From the Department of Biochemistry, the
¶¶ Department of Microbiology, and the
Central
Laboratory for Biomedical Research and Education, Yamaguchi University
School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi 755-8505, Japan, the ¶ Center for Gene Research, Yamaguchi University, Ube,
Yamaguchi 755-8505, Japan, and the
Department of Biochemistry and Molecular
Biology and National Science Foundation EPSCoR Oklahoma Laser Mass
Spectrometry Facility, University of Oklahoma Health Sciences Center,
Oklahoma City, Oklahoma 73190
Received for publication, October 17, 2000, and in revised form, January 2, 2001
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ABSTRACT |
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Chlamydiae proliferate only within
the infected host cells and are thought to be "energy parasites,"
because they take up ATP from the host cell as an energy source. In the
present study, we isolated from Chlamydia pneumoniae the
gene encoding adenylate kinase (AK). Using the enzyme produced in
Escherichia coli, its properties were characterized.
Km values for AMP and for ADP of the purified
C. pneumoniae AK (AKcpn) were each 330 µM,
which is significantly higher than the reported values of other AKs, whereas Km for ATP was 24 µM,
which was rather lower than others. AKcpn contains 1 g atom of
zinc/mol of 24,000-dalton protein. Mass spectrometric analysis of AKcpn
and analysis of properties of mutated AKcpn strongly suggested that
zinc is associated with four cysteine residues in the LID domain of the
enzyme. The apo-AKcpn that lost zinc retained AK activity, although
Km for AMP of apo-AKcpn increased about 2-fold and
Vmax decreased about one-half from that of
holo-AKcpn. The apo-AKcpn was more thermolabile and sensitive to
trypsin digestion than the holo-AKcpn. Moreover, the recovery in
vitro of the AK activity during the renaturation process of the
denatured apo-AKcpn was dependent on zinc. A mutated protein in which
cysteine residues in the LID domain were substituted by other amino
acids lost both zinc and enzyme activity. The mutated protein was more
sensitive to protease than the apo-AKcpn. These results indicate that
zinc in AKcpn, although not essential for the catalysis, stabilizes the
enzyme and probably plays a crucial role in proper folding of the
protein. Furthermore, the catalytic properties of AKcpn suggest a
distinctive regulatory mechanism in the metabolism compared with AKs in
other organisms.
Chlamydiae cause serious health problems in both humans and
animals. Chlamydia pneumoniae is a causative agent of
pneumonia and bronchitis (1) and may contribute to the pathogenesis of atherosclerosis (2). Despite its clinical importance, many aspects of
its biology and biochemistry have not yet been defined. Chlamydiae have
an obligatory intracellular lifestyle, growing only within host cells.
They are considered to be an "energy parasite," because they have
unique adenine nucleotide metabolism incorporating from host cells ATP
through ATP/ADP translocase and other high energy metabolites (3, 4).
Adenylate kinase (AK)1
catalyzes a reversible high energy phosphoryl transfer reaction between
ATP and AMP to generate ADP (5) and is considered to contribute to the
homeostasis of cellular adenine nucleotide composition (6). AK is
indispensable for the growth of Escherichia coli (7) and
Schizosaccharomyces pombe (8), suggesting that it is an
essential enzyme in single-cell organisms. Since chlamydiae have a
unique adenine nucleotide metabolism, their AK may have properties
distinct from other AKs.
In previous research, AK has been isolated from a variety of organisms.
The middle part of the protein is named the LID domain because of its
conformation. AKs are divided into two groups; the long and the short
forms with and without extra 27 amino acid residues, respectively, in
the LID domain (9, 10). Bacterial AKs and some vertebrate AK isozymes
such as AK2 and AK3 belong to the long form, whereas other vertebrate
AK isozymes such as AK1 are the short forms. In the long form AK
molecule, when substrates bind to the protein the LID domain comes from
a remote position in contact with the main body of the protein (11).
Some bacterial AKs were reported to contain zinc that contributes to
thermal stability of the protein (12, 13). Genetically engineered zinc-containing E. coli AK (AKeco) at the LID domain showed
enhanced thermal stability (14, 15). X-ray crystallography of AK from Bacillus stearothermophilus (AKbst) showed that zinc is held
by four cysteine residues in the LID domain (16). Thus, the LID domain
contributes to thermal stability of the protein by providing a
zinc-binding site. With respect to the biological importance of zinc in
enzymes other than as a catalytic role, zinc is also contained in zinc
finger that is thought to serve as a DNA-binding structure. Recently,
zinc has been reported to play a role in folding the zinc finger
proteins (17).
In the present study, we cloned the gene encoding AK of C. pneumoniae (AKcpn) and expressed it in E. coli. The
recombinant AK contained zinc that was bound by cysteine residues in
the LID domain of the enzyme molecule. From the kinetic analysis, AKcpn showed high Km for AMP and a low
Vmax value compared with AKeco. Analysis of the
apo-enzyme that had lost zinc and the mutant proteins, in which the
cysteine residues in the LID domain were substituted by other amino
acids, suggests that the zinc-containing LID domain contributed to
conformational stabilization and polypeptide folding of the AKcpn protein.
Materials--
P1,P5-Di(adenosine-5')-pentaphosphate
(Ap5A), p-(hydroxymercuri)benzenesulfonic acid
(PMPS), N-ethylmaleimide, and trypsin were purchased from
Sigma, pig AK1 (myokinase) from Roche Molecular Biochemicals (Basel,
Switzerland), 4-(2-pyridylazo)resorcinol (PAR) from Fluka (Buchs,
Switzerland). A standard sample for atomic absorption,
Zn(NO3)2 1 g/liter in 0.1 M
HNO3, and isopropyl- Bacterial Strains and Media--
E. coli was grown in
LB broth (Life Technologies, Inc.) or on LB agar plates (1.5% (w/v)
agar in LB broth) at 37 °C. E. coli CV2, a
temperature-sensitive AK mutant (7), was grown in LB broth at 30 °C.
When necessary, ampicillin (100 µg/ml) was added to the media. HEp-2
cells derived from human epidermoid carcinoma were infected with
elementary bodies (EB) of C. pneumoniae J138 (18), and the
bacteria were propagated in the cells using Iscove's modified
Dulbecco's medium (Life Technologies, Inc.) as described by
Matsushima et al. (19).
Preparation of EB of C. pneumoniae--
C.
pneumoniae-infected HEp-2 cells were harvested from 20 six-well
plastic dishes. After washing the cells with phosphate-buffered saline,
1 ml of a 0.05% trypsin, 0.53 mM EDTA solution was added to each well and incubated with the cells at 37 °C for 3 min. During
repeated washing with 1 ml of phosphate-buffered saline, detached cells
were collected by centrifugation at 3,000 × g and carefully suspended in 30 ml of 0.25 M sucrose, 10 mM sodium phosphate (pH 7.2), and 5 mM
L-glutamate (SPG buffer). The cells were subjected to the
freezing and thawing procedure; they were initially placed at
Enzyme Assay--
The enzyme activity of AK was measured as
described previously (20, 21). As a routine assay, ADP formation from
ATP and AMP was coupled with pyruvate kinase and lactate dehydrogenase reactions, and NADH oxidation was determined. For the assay of the
reverse reaction, formation of ATP from ADP was coupled with hexokinase
and glucose-6-phosphate dehydrogenase reactions, and NADP+ reduction was measured.
Protein Measurement--
Protein was measured according to
Bradford (22) using bovine serum albumin as a standard. The protein
concentrations thus determined with purified AKcpn were consistent with
the values calculated from the molar concentration of AKcpn determined
by amino acid analysis (23). The amino acid analysis was carried out
using the Millipore AccQ Tag System after hydrolysis of the protein in
6 N HCl at 120 °C for 24 h.
DNA Manipulations--
Restriction endonucleases, bacteriophage
T4 DNA polymerase, bacteriophage T4 polynucleotide kinase, Klenow
fragment of E. coli DNA polymerase I, Taq DNA
polymerase, and bacteriophage T4 DNA ligase were obtained from Takara
Shuzo (Kyoto, Japan) or New England BioLabs (Beverly, MA). The reaction
conditions with these enzymes were those suggested by the suppliers.
[ DNA Preparation from EB--
The EB suspension (300 µl) was
incubated in 25 mM EDTA, 0.5% SDS, 0.2 mg/ml proteinase K
at 56 °C for 1 h. TE-saturated phenol (300 µl) was added to
the suspension, and the mixture was shaken well. Then, 300 µl of
chloroform was added, and the mixture was shaken for 5 min, followed by
centrifugation at 12,000 × g for 10 min at 25 °C.
The upper layer and the interface were then placed in another tube and
mixed well with 400 µl of TE-saturated phenol/chloroform, followed by
centrifugation. The upper layer and the interface were mixed with 400 µl of chloroform and centrifuged. The upper layer was withdrawn and
mixed with 0.1 mg/ml RNase A and incubated at 37 °C for 1 h.
After extraction with TE-saturated phenol/chloroform four times, 2 M ammonium acetate and 70% ethanol were added to precipitate DNA, followed by centrifugation. After rinsing with 70%
ethanol, the precipitate was dissolved in 50 µl of TE buffer. Finally, a total of about 20 µg of genomic DNA was obtained.
Cloning of the AKcpn Gene--
Genomic DNA purified from
C. pneumoniae was broken into 2-3-kb DNA fragments by
sonication, ligated into the SmaI site of pUC18 to construct
a genomic library. The library was divided into 10 subgroups, each
containing about 1000 clones. A subgroup containing AK was
identified by PCR using the AKcpn specific primers. To
isolate the AK-positive clone, the library was introduced into E. coli CV2, and complemented clones were obtained on a plate incubated at 42 °C. Plasmid DNA from one of the clones was named pK238, which contained a 1.3-kb fragment.
Construction of Overproducer--
An AKcpn overproducer, pK253,
was constructed as follows. The plasmid pK238 was digested with
EcoRI and SalI to obtain a 1-kb fragment
containing the AKcpn gene. The fragment was cloned with pBluescript KS( Mutagenesis--
Mutagenesis of AKcpn was
carried out using the QuickChange site-directed mutagenesis kit
(Stratagene, La Jolla, CA). PCR reaction was performed with pK253 as a
template and the following synthetic DNA primer sets:
5'-GTGTTCAAGATTTCTTGCCCCCTCCGCTTCGCGTATCTACAACAC-3' and its
complementary DNA to obtain pK276 (AKcpn with double substitutions; C133A, C136A); 5'-GGACATACCGAAAGTCCAGACAGTCATGTGCCTTTGATAC-3' and its
complementary DNA to obtain pK273 (AKcpn with double substitutions; C149S, C152S); 5'-GTGTTCAAGATTTCTTAGCCCCTCCTGTTCGCG-3' and its complementary DNA to obtain pK279 (AKcpn with a substitution; C133S).
Using the primer sets for pK276, pK285 (AKcpn with quadruple substitutions; C133A, C136A, C149S, C152S) was constructed from pK273
as a template.
Overproduction of AKcpn in E. coli--
The
temperature-sensitive E. coli CV2 was used as the host cell
because of the low background AKeco activity. E. coli CV2 harboring pK253 was grown aerobically overnight at 37 °C in 10 ml of
LB broth supplemented with 100 µg/ml ampicillin. The whole culture
was then transferred to 2 liters of LB broth in the presence of
ampicillin, and the medium was further shaken until the cell density
reached A600 = ~0.6-0.8. Then, the culture
was chilled and shaken at 25 °C for 30 min, followed by the addition
of 0.4 mM
isopropyl- Purification of AKcpn--
Overproduced AKcpn was purified by
the procedure described by Bârzu and Michelson (26). The
cell-free extract (about 60 ml containing 360 mg of protein) was loaded
onto a 5-ml Affi-Gel Blue (Bio-Rad) column, equilibrated with 50 mM Tris-HCl (pH 7.5) at a flow rate of 15 ml/h. The column
was washed with 30 ml of the same buffer. AKcpn was eluted with 30 ml
of 50 mM Tris-HCl (pH 7.5) containing 0.05 mM
Ap5A, and 2-ml fractions were collected. The peak fractions
containing AKcpn (12-14 ml) were collected and directly applied to a
Sephadex G-100 (Amersham Pharmacia Biotech) column equilibrated with 50 mM Tris-HCl (pH 7.5). Eight milliliter fractions were
collected at a flow rate of 30 ml/h, and the peak fractions containing
AKcpn (32-40 ml) were pooled. To concentrate the solution, the pooled
fractions were dialyzed against a saturated ammonium sulfate solution,
and the samples were stored at 4 °C. Proteins from the purification
steps were resolved in 0.1% sodium dodecyl sulfate (SDS), 12.5%
polyacrylamide gel electrophoresis (PAGE) and stained with Coomassie
Brilliant Blue R-250.
Preparation and Use of Antibody Column--
Antisera against
AKcpn were obtained by three-time immunization of Japanese white
rabbits as previously described (27). Immunoblot analysis was carried
out as described previously (28) using anti-AKcpn serum diluted by
1:5,000-10,000. CNBr-activated Sepharose 4B (Amersham Pharmacia
Biotech) was used to prepare the antibody column for purification of
AKcpn as suggested by the supplier. The sample containing about 0.3 mg
of AKcpn or its mutant protein was loaded in a 0.5-ml bed volume of the
antibody column. The column was washed three times with 5 ml of 100 mM KCl, 3 mM NaCl, 2.5 mM
MgCl2, 1.25 mM EDTA, and 10 mM
HEPES (pH 7.3), then twice with 5 ml of 0.1% Triton X-100. When 50 mM glycine-HCl (pH 2.5) containing 0.1% Triton X-100 was
used to elute the wild-type AKcpn from the column, most of the protein
lost zinc. Therefore, the protein bound to the column was eluted with
50 mM glycine-HCl (pH 3.0) containing 0.1% Triton X-100.
Under these conditions, 73% of wild-type AKcpn held the zinc.
Preparation of Apo-AKcpn--
After addition of excess PMPS (500 µM) to the purified AKcpn (5-10 µM), zinc
ion was released from AKcpn upon binding of PMPS to cysteine residues,
and the released ion was trapped by 100 µM EDTA. After
standing at 25 °C for 10 min, 1 mM DTT was added to the
incubation mixture, and incubation was continued for another 10 min to
remove PMPS bound to the cysteine residues. The apo-enzyme preparation
was obtained after dialysis against 1,000-fold volume of 50 mM Tris-HCl (pH 7.5) to remove free PMPS, EDTA, and
DTT.
Determination of N-terminal Amino Acids--
The N-terminal
amino acid sequence of the purified AKcpn was determined using a
Shimazu PPSQ21 (Kyoto, Japan) amino acid sequencer as described
(29).
Zinc Analysis--
Atomic absorption was performed using 24 µM AKcpn solution with a Hitachi Z-6100 (Tokyo, Japan)
atomic absorption spectrophotometer. Zinc analysis using PMPS and PAR
was carried out using the method described by Hunt et al.
(30). Free zinc ion bound to PAR developed a red color with a maximal
absorption at 500 nm. The mercaptide bond formation was measured at
A250. The molar extinction coefficient of the
mercaptide bond was determined experimentally using a 100 µM cysteine solution and 0-200 µM PMPS in
50 mM Tris-HCl (pH 7.5). The value of 2.94 × 103/mol/cm was adopted to calculate the mercaptide bond concentration.
Mass Spectrometric Analysis--
The purified AKcpn (5 nmol) was
mixed with 5-fold PMPS (25 nmol), incubated at 25 °C for 10 min, and
dialyzed against 2 liters of 100 mM ammonium bicarbonate
(pH 8.2). The PMPS-treated AKcpn was digested with 4 µg/ml trypsin at
25 °C overnight; to remove salts in the sample, the peptides were
absorbed by ZipTipC18 (Millipore, Bedford, MA), washed, and then eluted
with 80% acetonitrile. A 1-µl aliquot of the sample was mixed with 1 µl of 10 mg/ml ferulic acid in 70% acetonitrile and 5% formic acid,
and the mixture was spotted on a sample plate for mass spectrometry.
Matrix-assisted laser desorption ionization time-of-flight mass
spectrometry (MALDI-TOF) was carried out using PerSeptive Biosystems
Voyager Elite (Framingham, MA).
Protease Sensitivity--
Samples containing 0.1 mg/ml protein
were incubated with 1 µg/ml trypsin at various temperatures for 0-30
min. AK activity was then assayed, and the activities of holo- and
apo-AKcpn were compared. Proteolytic products of the wild-type and the
mutant proteins were analyzed by SDS-PAGE.
Denaturation and Renaturation of AKcpn--
A 3-fold volume of 6 M guanidine-HCl (a final concentration of 4.5 M) was added to 6.0 µM enzyme dissolved in
1.25 µM EDTA and 50 µM DTT and incubated at
25 °C for 2 h. Then, the sample was diluted 10-fold with 50 mM Tris-HCl (pH 7.5) buffer in the presence of 0.2 mM DTT with or without ZnCl2. After incubation at 25 °C for 30 min, the AK activity was measured with the samples.
Cloning and Sequencing of the AKcpn Gene--
To clarify the
presence of the AK gene in C. pneumoniae, we
first carried out the PCR analysis with the bacterial genomic DNA using
primers designed from conserved regions of other AKs (31). A unique DNA
fragment of about 250 base pairs was obtained and sequenced. The
deduced amino acid sequence was homologous with other bacterial AKs,
suggesting that the C. pneumoniae genome contained at least
one AK gene. A genomic library of C. pneumoniae was constructed and divided into 10 subgroups, each containing about
1,000 clones. One subgroup of the library contained the AK
sequence as identified by PCR using primers newly designed from the
potential AK fragment obtained above. The subgroup was introduced into E. coli CV2 with a temperature-sensitive
mutation in the AK gene, and a clone that grew at 42 °C
was isolated. A plasmid was purified from the clone and named pK238
(Fig. 1A). The plasmid
contained a 1.3-kb insert at the SmaI site of pUC18. A DNA
sequence of 1.0-kb BamHI-EcoRI fragment in the
insert was determined (Fig. 1B) (DDBJ accession no.
AB022016). The fragment contained an open reading frame starting from
ATG that was not preceded by the Shine-Dalgarno sequence (SD) of
E. coli. However, the GTG codon, that was present 27 base
pairs upstream from the ATG codon in the same frame, accompanied SD.
This codon proved to be the start codon after determination of the
N-terminal amino acid sequence of the protein purified from E. coli (see below). The ribosome-binding site for translation
initiation of chlamydiae appears to be the same as that of E. coli (32). When we analyzed by immunoblot the protein existing in
the C. pneumoniae cells, the size of the native AKcpn was
similar to that of the recombinant protein expressed in E. coli.2 Therefore, GTG
should also be used for the start codon in C. pneumoniae.
The open reading frame contained 642 base pairs and encoded a protein
which had a sequence similar to that of AKbst, (35% identity). Thus,
the sequence was assigned to be the gene encoding AKcpn. Recent genomic
analysis of C. pneumoniae (18, 33) confirmed that the gene
we isolated was the unique AK gene in the genome. AKcpn showed high
similarity to Chlamydia trachomatis AK (AKctr, 45%
identity) and Rickettsia prowazekii AK (AKrpr, 34%
identity), suggesting that these pathogens are in close kinship. The
sequence containing four cysteine residues
(CXXC-Xn-CXXC) that was
predicted to be a zinc-binding motif was found in the LID domain of
AKcpn. The motif was also present in other AKs (Fig.
2). Among them, AKbst, AK from B. subtilis (AKbsu) and AK from Paracoccus denitrificans (AKpde) were previously shown to contain a zinc ion (12, 34, 35).
Therefore, other AKs carrying a zinc-binding motif are likely to have a
zinc ion.
Interestingly, AKcpn and AKctr have five and four cysteine residues,
respectively, other than the four cysteine residues in zinc-binding
motif in the LID domain. Other known AKs have less cysteine residues.
AKrpr, AKbst, and AKs from some Gram-positive and thermophilic bacteria
included only one or two residues residing outside the zinc-binding
motif. The reason for the high cysteine content in chlamydial AKs is unknown.
Purification of AKcpn--
AKcpn was overproduced in E. coli and purified to homogeneity. In a typical experiment,
starting from the crude extract of 20 g cells of E. coli CV2 harboring pK253 (350 mg of protein; specific activity,
7.0 units/mg protein), we obtained 3 mg of the purified enzyme
(specific activity, 350 units/mg protein) with a yield of 40% through
50-fold purification. From the protein profile obtained during the
Sephadex G-100 gel filtration, AKcpn was present as monomer under the
conditions used for purification. Fig. 3
shows SDS-PAGE of each purification step. In the lane of the purified
AKcpn preparation, a major band with an upper faint band was observed.
The major band corresponded to a 24-kDa protein that was consistent
with the molecular weight of AKcpn predicted from the nucleotide
sequence. The N-terminal amino acid sequence was determined with
proteins extracted from the major and faint bands, respectively. The
sequences were identical up to the 10th residue, and in both
preparations about 10% of the protein lost the N-terminal methionine
residue. The upper faint band was thought to be apo-AKcpn that had lost
the zinc ion (see below).
Kinetic Analysis of AKcpn--
Using purified AKcpn, substrate
specificity (Table I) and kinetic
properties (Table II) were determined.
AMP was a unique substrate as NMP in AKcpn. AKcpn did not use dAMP,
which is a good substrate for AKeco (36). However, both AKcpn and AKeco used all NTPs as a substrate, although NTP specificity was more relaxed
in AKcpn than that in AKeco. AKcpn exhibited extremely high
Km values for AMP and ADP compared with AKeco,
being 330 µM for both substrates. In contrast,
Km for ATP of AKcpn was lower than that of AKeco
with the value of 24 µM. Ki for
Ap5A of AKcpn was 4-fold higher than that of AKeco, which
was consistent with higher Km for AMP and/or ADP
of AKcpn.
AKcpn was not inhibited by N-ethylmaleimide, which inhibits
the AK1 activity by modifying a cysteine residue (data not shown), suggesting that cysteine residues of AKcpn are not directly involved in catalysis.
Zinc Content of AKcpn--
Atomic absorption spectroscopic
analysis of the purified 24.2 µM AKcpn revealed the
presence of a 16.5 µM zinc atom, indicating a content of
~0.7 g of zinc atoms/mol of protein. It was reported that non-heme
iron was present in the AKpde (35). However, iron was not detected in
AKcpn by atomic absorption spectroscopic analysis. Addition of PMPS to
AKcpn released the enzyme-bound zinc ion, which was quantified by
measuring A500 due to a chelate formation with
PAR (Fig. 4A). As varying
amounts of PMPS were added to the AKcpn solution (24.2 µM) in the presence of PAR, A500
linearly increased and reached a plateau value
(A500 = 1.64) at 3.7 mol of PMPS/mol of AKcpn.
When an excess of EDTA was added to the reaction mixture, the red color
disappeared. From the molar extinction coefficient of the zinc-chelated
PAR, the zinc concentration at the plateau was calculated to be 24.8 µM, which was very similar to the concentration of AKcpn.
When the mercaptide bond was measured at A250 in
the absence of PAR, and the findings were superimposed onto the figure
of the zinc titration experiments, a 3.5-fold molar excess of PMPS was
needed to reach the plateau. The plateau value of
A250 was 0.29, which was calculated to be
equivalent to four mercaptide bonds in each AKcpn molecule. These
findings indicate that the AKcpn molecule contains one zinc atom, which binds to four cysteine residues in the molecule.
Zinc-binding Site--
To identify the zinc-binding site in AKcpn,
we performed mass spectrometric analysis. 5-fold molar excess of PMPS
was added to AKcpn, and the PMPS-bound AKcpn was digested with trypsin. The tryptic peptides were then subjected to mass spectrometry. Upon
trypsin digestion, four cysteine residues in the zinc-binding motif
should be separated into two fragments, a and b
(Fig. 5), with molecular masses of 912 and 2282 Da, respectively. When one molecule of PMPS binds to a
cysteine residue, the molecular mass of the peptide should increase by
358 Da. Upon examination of peaks in mass spectrometry, peaks around
1270.5 and 1626.6 were unique with respect to the differences between
PMPS-treated and untreated AKcpn (Fig. 6,
A and B). These peaks are consistent with the
calculated molecular weights of the peptide a with one and
two PMPS molecules, respectively.
Due to the natural abundance of 13C, the peptide peaks
comprise major and minor components. A typical pattern of multiple
peaks around 1259.9 that were detected in both PMPS-treated and
untreated AKcpn is also shown in Fig. 6A. In contrast,
mercury included in PMPS gave additional multiplicity due to its
natural isotopes including those of atomic weights of 200 (23.10%) and
202 (29.65%) (see Fig. 6A, inset). Thus, the
multiple peaks are derived from an isotopic abundance of both carbon
and mercury atoms in the PMPS-bound peptides. The change in peak
pattern due to mercury isotopes was useful for identification of the
peaks originating from the PMPS-bound peptide in our analysis.
Therefore, it is expected that reagents containing mercury such as PMPS
can be generally used to analyze cysteine-containing peptides in mass spectrometry. From the above findings, it is concluded that at least
two cysteine residues located in the N-terminal part of the
zinc-binding motif of AKcpn were bound by PMPS.
Peaks of PMPS-bound peptide b were not detected at the
calculated positions. However, multiple peaks with a complex mass
pattern that would come from mercury isotopes were formed around 2372 and 2732 (data not shown). The mass difference between the two peaks
was 360, which approximately corresponded to the molecular weight of
PMPS. The molecular weights of these two peaks were smaller by 268 and
265 than the expected molecular weights of one- and two-PMPS-bound
peptide b, respectively. Since there is no other PMPS-bound
peptide corresponding to these molecular masses, the observed peaks
might be derived from the PMPS-bound peptide b. The nature
of derivation is unknown.
To further characterize the residues involved in zinc binding, we
constructed four AKcpn mutants in which cysteines in the LID domain
were changed to serine or alanine (Fig. 5). Although all four mutant
proteins were detected by Western blot analysis, the proteins were able
to neither complement E. coli CV2 (Table III) nor bind to the Affi-Gel Blue
column, suggesting that mutant proteins have no enzyme activity
in vivo and in vitro. Therefore, the anti-AKcpn
antibody column was used to purify the two mutant proteins, C133A,C136A
(pK276) and C149S,C152S (pK273). The zinc content in the mutant
proteins was analyzed using PMPS and PAR as previously described. Both
mutant proteins neither have enzyme activity nor contain zinc ions,
indicating that the protein lost zinc ion upon change of the cysteine
residues to other amino acids in the LID domain (Table III). These
results support our hypothesis that zinc binds to the cysteine residues
of the zinc-binding motif of AKcpn.
Preparation of Apo-AKcpn--
The AK activity of AKcpn was
measured after treatment with varying amounts of PMPS. Until 4.5-fold
excess PMPS was added to AKcpn in the presence of EDTA, the enzyme
activity did not significantly decrease despite of releasing
enzyme-bound zinc ions (Fig. 4, A and B).
However, PMPS at 15- or 76-fold molar excess made the enzyme inactive.
Excessive PMPS might have been associated with cysteine residues other
than those in the zinc-binding site, leading to conformational change
and inactivation of AKcpn. However, the activity of AKcpn was restored
on addition of DTT, which released the PMPS bound by the cysteine
residues. Zinc was not found in the AK treated as above (data not
shown). By this procedure using PMPS, EDTA, and DTT, we were able to
obtain the apo-enzyme that lost the bound zinc ion and retained the
enzyme activity.
Properties of Apo-AKcpn--
As mentioned above, zinc ion is not
essential for the catalytic activity of AKcpn. However, upon
examination of the kinetic properties, the Km
for AMP of the apo-enzyme was about 2-fold higher, and
Vmax was about one-half compared with those of
the holo-AKcpn (Table II). The findings suggested that the domain containing zinc ions affects both the binding of substrates and the
catalytic activity of the enzyme.
The thermal stability of apo-AKcpn was compared with the holo-enzyme.
After incubation at a given temperature for 30 min, AK activity was
determined (Fig. 7). The
half-inactivation temperature of apo-AKcpn was 42 °C, while that of
holo-AKcpn was 57 °C, indicating that the apo-enzyme is more labile
than the holo-enzyme.
Protease Sensitivity--
Trypsin sensitivities were compared
between holo- and apo-AKcpn at 42 °C (Fig.
8A). After a 10-min incubation
with trypsin, apo-AKcpn almost disappeared (lane
8), while holo-AKcpn remained (lane
3). After a 30-min incubation, apo-AKcpn was completely digested (lane 9), although holo-AKcpn still
remained (lane 4), indicating that apo-AKcpn was
more sensitive to trypsin than holo-enzyme at 42 °C. Similar
findings were obtained in trypsin treatment at 25 °C (data not
shown). Both holo- and apo-AKcpn were not digested by trypsin at
0 °C (Fig. 8B). The LID domain without zinc appeared to
be easily attacked by trypsin only at high temperature. However, the
mutant AKcpn protein of pK273 (C149S,C152S) disappeared after 30 min of
trypsin treatment at 0 °C, indicating that the mutant protein was
more sensitive to trypsin than the wild-type holo- and apo-AKcpn. These
findings indicate that replacement of amino acid residues and/or loss
of the zinc ion in the LID domain induced a conformation change in
AKcpn that leads to susceptibility to trypsin.
The apo-AKcpn migrated a little slowly compared with the holo-enzyme in
SDS-PAGE, although the findings were not perfectly reproducible.
Moreover, the mutant proteins of AKcpn that lacked zinc ions (Fig.
8B) migrated to the same position as the apo-AKcpn. Therefore, these suggested that the upper faint band observed in
SDS-PAGE of the purified enzyme (Fig. 3) may have been a spontaneously occurring apo-enzyme.
Renaturation of AKcpn with Zinc Ion--
After denaturation of
AKcpn in 4.5 M guanidine-HCl, renaturation was performed by
10-fold dilution with 50 mM Tris-HCl (pH 7.5) and 0.2 mM DTT (Fig. 9A).
The holo-enzyme restored about 20% of the enzyme activity after the
dilution (lane 2). Under the same conditions of
denaturation and renaturation, pig AK1 restored almost full activity,
as observed before denaturation (lane 7).
However, apo-AKcpn showed little recovery of the activity under both
the conditions (lanes 3 and 4). By
addition of 0.5 µM zinc chloride, renaturation of the
apo-enzyme was observed (lane 5). The
renaturation of holo-AKcpn was not affected by the presence of 0.5 µM zinc chloride (data not shown). When the effect of
various concentrations of zinc chloride on renaturation of apo-AKcpn
was assayed (Fig. 9B), the concentrations of zinc chloride greater than 5 µM prevented renaturation. Under the
present renaturation conditions, the addition of 0.5 µM
zinc to the apo-enzyme solution was most effective. Although the
maximum efficiency of the renaturation was at most 30%, we speculated
that binding of zinc to cysteine residues in the LID domain leads to
proper polypeptide folding of AKcpn.
In the present study, we isolated the gene encoding adenylate
kinase from C. pneumoniae (AKcpn) and characterized the
enzyme properties with proteins overproduced in E. coli. Two
distinctive features of AKcpn are found: (i) AKcpn contains zinc, and
(ii) AKcpn has high Km for AMP.
Our analysis using -SH titration, mass spectrometry and mutagenesis
indicates that the zinc in AKcpn is probably bound by four cysteine
residues of the zinc-binding motif in the LID domain that protrudes in
the middle of the molecule. The zinc-binding motif is found in AKs from
other bacteria (Fig. 2). It was shown that AKs from B. stearothermophilus (AKbst), B. subtilis (AKbsu), and
P. denitrificans (AKpde) contain zinc atoms. Zinc in AK has been thought to contribute to thermal stability of the protein from the
analysis of AKbst and AKbsu (12, 34). We developed a method to prepare
apo-AKcpn that lost zinc. The apo-AKcpn was inactivated by 80% during
incubation at 50 °C for 30 min, whereas holo-enzyme retained 70% of
the activity under the same conditions (Fig. 5). Thus, zinc contributes
to the thermal stability also in AKcpn. In addition, apo-AKcpn was more
sensitive to proteolysis by trypsin than holo-enzyme. Under our
experimental conditions at 42 °C where the apo-enzyme was completely
digested, the holo-enzyme did not significantly change (Fig.
8A). Another difference between apo- and holo-AKcpns was
their catalytic properties. Km value for AMP
increased approximately 2-fold in the apo-enzyme, while
Vmax decreased by about one-half in the
apo-enzyme. In the catalytic process, the LID domain is thought to move
upon substrate binding in an induced fit manner. Thus, it is concluded
that the LID domain upon binding zinc ion contributes to the
stabilization of the tertiary structure and flexibility of the protein.
Mutant AKcpn, in which cysteines in the zinc-binding motif were
substituted by other amino acids, lost not only zinc but also enzyme
activity. The mutant protein (C149S,C152S) was more sensitive to
trypsin than the wild-type enzyme. Loss of zinc-binding ability might
have brought about improper conformation during polypeptide folding in
protein synthesis in the cell. When we analyzed the renaturation
process of the guanidine-HCl denatured protein by monitoring the enzyme
activity of the renatured protein, renaturation of the apo-enzyme was
dependent on the presence of the zinc ion (Fig. 8). Under our
renaturation conditions, we obtained at most 30% recovery of the
enzyme. Thus, the zinc binding to the LID domain appears to assist
proper folding of AKcpn, which could be further promoted by chaperons
in vivo. Some monomeric proteins have observable
intermediates during the polypeptide folding (37). The folding of these
proteins starts from a specific subset of the native structure (a
folding nucleus), that provides further folding (37-40). If the
nucleus formation failed, proper protein folding would also fail and
the function of the protein would not be obtained. The present findings
suggest the possibility that the LID domain of AKcpn might be a folding
nucleus for correct conformation and that incorporation of zinc into
the LID domain might be an essential event for nucleation. Recent
studies have reported that zinc-finger proteins need zinc for their
native folding (17, 41). Moreover, foldings of some other zinc-binding proteins were dependent on presence of zinc (42, 43). Our observation,
the role of zinc in the folding process of AKcpn, supports a general
role of zinc in folding of zinc-containing proteins.
Chlamydiae show unique developmental stages during infection: dormant
elementary bodies and metabolically active reticulate bodies (44).
Reticulate bodies proliferate in the growing vacuole in the infected
cell. They have limited metabolic capabilities and show an obligate
dependence on the nutrient-rich cytoplasm of the host cell. Recent
genome-sequencing projects have confirmed the auxotrophic nature of
chlamydiae (33, 45). Regarding the metabolism of nucleotides,
chlamydiae appear to lack both the de novo and the salvage
pathways of purine and pyrimidine nucleotides. There is no homologue in
the chlamydial genome of the gene encoding phosphoribosylpyrophosphate
synthetase that is responsible for providing the key substance for
nucleotide synthesis. C. trachomatis was shown to be
auxotrophic for ribonucleoside triphosphate. Two NTP transport systems
have been characterized in the bacterium: one for ATP/ADP exchanges
transport and the other for ribonucleoside triphosphate/H+
symport (3).
Regarding ATP production, the presence of the genes encoding ATP
synthesis through Na+/H+ gradient across the
cytoplasmic membrane in C. trachomatis and C. pneumoniae was predicted (46). Moreover, the gene for glycolytic enzymes, with the capacity of substrate-level ATP production, is
thought to be present in the chlamydial cell. Interestingly, pyruvate
kinase, a rate-limiting enzyme in glycolysis, of C. trachomatis was not up-regulated allosterically by AMP and
fructose 1,6-bisphosphate in contrast to the property commonly found in
bacterial pyruvate kinase (47). Regarding other rate-limiting steps in
glycolysis, the reaction for glucose phosphorylation is absent in
chlamydiae; glucose 6-P is taken up through a hexose-P transporter (33, 45). Another interesting feature of chlamydia is that it probably uses
inorganic pyrophosphate (PPi)-dependent
phosphofructokinase in glycolysis (46). In contrast to
ATP-dependent phosphofructokinase, PPi-dependent phosphofructokinase catalyzes a
reversible reaction; the same enzyme probably serves for both
glycolysis and gluconeogenesis. It is difficult to consider that this
enzyme serves as a regulatory step in glycolytic pathway. Therefore, we
speculate that AMP does not play a regulatory role in chlamydia as
found in other organisms. Thus, metabolic regulation in chlamydiae may
use mechanisms different from those in other bacteria.
The present analysis on AKcpn showed that it had high
Km for AMP, 9-fold higher than that of AKeco, while
Km for ATP was low. Due to this property of AKcpn
and the mechanism of metabolic regulation mentioned above, the steady
state concentration of AMP in the chlamydial cell must be high. Indeed,
previous analysis of the chlamydial energy charge showed that it was
lower than that in other organisms (4, 48). For the ATP/ADP translocase to incorporate ATP, a sufficiently high concentration of ADP is necessary inside the cell. If AKcpn is localized at a particular site
near the translocase inside the cytoplasm and catalyzes the reaction
(AMP + ATP In conclusion, C. pneumoniae has a unique AK containing zinc
in the LID domain that contribute to stability and proper folding of
the protein and bearing a distinct catalytic property that is pertinent
to chlamydial energy metabolism.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-thiogalactopyranoside were obtained from Wako Pure Chemical (Osaka, Japan), and Urografin 76% from Nihon Schering (Osaka, Japan). All other chemicals were of
analytical grade.
80 °C for 30 min before being left at 25 °C. The cells were further disrupted with a glass-Teflon homogenizer by 15 strokes, followed by centrifugation at 800 × g for 10 min at
4 °C. The supernatant was poured into another tube, and 8 ml of 30%
(v/v) Urografin in 20 mM Tris-HCl (pH 7.5), and 150 mM KCl (TKCl buffer) was layered over the supernatant.
After centrifugation at 43,000 × g for 60 min at
4 °C, the supernatant was removed, and the precipitate was suspended
in 8 ml of SPG buffer. Then, 5 µg/ml DNase I and 10 mM
MgCl2 were added and this was followed by incubation for 30 min at 25 °C. Then, it was layered over discontinuous Urografin gradients (13 ml of 40%, 8 ml of 44%, and 5 ml of 52% Urografin in
TKCl buffer) and centrifuged at 43,000 × g for 60 min
at 4 °C. The EB band, located at the 44-52% Urografin interface,
was collected and diluted with three volumes of SPG buffer, followed by
additional centrifugation at 35,000 × g for 30 min at
4 °C. The EB pellet was suspended in 300 µl of 10 mM
Tris-HCl (pH 8.0), and 1 mM EDTA (TE buffer), centrifuged
at 12,000 × g for 10 min at 4 °C, and suspended
again in 300 µl TE buffer. The EB suspension was stored at
80 °C. The protein concentration of the EB suspension was about 1 mg/ml.
-32P]ATP was purchased from Amersham Pharmacia
Biotech (Uppsala, Sweden). DNA sequencing was carried out by the
dideoxy-chain termination method (24) using the double-stranded DNA
cycle sequencing system (Life Technologies, Inc.). Plasmid isolation
and other DNA manipulations were performed according to the methods
described by Sambrook et al. (25). DNA fragments produced by
PCR were cloned with a TA cloning kit (Invitrogen, Carlsbad, CA). DNA
in the sample was estimated from A260.
) (Stratagene) to obtain pK245. Then, the 1-kb BamHI fragment of pK245 was ligated into pUC18 to obtain
pK247. Finally, pK253 was constructed by ligating the 1-kb
EcoRI fragment of pK247 to EcoRI-digested
pKK223-3 (Amersham Pharmacia Biotech).
-D-thiogalactopyranoside. After further
incubation for 4 h, the cells were collected by centrifugation at
5,000 × g for 20 min at 4 °C. The cells were washed
once with 50 mM Tris-HCl (pH 7.5) and disrupted by
sonication in the same buffer. Cell debris was removed by
centrifugation at 30,000 × g for 20 min at 4 °C,
and the supernatant (60 ml) was saved as a crude extract.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (39K):
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Fig. 1.
Structure of pK238 (A) and
the DNA sequence of the AKcpn gene
(B). A, a physical map of pK238 (4.0 kb)
isolated from the genomic library of C. pneumoniae. The
plasmid contained the 1.3-kb fragment of genomic DNA. The AK-encoding
sequence is shaded. Arrows denote the
transcription direction. B, BamHI; E,
EcoRI; Sa, SalI; S,
SmaI; Amp, the ampicillin-resistant gene.
B, DNA sequence of the 1-kb
BamHI-EcoRI fragment of pK238 is presented after
removing the vector sequence. The open reading frame is
boxed. Candidates for the start codon are
underlined. The start GTG codon is accompanied by the SD
sequence (double underline). The stop codon is
shown as white letters in a black
box. The deduced amino acid residue is shown as
one letter under each codon. Four
circled cysteine residues constitute the zinc-binding motif
in the LID domain. The boxed GAATTC sequence represents the
EcoRI site.
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Fig. 2.
Alignment of amino acid sequences of LID
domains in bacterial AKs. The amino acid residues identical to
AKcpn are boxed. Stars indicate cysteine residues
of the zinc-binding motif. AKcpn, AKctr,
AKrpr, AKbst, AKbsu, AKpde,
AKhha, AKtma, AKafu, AKaae,
and AKeco represent AKs from C. pneumoniae,
C. trachomatis (GenBankTM accession no. AE001286), R. prowazekii (AJ235272), B. stearothermophilus (M88104),
B. subtilis (D00619), P. denitrificans (U64203),
Halobacterium halobium (D78200), Thermotoga
maritima (AE001798), Archaeoglobus fulgidus (AE001058),
Aquifex aeolicus (AE000672), and E. coli
(X03038), respectively. The protein-bound zinc was detected in AKcpn,
AKbst, AKbsu, and AKpde.
View larger version (66K):
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Fig. 3.
SDS-PAGE of fractions during the purification
of AKcpn. The arrowhead indicates the position of
AKcpn. Lane 1, crude extract (45 µg of
protein); lane 2, Affi-Gel Blue fraction (6.0 µg of protein); lane 3, Sephadex-G100 fraction
(4.0 µg of protein). M, protein size markers. Note that a
faint band is observed above the major band.
Substrate specificity of AKcpn
Kinetic properties of AKcpn and AKeco
View larger version (23K):
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Fig. 4.
Released zinc (A) and the
enzyme activity (B) of AKcpn in the presence of
PMPS. A, zinc ion released from AKcpn and the
mercaptide bond formation due to PMPS-binding were measured by
A500 (open square with
dotted line) and A250
(closed circle with solid
line) with and without PAR, respectively. When 5 mM EDTA was added after addition of excess PMPS,
A500 became almost zero in the presence of PAR
(open triangle). B, AKcpn and PMPS
were mixed in the given molar ratios, and the mixtures were incubated
for 10 min at 25 °C. AK activity was measured using the aliquots of
the incubation mixture. In two experiments (lanes
7 and 8), 25 mM DTT was added to the
incubation mixture after PMPS treatment, and incubation was continued
for an additional 10 min. Then, AK activity in each sample was measured
and expressed as relative activity to PMPS-untreated sample
(lane 1). Three independent series of experiments
gave similar results.
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Fig. 5.
Amino acid sequences of wild-type (pK253) and
mutant AKcpns. Stars indicate the zinc-binding motif in
the LID domain of wild-type AKcpn. Triangles represent the
sites of trypsin digestion. By complete digestion, four cysteine
residues of the zinc-binding motif are divided into two peptides,
a and b, with molecular weights of 912 and 2282, respectively. Mutation sites in mutant proteins are indicated below the
wild-type sequence (see Table III). Dots represent the same
amino acid as that of the wild-type enzyme.
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Fig. 6.
Mass spectrometric analysis of
trypsin-digested AKcpn. The purified AKcpn with and without PMPS
treatment was digested by trypsin, and the MALDI-TOF spectra of the
tryptic fragments were analyzed. Solid and dotted
lines represent PMPS-treated and non-treated AKcpn,
respectively. See "Experimental Procedures" for details. Spread
peaks around 1270.5 (A) and 1626.6 (B) appeared
only from PMPS-treated AKcpn and were assigned as peptide a
with one and two PMPS molecules, respectively (see Fig. 5). Peaks
around 1259.9 observed in both PMPS-treated and untreated AKcpn that
were unassigned are presented as reference. Inset in
panel A represents natural isotope abundance of
mercury.
Enzyme activity and zinc content of AKcpn mutant proteins
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Fig. 7.
Thermal stability of holo- and
apo-AKcpn. The 0.1 mg/ml holo- and apo-AKcpn in 50 mM
Tris-HCl (pH 7.5) was incubated for 30 min at a given temperature
(horizontal axis). Then, AK activities of these
aliquots were assayed as described previously (21). The AK activities
of holo- and apo-AKcpn (circle with solid
line and triangle with dotted
line, respectively) are shown as relative activities. Each
point represents the mean of three experimental findings with error
bars. A thin dotted line indicates the
50% original activity.
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Fig. 8.
Trypsin sensitivity of holo-, apo-, and
mutated AKcpns. A, 0.1 mg/ml holo- or apo-AKcpn was
incubated with 1 µg/ml trypsin at 42 °C for 0 min
(lanes 1 and 6), 3 min
(lanes 2 and 7), 10 min
(lanes 3 and 8), or 30 min
(lanes 4 and 9). After the incubation,
samples were loaded onto SDS-PAGE. Samples that were not treated with
trypsin (lanes 5 and 10) were also
run. An arrowhead indicates AKcpn. B, trypsin was
mixed with wild-type (WT) holo- and apo-AKcpn or mutant
(pK273) AKcpn as described above. The mixtures were incubated at
0 °C for 0 min (lanes 1, 3, and
5), 3 min (lane 6), 10 min
(lane 7), or 30 min (lanes
2, 4, and 8). Note that the positions
of apo- and mutant AKcpn are slightly higher than that of
holo-enzyme.
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Fig. 9.
Denaturation and renaturation of AKcpn.
A, AK activities of denatured and renatured AK are shown as
the activity relative to that before denaturation. Findings of
holo-AKcpn (lanes 1 and 2), apo-AKcpn
(lanes 3-5), and pig AK1 (lanes
6 and 7) are shown. ZnCl2 (0.5 µM) was added to the renaturing apo-AKcpn solution
(lane 5). Each bar represents the mean
of three experimental findings with error bars. B, effect of
zinc ion concentrations on renaturation of apo-AKcpn. Denatured
apo-AKcpn was diluted 10-fold (final concentration 0.15 µM) in the presence of various concentrations of zinc
chloride, and the AK activity was determined.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2ADP), the produced ADP would be efficiently used as the
substrate for ATP/ADP translocase.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Hiroshi Matsushima (Yamaguchi University School of Medicine) for providing the EB of C. pneumoniae J138. We thank Dr. Yasushi Kawata (Tottori University) for advising about experiments of protein renaturation, and Dr. Haruo Kobayashi (Kawasaki University of Medical Welfare) for advising about atomic absorption.
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FOOTNOTES |
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* This work was supported in part by Grant 97L00101 from the Japan Society for the Promotion of Science, Research for the Future Program and by Grants-in-aid 11877242, 12206067, and 11470133 from the Ministry of Education, Science and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number AB022016.
§ To whom correspondence should be addressed. Present address: Dept. of Microbiology, Yamaguchi University School of Medicine, 1-1-1 Minami-Kogushi, Ube, Yamaguchi 755-8505, Japan. Tel.: 81-836-22-2227; Fax: 81-836-22-2415; E-mail: koshirom@po.cc.yamaguchi-u.ac.jp.
** Present address: Dept. of Bioengineering, Faculty of Engineering, University of East Asia, Shimonoseki, Yamaguchi 751-8503, Japan.
§§ Supported by Grant EY06595 from the National Eye Institute.
Published, JBC Papers in Press, January 23, 2001, DOI 10.1074/jbc.M009461200
2 K. Miura, S. Inouye, K. Sakai, H. Takaoka, F. Kishi, M. Tabuchi, T. Tanaka, H. Matsumoto, M. Shirai, T. Nakazawa, and A. Nakazawa, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: AK, adenylate kinase; AKcpn, C. pneumoniae adenylate kinase; AKeco, E. coli adenylate kinase; AKbst, B. stearothermophilus adenylate kinase; PMPS, p-(hydroxymercuri)benzenesulfonic acid; Ap5A, P1,P5-di(adenosine-5')-pentaphosphate; PAR, 4-(2-pyridylazo)resorcinol; EB, elementary body; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; AKctr, C. trachomatis adenylate kinase; AKrpr, R. prowazekii adenylate kinase; AKbsu, B. subtilis adenylate kinase; AKpde, P. denitrificans adenylate kinase; SD, Shine-Dalgarno; kb, kilobase pair(s); DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis.
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