| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 Department of Biochemistry
2 Mass Spectrometry Research Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA
(RECEIVED October 11, 2005; FINAL REVISION January 11, 2006; ACCEPTED January 13, 2006)
 |
Abstract |
|---|
 TOP Abstract IntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Keywords: glycine N-methyltransferase; rat; phosphorylation
|
Introduction |
|---|
|
TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Although GNMT was first isolated from rabbit, most of the properties of GNMT were obtained using the enzyme from rat, either isolated from rat liver (Ogawa and Fujioka 1982) or recombinant enzyme (Ogawa et al. 1997). It was shown that GNMT is a tetrameric protein with active sites on each identical subunit. The molecular mass of the subunits was found to be 32.5 kDa. Some kinetic data on the GNMTs from other mammals, including human, were obtained and their cDNAs were sequenced (Ogawa et al. 1984, 1993; Aida et al. 1997; Chen et al. 1998). The GNMT proteins were found to be very similar, with identity of amino acid sequences of about 90% (Ogawa et al. 1993). Recombinant rat GNMT was crystallized as the apo-protein (Pattanayek et al. 1998), in complex with SAH and with SAM and acetate (as an analog of glycine) (Fu et al. 1996; Takata et al. 2003). Based on the crystal structures, a mechanism of enzyme action was proposed in which movements of the N-terminal part of the sequence plays a major role in the transfer of the methyl group from SAM to glycine (Takusagawa et al. 1999; Takata et al. 2003).
Study of the rat enzyme showed a difference in the kinetic parameters between recombinant protein and GNMT isolated from liver (Ogawa and Fujioka 1982; Ogawa et al. 1997). Moreover, it was found that the recombinant enzyme was not inhibited by concentrations of 5-CH3-H4Pte-Glu5 that inhibited the native rat liver enzyme (C. Wagner, unpubl.). It was proposed that a possible reason for the difference in kinetics was the N-terminal acetylation of the valine (Ogawa and Fujioka 1982) on the liver enzyme or other post-translational modifications of GNMT (phosphorylation, methylation, etc.) that are not present in the recombinant enzyme. It was found that GNMT could be phosphorylated in vitro by cAMP-dependent protein kinase, resulting in significantly increased enzyme activity (Wagner et al. 1989). In vivo phosphorylation of rat GNMT was recently described using a proteomics approach (Møller et al. 2003). Serine was identified as the site of phosphorylation, but its position was not localized. In this report, we present data on the identification of a number of phosphorylation sites of rat GNMT.
|
Results |
|---|
|
TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Most standard preparations of GNMT, that were routinely kept in the presence of 
-mercaptoethanol and DTT as reducing agents, showed multiple peaks in their mass spectra. In recombinant GNMT, in addition to the peak with a molecular mass of 32,420.7 Da, peaks of greater molecular mass (+75–79 Da) or multiples thereof were found probably as a result of -mercaptoethanol modification of the cysteine residues. In the case of the liver enzyme, multiple peaks were also found, but the lowest molecular mass peak was found to be 32,461.7 Da, which corresponds to full-size protein with an acetylated N-terminal residue.
LC-MS/MS analysis
The conclusion from initial QqTOF analyses of an intact liver GNMT sample was that the modified protein was not a significant fraction of the GNMT sample. Because published data (Møller et al. 2003) suggested that rat GNMT is phosphorylated in vivo, a more sensitive approach was used for identification of the phosphorylated site(s). In our work, two mass spectrometry approaches and different samples of GNMT were prepared using inhibitors of kinases and phosphatases.
The first approach used a full-scale LC-MS/MS analysis in order to analyze all possible sites of modification. After the most likely phosphorylated peptides were identified by analysis of Sequest and P-Mod algorithms, specific precursor ions were selected for MS/MS or MS/MS/MS analysis (of neutral loss of phosphoric acid ions) to verify the site of phosphorylation with improved spectral quality.
Analysis using Sequest showed that trypsin and chymotrypsin digestion of GNMT samples yielded a high coverage of amino acid sequence, not less than 88%. The liver and recombinant enzymes differ in an N-terminal acetylation and the presence of a number of peptides with possible modifications of liver GNMT. This difference in N-terminal acetylation was easily detected by LC-MS/MS analysis as a 42-Da increase of the mass of N-terminal peptide VDSVYR and the presence of all y- and b-ions after fragmentation of the peptide (Fig. 2).
The LC-MS/MS data revealed multiple phosphorylated peptides in all GNMT samples. Additional analysis of the data with another program (P-Mod) revealed phosphorylated tryptic peptides: 9–28, 60–89, 123–147, 176–190, and 240–271, with phosphorylated serine residues of S9, S71, S139, S182, and S241, respectively. A number of chymotryptic peptides containing the same serine residues as above were also detected (Fig. 3; Table 1).
To obtain additional data on the phosphorylated peptides, MS/MS analysis of selected precursor ions from the tryptic digest of GNMT samples was performed. The mass spectrometer was set up to acquire MS/MS spectra specifically of potentially phosphorylated peptides mentioned above by targeting the anticipated ions, rather than acquiring the data in a data-dependent fashion. All spectra that contained a phosphorylated residue were manually inspected for verification.
The most easily detected peptides were two phosphopeptides for which most of the b- and y-ions were found. Peptide 9–28, SLGVAAEGIPDQYADGEAAR, was found in the GNMT prepared by standard chromatography and in GNMT immunoprecipitated from hepatocytes. The phosphorylated peptide 176–190, NYDYILSTGCAPPGK, was found in all GNMT samples. Other peptides that were also detected by either the data-dependent analysis or targeted analysis are listed in Table 1.
Peptide 9–28 contains only one serine residue (S9). The presence of the 9–28 phosphorylated peptide was confirmed by the additional mass of 80 Da, and by the presence of most of the y- and b-ions in the MS/MS spectra, with a correspondingly 80-Da shift to account for the phosphorylation (Fig. 4). In addition, the spectrum contained a neutral loss of phosphoric acid (M2H+–H3PO4), which is an additional characteristic fragmentation that can occur in phosphorylated peptides. Interestingly, phosphorylation of S9 was detected only in GNMT prepared by standard chromatographic methods and in the preparation immunoprecipitated from hepatocytes. Peptide 176–190 was found in all GNMT samples by the presence of additional 80 Da mass units and by the presence of most of the b- and y-ions (Fig. 5).
Of particular interest were the peptides containing phosphorylated S139. The collision-induced fragmentation of this peptide clearly showed the presence of the phosphorylated serine residue (data not shown) in all samples except the standard liver GNMT preparation.
Analysis of rat GNMT by protein chemistry
In addition to mass spectrometric analysis, GNMT phosphorylation was studied using protein chemistry. To identify the phosphorylated residue produced by the isolated hepatocytes they were treated with radiolabeled [32P] sodium phosphate to incorporate [32P] into GNMT. After homogenization, GNMT was isolated by immuno-precipitation and SDS-electrophoresis. It was found that radioactive phosphate was incorporated into GNMT. Phosphoamino acid analysis showed that the only phosphorylated amino acid residue was serine (Fig. 6).
GNMT containing radioactive phosphate was used for determination of phosphopeptides by trypsin digestion and N-terminal sequencing. Identification of the specific phosphorylated serine residue(s) in that GNMT sample by peptide sequencing was complex. Incorporation of the radioactive [32P] into GNMT was low and greatly dependent on the different hepatocytes and crude extract preparations. Our results showed that radioactively labeled peptides were eluted in two or three peaks when the tryptic digest of GNMT was fractionated by reverse-phase chromatography on C18 columns. In one of those peaks a relatively pure peptide was eluted. Its N-terminal sequence was determined as NYDYIL, which corresponded to peptide 176–190, NYDYILSTGCAPPGK. This was confirmed by mass spectrometry. Sequencing of the peptides from another peak revealed the presence of a mixture of peptides, which we were unable to separate.
In vitro phosphorylation of GNMT by cAMP-dependent kinase
We reported earlier that rat GNMT could be phosphorylated in situ (Wagner et al. 1989), but the position of the phosphorylated serine was not determined. It was done later by N-terminal sequencing of the phosphorylated peptide (Yeo 1992). After tryptic digestion of the phosphorylated protein, the peptides were separated by C18 reverse-phase chromatography, and individual fractions of radiolabeled peptide were sequenced. It was found that the sequence of phosphorylated peptide was SLGVAADGIPEQYAGDAAR. This is the sequence of tryptic peptide 9–28, which means that the serine residue modified by cAMP-dependent kinase was S9. This also means that S9 phosphorylation of liver GNMT was a result of action of cAMP-dependent kinase.
|
Discussion |
|---|
|
TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
In the study of protein post-translational phosphorylation two things are critical: protein preparation and sensitivity of the analytical methods. Therefore, we used different methods for GNMT preparation to study effects of different treatments on the location of phosphate groups. Only by using LC-MS/MS mass spectrometry and powerful analytic software were we able to identify the specific serine residues, which are phosphorylated in rat GNMT.
Preparation of liver GNMT by standard chromatographic methods is designed to yield a large amount of purified protein, but takes from two to three weeks since it uses a number of column chromatographic steps. Analysis of a highly purified sample prepared in this way (Fig. 1) showed no major phosphorylated molecular species, but the presence of multiple phosphopeptides was well documented in other preparations (Table 1). Purification using phosphatase inhibitors and immunologic separation was designed to isolate small amounts of protein rapidly.
By using both approaches, we identified the major phosphorylation sites of rat GNMT as serine residues S9 and S182 and minor sites as serines S71 and S241. In all cases, the level of phosphorylation is low and the pattern of phosphorylation depends on the GNMT sample preparation. When precautions were taken to inhibit phosphatase action during GNMT preparation an additional phosphorylation was detected at S139.
The phosphorylated serine residues in rat GNMT are located within different sequence motifs, suggesting that different kinases used GNMT as substrate. A large database for substrate specificity for known kinases (Kennelly and Krebs 1991; Blom et al. 1999) allows us to speculate on the potential kinases that phosphorylated rat GNMT. For S9, located in the sequence YRTRS(9)LGVA, our experimental data showed that this residue is phosphorylated by cAMP-dependent protein kinase (PKA) in situ, for which the consensus motif is R-X(1–2)-S/T-X or R-XX-S/T (Kennelly and Krebs 1991). The most likely kinase that phosphorylates S241 may be a CaM II-dependent kinase for which the consensus sequence site of phosphorylation is R-XX-S/T-X. This is similar to the phosphorylation site of GNMT at Ser241 (FRLSYYPH). The question of whether these kinases are responsible for phosphorylation of GNMT in vivo remains to be answered.
A very interesting finding of this work is that almost all the phosphorylated serine residues that were found in the liver enzyme were found also in recombinant GNMT expressed in E. coli. The presence of serine/threonine/tyrosine kinases in prokaryotic organisms including E. coli is known (Shi et al. 1998). Activity of E. coli kinases on expressed mammalian protein is well documented (Du et al. 2005), but how this takes place is unknown.
The identification of the phosphorylation sites of rat GNMT was undertaken to elucidate a potential mechanism of regulation of conformation and activity of GNMT in vivo. Although the level of GNMT phosphorylation is low, it may be higher in vivo, and there is a possibility for regulation of the properties of enzyme by specific serine phosphorylation. GNMT is a tetrameric enzyme, the conformation and stability of which depends on the conformation of each monomer as they are tightly bound by multiple interactions (Fu et al. 1996; Pattanayek et al. 1998; Takata et al. 2003). The phosphorylation is not limited to rat liver, since we have also identified multiple phosphorylation sites in human GNMT expressed in H1299 cells (Z. Luka and C. Wagner, unpubl.), which suggests that it may have broad significance.
The most easily detected phosphorylated residues, serines S9 and S182, are located on the surface of the GNMT molecule. Phosphorylation of these residues could affect conformation and stability of the GNMT tetrameric structure. As shown in Figure 7, S9 and S182 are close enough in the crystal structure to predict a destabilizing effect of the phosphorylation on the quaternary structure. We previously have shown that stability of human GNMT tetramer determines activity of the enzyme (Luka and Wagner 2003).
Phosphorylation of S9 may have an impact on GNMT in two ways. This residue is located on the N-terminal part of each monomer, which is proposed to be critical for the enzymatic reaction (Takata et al. 2003). Therefore, phosphorylation of that residue could directly affect enzymatic activity of GNMT. Another possibile way of affecting GNMT action is by modulating folate binding. We have shown that in vitro phosphorylation of S9 by cAMP-dependent protein kinase decreases binding of 5-CH3-H4Pte-Glu5 (Wagner et al. 1989).
The other sites of serine phosphorylation found in this work also suggest regulation of enzyme activity. Serine 71 is located in the middle of a-helix 3 (residues 69–76), which is close to the active center, and it is possible that incorporation of a negative charge into the microenvironment could affect enzyme action. Serine 139 is located within the group of residues that directly participates in rat GNMT enzyme action according to a proposed mechanism (Takata et al. 2003). The G137 makes a hydrogen bond with the substrate glycine, while L136 and H142 form hydrogen bonds with SAM. One could expect a significant effect on the SAM and glycine binding in the active center as a result of the negative charge by phosphorylation of S139. The importance of the microenvironment in this particular portion of the GNMT structure was shown by discovery of a human mutant GNMT with a N140S substitution (Augoustides-Savvopoulou et al. 2003). Substitution of asparagine 140 in human GNMT, which is homologous to N138 in rat GNMT, almost completely inactivated the enzyme. It is important that all proposed mechanisms for regulation of rat GNMT activity by phosphorylation of different serine residues be tested experimentally by in vitro phosphorylation with specific kinases and by site-directed mutagenesis of phosphorylated serines. This work is currently underway.
|
Materials and methods |
|---|
|
TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
The standard native rat GNMT preparation was isolated from rat liver by a procedure routinely used in our laboratory for many years (Zamierowski and Wagner 1977; Suzuki and Wagner 1980). This included homogenization in isotonic sucrose, preparation of cytosol, and chromatography on gel-exclusion, anion and cation exchange columns, and hydroxylapatite. The homogenization buffer I contained 10 mM K-phosphate buffer (pH 7.0), 0.25 M sucrose, 5 mM EDTA, 10 mM -mercaptoethanol, 1 mM PMSF, 10 µg/mL leupeptin, 10 µg/mL aprotinin, 1 mM benzamidine, and 4 µg/mL soybean trypsin inhibitor. No specific inhibitors for phosphatases were used in this procedure. Pure enzyme usually had specific activities over 300 nmol of product/min/mg, but this varied, and specific activities of over 500 have been obtained.
In an attempt to preserve any evidence of native phosphorylation, two other rapid methods of preparing liver GNMT were employed using phosphatase inhibitors and immunoprecipitation with affinity purified anti-rat GNMT polyclonal antibodies. This was prepared by homogenizing rat liver in buffer II containing: 50 mM Tris-HCl (pH 7.5), 50 mM Na-fluoride, 20 mM glycerophosphate, 5 mM Na-pyrophosphate, 1 mM Na-orthovanadate, 2 µM okadaic acid, 2 µM microcystin LR, 1 mM dithiothreitol, 2 mM EDTA, 1 mM EGTA, 1 mM PMSF, 10 µM leupeptin, 1 µM calpain inhibitor, 1 µM pepstatin A, 1 µM aprotinin, and 10 mM -mercaptoethanol. The homogenate was centrifugated at 40,000g for 1 h and the supernatant was used for GNMT immunoprecipitation. This was done with protein ASepharose beads (Sigma). The protein beads (about 10 mg) were first incubated with anti-GNMT antibodies in 20 mM Na-phosphate buffer (pH 8.0) overnight. The liver supernatant (100 µL) then was added and incubated on ice for 30 min. Unbound proteins were removed with 50 mM Na-phosphate buffer (pH 8.0), and all bound proteins were removed from the Sepharose beads by boiling in the SDS sample buffer. The protein sample was fractionated by SDS electrophoresis, and the GNMT protein band was excised from the gel and subjected to in-gel digestion as described below.
We also purified GNMT from rat hepatocytes using immunoprecipitation. Rat liver hepatocytes were isolated by a modification of the procedure described by Horne et al. (1978). After the required surgical preparation, the liver was perfused first without recirculation at 14 mL/min for 7 min with Ca-free Krebs-Ringer HEPES (pH 7.4) equilibrated at 37°C with 100% oxygen. Then ~80 mL was recirculated and 20 mg of collagenase and 5 mg of soybean trypsin inhibitor added. The resulting suspension was shaken for 10 min at 37°C in a plastic Erlenmeyer flask aerated with a stream of 100% oxygen. The suspension was passed through nylon mesh and the viable (more dense) cells were collected by centrifugation at 50g for 1 min. The pellet was washed in incubation medium (Krebs-Ringer HEPES at pH 7.4, containing 2% gelatin and MEM-essential and nonessential amino acids, vitamins, and glutamine) and resuspended in homogenization buffer II of the same composition as used for preparation of liver GNMT by immunoprecipitation. GNMT was then prepared by the procedure used for preparation of immunoprecipitated liver GNMT.
Recombinant GNMT was prepared by expression of rat GNMT in E. coli using the recombinant plasmid prepared in Dr. Ogawa's laboratory according to a published protocol (Ogawa et al. 1997) with minor modifications.
Phosphorylation of GNMT in hepatocytes
The rat hepatocytes prepared as above (3 mL) were suspended in 3 volumes of modified Krebs-Ringer HEPES, containing no phosphate, 16 mM lactate, and 4 mM sodium pyruvate as an energy source, and 1.5% gelatin plus MEM essential and non-essential amino acids, vitamins, and 2 mM glutamine. GNMT may be considered as an enzyme involved in gluconeogenesis, since it is one of the principal enzymes in the pathway from methionine to pyruvate. Its activity is induced by starvation or by diabetes (Gorin and Rosenblum 1974), and unpublished experiments (C. Wagner and R.J. Cook) have shown that glucagon administration to intact rats results in elevated GNMT activity after 2 h. For this reason glucagon was included during incubation of the hepatocytes.
To label the proteins, 2 mCi of [32P] phosphate was added and the reaction was incubated for 1 h at 37°C. Then 100 nM glucagon and 10 µM 8-para-chloro-phenyl-thio-cAMP (cAMP analog) were added to enhance the in situ phosphorylation reaction. After 5 min incubation at 37°C the cells were washed and homogenized in three volumes of homogenization buffer. The homogenate was centrifugated at 100,000g for 1 h. GNMT was isolated from the supernatant by immunoprecipitation and SDS-electrophoresis. The GNMT band was visualized by radioautography, excised, and protein was extracted by electroelution. GNMT was hydrolyzed in 6 N HCl for the phosphoamino acid analysis and was digested with trypsin for the phosphopeptide sequence analysis.
Phosphopeptide sequence analysis
For phosphopeptide sequence analysis the cysteine residues of GNMT were carboxyamidomethylated in 8 M urea with iodoacetamide, and the modified protein was digested with trypsin in 2 M urea at 37°C for 36 h. The tryptic digest of GNMT containing [32P] was fractionated by reverse-phase HPLC on a SynChropak RP-P C-18 column or Rexchrom S5-100-ODS (25 cm x 4.6 mm) column (Regis Technologies) with monitoring at 214 nm. Radioactive peptides were sequenced at the Protein Analysis Core Laboratory of Vanderbilt University School of Medicine.
Identification of phosphorylated amino acid residues
For phosphoamino acid analysis carboxyamidomethylated GNMT was hydrolyzed in 6 N HCl at 110°C for 2 h under nitrogen. The hydrolysate was mixed with phosphorylated amino acid standards and analyzed by two-dimensional thin-layer electrophoresis on thin-layer cellulose plates as described elsewhere (Cooper et al. 1983). The first dimension was run at 1000 V in acetic acid:formic acid:water (78:25:897, v/v; pH 1.9) for 90 min and the second dimension was run at 1000 V in acetic acid:pyridine:water (50:5:945, v/v; pH 3.5) for 35 min. The internal standards were located by ninhydrin staining, and radiolabeled amino acid was visualized by autoradiography.
In vitro phosphorylation of GNMT by cAMP-dependent protein kinase
Rat liver GNMT was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase as described (Wagner et al. 1989). The reaction mixture contained 5 µmol of HEPES buffer (pH 7.4), 1 µmol of DTT, 0.5 µmol of MgCl2, 10 µmol of cold ATP, 10 µCi of [
-32P]ATP, 0.3 µg of catalytic subunit of protein kinase, and 10 µg of purified GNMT in a final volume of 100 µL. After incubation at 25°C for 1 h, the reaction was stopped by adding 100 µL of SDS sample buffer. After SDS electrophoresis the protein (GNMT) from the radioactive band was electroeluted and analyzed for phospho-amino acids and for phosphopeptides as above.
In-gel trypsin and chymotrypsin digestion for mass spectrometry analysis
The GNMT band in the gel after SDS electrophoresis and Coomassie staining was digested by trypsin or chymotrypsin according to a standard procedure for in-gel digestion (Ham 2005). Briefly, the gel fragment was washed in 50 µL of 50 mM ammonium bicarbonate, then in 50 µL of 50% acetonitrile–25 mM ammonium bicarbonate. After drying, the gel was placed into 50 mM ammonium bicarbonate–10 mM DTT and protein was reduced at 56°C for 15 min. Protein was carboxyamidomethylated in 20 mM iodoacetamide for 15 min and iodoacetamide was washed out.
Tryptic digestion was done with Promega Porcine trypsin in 50 mM ammonium bicarbonate at 37°C overnight. Peptides were extracted by 25 mM ammonium bicarbonate and one additional step with 50 µL of 5% formic acid–50% acetonitrile. Chymotryptic digestion was similar to tryptic digestion, with the exception that the digestion was performed for 5 h at room temperature. Sequencing grade chymotrypsin from Princeton Separations was used.
Intact protein analysis by electrospray mass spectrometry
The molecular mass of intact GNMT was measured using a QSTAR mass spectrometer (Applied Biosystems), having QqTOF geometry, equipped with an ESI source and a Finnigan TSQ700 (Thermo Electron) with an ESI source. The protein sample for QqTOF measurement was prepared in 25 mM ammonium bicarbonate (pH 7.9) and treated with TCEP. Protein samples without treatment with TCEP were analyzed in 20 mM Tris (pH 8.5), 1 mM EDTA, 1 mM sodium azide diluted 1:10 with 100/100/10 (v/v/v) of methanol/water/acetic acid.
GNMT analysis by LC-MS/MS
The LC-MS/MS analyses were performed on a ThermoFinnigan LTQ linear ion trap mass spectrometer equipped with a ThermoFinnigan Surveyor LC pump, NanoSpray source (Thermo Electron), and Xcalibur 1.4 instrument control and data analysis software. HPLC separation of the chymotryptic peptides was achieved with 100 µm x 11 cm C-18 capillary column (Monitor C18, 5 micron, 100 Å, Column Engineering), at 0.7 µL/min flow rate. Solvent A was H2O with 0.1% formic acid, and solvent B was acetonitrile containing 0.1% formic acid. The gradient program was: 0–3 min, linear gradient from 0%–5% B; 3–5 min, 5% B; 5–50 min, linear gradient to 50% B; 50–52 min, linear gradient to 80% B; 52–55 min, linear gradient to 90% B; 55–56 min, 90% B in solvent A. MS/MS scans were acquired using an isolation width of 3 m/z, an activation time of 30 msec, and activation Q of 0.250 and 30% normalized collision energy using 1 microscan and ion time of 100 for each MS/MS scan. The mass spectrometer was tuned prior to analysis using the synthetic peptide TpepK (AVAGKAGAR), so some parameters may have varied slightly from experiment to experiment, but typically the tune parameters were as follows: spray voltage of 1.8 kV, a capillary temperature of 150°C, a capillary voltage of 50 V, and tube lens 100 V. Initial analysis was performed using data-dependent scanning in which one full MS spectra, using a full mass range of 400–2000 amu, was followed by three MS/MS spectra. In the subsequent analysis, several specific precursor masses were selected for MS/MS analysis in a targeted fashion.
Protein potential modification sites were identified using the SEQUEST algorithm (Eng et al. 1994) and the SEQUEST Browser software (Thermo Electron). A list of peptides was created and each peptide was run through P-Mod software to check for possible chemical modifications (Hansen et al. 2005). The candidate modifications found by software were verified by visual inspection of corresponding spectra. Theoretical protein, peptide, and peptide fragment ion masses were generated using General Protein Mass Analysis for Windows version 6.01.1 (Lighthouse Data, Odense, Denmark) and Protein Prospector (http://prospector.ucsf.edu).
Protein analysis
The concentration of protein samples was determined by the BCA method (BCA Protein Assay Kit, Pierce) using bovine serum albumin as a standard. SDS electrophoresis was performed according to standard protocol in 10%–12% gels. Gels were stained with Bio-Safe Coomassie (Bio-Rad).
|
|
|
|
|
|
|
|
|
Footnotes |
|---|
Reprint requests to: Conrad Wagner, Department of Biochemistry, Medical Center, Vanderbilt University, 620 Light Hall, Nashville, TN 37232, USA; e-mail: Conrad.Wagner{at}vanderbilt.edu; fax: (615) 343-0407.
Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051906706.
|
Acknowledgments |
|---|
|
References |
|---|
|
TOPAbstractIntroductionResultsDiscussionMaterials and methodsReferences |
|---|
Augoustides-Savvopoulou P., Luka Z., Karyda S., Stabler S.P., Allen R.H., Patsiaoura K., Wagner C., Mudd S.H. 2003. Glycine N-methyltransferase deficiency: A new patient with a novel mutation J. Inherit. Metab. Dis. 26: 745–759.[Medline]
Balaghi M., Horne D.W., Wagner C. 1993. Hepatic one-carbon metabolism in early folate deficiency in rats Biochem. J. 291: 145–149.
Blom N., Gammeltoft S., Brunak S. 1999. Sequence- and structure-based prediction of eukaryotic protein phosphorylation sites J. Mol. Biol. 294: 1351–1362.[CrossRef][Medline]
Chen Y.M., Shiu J.Y., Tzeng S.J., Shih L.S., Chen Y.J., Lui W.Y., Chen P.H. 1998. Characterization of glycine-N-methyltransferase-gene expression in human hepatocellular carcinoma Int. J. Cancer 75: 787–793.[CrossRef][Medline]
Cook R.J. and Wagner C. 1984. Glycine N-methyltransferase is a folate binding protein of rat liver cytosol Proc. Natl. Acad. Sci. 81: 3631–3634.
Cooper J.A., Sefton B.M., Hunter T. 1983. Detection and quantification of phosphotyrosine in proteins Methods Enzymol. 99: 387–402.[Medline]
Du P., Loulakis P., Xie Z., Simons S.P., Geoghegan K.F. 2005. Tandem mass spectrometry of multiply phosphorylated forms of a "histidine-tag" derived from a recombinant protein kinase expressed in bacteria. Rapid Commun Mass Spectrom. 19: 547–551.
Eng J.K., McCormack A.L., Yates J.R. III. 1994. An approach to correlate tandem mass-spectral data of peptides with amino-acid-sequences in a protein database J. Am. Soc. Mass Spectrom. 5: 976–998.[CrossRef]
Farrar C. and Clarke S. 2002. Altered levels of S-adenosylmethionine and S-adenosylhomocysteine in the brains of L-isoaspartyl (D-Aspartyl) O-methyltransferase-deficient mice J. Biol. Chem. 277: 27856–27863.
Fu Z., Hu Y., Konishi K., Takata Y., Ogawa H., Gomi T., Fujioka M., Takusagawa F. 1996. Crystal structure of glycine N-methyltransferase from rat liver Biochemistry 35: 11985–11993.[CrossRef][Medline]
Gorin E. and Rosenblum S. 1974. Effect of triamcinolone treatment, starvation and diabetes on rat liver protein kinase activity Biochim. Biophys. Acta 343: 510–518.[Medline]
Ham A.-J. 2005. Proteolytic digestion protocols In The encyclopedia of mass spectromety Volume 2 Biological applications Part A: Peptides and proteins (eds. Caprioli R.M. and Gross M.L.) . pp. 10–17. Elsevier Ltd., Kidlington, Oxford, UK.
Hansen B.T., Davey S.W., Ham A.-J.L., Liebler D.C. 2005. P-Mod: An algorithm and software to map modifications to peptide sequences using tandem MS data. J Proteome Res. 4: 358–368.
Heady J.E. and Kerr S.J. 1973. Purification and characterization of glycine N- methyltransferase J. Biol. Chem. 248: 69–72.
Horne D.W., Briggs W.T., Wagner C. 1978. Transport of 5-methyl-tetrahydrofolic acid and folic acid in freshly isolated hepatocytes J. Biol. Chem. 253: 3529–3535.
Karasik A., Rothenberg P.L., Yamada K., White M.F., Kahn C.R. 1990. Increased protein kinase C activity is linked to reduced insulin receptor autophosphorylation in liver of starved rats J. Biol. Chem. 265: 10226–10231.
Kennelly P.J. and Krebs E.G. 1991. Consensus sequences as substrate specificity determinants for protein kinases and protein phosphatases J. Biol. Chem. 266: 15555–15558.
Luka Z. and Wagner C. 2003. Human glycine N-methyltransferase is unfolded by urea through compact monomer state Arch. Biochem. Biophys. 420: 153–160.[Medline]
Møller M.T.N., Samari H.R., Fengsrud M., Strømhaug P.E., Østvold C., Seglen P.O. 2003. Okadaic acid-induced, naringin-sensitive phosphorylation of glycine N-methyltransferase in isolated rat hepatocytes Biochem. J. 373: 505–513.[Medline]
Ogawa H. and Fujioka M. 1982. Purification and properties of glycine N-methyltransferase from rat liver J. Biol. Chem. 257: 3447–3452.
Ogawa H., Gomi T., Hori T., Fujioka M. 1984. Molecular cloning of cDNA for rat glycine methyltransferase Biochem. Biophys. Res. Commun. 124: 44–50.[Medline]
Ogawa H., Gomi T., Fujioka M. 1993. Mammalian glycine N-methyltransferases. Comparative kinetic and structural properties of the enzymes from human, rat, rabbit and pig livers Comp. Biochem. Physiol. B 106: 601–611.[CrossRef][Medline]
Ogawa H., Gomi T., Takata Y., Date T., Fujioka M. 1997. Recombinant expression of rat glycine N-methyltransferase and evidence for contribution of N-terminal acetylation to co-operative binding of S-adenosylmethionine Biochem. J. 327: 407–412.
Pattanayek R., Newcomer M.E., Wagner C. 1998. Crystal structure of apo- glycine N-methyltransferase (GNMT) Protein Sci. 7: 1326–1331.[Abstract]
Rowling M.J. and Schalinske K.L. 2003. Retinoic acid and glucocorticoid treatment induce hepatic glycine N-methyltransferase and lower plasma homocysteine concentrations in rats and rat hepatoma cells J. Nutr. 133: 3392–3398.
Shi L., Potts M., Kennelly P.J. 1998. The serine, threonine, and/or tyrosine- specific protein kinases and protein phosphatases of prokaryotic organisms: A family portrait FEMS Microbiol. Rev. 22: 229–253.[CrossRef][Medline]
Suzuki N. and Wagner C. 1980. Purification and characterization of a folate binding protein from rat liver cytosol Arch. Biochem. Biophys. 199: 236–248.[CrossRef][Medline]
Takata Y., Huang Y., Komoto J., Yamada T., Konishi K., Ogawa H., Gomi T., Fujioka M., Takusagawa F. 2003. Catalytic mechanism of glycine N-methyltransferase Biochemistry 42: 8394–8402.[CrossRef][Medline]
Takusagawa F., Ogawa H., Fujioka M. 1999. Glycine N-methyltransferase, a tetrameric enzyme In S-Adenosylmethionine-dependent methyltransferases: Structure and functions (eds. Cheng X. and Blumental R.M.) . pp. 93–122. World Scientific Publishing, Singapore.
Wagner C., Briggs W.T., Cook R.J. 1985. Inhibition of glycine N-methyltransferase activity by folate derivatives: Implications for regulation of methyl group metabolism Biochem. Biophys. Res. Commun. 127: 746–752.[CrossRef][Medline]
Wagner C., Decha-Umphai W., Corbin J. 1989. Phosphorylation modulates the activity of glycine N-methyltransferase, a folate binding protein. In vitro phosphorylation is inhibited by the natural folate ligand J. Biol. Chem. 264: 9638–9642.
Yeo E.-J. "Studies on a cytosolic folate-binding protein: Glycine N-methyltransferase." Ph.D. thesis 1992. Vanderbilt University, Nashville, TN.
Yeo E.J. and Wagner C. 1994. Tissue distribution of glycine N-methyltransferase, a major folate-binding protein of liver Proc. Natl. Acad. Sci. 91: 210–214.
Zamierowski M.M. and Wagner C. 1977. Identification of folate binding proteins of rat liver J. Biol. Chem. 252: 933–938.
CiteULike 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
