Surface NADH Oxidase of HeLa Cells Lacks Intrinsic Membrane Binding Motifs D. James Morre ´,* ,1 Dagmar Sedlak,* Xiaoyu Tang,* Pin-Ju Chueh,* Tao Geng,† and Dorothy M. Morre ´† *Department of Medicinal Chemistry and Molecular Pharmacology and †Department of Foods and Nutrition, Purdue University, West Lafayette, Indiana 47907-1333 Received February 20, 2001, and in revised form April 25, 2001; published online July 17, 2001 Disulfide-thiol interchange proteins with hydroqui- none (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 can- cer-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 discov- ered. Evidence for lack of involvement of a glyco- sylphosphatidylinositol-linkage was derived from the inability of treatment with a phosphatidylinositol-spe- cific 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 trun- cated form of recombinant tNOX. Incubation of cells or plasma membranes with 0.1 M sodium acetate, pH 5, at 37°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 disso- ciable receptor binding as a possible basis for their plasma membrane association. © 2001 Academic Press Key Words: cell surface NADH oxidase (NOX); tNOX; cancer; membrane anchors; HeLa cells. A cancer-associated and drug responsive hydroqui- none (NADH) oxidase (tNOX) of the HeLa cell sur- face has been described (1– 4) and cloned by our laboratory (5, AF207881). It is on the outside surface of the plasma membrane (6 – 8). Present, as well, are constitutive NOX proteins designated CNOX. CNOX proteins occur at the surfaces of both cancer and noncancer cells (9). These remain to be cloned and sequenced. The plasma membrane NADH oxidases of the cell surface have been considered to be peripheral. How- ever, studies to rule out an intrinsic nature have not been carried out previously. An external location for the cell surface NADH oxidase of HeLa cells is sup- ported by several lines of evidence. Included are stud- ies with vesicles of known absolute orientation (6), use of NADH oxidase-specific antisera (10) employment of impermeant conjugates of tNOX inhibitors (11) and response to the impermeant thiol reagent p-chloromer- curiphenylsulfonic acid (12). Also indicative of a cell surface location have been studies that demonstrate NADH oxidase activity with intact HeLa cells (13). As would be characteristic of extrinsic membrane proteins in general, the NOX proteins are shed from the cell surface where they appear in cell culture media (14, 15) or in sera (16, 17). The released proteins ap- pear to exhibit structural and functional characteris- tics similar to the cell-surface associated forms. In this report, we provide evidence for an association of the NOX proteins with the external surface of the plasma membranes of HeLa and other cell types and provide evidence for a lack of intrinsic binding oppor- tunities as well as the facile dissociation of the proteins by low pH treatment suggestive of extrinsic or periph- eral binding. 1 To whom correspondence and reprint requests should be ad- dressed. Fax: (765) 494-4007. E-mail: morre@pharmacy.purdue.edu. 0003-9861/01 $35.00 251 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. Archives of Biochemistry and Biophysics Vol. 392, No. 2, August 15, pp. 251–256, 2001 doi:10.1006/abbi.2001.2436, available online at http://www.idealibrary.com on