Download The demonstration of nickel in the urease of Helicobacter pylori by

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.
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