A rapid method for the purification of S-adenosylmethionine: Protein-carboxyl O-methyltransferase by affinity chromatography

Kim, Sangduk; Nochumson, Samuel; Chin, W.; Paik, Woon Ki

doi:10.1016/0003-2697(78)90059-3

Cited by 107 publications

(53 citation statements)

References 15 publications

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“…For affinity chromatography AdoHcy-Sepharose was prepared according to [21] using aminohexyl-Sepharose 4B from Pharmacia (Uppsala, Sweden). In order to maintain both charges o f the homocysteine moiety of the ligand, AdoHcy was not coupled directly by amide binding but through the a-amino group to the a-acetamido derivative of the aminohexylSepharose.…”

Section: Preparation Of S-adenosyl-l-homocysteine -Sephurosementioning

confidence: 99%

Purification, characterization, and kinetic mechanism of S‐adenosyl‐l‐methionine: vitexin 2″‐O‐rhamnoside 7‐O‐methyltransferase of Avena sativa L

Knogge

Weissenböck

1984

European Journal of Biochemistry

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An 0-methyltransferase catalyzing the transfer of the methyl group of S-adenosyl-L-methionine to the A-ring 7-hydroxyl group of vitexin 2"-O-rhamnoside has been isolated from oat primary leaves and purified 180-fold by protein fractionation with (NH,),S04 and chromatography on DEAE-cellulose and S-adenosyl-L-homocysteinesepharose. K, values for S-adenosyl-L-methionine and the flavonoid substrate were 1.6 pM and 15 pM, respectively. The lack of methyl transfer to biosynthetic intermediates suggests that the reaction is the last step in the biosynthetic pathway to the oat flavonoid 7-0-methylvitexin 2"-0-rhamnoside. Based on results obtained from kinetic inhibition studies and affinity chromatography a mono-iso Theorell-Chance mechanism is proposed with the nucleotide substrate binding before the flavonoid.Methylation of high and low-molecular-mass substances is a very abundant reaction in animal and plant tissues. The reversible covalent modification of proteins by methyltransfer via S-adenosyl-L-methionine as well as the methylation of DNA and RNAs are discussed as possible tools in cellular regulation [l -31. In plants methyl groups are very common substituents of secondary products. Several N-methyltransferases participate in alkaloid biosynthesis [4]. 0-Methyltransferases are involved in the biosynthesis of plant phenolics [5]. Particularly, numerous flavonoids are known which possess methoxyl groups at different positions of the A and B rings as well as in the 3 position of the heterocycle.Oat is frequently used as test object both in plant physiology and pathology. Several physiological reponses of the plant to light as well as one of the photoreceptors, the phytochrome system, are well known [6,7]. On the other hand, this plant is intensively investigated in the context of its rust diseases [8]. For both fields of research flavonoid biosynthesis could be of special interest.Flavonoids in general are suggested to have a function in the protection of plants against ultraviolet light [9]. Their biosynthesis, at least in part, is photo-controlled [lo]. In oat primary leaves three major flavonoid compounds accumulate : an isovitexin derivative and two structurally related vitexin derivatives one of which is methylated at the 7-hydroxyl group [Ill (Fig. 1). The latter is suggested to be a preformed antifungal substance [8].Whereas several enzymes have been isolated which catalyze the methyl transfer to the 3' position of 3',4'-dihydroxyflavonoids or to the 3 position of flavonols, only very few reports exist on para-methylation of B-ring (4') monoAbbreviations. MVR, 7-0-methylvitexin 2"-0-rhamnoside; VR, vitexin 2"-0-rhamnoside; AdoMet, S-adenosyl-L-methionine; AdoHcy, S-adenosyl-r -homocysteine; TLC, thin-layer chromatography.Enzyme. Flavonoid 0-methyltransferdse, AdoMet : vitexin 2"-0-rhamnoside 7-0-methyltransferase (EC 2.1.1 .-), R2= C-Glc-0-Rha, R3 = H : 7-0-methylvitexin 2-0-rhamnoside (MVR) ; R, = R2 = R3 = H : apigenin, R, = R, = H, R, = C-Glc : vitexin; R1 =CH3, R,=C-Glc, R,=H: 7-0-methylvitexin; R, ...

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Section: Preparation Of S-adenosyl-l-homocysteine -Sephurosementioning

confidence: 99%

Purification, characterization, and kinetic mechanism of S‐adenosyl‐l‐methionine: vitexin 2″‐O‐rhamnoside 7‐O‐methyltransferase of Avena sativa L

Knogge

Weissenböck

1984

European Journal of Biochemistry

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“…The isolated creamy white erythrocyte ghosts (5 mg protein/ml) were methylated by purified homologous protein methylase I1 [29] in the presence of labelled AdoMet as previously reported [ll]. Briefly, 1 mg of membrane proteins was incubated in presence of 40 nmol of S-adenosyl-L-[meth~il-~ Hlmethionine (specific activity 1.38 Ci/mmol), 140 units of purified protein methylase 11, 0.15 ml citratephosphate buffer (pH 6.2) in a final volume of 1 ml, for 60 min at 37 'C.…”

Section: Methylation Of Membrane Proteins In Vitromentioning

confidence: 99%

Increased methyl esterification of membrane proteins in aged red‐blood cells

Galletti

Ingrosso

Nappi

et al. 1983

European Journal of Biochemistry

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The enzymatic carboxyl methyl esterification of erythrocyte membrane proteins has been investigated in three different age-related fractions of human erythrocytes. When erythrocytes of different mean age, separated by density gradient centrifugation, were incubated under physiological conditions (pH 7.4, 37 "C) in the presence of ~-[metlzyl-~H]methionine, the precursor in vivo of the methyl donor S-adenosylmethionine, a fourfold increase in membrane-protein carboxyl methylation was observed in the oldest cells compared with the youngest ones.The identification of methylated species, based on comigration of radioactivity with proteins stained with Coomassie blue, analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, shows, in all cell fractions, a pattern similar to that reported for unfractionated erythrocytes. However in the membrane of the oldest erythrocytes the increase in methylation of the cytoskeletal proteins, bands 2.1 and 4.1, appears to be significantly more marked compared with that observed in the other methylated polypeptides. Furthermore the turnover rate of incorporated [3H]methyl groups in the membrane proteins of the oldest cells markedly increases during cell ageing. Particularly in band 4.1 the age-related increase in methyl esterification is accompanied by a significant reduction of the half-life of methyl esters.The activity of cytoplasmic protein methylase I1 does not change during cell ageing, while the isolated ghosts from erythrocytes of different age show an age-related increased ability to act as methyl-accepting substrates, when incubated in presence of purified protein methylase I1 and methyl-labelled S-adenosylmethionine, therefore the relevance of membrane structure in determining membrane protein methylation levels can be postulated.Finally the possible correlation of this posttranslational protein modification with erythrocyte ageing is discussed.The enzyme protein methylase I1 methyl esterifies free carboxyl groups of preformed proteins, using S-adenosylmethionine (AdoMet) as methyl donor [I -31. The protein carboxyl methyl esters formed are unstable and undergo enzymatic [4] and/or spontaneous hydrolysis [S -71 yielding methanol and the unmodified protein.The methyl esterification reaction, involving the reversible neutralization of protein negative charges, can be considered as an 'on-off mechanism by which a number of cellular events are regulated. The role of protein methylase I1 in the synaptic function, neurosecretory processes and bacterial and mammalian chemotaxis [3,8] has been reported by several laboratories. Furthermore it has been suggested that this enzyme could modulate the calcium-dependent cellular activities through the methyl esterification of calmodulin [9].Membrane proteins were shown to be the natural substrates for the cytosolic protein methylase I1 in the red blood cells [10,11]. In order to identify the methyl esterified polypeptides in the red blood cell membrane, a number of experimental approaches were employed, either in vitro [lo-1...

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“…Protein methylase I1 was purified 30001fold from fresh calf brain according to the procedure of Kim et al [24], using affinity chromatography on 6-aminohexyl-Sepharose (AH-Sepharose) covalently linked to S-adenosyl-L-homocysteine, followed by a Sephadex G-100 chromatography.…”

Section: Enzyme Purificationmentioning

confidence: 99%

“…Previous studies on substrate specificity showed that several pituitary polypeptides, including lutropin and corticotropin, were effective methyl acceptors in vitro [19]. It has recently been reported that erythrocyte membrane proteins are also good methyl substrates for the enzyme [20-211. The enzyme has been highly purified from various mammalian tissues, using conventional protein fractionation methods [22,23] ; recently a 3000-fold purifica-tion has been achieved by using S-adenosyl-L-homocysteine-linked Sepharose affinity chromatography [24].…”

mentioning

confidence: 99%

Studies on Substrate Specificity of S-Adenosylmethionine: Protein-Carboxyl Methyltransferase from Calf Brain

et al. 1980

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Kinetic properties of protein methylase I1 (S-adenosy1methionine:protein-carboxyl methyltransferase, EC 2.1.1.24), which methylates the free carboxyl groups of protein substrates, were studied with various analogs and derivatives of S-adenosyl-L-methionine (AdoMet) and S-adenosyl-Lhomocysteine (AdoHcy), using corticotropin as methyl-accepting polypeptide.Experiments with methyl-labelled structural analogs of AdoMet showed that the replacement of the amino groups of AdoMet by hydroxyl groups, as well as the removal of the carboxyl group, results in a loss of methylating ability of the molecule. Deaminated and decarboxylated derivatives were also inactive as inhibitors. Among the sulfonium analogs tested, only S-adenosyl-L-(2-amino-4-carboxymethy1thio)butyric acid exerted a significant inhibition.AdoHcy, the demethylated product of AdoMet, exerts a competitive inhibition on the reaction (Ki = 0.65 pM); the probable regulatory role of this inhibition will be discussed. The removal of the adenine amino group of the thioether resulted in a loss of the inhibitory effect. Experiments with the D-isomer of AdoHcy showed the relevance of the steric configuration of the a-carbon in the binding to the enzyme protein. 5'-Methylthioadenosine, a metabolic product of AdoMet, inhibited competitively the reaction (Ki = 41 pM) while 5'-methylthioadenosine derivatives, such as n-butylthioadenosine, isobutylthioadenosine and thioethanoladenosine, failed to exert any inhibition.Three synthetic polypeptides differing in amino acid composition and in chain length have also been tested as methyl-acceptor substrates and as inhibitors for the reaction. Only the copolymer of L-glutamic acid and L-tyrosine [poly (Glul,16, Tyrl)] ( M , = 22600) exerts a relevant non-competitive inhibition, while none of the tested synthetic polypeptides is active as substrate.Protein methylase I1 (S-adenosylmethionine : protein-carboxyl methyltransferase) catalyzes the transfer of the methyl group from S-adenosylmethionine (AdoMet) to free carboxyl groups of aspartyl and/or glutamyl residues in protein, yielding carboxyl methyl esters [l -31. In turn protein methyl esters undergo rapid and spontaneous hydrolysis into methanol at physiological pH and temperature [4,5]. The enzyme is widely distributed in nature and has been shown to be present in the cytosolic fraction of most mammalian tissues [6,7].

show abstract

A rapid method for the purification of S-adenosylmethionine: Protein-carboxyl O-methyltransferase by affinity chromatography

Cited by 107 publications

References 15 publications

Purification, characterization, and kinetic mechanism of S‐adenosyl‐l‐methionine: vitexin 2″‐O‐rhamnoside 7‐O‐methyltransferase of Avena sativa L

Purification, characterization, and kinetic mechanism of S‐adenosyl‐l‐methionine: vitexin 2″‐O‐rhamnoside 7‐O‐methyltransferase of Avena sativa L

Increased methyl esterification of membrane proteins in aged red‐blood cells

Studies on Substrate Specificity of S-Adenosylmethionine: Protein-Carboxyl Methyltransferase from Calf Brain

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