity is completely dissociated from the oxidase, eluting with molecules in the Mr range of 71,000-11,000. The Superose 12 elution profile of the material responsible for reconstitution coincides with that of membrane-derived phospholipid. Also, the reconstitutive capacity of total solubilized membrane and that of the Mr 71,000-11,000 region of the Superose eluate are recovered in a chloroform extract prepared by the method of Bligh and Dyer. It is concluded that loss of oxidase activity by gel filtration at a high octyl glucoside concentration is the consequence of delipidation. NADPH oxidase activity, revealed by reconstitution of Superose 12 fractions with exogenous phospholipid, correlates closely with the elution profile of cytochrome b559. Reconstitution of activity of delipidated oxidase can also be achieved with natural non-macrophage phospholipids and with synthetic phospholipids. Reconstitution of NADPH oxidase activity by lipids is governed by the following rules: (a) phospholipids are effective; lysophospholipids and neutral lipids are not; (b) phospholipids with polar heads represented by choline, ethanolamine, and serine, as well as cardiolipin, are effective; phosphatidylinositol is much less active; (c) phospholipids with unsaturated fatty acid residues are capable of reconstitution while saturated acyl residues do not confer activity; this specificity appears not to be related to the transition temperature of the phospholipids.
Shpungin
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Department of Human Microbiology, Sackler School of Medicine, Tel-Aviv, Israel.
Dotan
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I
Abo
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Pick
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E
D000645:Ammonium Sulfate; D000818:Animals; D002474:Cell-Free System; D002850:Chromatography, Gel; D003573:Cytochrome b Group / Q000502:physiology; D004789:Enzyme Activation / Q000187:drug effects; D005590:Fractional Precipitation; D005960:Glucosides; D006168:Guinea Pigs; D008264:Macrophages / Q000201:enzymology*; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D019255:NADPH Oxidases; D010743:Phospholipids / Q000502:physiology*; D045332:Photosystem II Protein Complex; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology*; D012995:Solubility; D013347:Subcellular Fractions; D013481:Superoxides / Q000378:metabolism*
D003573:Cytochrome b Group; D005960:Glucosides; D010743:Phospholipids; D045332:Photosystem II Protein Complex; D013481:Superoxides; C018619:octyl-beta-D-glucoside; D012967:Sodium Dodecyl Sulfate; C019313:cytochrome b559; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C022709:superoxide-forming enzyme; D000645:Ammonium Sulfate