Methyl Acceptor Research Articles

The Human Homologue of the Yeast Proteins Skb1 and Hsl7p Interacts with Jak Kinases and Contains Protein Methyltransferase Activity

To expand our understanding of the role of Jak2 in cellular signaling, we used the yeast two-hybrid system to identify Jak2-interacting proteins. One of the clones identified represents a human homologue of the Schizosaccaromyces pombe Shk1 kinase-binding protein 1, Skb1, and the protein encoded by the Saccharomyces cerevisiae HSL7 (histone synthetic lethal 7) gene. Since no functional motifs or biochemical activities for this protein or its homologues had been reported, we sought to determine a biochemical function for this human protein. We demonstrate that this protein is a protein methyltransferase. This protein, designated JBP1 (Jak-binding protein 1), and its homologues contain motifs conserved among protein methyltransferases. JBP1 can be cross-linked to radiolabeled S-adenosylmethionine (AdoMet) and methylates histones (H2A and H4) and myelin basic protein. Mutants containing substitutions within a conserved region likely to be involved in AdoMet binding exhibit little or no activity. We mapped the JBP1 gene to chromosome 14q11.2-21. In addition, JBP1 co-immunoprecipitates with several other proteins, which serve as methyl group acceptors and which may represent physiological targets of this methyltransferase. Messenger RNA for JBP1 is widely expressed in human tissues. We have also identified and sequenced a homologue of JBP1 in Drosophila melanogaster. This report provides a clue to the biochemical function for this conserved protein and suggests that protein methyltransferases may have a role in cellular signaling.

Recombinant human liver betaine-homocysteine S-methyltransferase: identification of three cysteine residues critical for zinc binding.

Betaine-homocysteine S-methyltransferase (BHMT; EC 2.1.1.5) catalyzes the transfer of an N-methyl group from betaine to homocysteine to produce dimethylglycine and methionine, respectively. The enzyme is found in the pathway of choline oxidation and is abundantly expressed in liver and kidney. We have recently shown that human BHMT is a zinc metalloenzyme [Millian, N. S., and Garrow, T. A. (1998) Arch. Biochem. Biophys. 356, 93-98]. To facilitate the rapid purification of human BHMT for further physical and mechanistic studies, including characterizing its metal binding properties, we have overexpressed the enzyme in E. coli as a fusion construct which facilitated its subsequent purification by a self-cleavable affinity tag system (IMPACT T7). Using this expression and purification system in conjunction with site-directed mutagenesis, we have identified Cys217, Cys299, and Cys300 as zinc ligands. Mutating any of these Cys residues to Ala results in the complete loss of activity and a significant reduction in the ability of the protein to bind zinc. Comparing the regions of BHMT amino acid sequence surrounding these Cys residues with similar amino acid sequences retrievable from protein databases, we have identified the following motif: G[ILV]NCX(20,100)[ALV]X(2)[ILV]GGCCX(3)PX(2)I, which we propose to be a signature for a family of zinc-dependent methyltransferases that utilize thiols or selenols as methyl acceptors. Some of the members of this family include the vitamin B(12)-dependent methionine synthases, E. coli S-methylmethionine-S-homocysteine methyltransferase, and A. bisulcatus S-methylmethionine-selenocysteine methyltransferase.

Solubilization and partial characterization of homogalacturonan-methyltransferase from microsomal membranes of suspension-cultured tobacco cells.

The transfer of a methyl group from S-adenosyl-L-methionine onto the carboxyl group of alpha-1,4-linked-galactosyluronic acid residues in the pectic polysaccharide homogalacturonan (HGA) is catalyzed by an enzyme commonly referred to as pectin methyltransferase. A pectin methyltransferase from microsomal membranes of tobacco (Nicotiana tabacum) was previously characterized (F. Goubet, L.N. Council, D. Mohnen [1998] Plant Physiol 116: 337-347) and named HGA methyltransferase (HGA-MT). We report the solubilization of HGA-MT from tobacco membranes. Approximately 22% of the HGA-MT activity in total membranes was solubilized by 0.65% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid containing 1 mM dithioerythritol. The addition of phosphatidylcholine and the methyl acceptors HGA or pectin (30% degree of esterification) to solubilized enzyme increased HGA-MT activity to 35% of total membrane-bound HGA-MT activity. Solubilized HGA-MT has a pH optimum of 7.8, an apparent K(m) for S-adenosyl-L-methionine of 18 microM, and an apparent V(max) of 0. 121 pkat mg(-1) of protein. The apparent K(m) for HGA and for pectin is 0.1 to 0.2 mg mL(-1). Methylated product was solubilized with boiling water and ammonium oxalate, two conditions used to solubilize pectin from the cell wall. The release of 75% to 90% of the radioactivity from the product pellet by mild base treatment showed that the methyl group was incorporated as a methyl ester rather than a methyl ether. The fragmentation of at least 55% to 70% of the radiolabeled product by endopolygalacturonase, and the loss of radioactivity from the product by treatment with pectin methylesterase, demonstrated that the bulk of the methylated product produced by the solubilized enzyme was pectin.

Properties of the methylcobalamin:H4folate methyltransferase involved in chloromethane utilization by Methylobacterium sp. strain CM4.

Methylobacterium sp. strain CM4 is a strictly aerobic methylotrophic proteobacterium growing with chloromethane as the sole carbon and energy source. Genetic evidence and measurements of enzyme activity in cell-free extracts have suggested a multistep pathway for the conversion of chloromethane to formate. The postulated pathway is initiated by a corrinoid-dependent methyltransferase system involving methyltransferase I (CmuA) and methyltransferase II (CmuB), which transfer the methyl group of chloromethane onto tetrahydrofolate (H4folate) [Vannelli et al. (1999) Proc. Natl Acad. Sci. USA 96, 4615-4620]. We report the overexpression in Escherichia coli and the purification to apparent homogeneity of methyltransferase II. This homodimeric enzyme, with a subunit molecular mass of 33 kDa, catalyzed the conversion of methylcobalamin and H4folate to cob(I)alamin and methyl-H4folate with a specific activity of 22 nmol x min-1 x (mg protein)-1. The apparent kinetic constants for H4folate were: Km = 240 microM, Vmax = 28.5 nmol x min-1 x (mg protein)-1. The reaction appeared to be first order with respect to methylcobalamin at concentrations up to 2 mM, presumably reflecting the fact that methylcobalamin is an artificial substitute for the methylated methyltransferase I, the natural substrate of the enzyme. Tetrahydromethanopterin, a coenzyme also present in Methylobacterium, did not serve as a methyl group acceptor for methyltransferase II. Purified methyltransferase II restored chloromethane dehalogenation by a cell free extract of a strain CM4 mutant defective in methyltransferase II.

Methylcobalamin:homocysteine methyltransferase from Methanobacterium thermoautotrophicum. Identification as the metE gene product.

Methanobacterium thermoautotrophicum is a methane-forming archaeon that grows on H2 and CO2 as sole carbon and energy source. Cell extracts of the methanogen were found to contain methylcobalamin: homocysteine methyltransferase activity which was purified 3000-fold to a specific activity of approximately 500 U.mg-1 protein. SDS/PAGE revealed the presence of a polypeptide with an apparent molecular mass of 34 kDa. Via its N-terminal amino acid sequence, the 34-kDa polypeptide was identified as the metE gene product. The metE gene was heterologously expressed in Escherichia coli. The overproduced protein was recovered in the inclusion body fraction and was found to be inactive. The protein could be partially solubilized by unfolding in 8 M urea and then refolding. The solubilized protein had a specific activity of 450 U.mg-1. It exhibited first-order kinetics with respect to methylcobalamin concentration and Michaelis-Menten kinetics with respect to L-homocysteine concentration (apparent Km 0.1 mM). The enzyme was specific for L-homocysteine as methyl acceptor. Methylcobalamin could be substituted with methylcobinamide as methyl donor.

Catechol-O-methyltransferase: variation in enzyme activity and inhibition by entacapone and tolcapone.

The aim of this investigation was to study the variation of catechol-O-methyltransferase (COMT) activity in the human liver, duodenal mucosa and renal cortex, and to investigate the inhibition of COMT by entacapone and tolcapone. This study included 87 samples of human liver, 94 samples of the duodenum and 72 samples of the renal cortex. The activity of COMT was measured with 3,4-dihydroxybenzoic acid (242 micromol x l(-1)), the methyl acceptor substrate, and adenosyl-L-methionine (44 micromol x l(-1)), the methyl donor substrate. The hepatic activity of COMT activity was significantly higher in men than in women, whereas it was not sex-dependent in the duodenum or renal cortex. The activity of COMT varied 4.4-fold in the liver of men, 2.6-fold in the duodenum and 5.3-fold in the renal cortex. The median estimates of COMT activity were 577, 499, 103 and 159 pmol x min(-1) x mg(-1) in the liver of men and women, in the duodenum and in the renal cortex, respectively. Entacapone and tolcapone were powerful inhibitors of COMT and their IC50 estimates were 151 and 773 nM (P = 0.008), respectively, in the liver; consistent results were obtained with the other tissues.

Changes in Formaldehyde Contents of Germinating Acorns of Quercus Cerris L. Under Low Temperature Stress Conditions

Acorns of Quercus cerris L., after saturation with water and storage at -20 degrees C, were studied for changes in their contents of endogenous formaldehyde and its potential precursor and generator compounds. For the measurement of formaldehyde, after conversion to formaldemethone and some methyl acceptor and donor substances, high performance liquid chromatography (HPLC) were used. First, the amount of formaldehyde was drastically decreased. Having reached a minimum value within three to five days of the beginning of low temperature storage, a higher steady-state than the control acorns was recorded. Trigonelline and gamma-amino-butyric acid in seedleaf extracts were identified by matrix assisted laser desorption-ionization mass spectrometry (MALDI-MS).

The effect of heat shock on the formaldehyde cycle in germinating acorns of European Turkey oak

The effect of heat shock (40 degrees C) on the formaldehyde cycle has been studied in European Turkey oak (Quercus cerris L.) acorns germinated to a 10% increase in mass. Hydroxy-methyl groups bonded to sulfur, oxygen and nitrogen atoms were made to react with dimedone and the derivative obtained (formaldemethone), which represented the endogenous formaldehyde level, was determined by high performance liquid chromatography. Qualitative alterations of methyl donors and acceptors in the response of acorns to the heat shock have been mapped by MALDI (matrix assisted laser desorption ionization) mass analysis. In the first experiment the acorns were prevented from withering by wrapping them in aluminium foil and in the second they were not. The relatively high temperature of the acorns wrapped in aluminium foil was the dominant stress effect and the role of withering was subsidiary. Alteration of the endogenous formaldehyde level in the seed-leaves reflected the phases of the stress syndrome. If the withering were not hindered, two local minima in the alteration of endogenous formaldehyde level were found. First, the increase in temperature decreased the endogenous formaldehyde level and after a local maximum a repeated local minimum was observed as a delayed response. It is presumed that the second minimum was induced by the decreasing water amount becoming more and more significant in the seed-leaves.

The O6-methylguanine-DNA methyltransferase from the hyperthermophilic archaeon Pyrococcus sp. KOD1: a thermostable repair enzyme.

The enzyme O6-methylguanine-DNA methyltransferase (MGMT) is the most common form of cellular defense against the biological effects of O6-methylguanine (O6-MeG) in DNA. Based on PCR amplification using primers derived from conserved amino acid sequences of MGMTs from 11 species, we isolated the DNA region coding for MGMT from the hyperthermophilic archaeon Pyrococcus sp. KOD1. The MGMT gene from KOD1 (mgtk) comprises 522 nucleotides, encoding 174 amino acid residues; its product shows considerable similarity to the corresponding mammalian, yeast and bacterial enzymes, especially around putative methyl acceptor sites. Phylogenetic analysis of MGMTs showed that archaeal MGMTs were grouped with their bacterial counterparts. The location of the MGMT gene on the KOD1 chromosome was also determined. The cloned KOD1 MGMT gene was overexpressed using the T7 RNA polymerase expression system, and the recombinant protein was purified by ammonium sulfate fractionation, heat treatment, ion-exchange chromatography and gel filtration chromatography. The purified recombinant protein was assayed for its enzyme activity by monitoring transfer of [3H]methyl groups from the substrate DNA to the MGMT protein; the activity was found to be stable at 90 degrees C for at least 30 min. When the mgtk gene was placed under the control of the lac promoter and expressed in the methyltransferase-deficient Escherichia coli strain KT233 (delta ada, delta ogt) cells, a MGMT was produced. The enzyme was functional in vivo and complemented the mutant phenotype, making the cells resistant to the cytotoxic properties of the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine.

ProteinN-Arginine Methylation in Adenosine Dialdehyde-Treated Lymphoblastoid Cells

Protein arginine methyltransferase was recently identified to be associated with some proteins in signal transduction pathways.N-Arginine methylation in RNA binding proteins with arginine- and glycine-rich RGG motifs is known to be the major protein methylation in cells. Considering that arginine methylation might be involved in certain human disorders, we used human lymphoblastoid cells that can be easily prepared from lymphocytes as a model system to study the methylation. Lymphoblastoid cells grown in the presence of 20 μM indirect methyltransferase inhibitor adenosine dialdehyde (AdOx) for 72 h appeared to accumulate high levels of hypomethylated proteins for the endogenous protein methyltransferase or recombinant glutathionS-transferase-fused yeast arginine methyltransferase (RMT1). Analysis of methyl-accept- ing polypeptides in AdOx-treated lymphoblastoid cells by SDS–PAGE and fluorography showed that many polypeptides between 29,000 and 90,000 Da were methylated by the endogenous methyltransferase. A few polypeptides could be methylated to a higher extent upon the addition of yeast GST–RMT1 fusion protein. A peptide (GGRGRGGGF) could compete for the majority of the methyl-accepting protein substrates in the AdOx-treated lymphoblastoid cell extracts, whether or not exogenous yeast RMT1 was included in the reaction. When the arginine residues in the peptide were replaced by lysine, no competition was observed. The results indicated that the protein methyl acceptors in lymphoblastoid cells share similar RGG motifs and that arginine residues should be the site of methylation.

The biosynthesis of 8-O-methylated sialic acids in the starfish Asterias rubens--isolation and characterisation of S-adenosyl-L-methionine:sialate-8-O-methyltransferase.

The unusual, 8-O-methylated sialic acids have only been found in glycoconjugates of certain species of starfish. Using a detergent-solubilised extract of a particulate fraction from gonads of Asterias rubens, a specific methylation of endogenous and exogenous glycoconjugate-bound sialic acids was detected with [14C]-S-adenosyl-L-methionine ([14C]AdoMet) as the methyl donor. For this test, a filtration assay was developed using glutardialdehyde-fixed horse erythrocyte membranes as methyl acceptor. The enzyme catalysing this reaction, the sialate-8-O-methyltransferase, was purified 22000-fold in a yield of 13% by ion-exchange chromatography and two cycles of affinity chromatography on S-adenosyl-L-homocysteine-Sepharose. Upon SDS/PAGE under reducing conditions, the purified sialate-8-O-methyltransferase revealed two bands with apparent molecular masses of 58 kDa and 62 kDa. Since no evidence for the presence of subunits was found, the relationship between these two species is unknown. The enzyme was optimally active over a broad range of pH (7.4-8.3) and at 37 degrees C. After EDTA treatment, restoration of the activity depended, in contrast to most methyltransferases, on Mn2+ or Co2+, the latter to a lesser extent. Although both, free N-acetylneuraminic acid and N-glycoloylneuraminic acid were methylated, sialic acids alpha-glycosidically bound to a number of oligosaccharides and glycoproteins were better substrates. In the presence of 20 microM AdoMet, apparent Km values of 299 microM and 44 microM were found for N-acetylneuraminic acid and N-acetylneuraminyl alpha2,3-lactose, respectively. Using N-acetylneuraminyl alpha2,3-lactose as acceptor, an apparent Km of 7.1 microM was found for S-adenosyl-L-methionine. Therefore, the sialate-8-O-methyltransferase is the first enzyme known to modify both free and glycoconjugate-bound sialic acids.

Evidence in favour of an oxidative N-demethylation of nicotine to nornicotine in tobacco cell cultures

Summary Nicotine N -demethylation to nornicotine has been studied in cell-free preparations from cell suspension cultures of Nicotiana tabacum , in attempt to explore the mechanism of this enzymatic reaction. Subcellular fractionation of the homogenate using isopycnic differential centrifugation, together with TEM, showed that most of the enzyme activity (2.8 × 10 −5 U) was present in the intermediate pellet whilst the maximum specific activity of 8.8 × 10 −5 U per mg protein appeared to be associated with the microsomal fraction. The addition of selected putative methyl group acceptors, i.e. putrescine, glycine and ethanolamine, to either dialysed or undialysed enzyme preparations, did not promote enzyme activity. In addition, studies involving Sephadex G-25 gel filtration showed that molecules smaller than 5,000 MW are unlikely to be involved in this reaction. The current findings appear to support an oxidative nicotine N -demethylation and, together with the results from previous studies, provide some tentative evidence for the involvement of cytochrome P-450 in nicotine N -demethylation. The possible mechanism is also discussed.

Tetrahydrofolate serves as a methyl acceptor in the demethylation of dimethylsulfoniopropionate in cell extracts of sulfate-reducing bacteria.

Tetrahydrofolate was shown to function as a methyl acceptor in the anaerobic demethylation of dimethylsulfoniopropionate to methylthiopropionate in cell extracts of the sulfate-reducing bacterium strain WN. Dimethylsulfoniopropionate-dependent activities were 0.56 micromol methyltetrahydrofolate min-1 (mg protein)-1 and were higher than required to explain the growth rate of strain WN on dimethylsulfoniopropionate. The reaction did not require ATP or reductive activation by titanium(III)-nitrilotriacetic acid. Preincubation of the extract under air significantly decreased the activity (35% loss in 3 h). Three other dimethylsulfoniopropionate-demethylating sulfate reducers, Desulfobacterium niacini, Desulfobacterium vacuolatum, and Desulfobacterium strain PM4, had dimethylsulfoniopropionate:tetrahydrofolate methyltransferase activities of 0.16, 0.05, and 0.24 micromol min-1 (mg protein)-1, respectively. No methyltransferase activity to tetrahydrofolate was found with betaine as a substrate, not even in extracts of betaine-grown cells of these sulfate reducers. Dimethylsulfoniopropionate demethylation in cell extracts of strain WN was completely inhibited by 0.5 mM propyl iodide; in the light, the inhibition was far less strong, indicating involvement of a corrinoid-dependent methyltransferase.

Rhizobium meliloti mutants deficient in phospholipid N-methyltransferase still contain phosphatidylcholine.

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes. In addition to this structural function, PC is thought to play a major role in lipid turnover and signalling in eukaryotic systems. In prokaryotes, only some groups of bacteria, among them the members of the family Rhizobiaceae, contain PC. To understand the role of PC in bacteria, we have studied Rhizobium meliloti 1021, which is able to form nitrogen-fixing nodules on its legume host plants and therefore has a very complex phenotype. R. meliloti was mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine, and potential mutants defective in phospholipid N-methyltransferase were screened by using a colony autoradiography procedure. Filters carrying lysed replicas of mutagenized colonies were incubated with S-adenosyl-L-[methyl-14C]methionine. Enzymatic transfer of methyl groups to phosphatidylethanolamine (PE) leads to the formation of PC and therefore to the incorporation of radiolabel into lipid material. Screening of 24,000 colonies for reduced incorporation of radiolabel into lipids led to the identification of seven mutants which have a much-reduced specific activity of phospholipid N-methyltransferase. In vivo labelling of mutant lipids with [14C]acetate showed that the methylated PC biosynthesis intermediates phosphatidylmonomethylethanolamine and phosphatidyldimethylethanolamine are no longer detectable. This loss is combined with a corresponding increase in the potential methyl acceptor PE. These results indicate that PC biosynthesis via the methylation pathway is indeed blocked in the mutants isolated. However, mass spectrometric analysis of the lipids shows that PC was still present when the mutants had been grown on complex medium and that it was present in the mutants in wild-type amounts. In vivo labelling with [methyl-14C]methionine shows that in phospholipid N-methyltransferase-deficient mutants, the choline moiety of PC is not formed by methylation. These findings suggest the existence of a second pathway for PC biosynthesis in Rhizobium.

Methyltransferase Activity of the Insect Orbivirus JKT-7400: Photoaffinity Labeling of a Minor Virion Protein, VP4, withS-Adenosylmethionine

JKT-7400 virus is an orbivirus originally isolated fromCulexmosquitoes. In earlier work we had described the viral structural proteins and presented evidence suggesting that a minor protein, VP6, located in the viral core was the viral guanylyltransferase. We now show that gradient-purified JKT-7400 virions possess a methyltransferase (MTase) activity which can use GTP or GDP as the methyl acceptor. The apparentKmof the MTase forS-adenosylmethionine (AdoMet) was 25 μM.Photoaffinity labeling experiments in which3H-[methyl]-AdoMet was incubated with virions or viral cores demonstrated labeling of VP4, a minor protein present in the viral core, suggesting that this protein is the viral MTase. Labeling of VP4 was inhibited by addition of unlabeled AdoMet orS-adenosylhomocysteine (AdoHcy).

Specificity of O -demethylation in extracts of the homoacetogenic Holophaga foetida and demethylation kinetics measured by a coupled photometric assay

The kinetics and specificity of O-demethylation were studied in cell-free extracts of the strictly anaerobic, methanethiol- and dimethylsulfide-producing homoacetogen Holophaga foetida strain TMBS4 with methanethiol and tetrahydrofolate (H4folate) as methyl acceptors. Extracts of cells grown with 3,4,5-trimethoxybenzoate contained an enzyme system that demethylated various phenyl methyl ethers with at least one ortho-positioned hydroxyl or methoxyl group (the ortho system) and also contained a decarboxylase. Extracts of cells grown with 3,5-dihydroxyanisole contained an enzyme system with a novel specificity that demethylated only the meta-hydroxylated compounds 3,5-dihydroxyanisole and 3-hydroxyanisole (the meta system) and lacked a decarboxylase. H4folate-dependent demethylation produced CH3-H4folate. For a photometric in vitro assay of the meta system, the NADPH-consuming phloroglucinol reductase (PR) reaction was coupled to the phloroglucinol-yielding demethylation of 3,5-dihydroxyanisole. The kinetics of the indicator enzyme PR were studied. The cell extract had a high and stable specific PR activity. PR was inhibited by phloroglucinol (substrate inhibition) and the substrate analogue 3,5-dihydroxyanisole. Doubling the PR activity of the coupled enzyme assay by additions of a PR-enriched fraction had no effect, showing that the PR activity supplied by cell extract did not limit reaction rates. Demethylation activity of the meta system with either methyl acceptor increased with the square of the protein concentration. With H4folate, the in vivo activity could be attained. Kinetic parameters for the methyl acceptors were determined.

Effects of alternative methyl group acceptors on the growth energetics of the O-demethylating anaerobe Holophaga foetida

The anaerobic bacterium Holophaga foetida can metabolize the methyl groups of methoxylated aromatic compounds either to acetate or to dimethyl sulphide. The effects of this metabolic flexibility were investigated under conditions of excess; substrate (batch culture) and substrate limitation (chemostat culture). Growth yield data suggest that transfer of the methyl groups to sulphide, in contrast to the homoacetogenic transfer to CO2, was not coupled to energy conservation. Under conditions of excess substrate, methyl groups were quantitatively transferred to sulphide. Growth yields decreased but growth rates increased upon the addition of sulphide during exponential growth in pH- and sulphide-regulated batch cultures. From the measured growth yields, the Gibbs free energy dissipation of catabolism plus anabolism ( ) was calculated using stoichiometric equations incorporating biomass formation (macrochemical equations). The observed increase in growth rate correlated well with an increase in , suggesting a relationship between growth kinetics and growth energetics. During steady-state growth in pH- and sulphide-regulated chemostat culture, a considerable fraction of the methyl groups was converted to acetate, despite the presence of sulphide. This resulted in similar growth yields and correspondingly similar values in the presence and absence of sulphide. Apparently, H. foetida uncouples catabolism and anabolism in batch culture under conditions of excess substrate to a greater extent than in the chemostat under substrate limitation, by transferring the methyl groups quantitatively to sulphide and thereby dissipating the Gibbs free energy change of the methyl transfer. The physiological significance of these findings could be that H. foetida adjusts the energetics of its metabolism to the growth conditions (i) to maximize the growth rate if substrate is available in excess or, (ii) to maximize the growth yield if substrate is limiting.

N-methyltransferase activities in caffeine biosynthesis: Biochemical characterization and time course during leaf development of Coffea arabica

In order to accurately measure and characterize the very labile methyltransferase activities catalysing the two last steps in caffeine biosynthesis (from 7-methylxanthine to theobromine, and from theobromine to caffeine by the 2nd and 3rd NMT activity, respectively), an assay was designed that renders the use of radioactivity superfluous. Since in the assay maximum velocity was achieved by sufficiently high concentrations of both the methyl donor and acceptor, sensitivity was equal to the conventional protocols using minute substance quantities of[methyl- 3H]- or[methyl- 14C]SAM of high specific radioactivity. For the above mentioned NMT activities of the coffee plant a maximum pH stability was found at pH 7.5. Temperature stability tests revealed an almost linear decrease by increasing temperature with the exception of the 3rd NMT that was relatively stable between 30 and 50°. The isoelectric point was located at pH 4.5 to 5.0 (2nd NMT), and the pH optima at 7 to 8 and 8.5 for the 2nd and 3rd NMT activity, respectively. The temperature optimum is broad and around 35° for both activities. Interestingly, the 2nd NMT activity has a lower K m for the methyl acceptor than the 3rd one, and this inversely for the methyl donor, SAM. Maximum relative and absolute methyltransferase activities coincide with leaf emergence, when, as expected, the chemical defence has highest priority and the purine alkaloids (PuA) are allocated at peak concentrations. The 2nd NMT activity was always considerably higher than the 3rd one. However, they frequently paralleled each other as they were components of one entity. The only transient accumulation of theobromine in the young leaflets suggestedly results from the competition of the two NMT activities for SAM and/or from a reduced supply of purine precursors in the preceding methylations.

Influence of osmotic stress on protein methylation in resealed erythrocytes.

Resealed erythrocytes are a useful means of targeting exogenous proteins or extending their activity. We tested human resealed erythrocytes as a model system for studying protein methyl esterification, a reaction involved in the processing of spontaneously deamidated/isomerized polypeptides. Our results show that resealed erythrocytes are still active in the metabolic processes that lead to the formation of methyl-esterified proteins. The methylation pattern of endogenous membrane proteins appeared to be similar to that of normal erythrocytes, with bands 2.1, 3, 4.1 and 4.2 as the major methyl acceptors. We detected methyl esterification of ovalbumin, as an exogenous substrate trapped within resealed erythrocytes. Methyl incorporation was almost completely inhibited by simultaneously loading red cells with adenosine and homocysteine thiolactone, in vivo precursors of the transmethylation inhibitor S-adenosylhomocysteine. We investigated the effects of repeated resealing procedures on methyl acceptability of endogenous membrane proteins. We found that methyl-incorporation levels increased, despite an apparent conserved protein composition of the membrane. This result suggests that osmotic stress to the membrane may be responsible for increased protein methylation due to the appearance of new sites or an increased accessibility of existing sites.

Protein methylation in cellular proliferation and differentiation: non-histone nuclear methyl acceptor protein(s) during 3'-methyl-4-dimethylaminoazobenzene-induced hepatocarcinogenesis

An accelerating effect of methyl-deficient diet (MDD) on hepatocarcinogenesis and methylation pattern of nuclear protein(s) by S-adenosylmethionine: protein arginine N-methyltransferase (protein methylase I, PM-I) have been studied with 3'-methyl-4-dimethyl- aminoazobenzene(MeDAB)-treated rats. The MDD+MeDAB-fed group produced typical cancer cells in the liver almost two weeks earlier than the control synthetic diet (CSD)+MeDAB-fed group. Protein methylase I (PM-I) activity in the livers of MDD alone fed rats began to increase at around 2 weeks after MDD-feeding, reaching a peak at 4 weeks and declining thereafter. When nuclei isolated either from normal livers or from cholangiocarcinoma cells were incubated with PM-I preparation from normal liver, 16 and 23-kDa nuclear proteins were the major methylated proteins, regardless of the source of the nuclei. However, when the above mentioned nuclei were incubated with PM-I preparations either from MDD alone fed livers or MDD+ MeDAB-induced cholangiocarcinoma cells, the methylation of 23-kDa protein was not detected. The result suggests that there is a hitherto-unknown PM-I specific to 23 kDa nuclear protein which was lost during methyl deficient diet feeding and hepatocarcinogenesis. The N-terminal 20 amino acids sequence of the 23-kDa protein was found to be ¹Gly-Val-Pro-Leu-?X-Arg-Leu-Phe-Asp-¹?His-Ala-Met-Leu-Gln-¹?Ala-His-Arg-Ala-His-²?Glu, having 94.7% sequence homology with human chorionic somatomammotropin precursor A and B.