Elastase controls the binding of the vitamin D-binding protein (Gc-globulin) to neutrophils: a potential role in the regulation of C5a co-chemotactic activity

The vitamin D-binding protein (DBP) binds to the plasma membranes of numerous cell types and mediates a diverse array of cellular functions. DBP bound to the surface of leukocytes serves as a co-chemotactic factor for C5a, significantly enhancing the chemotactic activity of pM concentrations of C5a. This study investigated the regulation of DBP binding to neutrophils as a possible key step in the process of chemotaxis enhancement to C5a. Using radioiodinated DBP as a probe, neutrophils released 70% of previously bound DBP into the extracellular media during a 60-min incubation at 37 degrees C. This was suppressed by serine protease inhibitors (PMSF, Pefabloc SC), but not by metallo- or thiol-protease inhibitors. DBP shed from neutrophils had no detectable alteration in its m.w., suggesting that a serine protease probably cleaves the DBP binding site, releasing DBP in an unaltered form. Cells treated with PMSF accumulate DBP vs time with over 90% of the protein localized to the plasma membrane. Purified neutrophil plasma membranes were used to screen a panel of protease inhibitors for their ability to suppress shedding of the DBP binding site. Only inhibitors to neutrophil elastase prevented the loss of membrane DBP-binding capacity. Moreover, treatment of intact neutrophils with elastase inhibitors prevented the generation of C5a co-chemotactic activity from DBP. These results indicate that steady state binding of DBP is essential for co-chemotactic activity, and further suggest that neutrophil elastase may play a critical role in the C5a co-chemotactic mechanism.     

Location of the epidermal growth factor binding site on the EGF receptor. A resonance energy transfer study

As a first step toward developing a structural map of key sites on the epidermal growth factor (EGF) receptor, we have used resonance energy transfer to measure the distance of closest approach between the receptor-bound growth factor molecule and lipid molecules at the surface of the plasma membrane. EGF, specifically labeled at its amino terminus with fluorescein 5-isothiocyanate, was used as an energy donor in these experiments, while either octadecylrhodamine B or octadecylrhodamine 101, inserted into plasma membranes isolated from human epidermoid carcinoma (A431) cells, served as the energy acceptors. The energy transfer measurements indicate that the amino terminus of the bound growth factor is about 67 A away from the plasma membrane. On the basis of the dimensions of the EGF molecule, this suggests that EGF binds to a site on its receptor that is a considerable distance (52-82 A) from the surface of these cells. Identical results were obtained under conditions where the receptor functions as an active tyrosine kinase, suggesting that the relative juxtaposition of the EGF binding domain to the membrane surface does not change with receptor autophosphorylation or with the activation of the receptor tyrosine kinase activity.     

Characterization of alpha-crystallin-plasma membrane binding

Alpha-crystallin, a large lenticular protein complex made up of two related subunits (alphaA- and alphaB-crystallin), is known to associate increasingly with fiber cell plasma membranes with age and/or the onset of cataract. To understand better the binding mechanism, we developed a sensitive membrane binding assay using lens plasma membranes and recombinant human alphaA- and alphaB-crystallins conjugated to a small fluorescent tag (Alexa350). Both alphaA and alphaB homopolymer complexes, as well as a reconstituted 3:1 heteromeric complex, bind to lens membranes in a specific, saturable, and partially irreversible manner that is sensitive to both time and temperature. The amount of alpha-crystallin that binds to the membrane increases under acidic pH conditions and upon removal of exposed intrinsic membrane protein domains but is not affected at high ionic strength, suggesting that alpha-crystallin binds to the fiber cell plasma membranes mainly through hydrophobic interactions. The binding capacity and affinity for the reconstituted 3:1 heteromeric complex were measured to be 3. 45 +/- 0.11 ng/microg of membrane and 4.57 +/- 0.50 x 10(-4) microg(-1) of membrane, respectively. The present membrane binding data support the hypothesis that the physical properties of a mixed alpha-crystallin complex may hold particular relevance for the function of alpha-crystallin within the lens.     

Localization of the dicarboxylate binding protein in the cell envelope of Escherichia coli K12

Examination of the localization of the dicarboxylate binding protein (DBP) in the cell envelope of Escherichia coli K12 reveals that this protein is present on the cell surface, and also in the inner and outer regions of the periplasmic space. The cell surface DBP is release by treating the cells with EDTA. This protein can be surface labeled by lactoperoxidase radioiodination, and by diazo[125I]iodosulfanilic acid in whole cells. It also binds tightly, but not covalently, to lipopolysaccharide. The DBP located in the outer region of the periplasmic space is released when the outer membrane is dissociated by EDTA-osmotic shock treatment. The DBP located in the inner region of the periplasmic space is released only when the EDTA-osmotic shocked cells are subjected to lysozyme treatment. At the moment, it is not certain whether this protein is bound to or trapped by the peptidoglycan network. This protein cannot be surface labeled in whole cells or in EDTA-osmotic shock treated cells; and it is not associated with lipopolysaccharide. Analysis of transport mutants indicates that these DBP are coded by the same gene.     

CD44 and annexin A2 mediate the C5a chemotactic cofactor function of the vitamin D binding protein

The vitamin D binding protein (DBP) is a plasma protein that significantly enhances the chemotactic activity of C5a and C5a(desArg) (cochemotactic activity). The objective of this study was to investigate how DBP mediates this process using neutrophils and U937 cells transfected with the C5a receptor (U937-C5aR cells) and comparing chemotaxis to C-activated serum (DBP dependent) vs purified C5a (DBP independent). Binding to the cell surface is essential for this protein to function as a chemotactic cofactor, and DBP binds to a chondroitin sulfate proteoglycan (CSPG) on neutrophil plasma membrane preparations. To determine whether a CSPG also functions to mediate cochemotactic activity, U937-C5aR cells were grown in chlorate to inhibit CSPG sulfation or treated with chondroitinase AC. Either treatment significantly inhibited chemotaxis only to C-activated serum. CD44 is a major cell surface CSPG on leukocytes, and functions to facilitate chemotaxis. Treatment of cells with anti-CD44 blocks chemotaxis of neutrophils and U937-C5aR cells to C-activated serum but not purified C5a. DBP binds to CD44 on the cell surface as evidenced by coimmunoprecipitation, confocal microscopy, and cell binding studies. Annexin A2 associates with CD44 in lipid rafts; therefore, its potential role in mediating cochemotactic activity was investigated. Results demonstrate that anti-A2 inhibits neutrophil and U937-C5aR chemotaxis specifically to C-activated serum, blocks DBP binding to cells, and colocalizes with anti-DBP on the cell surface. These results provide clear evidence that CD44 and annexin A2 mediate the C5a chemotactic cofactor function of DBP.     

The outer membrane protein OmpA of Mannheimia haemolytica A1 is involved in the binding of fibronectin

An enzyme-linked immunosorbent assay using bovine fibronectin as the substrate was used to demonstrate that Mannheimia haemolytica A1 binds to fibronectin. This binding to fibronectin was specific as no binding was observed with bovine fibrinogen. The binding to fibronectin was not observed if the M. haemolytica A1 cells were pretreated with trypsin or proteinase K, suggesting that it involved a protein molecule on the cell surface. Interestingly, the fibronectin-binding activity was found to be higher in an acapsular mutant compared with its parent strain. The fibronectin-binding protein was shown to be present in the outer membrane fraction of M. haemolytica A1. A 45 kDa outer membrane protein that binds to fibronectin was identified by Far-Western immunoblot analysis. This protein was purified and subjected to MS matrix-assisted laser desorption ionization time-of-flight analysis. The results identified it to be outer membrane OmpA based on comparison with the M. haemolytica A1 genomic sequence.     

The GIP receptor on pancreatic beta cell tumor: molecular identification by covalent cross-linking

125I-GIP binds reversibly to a high affinity binding site in crude plasma membranes prepared from a hamster pancreatic beta cell tumor. The treatment of labeled membranes with the cross-linker dithiobis (succinimidylpropionate) prevents, to a greater extent, the rapid dissociation of 125I-GIP-membrane complexes which is observed when 10(-6) M native GIP is added. Polyacrylamide gel electrophoresis of membrane proteins reveals a major 125I-GIP-protein complex of Mr 64,000. This labeling decreases when increasing concentrations (10(-9) -10(-6)M) of native GIP are added but is not altered by other peptide hormones (tested at 10(-6)M) including glucagon, VIP and insulin. The Mr 64,000 complex is not observed in tissues which have no specific binding sites for GIP such as intestinal epithelium. Assuming one molecule of 125I-GIP is bound per molecule of protein, one protein with Mr 59,000 is identified as the specific GIP binding site.     

Identification and characterization of human endothelial cell membrane binding sites for tissue plasminogen activator and urokinase

Cultured human endothelial cells synthesize and secrete two types of plasminogen activator, tissue plasminogen activator (t-PA) and urokinase (u-PA). Previous work from this laboratory (Hajjar, K.A., Hamel, N. M., Harpel, P. C., and Nachman, R. L. (1987) J. Clin. Invest. 80, 1712-1719) has demonstrated dose-dependent, saturable, and high affinity binding of t-PA to two sites associated with cultural endothelial cell monolayers. We now report that an isolated plasma membrane-enriched endothelial cell fraction specifically binds 125I-t-PA at a single saturable site (Kd 9.1 nM; Bmax 3.1 pmol/mg membrane protein). Ligand blotting experiments demonstrated that both single and double-chain t-PA specifically bound to a Mr 40,000 membrane protein present in detergent extracts of isolated membranes, while high molecular weight, low molecular weight, and single-chain u-PA associated with a Mr 48,000 protein. Both binding interactions were reversible and cell-specific and were inhibitable by pretreatment of intact cells with nanomolar concentrations of trypsin. The relevant binding proteins were not found in subendothelial cell matrix, failed to react with antibodies to plasminogen activator inhibitor type 1 and interacted with their respective ligands in an active site-independent manner. The isolated t-PA binding site was resistant to reduction and preserved the capacity for plasmin generation. In contrast, the isolated u-PA binding protein was sensitive to reduction, and did not maintain the catalytic activity of the ligand on the blot. The results suggest that in addition to sharing a matrix-associated binding site (plasminogen activator inhibitor type 1), both t-PA and u-PA have unique membrane binding sites which may regulate their function. The results also provide further support for the hypothesis that plasminogen and t-PA can assemble on the endothelial cell surface in a manner which enhances cell surface generation of plasmin.     

Identification of the hemoglobin binding sites on the inner surface of the erythrocyte membrane

The hemoglobin binding sites on the inner surface of the erythrocyte membrane were identified by measuring the fraction of hemoglobin released following selective proteolytic or lipolytic enzyme digestion. In addition, binding stoichiometry to and fractional hemoglobin release from inside-out vesicle preparations of human and rabbit membranes were compared since rabbit membranes differ significantly from human membranes only in that they lack glycophorin. Our results show that rabbit inside-out vesicles bind about 65% less human or rabbit hemoglobin under conditions of optimal and stoichiometric binding, despite being otherwise similar in composition. We suggest that this difference is either directly or indirectly due to the absence of glycophorin in rabbit membranes. Further supportive evidence includes demonstrating (a) that neuraminidase treatment of human membranes did not affect hemoglobin binding and (b) that reconstitution of isolated glycophorin into phospholipid vesicles increased the hemoglobin binding capacity in a manner proportional to the fraction of glycophorin molecules oriented with their cytoplasmic sides exposed to the exterior of the vesicle. Proteolysis of human inside-out vesicles either before or after addition of hemoglobin reduced the binding capacity by about 25%. This is consistent with the known proportion of total hemoglobin binding sites involving band 3 protein and the selective lability of the cytoplasmic aspect of band 3 protein to proteolysis. Phospholipid involvement in hemoglobin binding was determined using various phospholipase C preparations which differ in their reactivity profiles. Approximately 38% of the bound hemoglobin was released upon cleavage of phospholipid headgroups. These results suggest that the predominant sites of binding for hemoglobin on the inner surface of the red cell membrane are the two major integral membrane glycoproteins.     

Identification of the D-glucose-inhibitable cytochalasin B binding site as the glucose transporter in rat diaphragm plasma and microsomal membranes

[3H]Cytochalasin B binding and its competitive inhibition by D-glucose have been used to identify, the glucose transporter in plasma and microsomal membranes prepared from intact rat diaphragm. Scatchard plot analysis of [3H]cytochalasin B binding yields a binding site with a dissociation constant of roughly 110 nM. Since the inhibition constant of cytochalasin B for D-glucose uptake by diaphragm plasma membranes is similar to this value, this site is identified as the glucose transporter. Plasma membranes prepared from diaphragms bind approx. 17 pmol of cytochalasin B/mg of membrane protein to the D-glucose-inhibitable site. If 280 nM (40000 microunits/ml) insulin is present during incubation, cytochalasin B binding is increased roughly 2-fold without alteration in the dissociation constant of this site. In addition, membranes in the microsomal fraction contain 21 pmol of D-glucose-inhibitable cytochalasin B binding sites/mg of membrane protein. In the presence of insulin during incubation the number of these sites in the microsomal fraction is decreased to 9 pmol/mg of membrane protein. These results suggest that rat diaphragm contain glucose transporters with characteristics identical to those observed for the rat adipose cell glucose transporter. In addition, insulin stimulates glucose transport in rat diaphragm through a translocation of functionally identical glucose transporters from an intracellular membrane pool to the plasma membrane without an alteration in the characteristics of these sites.     

SecA protein, a peripheral protein of the Escherichia coli plasma membrane, is essential for the functional binding and translocation of proOmpA.

We have reconstituted protein translocation across plasma membrane vesicles of Escherichia coli using purified proOmpA and trigger factor, a 63 kd soluble protein. Treatment of membrane vesicles with urea inactivates them for translocation unless a factor present in cytoplasmic extracts is added during the translocation reaction. Sedimentation analysis showed that the stimulatory activity is of distinctly higher mol. wt than trigger factor. Cytoplasmic extracts from a strain that greatly overproduces the SecA protein are highly enriched in the stimulatory activity for untreated membranes and restore translocation to urea-treated membranes, suggesting that this protein is the stimulatory factor. This assay was used to monitor the isolation of SecA protein from the overproducing strain. The purified protein is soluble, yet binds peripherally to membranes with high affinity and supports translocation. Using pure proOmpA, SecA protein, trigger factor and urea-treated membranes, the protein export process was resolved into binding and translocation steps. We find that proOmpA binds to membrane vesicles with or without SecA protein, but that translocation only occurs when SecA was bound prior to proOmpA.     

Anion transport inhibitor binding to band 3 in red blood cell membranes

The inhibitor of anion exchange 4,4''-dibenzoamido-2,2''-disulfonic stilbene (DBDS) binds to band 3, the anion transport protein in human red cell ghost membranes, and undergoes a large increase in fluorescence intensity when bound to band 3. Equilibrium binding studies performed in the absence of transportable anions show that DBDS binds to both a class of high-affinity (65 nM) and low-affinity (820 nM) sites with stoichiometry equivalent to 1.6 nmol/mg ghost protein for each site, which is consistent with one DBDS site on each band 3 monomer. The kinetics of DBDS binding were studied both by stopped-flow and temperature-jump experiments. The stopped-flow data indicate that DBDS binding to the apparent high-affinity site involves association with a low-affinity site (3 microM) followed by a slow (4 s-1) conformational change that locks the DBDS molecule in place. A detailed, quantitative fit of the temperature-jump data to several binding mechanisms supports a sequential-binding model, in which a first DBDS molecule binds to one monomer and induces a conformational change. A second DBDS molecule then binds to the second monomer. If the two monomers are assumed to be initially identical, thermodynamic characterization of the binding sites shows that the conformational change induces an interaction between the two monomers that modifies the characteristics of the second DBDS binding site.     

The appendage domain of the AP-2 subunit is not required for assembly or invagination of clathrin-coated pits

Coated pits contain a resident membrane molecule(s) that binds clathrin AP-2 with high affinity. AP-2 binding to this site is likely to be the first step in coated pit assembly because this subunit functions as a template for the polymerization of clathrin into flat polygonal lattices. Integral membrane proteins involved in receptor mediated endocytosis cluster in the newly assembled pits as they invaginate and bud from the membrane. The AP-2 subunit is a multi-domain, molecular complex that can be separated by proteolysis into a brick-shaped core and ear-like appendage domains. We have used this property to identify the domain involved in the various stages of coated pit assembly and budding. We found that the core of AP-2 is the domain that binds both to membranes and to triskelions during assembly. Triskelions are perfectly capable of forming lattices on the membrane bound cores. Clathrin lattices bound only to core domains were also able to invaginate normally. Limited proteolysis was also useful for further characterizing the AP-2 binding site. Elastase treatment of the inside membrane surface released a peptide fraction that is able to bind AP-2 in solution and prevent it from interacting with membranes. Affinity purification of binding activity yielded a collection of peptides that was dominated by a 45-kD species. This is the candidate peptide for containing the AP-2-binding site. Therefore, the appendage domain does not directly participate in any of the assembly or invagination events required for coated pit function.     

Properties of erythrocyte membrane binding and autolytic activation of calcium-activated neutral protease

The binding of a calcium-activated neutral protease (CANP) with high calcium sensitivity (muCANP) to erythrocyte membranes and its subsequent autolytic activation on the membranes were analyzed by an immunoblot technique. In the presence of calcium ions, muCANP bound to the erythrocyte membranes as a heterodimer of 79- and 28-kDa subunits and was converted quickly on the membranes to an active form with a 76-kDa large subunit. The active form was then released from the membranes to the soluble fraction. These sequential reactions, however, were not specific to inside-out vesicles, but occurred also, except for some Ca2+-independent binding, on right side-out vesicles. A rapid degradation of some membrane proteins was observed after binding of muCANP to the membranes. The binding of muCANP to erythrocyte membranes was inhibited by substrates and the endogenous CANP inhibitor, which is also a suicide substrate. These results strongly suggest that muCANP binds to membranes by recognition of membrane proteins as substrates and not at a special site for activation. Thus, a possible mechanism for muCANP activation on membranes is that muCANP first binds to substrates on membranes, is activated, and then degrades the substrates to deform the membrane structures.     

Plasma membrane association of Acanthamoeba myosin I

Myosin I accounted for approximately 2% of the protein of highly purified plasma membranes, which represents about a tenfold enrichment over its concentration in the total cell homogenate. This localization is consistent with immunofluorescence analysis of cells that shows myosin I at or near the plasma membrane as well as diffusely distributed in the cytoplasm with no apparent association with cytoplasmic organelles or vesicles identifiable at the level of light microscopy. Myosin II was not detected in the purified plasma membrane fraction. Although actin was present in about a tenfold molar excess relative to myosin I, several lines of evidence suggest that the principal linkage of myosin I with the plasma membrane is not through F- actin: (a) KI extracted much more actin than myosin I from the plasma membrane fraction; (b) higher ionic strength was required to solubilize the membrane-bound myosin I than to dissociate a complex of purified myosin I and F-actin; and (c) added purified myosin I bound to KI- extracted plasma membranes in a saturable manner with maximum binding four- to fivefold greater than the actin content and with much greater affinity than for pure F-actin (apparent KD of 30-50 nM vs. 10-40 microM in 0.1 M KCl plus 2 mM MgATP). Thus, neither the MgATP-sensitive actin-binding site in the NH2-terminal end of the myosin I heavy chain nor the MgATP-insensitive actin-binding site in the COOH-terminal end of the heavy chain appeared to be the principal mechanism of binding of myosin I to plasma membranes through F-actin. Furthermore, the MgATP- sensitive actin-binding site of membrane-bound myosin I was still available to bind added F-actin. However, the MgATP-insensitive actin- binding site appeared to be unable to bind added F-actin, suggesting that the membrane-binding site is near enough to this site to block sterically its interaction with actin.     

Identification of the d-glucose-inhibitable cytochalasin B binding site as the glucose transporter in rat diaphragm plasma and microsomal membranes

[³H]Cytochalasin B binding and its competitive inhibition by d-glucose have been used to identify the glucose transporter in plasma and microsomal membranes prepared from intact rat diaphragm. Scatchard plot analysis of [³H]cytochalasin B binding yields a binding site with a dissociation constant of roughly 110 nM. Since the inhibition constant of cytochalasin B for d-glucose uptake by diaphragm plasma membranes is similar to this value, this site is identified as the glucose transporter. Plasma membranes prepared from diaphragms bind approx. 17 pmol of cytochalasin B/mg of membrane protein to the d-glucose-inhibitable site. If 280 nM (40 000 μunits/ml) insulin is present during incubation, cytochalasin B binding is increased roughly 2-fold without alteration in the dissociation constant of this site. In addition, membranes in the microsomal fraction contain 21 pmol of d-glucose-inhibitable cytochalasin B binding sites/mg of membrane protein. In the presence of insulin during incubation the number of these sites in the microsomal fraction is decreased to 9 pmol/mg of membrane protein. These results suggest that rat diaphragm contain glucose transporters with characteristics identical to those observed for the rat adipose cell glucose transporter. In addition, insulin stimulates glucose transport in rat diaphragm through a translocation of functionally identical glucose transporters from an intracellular membrane pool to the plasma membrane without an alteration in the characteristics of these sites.     

Mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation

The clathrin-coated pit lattice is held onto the plasma membrane by an integral membrane protein that binds the clathrin AP-2 subunit with high affinity. In vitro studies have suggested that this protein controls the assembly of the pit because membrane bound AP-2 is required for lattice assembly. If so, the AP-2 binding site must be a resident protein of the coated pit and recycle with other receptors that enter cells through this pathway. Proper recycling, however, would require the switching off of AP-2 binding to allow the binding site to travel through the endocytic pathway unencumbered. Evidence for this hypothesis has been revealed by the cationic amphiphilic class of drugs (CAD), which have previously been found to inhibit receptor recycling. Incubation of human fibroblasts in the presence of these drugs caused clathrin lattices to assemble on endosomal membranes and at the same time prevented coated pit assembly at the cell surface. These effects suggest that CADs reverse an on/off switch that controls AP-2 binding to membranes. We conclude that cells have a mechanism for switching on and off AP-2 binding during the endocytic cycle.     

Cell-cycle-dependent binding kinetics for the early endosomal tethering factor EEA1

Early endosomal antigen 1 (EEA1) is a cytosolic protein that specifically binds to early endosomal membranes where it has a crucial role in the tethering process leading to homotypic endosome fusion. Green fluorescent protein-tagged EEA1 (EEA1-GFP) was bound to the endosomal membrane throughout the cell cycle, and measurements using fluorescent recovery after photobleaching showed two fractions: one rapidly exchanging with the cytosolic pool, and the other with a long half-life. The exchange consists of a release and binding process, and we have separated these two by using GFP and photoactivable GFP. The release rate was identical to the exchange rate, showing that the dissociation characteristics determine the cycling of this molecule. During mitosis, we found that the dissociation rate was markedly accelerated and, in addition, the long-lived fraction was markedly reduced. This indicates that a fusion arrest in mitosis is not the result of EEA1 not binding to early endosomes, but rather due to the marked shift in membrane-binding characteristics. This might be a general mechanism to fine-tune and control tethering and fusion of early endosomes.     

A 39-kD plasma membrane protein (IP39) is an anchor for the unusual membrane skeleton of Euglena gracilis

The major integral plasma membrane protein (IP39) of Euglena gracilis was radiolabeled, peptide mapped, and dissected with proteases to identify cytoplasmic domains that bind and anchor proteins of the cell surface. When plasma membranes were radioiodinated and extracted with octyl glucoside, 98% of the extracted label was found in IP39 or the 68- and 110-kD oligomers of IP39. The octyl glucoside extracts were incubated with unlabeled cell surface proteins immobilized on nitrocellulose (overlays). Radiolabel from the membrane extract bound one (80 kD) of the two (80 and 86 kD) major membrane skeletal protein bands. Resolubilization of the bound label yielded a radiolabeled polypeptide identical in Mr to IP39. Intact plasma membranes were also digested with papain before or after radioiodination, thereby producing a cytoplasmically truncated IP39. The octyl glucoside extract of truncated IP39 no longer bound to the 80-kD membrane skeletal protein in the nitrocellulose overlays. EM of intact or trypsin digested plasma membranes incubated with membrane skeletal proteins under stringent conditions similar to those used in the nitrocellulose overlays revealed a partially reformed membrane skeletal layer. Little evidence of a membrane skeletal layer was found, however, when plasma membranes were predigested with papain before reassociation. A candidate 80-kD binding domain of IP39 has been tentatively identified as a peptide fragment that was present after trypsin digestion of plasma membranes, but was absent after papain digestion in two-dimensional peptide maps of IP39. Together, these data suggest that the unique peripheral membrane skeleton of Euglena binds to the plasma membrane through noncovalent interactions between the major 80-kD membrane skeletal protein and a small, papain sensitive cytoplasmic domain of IP39. Other (62, 51, and 25 kD) quantitatively minor peripheral proteins also interact with IP39 on the nitrocellulose overlays, and the possible significance of this binding is discussed.     

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.