Surface NADH oxidase of HeLa cells lacks intrinsic membrane binding motifs.
Disulfide-thiol interchange proteins with hydroquinone (NADH) oxidase activities (designated NOX for plasma membrane-associated NADH oxidases) occur as extrinsic membrane proteins associated with the plasma membrane at the outer cell surface. The cancer-associated NOX protein, designated tNOX, has been cloned. The 34-kDa plasma membrane-associated form of the protein contains no strongly hydrophobic regions and is not transmembrane. No myristoylation or phosphatidylinositol anchor motifs were discovered. Evidence for lack of involvement of a glycosylphosphatidylinositol-linkage was derived from the inability of treatment with a phosphatidylinositol-specific phospholipase C or with nitrous acid at low pH to release the NOX protein from the surface of HeLa cells or from plasma membranes isolated from HeLa cells. Binding of NOX protein to the plasma membrane via amino acid side chain modification or by attachment of fatty acids also is unlikely based on use of specific fatty acid antisera to protein bound fatty acids and as a result of binding to the cancer cell surface of a truncated form of recombinant tNOX. Incubation of cells or plasma membranes with 0.1 M sodium acetate, pH 5, at 37 degrees C for 1 h, was sufficient to release tNOX from the HeLa cell surface. Release was unaffected by protease inhibitors or divalent ions and was not accelerated by addition of cathepsin D. The findings suggest dissociable receptor binding as a possible basis for their plasma membrane association.
Morré
D J
DJ
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana 47907-1333, USA. morre@pharmacy.purdue.edu
Sedlak
D
D
Tang
X
X
Chueh
P J
PJ
Geng
T
T
Morré
D M
DM
D015153:Blotting, Western; D002402:Cathepsin D / Q000494:pharmacology; D002455:Cell Division; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009097:Multienzyme Complexes / Q000737:chemistry*; D009227:Myristic Acids / Q000737:chemistry; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D000074624:NADPH Oxidase 1; D009608:Nitrous Acid / Q000494:pharmacology; D017830:Octoxynol; D010716:Phosphatidylinositols / Q000737:chemistry; D010740:Phospholipases / Q000378:metabolism; D011092:Polyethylene Glycols / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry; D013696:Temperature; D013997:Time Factors
D003902:Detergents; D009097:Multienzyme Complexes; D009227:Myristic Acids; D010716:Phosphatidylinositols; D011480:Protease Inhibitors; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D000074624:NADPH Oxidase 1; D010740:Phospholipases; D002402:Cathepsin D; D009608:Nitrous Acid