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Affinity Purification of Helicobacter pyloriUrease

1998; Elsevier BV; Volume: 273; Issue: 29 Linguagem: Inglês

10.1074/jbc.273.29.18130

1083-351X

Faustino C. Icatlo, Masahiko Kuroki, Chizu Kobayashi, Hideaki Yokoyama, Yutaka Ikemori, Tomomi Hashi, Yoshikatsu Kodama,

Galectins and Cancer Biology

A simple, reproducible and high yield method ofHelicobacter pylori urease enzyme purification was developed using a heparinoid (Cellufine sulfate) affinity gel. The purification method involved two sequential steps using the same gel that takes advantage of the differential affinity of urease to the heparinoid at two levels of hydrogen ion concentration. SDS-polyacrylamide gel electrophoresis analysis of affinity-purified urease revealed two major protein bands with about 62- and 30-kDa molecular mass. When whole cell lysates of clinical and laboratory strains of H. pylori were probed by Western blot, anti-urease hyperimmune serum produced by affinity-purified urease in rabbit recognized only the two bands corresponding to the urease A and B subunits. To probe the molecular relevance of affinity gel adherence to mucin adherence, the purified urease was derivatized withN-hydroxysuccinimidobiotin and used in adherence assays. Competitive inhibition tests revealed commonality of urease receptors among gastric mucin, heparin, and heparinoid. Composite data on adherence kinetics modulated by pH, salt, incubation time, and concentration of urease or mucin were indicative of conformation-dependent ligand-receptor interaction. A simple, reproducible and high yield method ofHelicobacter pylori urease enzyme purification was developed using a heparinoid (Cellufine sulfate) affinity gel. The purification method involved two sequential steps using the same gel that takes advantage of the differential affinity of urease to the heparinoid at two levels of hydrogen ion concentration. SDS-polyacrylamide gel electrophoresis analysis of affinity-purified urease revealed two major protein bands with about 62- and 30-kDa molecular mass. When whole cell lysates of clinical and laboratory strains of H. pylori were probed by Western blot, anti-urease hyperimmune serum produced by affinity-purified urease in rabbit recognized only the two bands corresponding to the urease A and B subunits. To probe the molecular relevance of affinity gel adherence to mucin adherence, the purified urease was derivatized withN-hydroxysuccinimidobiotin and used in adherence assays. Competitive inhibition tests revealed commonality of urease receptors among gastric mucin, heparin, and heparinoid. Composite data on adherence kinetics modulated by pH, salt, incubation time, and concentration of urease or mucin were indicative of conformation-dependent ligand-receptor interaction. Underscoring the significance of urease in Helicobacter pylori gastro-duodenal infection are the observations that urease is essential for colonization in animal models (1Eaton K.A. Krakowka S. Infect. Immun. 1994; 62: 3604-3607Crossref PubMed Google Scholar, 2Tsuda M. Karita M. Morshed M.G. Okita K. Nakazawa T. Infect. Immun. 1991; 62: 3586-3589Crossref Google Scholar) and that native (3Marchetti M. Arico B. Burroni A.A. Figura N. Rappuoli R. Ghiara P. Science. 1995; 267: 1655-1658Crossref PubMed Scopus (535) Google Scholar) or recombinant urease subunits (4Lee C.K. Weltzin R. Thomas Jr., W.D. Kleanthous H. Ermak T.H. Soman G. Hill J.E. Ackerman S.K. Monath T.P. J. Infect. Dis. 1995; 172: 161-172Crossref PubMed Scopus (203) Google Scholar, 5Michetti P. Corthezy-Theulaz I. Dovin C. Haas R. Vaney A.C. Heitz M. Bille J. Kraehenbuhl J.P. Saraga E. Blum A.L. Gastroenterology. 1994; 107: 1002-1011Abstract Full Text PDF PubMed Scopus (217) Google Scholar, 6Ferrero R.L. Thiberge J.M. Huerre M. Labigne A. Infect. Immun. 1994; 62: 4981-44989Crossref PubMed Google Scholar) could protect animals from experimental challenge when used as a vaccine. To date, urease is the first vaccine candidate to be tested in human clinical trials (7Haas R. Meyer T.F. Biologicals. 1997; 25: 175-177Crossref PubMed Scopus (7) Google Scholar). Although urease is biosynthesized and localized within the cytosolic compartment of H. pylori, it ultimately accumulates on the bacterial surface through a proposed fourth mechanism of protein export to a subcellular compartment (8Phadnis S.H. Parlow M.H. Levy M. Ilver D. Caulkins C.M. Connors J.B. Dunn B.E. Infect. Immun. 1996; 64: 905-912Crossref PubMed Google Scholar, 9Dunn B.E. Vakil N.B. Scheinder B.G. Miller M.M. Zitzer J.B. Peutz T. Phadnis S.H. Infect. Immun. 1997; 65: 1181-1188Crossref PubMed Google Scholar). According to this model, H. pylori cells undergo spontaneous autolysis, presumably driven by a yet unidentified autolysin gene. The subsequent release of cellular debris brings the urease enzyme to the surface of other bacterial cells with which it gets in contact in vivo or in vitro. A separate study, however, has suggested that autolysis may only be a minor event compared with active secretion of the enzyme (10Vanet A. Labigne A. Infect. Immun. 1998; 66: 1023-1027Crossref PubMed Google Scholar). Regardless, the surface localization of urease protein potentially confers a central pathogenic role for this cytosolic protein. Urease may influence H. pylori nutrition via utilization of urea-derived ammonia for protein synthesis (11Williams C.L. Preston T. Hossack M. Slater C. McColl K.E.L. FEMS Immunol. Med. Microbiol. 1996; 13: 87-94Crossref PubMed Google Scholar) and induction of proinflammatory cytokines (12Harris P.R. Mobley H.L.T. Perez-Perez G.I. Blaser M.J. Smith P.D. Gastroenterology. 1996; 111: 419-425Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar) and promote bacterial survival in acidic medium by generating ammonia within its immediate mucinous pericellular space. Urease is one of the most abundant proteins of H. pylori; therefore, understanding the host-urease interactive events may yet hold the key to a rational and efficacious mode of treatment and prevention of colonization. To date, however, the fundamental mechanism underlying the essential role of urease in the colonization of the gastric mucosa remains unknown (13Labigne A. de Reuse H. Infectious Agents and Disease. 5. Lippincott-Raven Publishers, Philadelphia1996: 191-202Google Scholar). A major deterrent in elucidating this essential role is the lack of a simple, reliable, and high yield method of purification of this enzyme. Easy access to highly purified urease proteins by investigators should therefore facilitate advances in therapeutic or preventive approaches based on the abrogation of known or yet undetermined biological activity of thisHelicobacter protein. Previous purification methods of native or recombinant urease invariably required two or several steps involving conventional size exclusion and cation exchange (14Evans Jr., D.J. Evans D.G. Kirkpatrick S.S. Graham D.Y. Microb. Pathog. 1991; 10: 15-26Crossref PubMed Scopus (115) Google Scholar) or fast performance liquid chromatography (FPLC) 1The abbreviations used are: FPLC, fast protein liquid chromatography; BHI, brain heart infusion; FBS, fetal bovine serum; ELISA, enzyme-linked immunosorbent assay; PAGE, polyacrylamide gel electrophoresis. combining cation exchange, size exclusion chromatography, and sometimes the conventional hydrophobic interaction gel (4Lee C.K. Weltzin R. Thomas Jr., W.D. Kleanthous H. Ermak T.H. Soman G. Hill J.E. Ackerman S.K. Monath T.P. J. Infect. Dis. 1995; 172: 161-172Crossref PubMed Scopus (203) Google Scholar, 15Dunn B.E. Campbell G.P. Perez-Perez G.I. Blaser M.J. J. Biol. Chem. 1990; 265: 9464-9469Abstract Full Text PDF PubMed Google Scholar, 16Hu L.-T. Mobley L.T. Infect. Immun. 1990; 58: 992-998Crossref PubMed Google Scholar). FPLC requires sophisticated instrumentation and expensive columns usually inaccessible to small research laboratories, and the multiplicity of chromatographic steps tends to compromise the recovery rate in terms of both protein and catalytically active enzyme. Immunoaffinity purification has been performed using monoclonal antibody (7Haas R. Meyer T.F. Biologicals. 1997; 25: 175-177Crossref PubMed Scopus (7) Google Scholar) that requires a time-consuming antibody-coupling step to the support matrix and with the column so designed not usable over an extended period of time. The simplest method used so far was the procedure involving conventional agarose gel filtration and DEAE-cellulose ion exchange (14Evans Jr., D.J. Evans D.G. Kirkpatrick S.S. Graham D.Y. Microb. Pathog. 1991; 10: 15-26Crossref PubMed Scopus (115) Google Scholar). As a first step to facilitate further studies on the pathogenic mechanism of urease, we developed a simpler method of purifying the native enzyme from crude bacterial extract. In this study, we took a step further by using a single type of gel for a two-stage conventional affinity chromatography. The gel, a heparinoid consisting of cellulose matrix with sulfate esters as functional groups, has been shown to be useful for purification of a wide range of viruses, enzymes, and other biomolecules that interact with a heparinoid ligand. Lipopolysaccharide does not bind to the sulfated polymer, making this material suitable for detoxification of biological substances (Amicon product manual). Binding to the heparinoid gel prompted us to use the urease protein thus purified in adherence assays, using other sulfoconjugates as receptor substrate. The observed high affinity binding to heparin and mucin was discussed in relation to the possible adhesin function byH. pylori urease. Fresh clinicalH. pylori strains 130, 132, and 135, isolated as described previously (17Kabir A.M.A. Aiba Y. Takagi A. Kamiya S. Miwa T. Koga Y. Gut. 1997; 41: 49-55Crossref PubMed Scopus (297) Google Scholar), and NCTC 11637 were used for urease purification experiments (number 130) and for Western blot analysis (all strains). The strains were obtained from Dr. Yuji Aiba (Department of Internal Medicine IV, School of Medicine, Tokai University, Isehara, Kanagawa, Japan). Stock culture of each strain was prepared by initial passage in brain heart infusion (BHI, Difco) agar plate containing 5% defibrinated horse blood (4 days, 37 °C) and then passaged to 20-mlBrucella broth (Difco) in 100-ml bottle (plus 0.2% β-cyclodextrin and 10% fetal bovine serum (FBS)). Bottles were incubated overnight with gyratory shaking at 37 °C. The gas phase inside the bottle for all broth cultures in this study was replaced with a mixture of 80% nitrogen, 10% hydrogen, and 10% carbon dioxide prior to incubation. Bacterial cell pellets were collected and stored as stock culture at −80 °C in 20% glycerol or 10% skim milk containing 2% sodium glutamate. For mass urease production, a stock culture of strain 130 was thawed, passaged to 20-ml Brucellabroth, and incubated for 24 h at 37 °C with gyratory shaking. Five ml of the resulting bacterial broth suspension were inoculated to 1-liter bottles containing 200-ml Brucella broth medium with 5% FBS. After overnight incubation with gyratory shaking, 5 ml of the suspension was again passaged to 1-liter bottles containingBrucella broth with 2–5% FBS. The bottles were incubated for 48 h at 37 °C using a bidirectional shaker at 75 rpm (Takasaki Scientific Instruments Corp.). The cell biomass was collected by centrifugation at 12,000 × g for 20 min. To compare urease yield according to cultivation medium composition, BHI broth instead of Brucella broth was used under exactly the same cultivation conditions. H. pylori whole cells used in adherence assays were propagated once from stock culture by inoculation of Brucella broth with 2% FBS and harvested 24 h later. The whole cells were immediately biotinylated as described previously (18Ofek I. Courtney H. Schifferli D.M. Beachey E.H. J. Clin. Microbiol. 1986; 24: 512-516Crossref PubMed Google Scholar). The cell biomass pelleted during harvest was collected by scraping with a sterile metal spatula and directly transferring the cell pellet to 10-ml polypropylene tubes. About 1 g or less ofH. pylori cells (wet weight) was transferred to each tube, and the material was spread as thinly as possible on the tube wall and stocked at −80 °C until use. To extract urease, the cell pellet was thawed at room temperature and then vortexed with about 6.5 ml of sterile distilled water per tube for a total of 20 s with brief stops every 5 s. Cells from the mixture were removed by centrifugation at 15,000 × g for 30 min, and the supernatant was filtered using a 0.22 Millex GV Millipore filter. The filtered sample or crude urease extract was added with a 10× concentration of 20 mm phosphate, 1 mm EDTA buffer, 1 mm 2-mercaptoethanol (pH 6.5) at a volume ratio of 1:10 to the total crude urease extract volume. The resulting buffered extract was adjusted to pH 6.5. The enzymatic activity of urease was quantified using a previously described method based on jackbean urease standard (19Nagata K. Mizuda T. Tonokatu Y. Fukuda Y. Okamura H. Hayashi T. Shimoyama T. Tamura T. Infect. Immun. 1992; 60: 4826-4831Crossref PubMed Google Scholar). Results were expressed in units, with 1 enzyme unit equivalent to 1 μmol of ammonia liberated from urea per min per mg of protein. For preliminary optimization experiments, Cellufine sulfate (Amicon) and heparin-agarose (Sigma) were tested for urease affinity adherence at different pH. Equal volumes of 0.8 ml Cellufine sulfate, cellulose, heparin-agarose, or beaded agarose preadjusted to desired pH in duplicate into glass tubes were equilibrated with the adhesion medium (20 mm phosphate, 0.05% Tween 20). One ml of crude urease extract prediluted 10 times with phosphate buffer at different pH was dispensed to each tube containing gel that had been pre-equilibrated to the same pH as the urease sample. To determine the effect of physiological salt concentration on binding affinity, another set of tubes similarly prepared contained 0.15 m NaCl in the adhesion medium. The tubes were then shaken in slanted position at room temperature for 15 min using a bidirectional shaker. The gels were centrifuged at low speed and were washed three times with 10 gel volumes of PE buffer (20 mm phosphate, 1 mm EDTA) with pH similar to the gel pH. After aspirating the last wash buffer, bound urease was eluted with 2 ml/tube of 2 m NaCl in PE buffer, pH 7.0. After pelleting the gels, urease enzyme activity in the bound and unbound fractions was measured as described above. Cellufine sulfate was subsequently selected over heparin-agarose based on its generally higher affinity and higher pH optimum. In a typical experiment, the step A gel column (14 mm diameter) containing 155 mm of Cellufine sulfate bed height was equilibrated with PE buffer, pH 6.5 (PE65). About 5.5 ml of crude urease extract was then applied to the step A column at a flow rate of 9 ml/min. Elution was done with PE65, and the eluate was fractionated with an automatic fraction collector (Atto) (1.25 ml/fraction). Protein concentration in the void volume was measured continuously with a UV monitor (280 nm). Urease eluting with the void volume as the first peak was confirmed by enzyme measurement as described above. For analytical purposes, the whole sample input was fractionated, resulting in a total of three major peaks with the urease enzyme fraction coincident with the first peak. For routine urease production purposes, elution was terminated after the first peak and the tail of the second peak were eluted. Fractions were immediately analyzed quantitatively for enzyme activity. Urease-containing fractions were then selected, pooled, adjusted to pH 5.5, and adsorbed to the step B column (14-mm diameter) with about 63 mm of Cellufine sulfate bed height that had been preequilibrated with PE buffer, pH 5.5. The same flow rate as in step A was maintained followed by an extensive wash. For biotin labeling of urease and for immunizations, 20 mm phosphate buffer, pH 5.5, was used (PO55) for washing the step B column. In all experiments, gel-bound urease was eluted using 20 mm phosphate buffer, pH 7.4, containing 0.15 m NaCl (PO74). Eluted fractions collected in 2.5-ml volumes were quantitatively analyzed for enzyme activity and protein content before storage at −80 or −135 °C. In some experiments, the peak fractions were immediately labeled with biotin (see procedure below). Some affinity-purified urease samples were quantitatively checked for remaining enzymatic activity before and after a single freeze-thaw cycle. To determine whether a single step method will result in selective binding of urease to the Cellufine sulfate gel, the crude urease extract was adjusted to pH 5.5, immediately adsorbed to the step B gel, and eluted as above. Quantitative protein analysis was performed using the Bradford test reagent (Bio-Rad). The percentage urease content of crude extract or partially purified samples (step A pooled eluate) was determined by analyzing the integrated densities of protein bands in SDS-PAGE gels using an image analyzer (Aspect, Mitani Corporation, Fukui-ken, Japan) system with software installed on a NEC PC-9801RX personal computer. The total urease content was calculated by adding the urease A and B subunit percentage integrated densities. The percentage obtained was used to calculate the total urease protein contained in a crude or partially purified test sample based on the total sample protein obtained by the Bradford test (mg/ml). Step A protein recoveries were based on the urease protein (about 29.5–32%) contained in the original crude extract, while step B recoveries (final product) were based on both the urease protein contained in the crude extract and the total protein content of the crude extract. Percentage enzyme recovery calculation was based on the sum total of enzyme activity (units/ml) recovered in all positive fractions just after elution from the step A or B column compared with the original enzyme activity just before purification. Anti-urease rabbit serum was produced using 100 mg of affinity-purified urease as antigen mixed with an equal volume of a block copolymer adjuvant CRL89–41 (TiterMax Classic, Cytrx Corp.). The resulting emulsion was administered subcutaneously to a 2.5-kg Japanese White rabbit at four different sites. At 3, 4, and 5 weeks after primer immunization, the same amount of urease protein with the same adjuvant was administered via the same route. The rabbit was exsanguinated 3 weeks after the last booster, and total serum was collected followed by antibody titration (enzyme-linked immunosorbent assay (ELISA)) and storage at −30 °C. Anti-whole cell serum was prepared by immunizing a rabbit using whole cell homogenate of H. pylori number 130. The antigen was prepared by disrupting broth cultivated H. pylori number 130 cells using a glass bead homogenizer (MSK, B. Braun Flexible Biotechnology). A sample of whole cell homogenate was plated onto BHI blood agar plates to confirm cell disruption. The antigen was dispensed in aliquots and stored at −80 °C until use. For primer immunization, about 3 mg of the lysed cell suspension was mixed with an equal volume of complete Freund adjuvant. The emulsified antigen was injected subcutaneously at weeks 2, 3, and 4 postprimer. In the fifth week, the same amount of antigen was injected intravenously via ear vein without adjuvant. The rabbit was exsanguinated 2 weeks after the last booster. Serum was collected for ELISA titration and stored as hyperimmune anti-whole cell serum at −30 °C until use. The antigenicity of affinity-purified urease was assessed by checking the antibody response of immunized rabbit using the whole cell lysate or purified urease as antigen coating for ELISA antibody titration. Assay plates were prepared by coating 96-well Immulon 2 plates (Dynatech Laboratories, Inc.) with either whole cell lysate of strain 130 H. pylori or affinity-purified urease. The whole cell lysate was prepared by dissolving whole H. pylori cells in 1% SDS, and unsolubilized cells were removed by centrifugation. For the assay, the solubilized whole cell antigen or affinity-purified urease was diluted to a final concentration of 5 μg/ml using carbonate-bicarbonate buffer, pH 9.6, as diluent. About 100 μl of this preparation was dispensed per well, and plates were incubated overnight at 4 °C. The plates were washed three times with PBS containing 0.05% Tween (PBS-Tween) and blocked with 3% bovine serum albumin, fraction V (Sigma) for a 1-h incubation at 37 °C. Plates were washed three times with PBS-Tween, and 100 μl of the test rabbit serum prediluted 100 times with PBS-Tween was dispensed to each well. After 1 h at 37 °C, wells were washed three times with PBS-Tween and 100 μl of horseradish peroxidase-goat anti-rabbit IgG conjugate (Cappel) in suitable dilution was dispensed to each well. After 30 min of incubation at 37 °C, plates were washed five times with PBS-Tween. Color reaction was developed withortho-phenylenediamine dihydrochloride and was stopped with 3 n H2SO4. Absorbance was read at 490 nm. Two wells/plate not coated with the antigen but treated similarly as the test wells were used as the blank whose OD was subtracted from all test well readings. Titers of test sera were determined based on the regression of a standard calibration curve co-processed with test samples. To verify the mechanism of adherence by urease to the affinity gels, affinity-purified urease was biotinylated just after elution from the step B column for use in an adherence assay. The purified urease was labeled via amino groups with N-hydroxysuccinimide biotin (Sigma) for 180 min (room temperature) at different (1:1 to 128:1) biotin:protein molar ratio based on the monomeric mass of urease. The labeled urease samples were monitored for enzyme activity at different incubation time points for up to 180 min. Labeling was quenched by 20-fold dilution of the sample with PO55 buffer, and the solution was readjusted to pH 5.5 and readsorbed to and eluted from the step B affinity gel as described above. To detect, quantify, and determine the lower detection limit for biotin-labeled urease fractions, samples were coated on ELISA plates and detected using streptavidin-horseradish peroxidase conjugate (Zymed). Urease labeled at a 1:32 biotin:protein molar ratio was found to have a lower limit of detection at about 10 ng/ml. In another instance, biotinylated urease was blotted onto nitrocellulose membrane after SDS-PAGE, and the urease A and B subunits were detected with streptavidin-horseradish peroxidase conjugate as outlined below to confirm specificity of labeling as well as detection of both subunits A and B at the biotin:protein molar ratio used. Peak fractions of labeled urease eluted from the step B column were stored at −135 °C. SDS-PAGE (10% polyacrylamide gel) (20Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207460) Google Scholar) under reducing conditions was used to analyze the purification process. To confirm the purity of affinity-purified urease protein, two approaches were followed. First, Western blot was conducted with whole cell lysates of H. pylori strains NCTC 11637, numbers 130, 132, and 135 as antigens probed by anti-urease rabbit serum. Second, crude urease extract of H. pyloristrain 130 was probed in a Western blot with rabbit anti-whole cell serum. In this step, an unfixed duplicate gel was used to transfer the electrophoresed protein to nitrocellulose membrane by the standard procedure of Towbin (21Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-5354Crossref PubMed Scopus (44931) Google Scholar). The blotted proteins were then probed with the appropriate rabbit serum in a suitable dilution, and the rabbit IgG complexed with specific antigen was probed with anti-rabbit IgG-horseradish peroxidase conjugate (Cappel). Blots were developed with 4-chloro-1-naphthol, and the reaction was stopped with distilled water. Crude gastric mucus was obtained from the stomach of a 2-month-old weanling pig. To prepare crude mucus, the lumen of isolated stomach was perfused with PBS, pH 7.4, containing 0.1m phosphate, 0.15 m NaCl, 5 mm N-ethylmaleimide, 1 mm phenylmethylsulfonyl fluoride, and 1 mm EDTA. After exposing the luminal wall through a greater curvature incision, the mucosal surface was scraped using a metal spatula with a minimum amount of the perfusion buffer. The scraped material was homogenized with a Polytron homogenizer (Kinematica Gmbh, Switzerland) at 12,000 rpm for 5 min. The sample was centrifuged sequentially at 15,000 × g and 25,000 × g, both at 4 °C, with the supernatant collected in both instances followed by dialysis in distilled water and freeze-drying. Preparation of purified mucin from crude gastric or duodenal mucus followed a previously described procedure (22Davies J. Carlstedt I. Nilsson A.-K. Hakansson A. Sabharwal H. van Alphen L. van Ham M. Svanborg C. Infect. Immun. 1995; 63: 2485-2492Crossref PubMed Google Scholar) involving two cycles of equilibrium density gradient centrifugation for 48 h. Periodic acid-Schiff-positive fractions from the resulting cesium chloride gradient were pooled and freeze-dried. The sample was then diluted in an appropriate volume of 0.1 m phosphate buffer, 0.1 m NaCl, pH 6.8, and passed through a Sepharose CL-4B column preequilibrated in the same buffer. Fractions showing a single band of glycoprotein (∼66 kDa) as revealed in Coomassie Brilliant Blue- and periodic acid-Schiff-stained duplicate SDS-PAGE gels (run under reducing conditions) were pooled, dialyzed in PBS, pH 6.8, and stored in aliquots at −80 °C until use in an adherence assay. Purified swine gastric mucin was labeled withN-hydroxysuccinimidobiotin (734:1 biotin:protein molar ratio based on a mucin molecular weight of 2 × 106) with the reaction carried out at room temperature for 1 h. Unbound biotin was removed using a 15-ml capacity Centricon-10 concentrator (Amicon) and stocked at −80 °C until use. Duodenal mucin was purified from the duodenum of the same weanling pig using the same procedure as above but was not labeled. For adherence and competitive inhibition assays, the ELISA format was used. Unless specified, gastric mucin was used for all procedures involving mucin. The adhesion medium consisted of 20 mm phosphate, 0.15 m NaCl, and 0.05% Tween 20 preadjusted to the desired pH. All tests were done in duplicate with two wells/sample/test. Wells were coated with substrate or ligand using 50 μl/well. Two blank wells in each plate not coated with receptor substrate but treated similarly as test wells were used to normalize optical density readings to base line. All quantitative analyses of test sample urease concentration that remained bound to the wells were based on the regression of a calibration curve generated by a known standard co-processed with the test samples. For all adherence assays, Immulon 2 ELISA plates were coated overnight at 4 °C with 250 μg/ml substrate (unless stated otherwise) consisting of either mucin or crude mucus in neutral PBS or of heparin (Eastman Kodak Co.) or melted agarose (Seakem) in pH 9.6 carbonate-bicarbonate buffer. Plates were blocked with 3% bovine serum albumin for 1 h and washed three times with PBS-Tween before all assay procedures. To confirm the effect of pH on adherence, about 2.5 μg/ml biotinylated urease (32:1 biotin:protein molar ratio) in adhesion medium with a pH of 2.0–8.0 was incubated in wells precoated with mucin, heparin, crude gastric mucus, duodenal mucin, or melted agarose for 1 h at 37 °C. Unbound urease was removed by washing five times with 200 μl of the adhesion medium having the same pH as the test well. The remaining bound urease was heat-fixed for 10 min at 65 °C (water bath). Wells were washed three times with PBS-Tween, pH 7.4, probed with streptavidin-horseradish peroxidase, and processed as in ELISA. To determine saturability of receptors, a 2-fold dilution of labeled urease was allowed to adhere to mucin-coated wells for 1 h at 37 °C (at pH 4.0, since labeled urease exhibited peak adherence at around this pH), and bound urease was probed by ELISA. In another experiment, ELISA wells were precoated overnight with a constant amount (5 μg/ml) of native urease (using pH 9.6 carbonate-bicarbonate coating buffer), and a 2-fold dilution of labeled mucin was allowed to adhere at pH 4.0 for 1 h at 37 °C followed by ELISA as described above. To determine the kinetics of adherence to mucin and heparin over time, biotinylated urease was prediluted at pH 4.0 and 7.0 (control) adhesion medium and incubated in mucin- or heparin-coated wells over a 5-h period. At several time points within this period, wells were washed five times with the adhesion medium having the same pH as the test well. Adherent urease was probed and quantified by ELISA as above. To probe the molecular make-up of urease receptor, a competitive inhibition assay was performed essentially following the same receptor-based ELISA using plate-immobilized heparin (250 μg/ml). Urease was mixed with 2-fold decreasing concentration of mucin, heparin-agarose, beaded agarose (Sepharose CL-4B, Amersham Pharmacia Biotech), Cellufine sulfate, or cellulose (Takara) at pH 4.0 and incubated for 60 min at 37 °C with gentle shaking. The mixtures were transferred to the heparin-coated wells and incubated with gentle shaking at 37 °C for only 30 min to avoid nonspecific adherence by heparinoid gel. The unbound urease with inhibitor was removed from each well by washing five times with pH 4.0 adhesion medium while the remaining urease was heat-fixed, and plates were processed by ELISA. To determine whether urease can prevent adherence of whole H. pylori cells, heparin or mucin was used as substrate (both precoated at 125 μg/ml) in an ELISA-based assay. A 2-fold dilution series of native urease at pH 4.0 with an initial concentration of about 380 mg/ml was allowed to adsorb to immobilized heparin or mucin for 1 h at 37 °C. A constant amount of biotinylated whole bacterial cells (10-fold dilution of 0.7 OD at 550 nm or about 1.43 × 107 colony-forming units/well) was dispensed to each well and incubated for anoth