- Email: [email protected]
Purification and characterization of protein methylase II from Helicobacter pylori
FEMS Microbiology Letters 195 (2001) 53^58
www.fems-microbiology.org
Puri¢cation and characterization of protein methylase II from Helicobacter pylori Young Man Kim a , Seong Hoon Ahn a , Dong Wan Seo a;1 , Yong Kee Kim a , Jeung Whan Han a , Sungyoul Hong b , Sangduk Kim c , Woon Ki Paik d , Hyang Woo Lee a; * b
a Department of Pharmacy, College of Pharmacy, Sungkyunkwan University, Suwon 440-746, South Korea Department of Genetic Engineering, College of Life Science and Natural Resources, Sungkyunkwan University, Suwon 440-746, South Korea c Graduate School of Biotechnology, Korea University, Seoul 136-701, South Korea d Department of Biochemistry and Molecular Biology, School of Medicine, Ajou University, Suwon 442-749, South Korea
Received 1 September 2000; received in revised form 5 December 2000 ; accepted 12 December 2000
Abstract Protein methylase II (AdoMet:protein-carboxyl O-methyltransferase, EC 2.1.1.24) was identified and purified 115-fold from Helicobacter pylori through Q-Sepharose ion exchange column, AdoHcy-Sepharose 4B column, and Superdex 200 HR column chromatography using FPLC. The purified preparation showed two protein bands of about 78 kDa and 29 kDa molecular mass on SDS^PAGE. On nondenaturing gel electrophoresis, the enzyme migrated as a single band with a molecular mass of 410 kDa. In addition, MALDI-TOF-MS analysis and Superdex 200 HR column chromatography of the purified enzyme showed a major mass signal with molecular mass values of 425 kDa and 430 kDa, respectively. Therefore, the above results led us to suggest that protein methylase II purified from H. pylori is composed of four heterodimers with 425 kDa (4U(78+29) = 428 kDa). This magnitude of molecular mass is unusual for protein methylases II so far reported. The enzyme has an optimal pH of 6.0, a Km value of 5.0U1036 M for S-adenosyl-L-methionine and a Vmax of 205 pmol methyl-14 C transferred min31 mg31 protein. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Puri¢cation ; Protein methylase II; Helicobacter pylori
1. Introduction Protein methylation occurs ubiquitously in nature, in organisms ranging from prokaryotic to eukaryotic, and involves N-methylation of arginine, lysine, histidine, alanine, proline, and glutamine, O-methylation of glutamic acid and aspartic acid, and S-methylation of cysteine and methionine [1^4]. The methyl donor for the reaction is S-adenosyl-L-methionine (AdoMet) and the demethylated product is S-adenosyl-L-homocysteine (AdoHcy). Protein methylase II (EC 2.1.1.24), one of the carboxyl O-methylating enzymes, can methylate side-chain carboxyl * Corresponding author. Tel. : +82 (31) 290-7702; Fax: +82 (31) 290-7722; E-mail: [email protected] 1 Present address: Extracellular Matrix Pathology Section, Laboratory of Pathology, Division of Clinical Sciences, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
groups of proteins with S-adenosyl-L-methionine [5^7]. The enzyme was ¢rst described by Axelrod and Daly in 1965 as a methanol forming enzyme in pituitary extracts [5], and subsequently Kim and Paik isolated a methyltransferase from calf thymus to catalyze carboxymethylation of proteins [6,8]. Protein methylase II has since been puri¢ed from various mammalian sources including brain, erythrocytes and testis [9^11]. In addition to mammalian sources, activities of protein methylase II have been detected in bacteria [12,13]. It is now clear that at least four distinct types of protein-carboxyl methyltransferases exist [7], and their suggested biochemical function includes bacterial chemotaxis [12], protein repair [13,14], and signal transduction [15]. However, the overall de¢nitive physiological signi¢cance of protein methylase II remains to be clari¢ed. In 1984 Marshall and Warren discovered and succeeded in isolating Helicobacter pylori from stomach [18]. The ¢rst link between H. pylori and human disease was the
0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 5 4 5 - 0
FEMSLE 9759 23-1-01
54
Y.M. Kim et al. / FEMS Microbiology Letters 195 (2001) 53^58
observation that this organism was almost invariably associated with chronic super¢cial gastritis [19]. In the absence of ulcer inducing medications, duodenal ulcer disease occurred almost exclusively among persons infected with H. pylori. In addition to the possible link of H. pylori to peptic ulcers, it was demonstrated that concurrent or previous H. pylori infection is associated with a 2.7^12fold-increased risk of gastric cancer [20]. Because gastric adenocarcinoma is one of the most common and deadly carcinomas worldwide, eradication or prevention of H. pylori infection might reduce the incidence of this cancer. In the present study, protein-carboxyl O-methyltransferase has been identi¢ed and puri¢ed from H. pylori. Unlike other protein-carboxyl O-methyltransferases, H. pylori protein methylase II has a distinct characteristic of molecular structure composed of four subunits of heterodimers, having a molecular mass of 425 kDa.
mined by Bradford's method [22], using bovine serum albumin as a standard. 2.3. Puri¢cation of protein methylase II from H. pylori
2.2. Assay for protein methylase II
H. pylori collected from plates (wet weight 30 g) were suspended in four volumes of phosphate/phenylmethylsulfonyl £uoride (PMSF) bu¡er containing 0.1 M potassium phosphate, 1.0 mM EDTA, 0.1 mM PMSF, 5% glycerol and 0.24 mM L-mercaptoethanol (pH 7.0). The suspension was sonicated for 10 min, and the sonicate was centrifuged at 105 000Ug for 1 h at 4³C. The cell-free extract was brought to 90% saturation with ¢nely powdered ammonium sulfate. The suspension was stirred for 30 min and then centrifuged for 15 min at 105 000Ug. The precipitate was resuspended in 10 ml bu¡er A (20 mM Tris^HCl bu¡er, pH 8.0, containing 1 mM EDTA, 0.1 mM EGTA, 0.24 mM L-mercaptoethanol, 0.1 mM PMSF, and 5% glycerol), dialyzed (10 kDa MW cut-o¡) against 4 l of bu¡er A overnight at 4³C, and applied to a QSepharose column (i.d. 26 mmU10 cm, Amersham Pharmacia Biotech, USA). The enzyme was eluted with a linear gradient of bu¡er A containing 0^0.5 M NaCl at a £ow rate of 1 ml min31 . The active fractions having methylase activity were pooled and concentrated with ultra¢ltration membrane (YM 10; cut-o¡ of 10 kDa ; Amicon, USA) to remove residual salt. The concentrate was subjected to an a¤nity column. AdoHcy-Sepharose 4B a¤nity column (i.d. 28 mmU3.4 cm), prepared by the method of Kim et al. [23], was equilibrated with bu¡er B (5 mM sodium phosphate, 5 mM EDTA, 2.4 mM L-mercaptoethanol, pH 6.2) at a £ow rate of 1.8 ml min31 . The enzyme was eluted with 0.5 mM AdoMet in bu¡er B. Fractions (1.8 ml) were collected into test tubes containing 0.2 ml of 50 mM sodium borate, 5% glycerol and 50 mM EDTA (pH 9.3) to
Enzyme activity was determined by a modi¢ed method of Kim and Paik [5]. The reaction mixture containing 10 mM sodium phosphate bu¡er (pH 7.0), 0.6 mg of histone (calf thymus type II-As, Sigma Chemical Co., USA), and appropriate amounts of enzyme preparation in a total volume of 0.09 ml was preincubated at 37³C for 5 min, and was further incubated for 15 min after the addition of 0.01 ml of 25 WM Ado[methyl-14 C]Met (130 dpm pmol31 , Amersham Pharmacia Biotech, USA). At the end of incubation, 0.1 ml of 125 mM sodium borate bu¡er (pH 11.0) was added to the incubation mixture, and the mixture was further incubated for 5 min at 37³C. 1 ml of isoamyl alcohol was added to the mixture, the mixture was vortexed and centrifuged at 10 000Ug for 5 min at room temperature, and the radioactivity of the supernatant (0.7 ml) was determined. One enzyme unit is de¢ned as the amount of enzyme that catalyzes the transfer of 1 pmol methyl-14 C to the methyl acceptor substrate min31 at pH 7.0 and 37³C. Speci¢c activity is de¢ned as units of enzyme per mg of protein. Protein concentration was deter-
Fig. 1. Gel permeation chromatography on a Superdex 200 HR column. The concentrated enzyme preparation (600 Wg) was loaded on a Superdex 200 HR column (i.d. 10 mmU30 cm) and eluted with 20 mM Tris^ HCl (pH 8.0) at a £ow rate of 0.4 ml min31 . The solid line represents OD280 and closed circles the enzyme activity.
2. Materials and methods 2.1. Strains and culture conditions H. pylori NCTC11637 was purchased from the National Collection of Type Cultures (NCTC, UK). H. pylori (Sydney strain) and clinically isolated H. pylori were kindly provided by Daewoong Pharmaceutical Co., Korea. H. pylori NCTC11637, H. pylori (Sydney strain), and clinically isolated H. pylori were cultivated by agar plate which was prepared using blood agar base No. 2 (Difco, USA) with antibiotics (vancomycin, bacitracin, amphotericin, polymyxin B, and nalidixic acid) with 10% blood [21]. Plates were incubated for 3 days at 37³C in 10% CO2 .
FEMSLE 9759 23-1-01
Y.M. Kim et al. / FEMS Microbiology Letters 195 (2001) 53^58
55
Table 1 Puri¢cation of protein methylase II from H. pylori Puri¢cation step
Volume (ml)
Protein (mg ml31 ) Total protein (mg)
Speci¢c activity (U mg31 ) Total activity (Ua )
Yield (%)
Supernatant after sonication (NH4 )2 SO4 precipitate Q-Sepharose AdoHcy-Sepharose 4B a¤nity Superdex 200 HR gel permeation
109 88 37.5 1.9 1
12.3 13.8 3.8 0.3 0.04
1.6 1.6 6.3 122 184
100 86 40 3.3 0.3
1341 1214 143 0.6 0.04
2246 1942 901 73 7.4
Data represent a typical puri¢cation procedure, which was repeated three times with similar results. a Units: pmol methyl-14 C transferred min31 .
stabilize the eluted enzyme. The fractions eluted with AdoMet were concentrated by ultra¢ltration membrane (YM 10; Amicon, USA) [23]. The above concentrated enzyme preparation was applied onto a Superdex 200 HR column (i.d. 1.0U30 cm, Amersham Pharmacia Biotech, USA) previously equilibrated with bu¡er A containing 0.15 M NaCl. The column was then eluted with bu¡er A at a £ow rate of 0.4 ml min31 (Fig. 1). The fractions containing protein methylase II activity were pooled, concentrated and residual salts were removed by ultra¢ltration membrane (YM 10; Amicon, USA). The puri¢ed enzyme was stored at 320³C until use.
subjected to SDS^PAGE and electroblotted on PVDF membrane. The membrane was dried, and the protein methylase II band was sequenced. N-terminal amino acid sequence analysis was performed with an Applied Biosystems Procise Protein Sequencer (Perkin-Elmer Applied Biosystems, USA), equipped with an on-line analyzer for phenylthiohydantoin-derivatized amino acids.
2.4. Estimation of molecular mass of the puri¢ed protein methylase II
The presence of protein methylase II in three strains (NCTC11637, Sydney strain, clinical isolates) of H. pylori was identi¢ed. The speci¢c activity of each strain was 0.5, 1.7, and 2.2 pmol Ado[methyl-14 C]Met transferred min31 mg31 protein, respectively. Although the clinically isolated strain had the highest speci¢c activity, it was very di¤cult to culture for the following experiment. Also, because
The molecular mass of the puri¢ed protein methylase II was determined by SDS polyacrylamide gel electrophoresis (SDS^PAGE), non-denaturing PAGE, matrix-assisted laser desorption/ionization time-of-£ight mass spectrometry (MALDI-TOF-MS; Perseptive Analytical Elite, USA) analysis, and gel permeation chromatography. SDS^ PAGE was performed according to the method of Laemmli [24] and non-denaturing PAGE was performed according to the method of Bhown and Bennett [25]. The molecular mass of the puri¢ed enzyme was determined using MALDI-TOF-MS. The puri¢ed protein methylase II from H. pylori was ¢rst dissolved in 0.1% tri£uoroacetic acid, and then was added to the matrix solution (saturated dihydrobenzoic acid in 1:3:2 formic acid:water:2-propanol), so that the ¢nal protein concentration of the mixture was less than 0.1 mg ml31 . An aliquot of matrix/protein solution (0.5 Wl) was then applied to a metal probe tip and dried [26,27]. Also, the molecular mass of the puri¢ed enzyme was determined by gel permeation on a Superdex 200 HR column. The relative elution volume of protein methylase II was compared with that of standard markers (vitamin B-12, 1.35 kDa; myoglobulin, 17 kDa ; ovalbumin, 44 kDa ; Q-globulin, 158 kDa ; thyroglobulin, 670 kDa). 2.5. N-terminal sequencing of the puri¢ed protein methylase II The puri¢ed protein methylase II from H. pylori was
3. Results 3.1. Identi¢cation of protein methylase II from H. pylori
Fig. 2. Determination of molecular mass of the enzyme by SDS^PAGE and non-denaturing PAGE. A: M represents molecular mass standard markers including myosin (205 kDa), L-galactosidase (116 kDa), phosphorylase b (97.4 kDa), bovine serum albumin (66 kDa), egg ovalbumin (45 kDa), and carbonic anhydrase (29 kDa). A and G represent enzyme preparations puri¢ed by AdoHcy-Sepharose a¤nity and Superdex 200 HR column chromatography, respectively. B: M represents molecular mass standards used: apoferritin (443 kDa), urease (272 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), and chicken egg albumin (45 kDa). G represents enzyme preparation puri¢ed by Superdex 200 HR column chromatography.
FEMSLE 9759 23-1-01
56
Y.M. Kim et al. / FEMS Microbiology Letters 195 (2001) 53^58
Fig. 3. Analysis of protein methylase II by MALDI-TOF-MS.
strain NCTC11637 has a low speci¢c activity, we selected the Sydney strain for puri¢cation of protein methylase II. As shown in Table 1, the enzyme was puri¢ed by Q-Sepharose ion exchange, AdoHcy-Sepharose 4B a¤nity and Superdex 200 HR column chromatography, and the ¢nal preparation had a speci¢c activity of 184 pmol Ado[methyl-14 C]Met transferred min31 mg31 protein with 115-fold puri¢cation and 0.3% yield.
The molecular mass of the puri¢ed enzyme preparation was also analyzed by Superdex 200 HR gel permeation and found to be approximately 430 kDa (data not shown), which was very close to the value of 410 kDa determined by non-denaturing gel electrophoresis (Fig. 2B). As shown in Fig. 3, MALDI-TOF-MS analysis of native protein methylase II showed a major mass signal with a molecular mass value of 425 073 Da.
3.2. Molecular mass and subunit size of protein methylase II from H. pylori
3.3. N-terminal analyses of the puri¢ed protein methylase II from H. pylori
The puri¢ed protein methylase II preparation was resolved into two bands on SDS^PAGE, having molecular masses of 78 and 29 kDa (Fig. 2A). On the other hand, the enzyme migrated as a single band with a molecular mass of 410 kDa on non-denaturing gel electrophoresis (Fig. 2B).
As shown in Table 2, N-terminal sequences of each subunit of protein methylase II from H. pylori were analyzed using the GenBank database system. The N-terminal sequence (1^12) of the 78-kDa subunit was similar to that of chlorohydrolase in strains 26695 and J99 of H. pylori with 91% identity, and the N-terminal sequence (1^20) of the
Table 2 N-terminal analyses of the puri¢ed protein methylase II from H. pylori Subunit
N-terminal sequence
Identity (%)
78 kDa Chlorohydrolase Chlorohydrolase 29 kDa Glutamine synthetase Glutamine synthetase
MQEIIGACLV FL MQEIIGASLV FL MQEIIGASLV FL MIVRTQMCEC KIKRFFEFCK MIVRTQNSES KIKEFFEFCK MVVRTQNSES KIKEFFEFCK
FEMSLE 9759 23-1-01
11/12 (91%) 11/12 (91%) 16/20 (80%) 15/20 (75%)
H. pylori strain Sydney 26695 J99 Sydney 26695 J99
Y.M. Kim et al. / FEMS Microbiology Letters 195 (2001) 53^58
57
4. Discussion
Fig. 4. Lineweaver^Burk plot of Ado[methyl-14 C]Met on protein methylase II from H. pylori. The reaction mixture containing 13.5Wg of the puri¢ed protein methylase II and 1 mg of histone was incubated under the condition described in Section 2. Km for AdoMet was 5.0U1036 M and Vmax was 205 pmol min31 mg31 protein.
29-kDa subunit was similar to that of glutamine synthetase in strains 26695 and J99 of H. pylori with 80% and 75% identity, respectively. However, the molecular masses of chlorohydrolase (45.5 kDa) and glutamine synthetase (54.6 kDa) are signi¢cantly di¡erent from those of the subunits of puri¢ed protein methylase II. And there are no sequence homologies between these two enzymes and other protein methylases, indicating that protein methylase II puri¢ed from H. pylori is a novel enzyme, distinguished from chlorohydrolase, glutamine synthetase, and other protein methylases. 3.4. Characteristics of protein methylase II from H. pylori There was a linear relationship between the amount of substrate protein (histone 0^64 mg) and enzyme activity (data not shown). The Michaelis constants (Km value) and the maximum reaction velocity for AdoMet were determined by Lineweaver^Burk plot: Km and Vmax values were 5.0U1036 M and 205 pmol Ado[methyl-14 C]Met min31 mg31 enzyme protein, respectively (Fig. 4). Table 3 shows that histone and hemoglobin served as the best in vitro substrates for the puri¢ed protein methylase II. The enzyme had optimum pH of 6.0 (data not shown).
Protein methylase II has been found in organisms ranging from prokaryotes to eukaryotes [1^4,28]. In the present study, the enzyme was puri¢ed from H. pylori by means of Q-Sepharose, AdoHcy-Sepharose 4B a¤nity and Superdex 200 HR chromatography. The ¢nal puri¢ed enzyme preparation had a speci¢c activity of 184 pmol methyl-14 C min31 mg31 protein, representing a 115-fold puri¢cation with a yield of 0.3% (Table 1). This extent of puri¢cation is in the range of enzyme puri¢cations from other sources : 1160-fold puri¢cation from Escherichia coli with a yield of 0.067% [12] and 13-fold puri¢cation from Salmonella typhimurium with 13% yield [14]. Purity and molecular mass of the puri¢ed enzyme was determined by SDS^PAGE, non-denaturing PAGE, MALDI-TOF-MS and gel permeation chromatography. The molecular mass of the enzyme determined by nondenaturing PAGE, MALDI-TOF-MS and gel permeation chromatography gave values of 410, 425, and 430 kDa, respectively. On SDS^PAGE, however, H. pylori protein methylase II was found to be composed of a heterodimer of 78 and 29 kDa (Fig. 2A). It is, therefore, highly likely that this enzyme appears to be composed of four heterodimers (4U(78+29) = 428 kDa). The molecular mass of protein methylase II (AdoMet:protein-carboxyl O-methyltransferases) isolated from various organisms ranged from 24 to 33 kDa [6,8^11], however, the enzyme of Streptomyces fradiae comprised two identical subunits of 32 kDa [29]. Thus, the enormous size as well as the composition are quite unusual for protein methyltransferases in general. Among various substrates for H. pylori protein methylase II (Table 3), histone was found to be the most e¤cient substrate. N-terminal sequences of each subunit of protein methylase II from H. pylori were compared to the deduced amino acid sequence of H. pylori in GenBank. The Nterminal amino acid sequences of the 78-kDa and 29kDa subunits were very close to those of chlorohydrolase and glutamine synthetase in strains 26695 and J99 of H. pylori. However, the molecular masses of chlorohydrolase (45.5 kDa) and glutamine synthetase (54.6 kDa) are signi¢cantly di¡erent from those of the subunits of puri¢ed
Table 3 Substrate speci¢city of protein methylase II from H. pylori Substrate proteina
Speci¢c activity (pmol min31 mg31 )
Relative activity (%)
Histone Hemoglobin Myelin basic protein Albumin Cytochrome c Gelatin Q-Globulin
180 174 152 142 102 89 59
100 97 84 79 57 49 33
The incubation and assay conditions were as described in Section 2. a The amount of substrate used was 0.6 mg.
FEMSLE 9759 23-1-01
58
Y.M. Kim et al. / FEMS Microbiology Letters 195 (2001) 53^58
protein methylase II, showing no identity between protein methylase II from the Sydney strain of H. pylori and the other two enzymes. The discrepancy in molecular mass between protein methylase II puri¢ed from the Sydney strain of H. pylori and the other two enzymes deduced from GenBank data of strains 26695 and J99 of H. pylori might be attributed to a di¡erence in genotype between strains. Also, similar sequences found in chlorohydrolase and glutamine synthetase have never been observed in other protein methylases. Therefore, protein methylase II from the Sydney strain of H. pylori appears to be a novel enzyme distinguished from other protein methylases. The biological signi¢cance of the presence of protein methylase II in H. pylori is not obvious at present. However, the enzyme with the unusual characteristic of such a large molecular mass (425 kDa) with eight heterosubunits is suggestive of its complex function, and its function should be ¢nely modulated in the stomach. Obviously, further studies on the exact amino acid sequence of protein methylase II of the Sydney strain of H. pylori, its location in the genome and the biological signi¢cance of H. pylori protein methylase II are needed. 5. Uncited references
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[16,17]
[19]
Acknowledgements
[20]
This work forms part of the thesis of Y.M.K. presented to the Graduate Board of Sungkyunkwan University, Seoul, Korea in partial ful¢llment of the requirements for the Ph.D. degree.
[21]
[22]
[23]
References [1] Ambler, R.P. and Rees, M.W. (1959) O-N-Methyl-L-lysine in bacterial £agella protein. Nature 184, 56^57. [2] Paik, W.K. and Kim, S. (1980) Protein Methylation : A Series of Monographs. John Wiley, New York. [3] Selmer, T., Kahnt, J., Goubeaud, M., Shima, S., Grabarse, W., Ermler, U. and Thauer, R.K. (2000) The biosynthesis of methylated amino acids in the active site region of methyl-coenzyme M reductase. J. Biol. Chem. 275, 3755^3760. [4] Chen, D., Ma, H., Hong, H., Koh, S.S., Huang, S.M., Schurter, B.T., Aswad, D.W. and Stallcup, M.R. (1999) Regulation of transcription by a protein methyltransferase. Science 284, 2174^2177. [5] Axelrod, J. and Daly, J. (1965) Pituitary gland: Enzymic formation of methanol from S-adenosyl-L-methionine. Science 150, 892^893. [6] Kim, S. and Paik, W.K. (1970) Puri¢cation and properties of protein methylase II. J. Biol. Chem. 245, 1806^1813. [7] Clarke, S. (1985) Protein carboxyl methyltransferases : two distinct classes of enzymes. Annu. Rev. Biochem. 54, 479^506. [8] Kim, S. (1973) Puri¢cation and properties of protein methylase II. Arch. Biochem. Biophys. 157, 476^484. [9] Aswad, D.W. and Deight, E.A. (1983) Puri¢cation and characteriza-
[24] [25]
[26]
[27]
[28]
[29]
tion of two distinct isozymes of protein carboxylmethylase from bovine brain. J. Neurochem. 40, 1718^1726. Gilbert, J.M., Fowler, A., Bleibaum, J. and Clarke, S. (1988) Puri¢cation of homologous protein carboxyl methyltransferase isozymes from human and bovine erythrocytes. Biochemistry 27, 5227^5233. Jung, K., Kwon, M., Lee, H.Y., Lee, H.W. and Hong, S. (1995) Puri¢cation and characterization of protein methylase II from porcine testis. J. Biochem. Mol. Biol. 28, 149^154. Fu, J.C., Ding, L. and Clarke, S. (1991) Puri¢cation, gene cloning, and sequence analysis of an L-isoaspartyl protein carboxyl methyltransferase from Escherichia coli. J. Biol. Chem. 266, 14562^14572. Simms, S.A. and Subbaramaiah, K. (1991) The kinetic mechanism of S-adenosyl-L-methionine:glutamylmethyltransferase from Salmonella typhimurium. J. Biol. Chem. 266, 12741^12746. Simms, S.A., Stock, A.M. and Stock, J.B. (1987) Puri¢cation and characterization of the S-adenosylmethionine:glutamyl methyltransferase that modi¢es membrane chemoreceptor proteins in bacteria. J. Biol. Chem. 262, 8537^8543. MacLaren, D.C. and Clarke, S. (1995) Expression and puri¢cation of a human recombinant methyltransferase that repairs damaged proteins. Protein Expr. Purif. 6, 99^108. Brennan, T.V., Anderson, J.W., Jia, Z., Waygood, E.B. and Clarke, S. (1994) Repair of spontaneously deamidated HPr phosphocarrier protein catalyzed by the L-isoaspartate-(D-aspartate) O-methyltransferase. J. Biol. Chem. 269, 24586^24595. Stephenson, R.C. and Clarke, S. (1990) Identi¢cation of a C-terminal protein carboxyl methyltransferase in rat liver membranes utilizing a synthetic farnesyl cysteine-containing peptide substrate. J. Biol. Chem. 265, 16248^16254. Marshall, B.J. and Warren, J.R. (1984) Unidenti¢ed curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet 1, 1311^1315. Blaser, M.J. (1992) Hypotheses on the pathogenesis and natural history of Helicobacter pylori-induced in£ammation. Gastroenterology 102, 720^727. Ernst, P. (1999) The role of in£ammation in the pathogenesis of gastric cancer (Review). Aliment. Pharmacol. Ther. 1, 13^18. Morshed, M.G., Islam, M.S., Zinnah, F., Rumi, M.A., Ahmad, M.M. and Huq, F. (1995) Growth of Helicobacter pylori in candle jar. Lancet 346, 511. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248^254. Kim, S., Nochumson, S., Chin, W. and Paik, W.K. (1978) A rapid method for the puri¢cation of S-adenosylmethionine:protein-carboxyl O-methyl-transferase by a¤nity chromatography. Anal. Biochem. 84, 415^422. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680^685. Bhown, A.S. and Bennett, J.C. (1983) High-sensitivity sequence analysis of proteins recovered from sodium dodecyl sulfate gels. Methods Enzymol. 91, 450^455. Karas, M. and Hillenkamp, F. (1988) Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal. Chem. 60, 2299^2301. Reiber, D.C., Brown, R.S., Weinberger, S., Kenny, J. and Bailey, J. (1998) Unknown peptide sequencing using matrix-assisted laser desorption/ionization and in-source decay. Anal. Chem. 70, 1214^ 1222. Paik, W.K. and Kim, S. (1990) Reevaluation of the enzymology of protein methylation. In: Protein Methylation (Paik, W.K. and Kim, S., Eds.), pp. 23^31. CRC Press, Boca Raton, FL. Bauer, N.J., Kreuzman, A.J., Dotzlaf, J.E. and Yeh, W.K. (1988) Puri¢cation, characterization, and kinetic mechanism of S-adenosylL-methionine :macrocin O-methyltransferase from Streptomyces fradiae. J. Biol. Chem. 263, 15619^15625.
FEMSLE 9759 23-1-01
www.fems-microbiology.org
Puri¢cation and characterization of protein methylase II from Helicobacter pylori Young Man Kim a , Seong Hoon Ahn a , Dong Wan Seo a;1 , Yong Kee Kim a , Jeung Whan Han a , Sungyoul Hong b , Sangduk Kim c , Woon Ki Paik d , Hyang Woo Lee a; * b
a Department of Pharmacy, College of Pharmacy, Sungkyunkwan University, Suwon 440-746, South Korea Department of Genetic Engineering, College of Life Science and Natural Resources, Sungkyunkwan University, Suwon 440-746, South Korea c Graduate School of Biotechnology, Korea University, Seoul 136-701, South Korea d Department of Biochemistry and Molecular Biology, School of Medicine, Ajou University, Suwon 442-749, South Korea
Received 1 September 2000; received in revised form 5 December 2000 ; accepted 12 December 2000
Abstract Protein methylase II (AdoMet:protein-carboxyl O-methyltransferase, EC 2.1.1.24) was identified and purified 115-fold from Helicobacter pylori through Q-Sepharose ion exchange column, AdoHcy-Sepharose 4B column, and Superdex 200 HR column chromatography using FPLC. The purified preparation showed two protein bands of about 78 kDa and 29 kDa molecular mass on SDS^PAGE. On nondenaturing gel electrophoresis, the enzyme migrated as a single band with a molecular mass of 410 kDa. In addition, MALDI-TOF-MS analysis and Superdex 200 HR column chromatography of the purified enzyme showed a major mass signal with molecular mass values of 425 kDa and 430 kDa, respectively. Therefore, the above results led us to suggest that protein methylase II purified from H. pylori is composed of four heterodimers with 425 kDa (4U(78+29) = 428 kDa). This magnitude of molecular mass is unusual for protein methylases II so far reported. The enzyme has an optimal pH of 6.0, a Km value of 5.0U1036 M for S-adenosyl-L-methionine and a Vmax of 205 pmol methyl-14 C transferred min31 mg31 protein. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Puri¢cation ; Protein methylase II; Helicobacter pylori
1. Introduction Protein methylation occurs ubiquitously in nature, in organisms ranging from prokaryotic to eukaryotic, and involves N-methylation of arginine, lysine, histidine, alanine, proline, and glutamine, O-methylation of glutamic acid and aspartic acid, and S-methylation of cysteine and methionine [1^4]. The methyl donor for the reaction is S-adenosyl-L-methionine (AdoMet) and the demethylated product is S-adenosyl-L-homocysteine (AdoHcy). Protein methylase II (EC 2.1.1.24), one of the carboxyl O-methylating enzymes, can methylate side-chain carboxyl * Corresponding author. Tel. : +82 (31) 290-7702; Fax: +82 (31) 290-7722; E-mail: [email protected] 1 Present address: Extracellular Matrix Pathology Section, Laboratory of Pathology, Division of Clinical Sciences, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
groups of proteins with S-adenosyl-L-methionine [5^7]. The enzyme was ¢rst described by Axelrod and Daly in 1965 as a methanol forming enzyme in pituitary extracts [5], and subsequently Kim and Paik isolated a methyltransferase from calf thymus to catalyze carboxymethylation of proteins [6,8]. Protein methylase II has since been puri¢ed from various mammalian sources including brain, erythrocytes and testis [9^11]. In addition to mammalian sources, activities of protein methylase II have been detected in bacteria [12,13]. It is now clear that at least four distinct types of protein-carboxyl methyltransferases exist [7], and their suggested biochemical function includes bacterial chemotaxis [12], protein repair [13,14], and signal transduction [15]. However, the overall de¢nitive physiological signi¢cance of protein methylase II remains to be clari¢ed. In 1984 Marshall and Warren discovered and succeeded in isolating Helicobacter pylori from stomach [18]. The ¢rst link between H. pylori and human disease was the
0378-1097 / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S 0 3 7 8 - 1 0 9 7 ( 0 0 ) 0 0 5 4 5 - 0
FEMSLE 9759 23-1-01
54
Y.M. Kim et al. / FEMS Microbiology Letters 195 (2001) 53^58
observation that this organism was almost invariably associated with chronic super¢cial gastritis [19]. In the absence of ulcer inducing medications, duodenal ulcer disease occurred almost exclusively among persons infected with H. pylori. In addition to the possible link of H. pylori to peptic ulcers, it was demonstrated that concurrent or previous H. pylori infection is associated with a 2.7^12fold-increased risk of gastric cancer [20]. Because gastric adenocarcinoma is one of the most common and deadly carcinomas worldwide, eradication or prevention of H. pylori infection might reduce the incidence of this cancer. In the present study, protein-carboxyl O-methyltransferase has been identi¢ed and puri¢ed from H. pylori. Unlike other protein-carboxyl O-methyltransferases, H. pylori protein methylase II has a distinct characteristic of molecular structure composed of four subunits of heterodimers, having a molecular mass of 425 kDa.
mined by Bradford's method [22], using bovine serum albumin as a standard. 2.3. Puri¢cation of protein methylase II from H. pylori
2.2. Assay for protein methylase II
H. pylori collected from plates (wet weight 30 g) were suspended in four volumes of phosphate/phenylmethylsulfonyl £uoride (PMSF) bu¡er containing 0.1 M potassium phosphate, 1.0 mM EDTA, 0.1 mM PMSF, 5% glycerol and 0.24 mM L-mercaptoethanol (pH 7.0). The suspension was sonicated for 10 min, and the sonicate was centrifuged at 105 000Ug for 1 h at 4³C. The cell-free extract was brought to 90% saturation with ¢nely powdered ammonium sulfate. The suspension was stirred for 30 min and then centrifuged for 15 min at 105 000Ug. The precipitate was resuspended in 10 ml bu¡er A (20 mM Tris^HCl bu¡er, pH 8.0, containing 1 mM EDTA, 0.1 mM EGTA, 0.24 mM L-mercaptoethanol, 0.1 mM PMSF, and 5% glycerol), dialyzed (10 kDa MW cut-o¡) against 4 l of bu¡er A overnight at 4³C, and applied to a QSepharose column (i.d. 26 mmU10 cm, Amersham Pharmacia Biotech, USA). The enzyme was eluted with a linear gradient of bu¡er A containing 0^0.5 M NaCl at a £ow rate of 1 ml min31 . The active fractions having methylase activity were pooled and concentrated with ultra¢ltration membrane (YM 10; cut-o¡ of 10 kDa ; Amicon, USA) to remove residual salt. The concentrate was subjected to an a¤nity column. AdoHcy-Sepharose 4B a¤nity column (i.d. 28 mmU3.4 cm), prepared by the method of Kim et al. [23], was equilibrated with bu¡er B (5 mM sodium phosphate, 5 mM EDTA, 2.4 mM L-mercaptoethanol, pH 6.2) at a £ow rate of 1.8 ml min31 . The enzyme was eluted with 0.5 mM AdoMet in bu¡er B. Fractions (1.8 ml) were collected into test tubes containing 0.2 ml of 50 mM sodium borate, 5% glycerol and 50 mM EDTA (pH 9.3) to
Enzyme activity was determined by a modi¢ed method of Kim and Paik [5]. The reaction mixture containing 10 mM sodium phosphate bu¡er (pH 7.0), 0.6 mg of histone (calf thymus type II-As, Sigma Chemical Co., USA), and appropriate amounts of enzyme preparation in a total volume of 0.09 ml was preincubated at 37³C for 5 min, and was further incubated for 15 min after the addition of 0.01 ml of 25 WM Ado[methyl-14 C]Met (130 dpm pmol31 , Amersham Pharmacia Biotech, USA). At the end of incubation, 0.1 ml of 125 mM sodium borate bu¡er (pH 11.0) was added to the incubation mixture, and the mixture was further incubated for 5 min at 37³C. 1 ml of isoamyl alcohol was added to the mixture, the mixture was vortexed and centrifuged at 10 000Ug for 5 min at room temperature, and the radioactivity of the supernatant (0.7 ml) was determined. One enzyme unit is de¢ned as the amount of enzyme that catalyzes the transfer of 1 pmol methyl-14 C to the methyl acceptor substrate min31 at pH 7.0 and 37³C. Speci¢c activity is de¢ned as units of enzyme per mg of protein. Protein concentration was deter-
Fig. 1. Gel permeation chromatography on a Superdex 200 HR column. The concentrated enzyme preparation (600 Wg) was loaded on a Superdex 200 HR column (i.d. 10 mmU30 cm) and eluted with 20 mM Tris^ HCl (pH 8.0) at a £ow rate of 0.4 ml min31 . The solid line represents OD280 and closed circles the enzyme activity.
2. Materials and methods 2.1. Strains and culture conditions H. pylori NCTC11637 was purchased from the National Collection of Type Cultures (NCTC, UK). H. pylori (Sydney strain) and clinically isolated H. pylori were kindly provided by Daewoong Pharmaceutical Co., Korea. H. pylori NCTC11637, H. pylori (Sydney strain), and clinically isolated H. pylori were cultivated by agar plate which was prepared using blood agar base No. 2 (Difco, USA) with antibiotics (vancomycin, bacitracin, amphotericin, polymyxin B, and nalidixic acid) with 10% blood [21]. Plates were incubated for 3 days at 37³C in 10% CO2 .
FEMSLE 9759 23-1-01
Y.M. Kim et al. / FEMS Microbiology Letters 195 (2001) 53^58
55
Table 1 Puri¢cation of protein methylase II from H. pylori Puri¢cation step
Volume (ml)
Protein (mg ml31 ) Total protein (mg)
Speci¢c activity (U mg31 ) Total activity (Ua )
Yield (%)
Supernatant after sonication (NH4 )2 SO4 precipitate Q-Sepharose AdoHcy-Sepharose 4B a¤nity Superdex 200 HR gel permeation
109 88 37.5 1.9 1
12.3 13.8 3.8 0.3 0.04
1.6 1.6 6.3 122 184
100 86 40 3.3 0.3
1341 1214 143 0.6 0.04
2246 1942 901 73 7.4
Data represent a typical puri¢cation procedure, which was repeated three times with similar results. a Units: pmol methyl-14 C transferred min31 .
stabilize the eluted enzyme. The fractions eluted with AdoMet were concentrated by ultra¢ltration membrane (YM 10; Amicon, USA) [23]. The above concentrated enzyme preparation was applied onto a Superdex 200 HR column (i.d. 1.0U30 cm, Amersham Pharmacia Biotech, USA) previously equilibrated with bu¡er A containing 0.15 M NaCl. The column was then eluted with bu¡er A at a £ow rate of 0.4 ml min31 (Fig. 1). The fractions containing protein methylase II activity were pooled, concentrated and residual salts were removed by ultra¢ltration membrane (YM 10; Amicon, USA). The puri¢ed enzyme was stored at 320³C until use.
subjected to SDS^PAGE and electroblotted on PVDF membrane. The membrane was dried, and the protein methylase II band was sequenced. N-terminal amino acid sequence analysis was performed with an Applied Biosystems Procise Protein Sequencer (Perkin-Elmer Applied Biosystems, USA), equipped with an on-line analyzer for phenylthiohydantoin-derivatized amino acids.
2.4. Estimation of molecular mass of the puri¢ed protein methylase II
The presence of protein methylase II in three strains (NCTC11637, Sydney strain, clinical isolates) of H. pylori was identi¢ed. The speci¢c activity of each strain was 0.5, 1.7, and 2.2 pmol Ado[methyl-14 C]Met transferred min31 mg31 protein, respectively. Although the clinically isolated strain had the highest speci¢c activity, it was very di¤cult to culture for the following experiment. Also, because
The molecular mass of the puri¢ed protein methylase II was determined by SDS polyacrylamide gel electrophoresis (SDS^PAGE), non-denaturing PAGE, matrix-assisted laser desorption/ionization time-of-£ight mass spectrometry (MALDI-TOF-MS; Perseptive Analytical Elite, USA) analysis, and gel permeation chromatography. SDS^ PAGE was performed according to the method of Laemmli [24] and non-denaturing PAGE was performed according to the method of Bhown and Bennett [25]. The molecular mass of the puri¢ed enzyme was determined using MALDI-TOF-MS. The puri¢ed protein methylase II from H. pylori was ¢rst dissolved in 0.1% tri£uoroacetic acid, and then was added to the matrix solution (saturated dihydrobenzoic acid in 1:3:2 formic acid:water:2-propanol), so that the ¢nal protein concentration of the mixture was less than 0.1 mg ml31 . An aliquot of matrix/protein solution (0.5 Wl) was then applied to a metal probe tip and dried [26,27]. Also, the molecular mass of the puri¢ed enzyme was determined by gel permeation on a Superdex 200 HR column. The relative elution volume of protein methylase II was compared with that of standard markers (vitamin B-12, 1.35 kDa; myoglobulin, 17 kDa ; ovalbumin, 44 kDa ; Q-globulin, 158 kDa ; thyroglobulin, 670 kDa). 2.5. N-terminal sequencing of the puri¢ed protein methylase II The puri¢ed protein methylase II from H. pylori was
3. Results 3.1. Identi¢cation of protein methylase II from H. pylori
Fig. 2. Determination of molecular mass of the enzyme by SDS^PAGE and non-denaturing PAGE. A: M represents molecular mass standard markers including myosin (205 kDa), L-galactosidase (116 kDa), phosphorylase b (97.4 kDa), bovine serum albumin (66 kDa), egg ovalbumin (45 kDa), and carbonic anhydrase (29 kDa). A and G represent enzyme preparations puri¢ed by AdoHcy-Sepharose a¤nity and Superdex 200 HR column chromatography, respectively. B: M represents molecular mass standards used: apoferritin (443 kDa), urease (272 kDa), alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), and chicken egg albumin (45 kDa). G represents enzyme preparation puri¢ed by Superdex 200 HR column chromatography.
FEMSLE 9759 23-1-01
56
Y.M. Kim et al. / FEMS Microbiology Letters 195 (2001) 53^58
Fig. 3. Analysis of protein methylase II by MALDI-TOF-MS.
strain NCTC11637 has a low speci¢c activity, we selected the Sydney strain for puri¢cation of protein methylase II. As shown in Table 1, the enzyme was puri¢ed by Q-Sepharose ion exchange, AdoHcy-Sepharose 4B a¤nity and Superdex 200 HR column chromatography, and the ¢nal preparation had a speci¢c activity of 184 pmol Ado[methyl-14 C]Met transferred min31 mg31 protein with 115-fold puri¢cation and 0.3% yield.
The molecular mass of the puri¢ed enzyme preparation was also analyzed by Superdex 200 HR gel permeation and found to be approximately 430 kDa (data not shown), which was very close to the value of 410 kDa determined by non-denaturing gel electrophoresis (Fig. 2B). As shown in Fig. 3, MALDI-TOF-MS analysis of native protein methylase II showed a major mass signal with a molecular mass value of 425 073 Da.
3.2. Molecular mass and subunit size of protein methylase II from H. pylori
3.3. N-terminal analyses of the puri¢ed protein methylase II from H. pylori
The puri¢ed protein methylase II preparation was resolved into two bands on SDS^PAGE, having molecular masses of 78 and 29 kDa (Fig. 2A). On the other hand, the enzyme migrated as a single band with a molecular mass of 410 kDa on non-denaturing gel electrophoresis (Fig. 2B).
As shown in Table 2, N-terminal sequences of each subunit of protein methylase II from H. pylori were analyzed using the GenBank database system. The N-terminal sequence (1^12) of the 78-kDa subunit was similar to that of chlorohydrolase in strains 26695 and J99 of H. pylori with 91% identity, and the N-terminal sequence (1^20) of the
Table 2 N-terminal analyses of the puri¢ed protein methylase II from H. pylori Subunit
N-terminal sequence
Identity (%)
78 kDa Chlorohydrolase Chlorohydrolase 29 kDa Glutamine synthetase Glutamine synthetase
MQEIIGACLV FL MQEIIGASLV FL MQEIIGASLV FL MIVRTQMCEC KIKRFFEFCK MIVRTQNSES KIKEFFEFCK MVVRTQNSES KIKEFFEFCK
FEMSLE 9759 23-1-01
11/12 (91%) 11/12 (91%) 16/20 (80%) 15/20 (75%)
H. pylori strain Sydney 26695 J99 Sydney 26695 J99
Y.M. Kim et al. / FEMS Microbiology Letters 195 (2001) 53^58
57
4. Discussion
Fig. 4. Lineweaver^Burk plot of Ado[methyl-14 C]Met on protein methylase II from H. pylori. The reaction mixture containing 13.5Wg of the puri¢ed protein methylase II and 1 mg of histone was incubated under the condition described in Section 2. Km for AdoMet was 5.0U1036 M and Vmax was 205 pmol min31 mg31 protein.
29-kDa subunit was similar to that of glutamine synthetase in strains 26695 and J99 of H. pylori with 80% and 75% identity, respectively. However, the molecular masses of chlorohydrolase (45.5 kDa) and glutamine synthetase (54.6 kDa) are signi¢cantly di¡erent from those of the subunits of puri¢ed protein methylase II. And there are no sequence homologies between these two enzymes and other protein methylases, indicating that protein methylase II puri¢ed from H. pylori is a novel enzyme, distinguished from chlorohydrolase, glutamine synthetase, and other protein methylases. 3.4. Characteristics of protein methylase II from H. pylori There was a linear relationship between the amount of substrate protein (histone 0^64 mg) and enzyme activity (data not shown). The Michaelis constants (Km value) and the maximum reaction velocity for AdoMet were determined by Lineweaver^Burk plot: Km and Vmax values were 5.0U1036 M and 205 pmol Ado[methyl-14 C]Met min31 mg31 enzyme protein, respectively (Fig. 4). Table 3 shows that histone and hemoglobin served as the best in vitro substrates for the puri¢ed protein methylase II. The enzyme had optimum pH of 6.0 (data not shown).
Protein methylase II has been found in organisms ranging from prokaryotes to eukaryotes [1^4,28]. In the present study, the enzyme was puri¢ed from H. pylori by means of Q-Sepharose, AdoHcy-Sepharose 4B a¤nity and Superdex 200 HR chromatography. The ¢nal puri¢ed enzyme preparation had a speci¢c activity of 184 pmol methyl-14 C min31 mg31 protein, representing a 115-fold puri¢cation with a yield of 0.3% (Table 1). This extent of puri¢cation is in the range of enzyme puri¢cations from other sources : 1160-fold puri¢cation from Escherichia coli with a yield of 0.067% [12] and 13-fold puri¢cation from Salmonella typhimurium with 13% yield [14]. Purity and molecular mass of the puri¢ed enzyme was determined by SDS^PAGE, non-denaturing PAGE, MALDI-TOF-MS and gel permeation chromatography. The molecular mass of the enzyme determined by nondenaturing PAGE, MALDI-TOF-MS and gel permeation chromatography gave values of 410, 425, and 430 kDa, respectively. On SDS^PAGE, however, H. pylori protein methylase II was found to be composed of a heterodimer of 78 and 29 kDa (Fig. 2A). It is, therefore, highly likely that this enzyme appears to be composed of four heterodimers (4U(78+29) = 428 kDa). The molecular mass of protein methylase II (AdoMet:protein-carboxyl O-methyltransferases) isolated from various organisms ranged from 24 to 33 kDa [6,8^11], however, the enzyme of Streptomyces fradiae comprised two identical subunits of 32 kDa [29]. Thus, the enormous size as well as the composition are quite unusual for protein methyltransferases in general. Among various substrates for H. pylori protein methylase II (Table 3), histone was found to be the most e¤cient substrate. N-terminal sequences of each subunit of protein methylase II from H. pylori were compared to the deduced amino acid sequence of H. pylori in GenBank. The Nterminal amino acid sequences of the 78-kDa and 29kDa subunits were very close to those of chlorohydrolase and glutamine synthetase in strains 26695 and J99 of H. pylori. However, the molecular masses of chlorohydrolase (45.5 kDa) and glutamine synthetase (54.6 kDa) are signi¢cantly di¡erent from those of the subunits of puri¢ed
Table 3 Substrate speci¢city of protein methylase II from H. pylori Substrate proteina
Speci¢c activity (pmol min31 mg31 )
Relative activity (%)
Histone Hemoglobin Myelin basic protein Albumin Cytochrome c Gelatin Q-Globulin
180 174 152 142 102 89 59
100 97 84 79 57 49 33
The incubation and assay conditions were as described in Section 2. a The amount of substrate used was 0.6 mg.
FEMSLE 9759 23-1-01
58
Y.M. Kim et al. / FEMS Microbiology Letters 195 (2001) 53^58
protein methylase II, showing no identity between protein methylase II from the Sydney strain of H. pylori and the other two enzymes. The discrepancy in molecular mass between protein methylase II puri¢ed from the Sydney strain of H. pylori and the other two enzymes deduced from GenBank data of strains 26695 and J99 of H. pylori might be attributed to a di¡erence in genotype between strains. Also, similar sequences found in chlorohydrolase and glutamine synthetase have never been observed in other protein methylases. Therefore, protein methylase II from the Sydney strain of H. pylori appears to be a novel enzyme distinguished from other protein methylases. The biological signi¢cance of the presence of protein methylase II in H. pylori is not obvious at present. However, the enzyme with the unusual characteristic of such a large molecular mass (425 kDa) with eight heterosubunits is suggestive of its complex function, and its function should be ¢nely modulated in the stomach. Obviously, further studies on the exact amino acid sequence of protein methylase II of the Sydney strain of H. pylori, its location in the genome and the biological signi¢cance of H. pylori protein methylase II are needed. 5. Uncited references
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[16,17]
[19]
Acknowledgements
[20]
This work forms part of the thesis of Y.M.K. presented to the Graduate Board of Sungkyunkwan University, Seoul, Korea in partial ful¢llment of the requirements for the Ph.D. degree.
[21]
[22]
[23]
References [1] Ambler, R.P. and Rees, M.W. (1959) O-N-Methyl-L-lysine in bacterial £agella protein. Nature 184, 56^57. [2] Paik, W.K. and Kim, S. (1980) Protein Methylation : A Series of Monographs. John Wiley, New York. [3] Selmer, T., Kahnt, J., Goubeaud, M., Shima, S., Grabarse, W., Ermler, U. and Thauer, R.K. (2000) The biosynthesis of methylated amino acids in the active site region of methyl-coenzyme M reductase. J. Biol. Chem. 275, 3755^3760. [4] Chen, D., Ma, H., Hong, H., Koh, S.S., Huang, S.M., Schurter, B.T., Aswad, D.W. and Stallcup, M.R. (1999) Regulation of transcription by a protein methyltransferase. Science 284, 2174^2177. [5] Axelrod, J. and Daly, J. (1965) Pituitary gland: Enzymic formation of methanol from S-adenosyl-L-methionine. Science 150, 892^893. [6] Kim, S. and Paik, W.K. (1970) Puri¢cation and properties of protein methylase II. J. Biol. Chem. 245, 1806^1813. [7] Clarke, S. (1985) Protein carboxyl methyltransferases : two distinct classes of enzymes. Annu. Rev. Biochem. 54, 479^506. [8] Kim, S. (1973) Puri¢cation and properties of protein methylase II. Arch. Biochem. Biophys. 157, 476^484. [9] Aswad, D.W. and Deight, E.A. (1983) Puri¢cation and characteriza-
[24] [25]
[26]
[27]
[28]
[29]
tion of two distinct isozymes of protein carboxylmethylase from bovine brain. J. Neurochem. 40, 1718^1726. Gilbert, J.M., Fowler, A., Bleibaum, J. and Clarke, S. (1988) Puri¢cation of homologous protein carboxyl methyltransferase isozymes from human and bovine erythrocytes. Biochemistry 27, 5227^5233. Jung, K., Kwon, M., Lee, H.Y., Lee, H.W. and Hong, S. (1995) Puri¢cation and characterization of protein methylase II from porcine testis. J. Biochem. Mol. Biol. 28, 149^154. Fu, J.C., Ding, L. and Clarke, S. (1991) Puri¢cation, gene cloning, and sequence analysis of an L-isoaspartyl protein carboxyl methyltransferase from Escherichia coli. J. Biol. Chem. 266, 14562^14572. Simms, S.A. and Subbaramaiah, K. (1991) The kinetic mechanism of S-adenosyl-L-methionine:glutamylmethyltransferase from Salmonella typhimurium. J. Biol. Chem. 266, 12741^12746. Simms, S.A., Stock, A.M. and Stock, J.B. (1987) Puri¢cation and characterization of the S-adenosylmethionine:glutamyl methyltransferase that modi¢es membrane chemoreceptor proteins in bacteria. J. Biol. Chem. 262, 8537^8543. MacLaren, D.C. and Clarke, S. (1995) Expression and puri¢cation of a human recombinant methyltransferase that repairs damaged proteins. Protein Expr. Purif. 6, 99^108. Brennan, T.V., Anderson, J.W., Jia, Z., Waygood, E.B. and Clarke, S. (1994) Repair of spontaneously deamidated HPr phosphocarrier protein catalyzed by the L-isoaspartate-(D-aspartate) O-methyltransferase. J. Biol. Chem. 269, 24586^24595. Stephenson, R.C. and Clarke, S. (1990) Identi¢cation of a C-terminal protein carboxyl methyltransferase in rat liver membranes utilizing a synthetic farnesyl cysteine-containing peptide substrate. J. Biol. Chem. 265, 16248^16254. Marshall, B.J. and Warren, J.R. (1984) Unidenti¢ed curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet 1, 1311^1315. Blaser, M.J. (1992) Hypotheses on the pathogenesis and natural history of Helicobacter pylori-induced in£ammation. Gastroenterology 102, 720^727. Ernst, P. (1999) The role of in£ammation in the pathogenesis of gastric cancer (Review). Aliment. Pharmacol. Ther. 1, 13^18. Morshed, M.G., Islam, M.S., Zinnah, F., Rumi, M.A., Ahmad, M.M. and Huq, F. (1995) Growth of Helicobacter pylori in candle jar. Lancet 346, 511. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248^254. Kim, S., Nochumson, S., Chin, W. and Paik, W.K. (1978) A rapid method for the puri¢cation of S-adenosylmethionine:protein-carboxyl O-methyl-transferase by a¤nity chromatography. Anal. Biochem. 84, 415^422. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680^685. Bhown, A.S. and Bennett, J.C. (1983) High-sensitivity sequence analysis of proteins recovered from sodium dodecyl sulfate gels. Methods Enzymol. 91, 450^455. Karas, M. and Hillenkamp, F. (1988) Laser desorption ionization of proteins with molecular masses exceeding 10,000 daltons. Anal. Chem. 60, 2299^2301. Reiber, D.C., Brown, R.S., Weinberger, S., Kenny, J. and Bailey, J. (1998) Unknown peptide sequencing using matrix-assisted laser desorption/ionization and in-source decay. Anal. Chem. 70, 1214^ 1222. Paik, W.K. and Kim, S. (1990) Reevaluation of the enzymology of protein methylation. In: Protein Methylation (Paik, W.K. and Kim, S., Eds.), pp. 23^31. CRC Press, Boca Raton, FL. Bauer, N.J., Kreuzman, A.J., Dotzlaf, J.E. and Yeh, W.K. (1988) Puri¢cation, characterization, and kinetic mechanism of S-adenosylL-methionine :macrocin O-methyltransferase from Streptomyces fradiae. J. Biol. Chem. 263, 15619^15625.
FEMSLE 9759 23-1-01