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FEMS Microbiology Letters 77 (1991) 51-54 Published by Elsevier 51 FEMSLE 04234 The demonstration of nickel in the urease of Helicobacter pylori by atomic absorption spectroscopy P.R. H a w t i n 1 H . T . Delves 2 a n d D . G . N e w e l l 3 i PHLS, Southampton General Hospital, Southampton, 2 Department of Clinical Biochemistry, University of Southampton, Southampton, and 3 Division of Pathology, PHLS Centre for Applied Microbiology and Research, Porton Down, Salisbury, U.I( Received 9 July 1990 Revision received 24 July 1990 Accepted 31 July 1990 Key words: Helicobacterpylori; Urease; Nickel 1. SUMMARY Fast protein liquid chromatography and S D S P A G E have been used to isolate and purify Helicobacter pylori urease. A nickel component of the urease was detected in the purified proteins by atomic absorption spectroscopy. The nickel was present only in the 61 kDa polypeptide and in the ratio of between five and six atoms to one molecule of urease, suggesting a hexameric structure. These results are discussed in relation to other bacterial ureases and urease activity at low pH. 2. I N T R O D U C T I O N Helicobacter pylori is a Gram-negative, spiral organism which colonises the gastric mucosa of humans and some non-human primates. There is a close association between the presence of the Correspondence to: P.R. Hawtin, PHLS, Level B, South Academic Block, Southampton General Hospital, Tremona Road, Southampton, SO9 4XY, U.K. organism, gastritis and duodenal ulceration. A major characteristic of H. pylori is the production of abundant urease. This urease has attracted much attention due to its high activity, antigenicity and its use in diagnostic tests. The complete D N A sequence of the urease gene is now known [1]. Two polypeptides of 61 and 28 kDa have been shown to be essential for enzyme activity [2]. A third polypeptide (56 kDa) co-purifies with the urease by size-exclusion chromatographic techniques [2]. Although the urease differs from other bacterial ureases in its polypeptide composition it maintains considerable sequence homology with these same enzymes [1]. There is also antigenic conservation between the ureases of different gastric spiral organisms supporting the possibility of a common evolutionary origin for these bacteria [31. Many ureases contain nickel in varying amounts, which is usually essential to enzyme activity [4]. The aim of this study was to demonstrate the presence of nickel in H. pylori urease, to determine the ratio of nickel atoms per molecule of enzyme and to establish the distribution of the nickel in the two polypeptides. 0378-1097/91/$03.50 © 1991 Federation of European Microbiological Societies 52 3. MATERIALS A N D M E T H O D S 3.1. Fast protein liquid chromatography (FPLC) A sonicate was prepared from H. pylori N C T C 11638 and was fractionated by size exclusion gel filtration using a 200 ~1 injection loop on a Superose 6 FPLC column (Pharmacia Ltd.) as previously described [2]. The urease activity was estimated by double diluting, in phosphate-buffered saline. Briefly, 100 /zl of the fraction (starting at 1 : 10 dilution) was incubated at room temperature with 100 /~1 of 100 mM urea containing 0.2% (w/v) phenol red. After 10 rain the absorbance was read at 540 nm. 3.2. Protein estimation Protein estimation was by the method of Lowry et al. [5] using bovine serum albumin as the standard. 3.3. SDS-polyacrylamide gel electrophoresis (SDSPAGE) Linear gradient (10-25% w / v ) S D S - P A G E was performed as described by Lambden et al. [6] and the protein bands visualised by Coomassie brilliant blue staining. The major bands were carefully cut out with a razor blade and collected into ug/[ a microfuge tube for further SDS PAGE and atomic absorption spectroscopy. Relative molecular masses were estimated from standards (Sigma Ltd.) containing a-lactalbumin, trypsin inhibitor, trypsinogen, carbonic anhydrase, glyceraldehyde3-phosphate dehydrogenase, egg albumin and bovine serum albumin. 3.4. Atomic absorption spectroscopy Nickel concentrations in FPLC fractions and S D S - P A G E gel pieces were measured by electrothermal atomisation and atomic absorption spectroscopy using a Perkin Elmer Zeeman 5000 AA instrument with a HGA500 furnace and a Model 3600 data station. Integrated absorbance signals at 232.0 nm from 10 #1 injections of 1 : 3 aqueous dilutions of the FPLC fractions were compared with those from aqueous nickel standards for quantitation. 4. RESULTS 4.1. The isolation and purification of urease The urease activity from fractionated sonicated supernatant was concentrated in fractions 14 and [mg/ml](A540) Nickel 120 0,6 100 0.5 80 0.4 60 0.3 40 0.2 20 0.1 Eluent 10 11 12 14 13 Fraction 0.0 15 16 17 18 19 No. Fig. 1. FPLC separation of H. pylori sonicate supernatant. The protein concentration (rng/ml) ( + . . . . . . (zx zx), and nickel concentration (/~g/l) ( ~ ) of each fraction is shown. + ), urease activity (A540) 53 123 4 66 61 56 56 m 45 i!~iiiii~i! ~i kDa ...... 36 kDa kDa 29 28 28 .... 24 ~iiii~, 21 (0.166/0.353 mg ml ~) of the total protein in fraction 15. Thus, the best estimate gave 5.21 nickel atoms per molecule of urease. Nickel was detected in the 61 kDa polypeptide only, when the S D S - P A G E gel slice containing this polypeptide was subjected to atomic absorption spectroscopy. No nickel was detected in the gel slices containing the 56 or 28 kDa polypeptides or in a blank area of gel. 5. DISCUSSION (a) (b) Fig. 2. S D S - P A G E of (a) FPLC fraction 15; (b) gel slices containing the major polypeptides of FPLC fraction 15: lane 1, 61 kDa polypeptide; lane 2, 56 kDa polypeptide; lane 3, 28 kDa polypeptide; and lane 4, molecular mass markers. 15 with a small amount detected in fraction 16 (Fig. 1). Fraction 15 comprised three major polypeptides, of apparent molecular mass 61, 56, and 28 kDa which represented 25, 18 and 22% respectively of the total protein deduced by scanning gel densitometry of the Coomassie blue stained gel (Fig. 2a). S D S - P A G E gel slices, containing each of the major polypeptides, were again run on S D S - P A G E to check purity (Fig. 2b). 4.2. Identification of nickel The nickel content of FPLC fractions 10 to 19, as determined by atomic absorption spectroscopy, was confined to fractions 14, 15 and 16 (Fig. 1). The concentration of nickel in each of these fractions was 41, 100 and 17/~g 1-~ respectively. On the basis of the previously established molecular mass (510 kDa) of H. pylori urease [2], the calculation of the number of atoms of nickel per molecule of urease was performed on the nickel concentration of fraction 15. This fraction showed the highest specific activity in relation to nickel. The native urease consists of the 61 and 28 kDa polypeptides only [2] which represented 47% Nickel has been identified as an integral part of H. pylori urease by atomic absorption spectroscopy. This finding is consistent with the presence of nickel reported in other bacterial ureases including medically important bacteria such as Klebsiella aerogenes [4]. The presence of nickel in H. pylori urease would be expected because of the inhibition of enzyme activity by acetohydroxamic acid [7]. It is known that the hydroxamate moiety binds to this metal ion [4]. Mooney et al. [81 have shown that acetohydroxamic acid abolished the urea/urease dependent acid resistance of H. pylori at pH 1 and 2, and reduced growth at low p H in the presence of urea without inhibitor. On the basis of these results Mooney et al. have suggested that this urease inhibitor could be used for anti-H. pylori therapy. However, the nickel component of Arthrobacter oxydans urease can be released under such acidic conditions, leading to irreversible loss of activity [9]. Moreover, the activity of H. pylori urease is irreversibly lost below the pH of 4.5 [10] indicating a similar mechanism. Therefore, interpretation of the action of urease inhibitors at very low pH may be difficult. H. pylori urease is inhibited by the divalent cation chelator ethylenediamine-tetraacetic acid. Nevertheless, twice the amount of chelator was required for the same degree of inhibition as Proteus mirabilis urease [7]. This suggests that the nickel is strongly bound in the H. pylori enzyme which is supported by the nickel remaining in the urease after denaturation and electrophoresis. The estimate of five atoms of nickel per molecule of urease would appear to be low. The 61 and 28 kDa polypeptides are in approximately 54 equimolar amounts producing a deduced subunit mass of 89 kDa, and as the molecular mass of the urease is greater than 500 kDa, then the suggested structure is hexameric. This would indicate that the probable number of nickel atoms is six. These results support the model of six copies of each of the two polypeptides in the native enzyme suggested by Hu and Mobley [11]. The detection of nickel in the 61 kDa polypeptide is consistent with the identification of a histidine-rich region, in the amino acid sequence of H. pylori urease, which is thought to contain the nickel-binding active site [1]. We have previously reported that a monoclonal antibody (CPll), which recognised the 28 kDa polypeptide by Western blotting, inhibited and captured the active urease, may, therefore, be directed against an epitope at or near the active site [2]. However, the detection of the nickel in the 61 kDa polypeptide would suggest that the enzyme active site is in this larger polypeptide. If this is the case, the monoclonal antibody may either spatially occlude the substrate binding site or distort the 3-dimensional conformation of the protein in the antigen/antibody complex. Both mechanisms could reduce enzyme activity. Our results confirm that nickel is an integral part of H. pylori urease and is, therefore, probably essential for urease activity. The effect of low gastric pH on the nickel in H. pylori urease should be investigated further before urease inhibitors are considered for anti-H, pylori therapy. REFERENCES [1] Clayton, C.L., Pallen, M.J., Kleanthous, H., Wren, B.W. and Tabaqchali, S. (1990) Nucleic Acids Res. 18, 362. [2] Hawtin, P.R., Stacey, A.R. and Newell, D.G. (1990) J. Gen. Microbiol., in press. [3] Newell, D.G., Lee, A., Hawtin, P.R., Hudson, M.J., Stacey, A.R. and Fox, J. (1989) FEMS Microbiol. Lett. 65, 183186. [4] Hausinger, R.P. (1987) Microbiol. Rev. 51, 22-42. [5] Lowry, O.H., Rosebourgh, N.J., Farr, L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. [6] Lambden, P.R., Heckels, J.E., James, L.T., and Watt, P.J. (1979) J. Gen. Microbiol. 114, 305-312. [7] Mobley, H.L.T., Cortesia, M.J., Rosenthal, L.E. and Jones, B.D. (1988) J. Clin. Microbiol. 26, 831-836. [8] Mooney, C., Munster, D.J., Bagshaw, P.F. and Allardyce, R.A. (1990) Lancet i, 1232. [9] Mobley, H.L.T. and Hausinger, R.P. (1989) Microbiol. Rev. 53, 85-108. [10] Taylor, M.B., Goodwin, C.S. and Karim, Q.N. (1988) FEMS Microbiol. Lett. 55, 259-262. [11] Hu, L-T. and Mobley, H.L.T. (1990) Infect. 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