Presence Of S-adenosylmethionine Research Articles

Characterization of the DNA methylase activity of the restriction enzyme from Escherichia coli K.

The restriction endonuclease from Escherichia coli K is a multifunctional protein which efficiently methylates heteroduplex DNA (one strand modified and one strand unmodified) in the presence of S-adenosylmethionine (AdoMet), ATP, and Mg2+. The methylase activity is catalytic, and seems to modify different heteroduplex host specificity sites for E. coli K with equal efficiency. In the methylase reaction, both AdoMet and ATP (or its imido analog) act as allosteric effectors, but AdoMet also serves as a methyl donor. Preincubation of the enzyme with AdoMet eliminates the lag period observed in DNA methylation. The rate of enzyme activation was determined using the AdoMet analog Sinefungin. The result are consistent with the hypothesis that the early steps of AdoMet binding and enzyme activation are common to both restriction and modification reactions.

The conversion of cephalosporins to 7 alpha-methoxycephalosporins by cell-free extracts of Streptomyces clavuligerus.

In the presence of S-adenosylmethionine, 2-oxoglutarate, Fe2+ and a reducing agent, cell-free extracts of Streptomyces clavuligerus convert cephalosporin C and O-carbamoyldeacetylcephalosporin C into 7 alpha-methoxy derivatives. No synthesis of a 7 alpha-methoxy derivative of deacetylcephalosporin C was detected in the system used, and the 7 alpha-methoxy derivative of deacetoxycephalosporin C was produced only in relatively small amounts. It appears that the 7 alpha-methoxy group is introduced after the cephalosporin ring system has been formed and that its introduction may represent the final step in a biosynthetic pathway.

Formation of 3-hydroxy-4-methoxyphenylalanine from 3,4-dihydroxyphenylalanine by rat liver homogenate.

The O-methylation of 3, 4-dihydrcxyphenylalanine with rat liver homogenate was investigated in the presence of S-adenosylmethionine and MgCl2 in vitro. By catechol-O-methyltransferase, it was found that 3, 4-dihydroxyphenylalanine was methylated to 3-hydroxy-4-methoxyphenylalanine.

Steps in the reaction mechanism of the Escherichia coli plasmid P15-specific restriction endonuclease

The restriction endonuclease coded by the Escherichia coli plasmid P15 cleaves unmodified DNA in the presence of ATP and magnesium ions. This reaction is stimulated by the addition of S-adenosylmethionine. Both ATP and S-adenosylmethionine behave as allosteric effectors. The enzyme forms a complex with unmodified DNA in the absence of S-adenosylmethionine and ATP. Neither the rate of complex formation nor its stability is significantly affected by S-adenosylmethionine. The reaction of ATP with this complex is a late step in the reaction sequence prior to DNA cleavage and is affected by the presence of S-adenosylmethionine.

Endo R DpnI restriction of Escherichia coli DNA synthesized in vitro. Evidence that the ends of Okazaki pieces are determined by template deoxynucleotide sequence

A restriction enzyme from Diplococcus pneumoniae, Endo R DpnI, cuts methylated DNA on cellophane discs into pieces which are about the same size as Okazaki pieces. DNA was synthesized in vitro on cellophane discs in the presence of β-nicotinamide mononucleotide to prevent joining of Okazaki pieces. This DNA was methylated by the addition of S-adenosyl methionine to the reaction mixture. When Endo R DpnI was used to cut methylated DNA made in vitro in the presence of S-adenosyl methionine and β-nicotinamide mononucleotide, no decrease in sedimentation of the Okazaki pieces was observed. Control experiments demonstrated that Okazaki pieces were methylated in vitro and that Endo R DpnI was capable of cutting double-stranded DNA containing methylated Okazaki pieces, that is, synthesized in β-nicotinamide mononucleotide and S-adenosyl methionine. These results are interpreted to mean that the ends of Okazaki pieces are non-randomly distributed with respect to 6-methyl adenine residues.

A 24-methylene lophenol C-28 methyltransferase from suspension cultures of bramble cells

Microsomes from suspension cultures of bramble ( Rubus fruticosus) were incubated in the presence of S-adenosyl methionine and various Δ-24(25) and Δ-24(28) sterols. Cycloartenol, a δ-24 9β,19β-cyclopropyl sterol, is efficiently methylated, giving 24-methylene cycloartanol. Some 24-methylene sterols were tested as substrates for a second methylation. Whereas 24-methylene cycloartanol, cycloeucalenol and obtusifoliol were not transformed, 24-methylene lophenol was efficiently methylated giving 24-ethylidene lophenol, a sterol bearing two extra carbon atoms at C-24. In the same conditions 24-methylene cholesterol was a poor substrate. These results are discussed in the context of biosynthesis of sterols in organisms belonging to photosynthetic phyla.

Mechanism of formation of reovirus mRNA 5'-terminal blocked and methylated sequence, m7GpppGmpC.

Blocked and methylated 5' termini of reovirus mRNA are formed by viral cores at an early stage of transcription. Cores incubated in a complete transcription reaction mixture for 30 s, or in a mixture lacking UTP and ATP for a longer time, synthesize the "cap" structure, m7GpppGmpC. The dinucleotide ppGpC functions as substrate for a core-associated guanylyltransferase and is converted to GpppGpC by addition of pG from GTP. For optimal conversion, both the diphosphate terminus and phosphodiester bond are required. pGpC is not a substrate, but pppGpC is utilized after removal of the gamma-phosphate by a core nucleotide phospohydrolase. Methyltransferases also present in cores transfer methyl groups sequentially from S-adenosylmethionine (AdoMet) to the N7-position of the 5'-terminal guanosine followed by the 2'-OH of the penultimate guanosine. GpppGpC is hydrolyzed by cores in the presence of pyrophosphate to ppGpC, the predominant 5'-terminal structure of reovirus mRNA made in the absence of S-adenosylmethionine. N7-methylation prevents pyrophosphorolysis of m7GpppGpC, which may explain the increased proportion of blocked, methylated 5' termini in viral mRNA synthesized in the presence of S-adenosylmethionine. On the basis of these findings, the following reaction series is proposed for the synthesis of reovirus mRNA caps. In the series, AdoHcy is the abbreviation for S-adenosylhomocysteine (see article)9

Macromolecule synthesis in a mutant of Saccharomyces cerevisiae inhibited by S-adenosyimethionine.

Saccharomyces cerevisiae strain 83384-B3 carries the sai-1 mutation which confers sensitivity to S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH). It was shown that the mutant is impermeable to precursors of ribonucleic acid (RNA) and protein during inhibition by SAM (0.2 mM). Inhibition of uptake of adenine and uracil was nearly complete 3 h after growth in the presence of SAM and the uptake of leucine was at least 10-fold lower. The incorporation of 3H-adenine into ribosomal RNA, transfer RNA and heterodisperse RNA, believed to be messenger, was reduced 10-fold when measured after 1 h inhibition. The inhibition of growth was completely reversed by methionine (2.0 mM) in cells previously exposed to SAM for 90 min. The polysome content in cells inhibited by SAM was 25% less than the control after 4 h inhibition. Ribosome synthesis increased only about 40% in the presence of SAM and about 5-fold in the control over an 8 h period. All classes of RNA were synthesized during inhibition.

Ribosome binding to reovirus mRNA in protein synthesis requires 5′ terminal 7-methylguanosine

Purified reovirus synthesizes in vitro a mixture of mRNA molecules that contain 5′ terminal structures of the type ppG…, GpppG…, and m 7GpppG m, the relative amount of each depending upon the presence of S-adenosylmethionine (SAM) or S-adenosylhomocysteine (SAH) in the transcription incubation mixture. Reovirus mRNAs containing 5′ termini of the type ppG… and GpppG… can be specifically modified by wheat germ extracts in the presence of SAM to yield the structure m 7GpppG… (Muthukrishnan et al., 1975). The mRNA methylase activity is ribosome-independent and is recovered almost entirely in the high speed supernatant fraction of wheat germ extracts. Its activity is inhibited by aurintricarboxylic acid. Ribosome binding experiments with reovirus mRNA and wheat germ extract indicate that only those mRNA molecules containing 5′ terminal m 7G are capable of participating in the initiation of protein synthesis and subsequent polysome formation. mRNA with unmethylated 5′ termini do not form a complex with 40S ribosomal subunits. Discrimination between unmethylated and methylated reovirus mRNA, active in protein synthesis, apparently occurs at or before the formation of 40S-mRNA complexes. T1 or pancreatic RNAase digestion of methylated mRNA bound to 80S ribosomes yields 5′ terminal fragments of apparent chain lengths about 32 and 36 nucleotides, respectively. A portion of the T1 RNAase-resistant fragments rebinds to ribosomes to form a nuclease-resistant complex. In contrast, the shorter 5′ terminal oligonucleotide m 7GpppG mpCpUp(Np) 3Gp derived by RNAase T1 digestion of purified reovirus mRNA does not bind to ribosomes. The results suggest that 5′ terminal m 7G may function as the primary recognition signal for ribosome binding and that wheat germ ribosomes bind very close to the 5′ termini of some of the species of methylated reovirus mRNAs.

Modification of the 5'-terminus of mRNA by soluble guanylyl and methyl transferases from vaccinia virus.

RNA guanylyl and methyl transferases have been solubilized from vaccinia virus cores. The guanylyl transferase specifically adds a GMP residue to the 5'-terminus of unmethylated vaccinia virus mRNA to form the structures G(5')ppp(5')Gp- and G(5')ppp(5')Ap-. Studies with [alpha-32P]GTP and [beta, gamma-32P]GTP indicated that only the alpha-phosphate is transferred. In the presence of S-adenosylmethionine, the methyl transferases convert the blocked 5'-termini to m7G(5')ppp(5')Gmp- and m7G(5')ppp(5')Amp-. Similarly, the enzymes can modify synthetic poly(A) to form the structure m7G(5')ppp(5')Amp-.

The role of S-adenosylmethionine in the cleavage of deoxyribonucleic acid by the restriction endonuclease from Escherichia coli K

The restriction endonuclease from Escherichia coli K specifically cleaves foreign DNA in the presence of S-adenosylmethionine, ATP, and Mg2+. The role of S-adenosylmethionine in this reaction has been studied by following the specific binding of the enzyme to unmodified DNA. The results indicate that S-adenosylmethionine acts as an allosteric effector. However, the rate-limiting step in the activation of the enzyme is not the binding of the effector itself, but an event subsequent to it. The interaction of the S-adenosylmethionine with two mutant K restriction endonucleases isolated previously has also been investigated. One of them, which is defective in restriction, can be activated in a manner similar to the wild type enzyme, while the other one, which lacks both restriction and modification activities (due to a mutation in the subunit responsible for DNA recognition), shows no such effect.

Methylation-dependent translation of viral messenger RNAs in vitro.

Methylated reovirus and vesicular stomatitis virus mRNAs, synthesized in vitro in the presence of S-adenosylmethionine by the virion-associated polymerases (RNA nucleotidyltransferases, EC 2.7.7.6), stimulate protein synthesis by wehat germ extracts to a greater extent than unmethylated mRNAs. Addition of S-adenosylmethionine to a cell-free extract programmed with unmethylated mRNA stimulates protein synthesis and results in methylation of the mRNA. An inhibitor of mRNA methylation. S-adenosylhomocysteine, blocks translation of unmethylated, but not of methylated, mRNAs. Aurintricarboxylic acid, which inhibits polypepetide chain initiation, also prevents mRNA methylation by wheat germ extracts. In contrast, sparsomycin, which inhibits polypeptide chain elongation, does not reduce mRNA methylation. The results indicate that methylation of viral mRNA is required for translation in vitro and suggest that mRNA methylation occurs at the initiation step of protein synthesis.

M7G5'ppp5'GmptcpUp at the 5' terminus of reovirus messenger RNA.

In the presence of S-adenosyl methionine the 5' terminal guanosine residue of in vitro synthesized reovirus mRNA becomes methylated at the 2'-OH position. In addition, 7-methyl guanylic acid is condensed covalently at the 5' terminus resulting in the formation of a 5' to 5' triphosphate bridge. Analysis of the 5' terminal sequence of methylated reovirus mRNA revealed that it has the structure m7G5'ppp5'GmpCpUp.

Reovirus messenger RNA contains a methylated, blocked 5'-terminal structure: m-7G(5')ppp(5')G-MpCp-.

Reovirus mRNA synthesized in vitro by the virus-associated RNA polymerase in the presence of S-adenosylmethionine contains blocked, methylated 5'-termini with the structure, m-7G(5')ppp(5')G-MpCp. The functional significance and possible mechanism of formation of this novel 5'-5' terminal nucleotide linkage are discussed.

Interaction between oestrogens and neurotransmitters at the hypophysial-hypothalamic level

When 2-hydroxylated oestrogens are incubated with the 150,000 g supernatant of rat brain in the presence of S-adenosylmethionine, the isomeric 2- and 3- monomethyl ethers are formed. The enzymic methylation of 2-hydroxyoestradiol-17β and 2-hydroxy-17α-ethinyloestradiol-17β in brain tissue is catalysed by a S-adenosylmethionine: catechol O-methyltransferase (EC 2.1.1.6) which exhibits similar kinetics as the catechol 0-methyltransferase of human liver. The K m values were 19 μM for 2-hydroxyoestradiol-17β and 36 μM for 2-hydroxy-17α-ethinyloestradiol-17β; the K m values for catechol amines were about 10 times higher and amounted to 320 μM for adrenaline, 270 μM for noradrenaline and 240 μM for dopamine. These findings indicated that the methylation of catechol amines by the brain catechol O-methyltransferase may be inhibited by catechol oestrogens. Accordingly, the effect of 2-hydroxyoestradiol-17β and 2-hydroxy-17α-ethinyloestradiol-17β on the methylation of adrenaline, noradrenaline and dopamine has been studied. The following K i values were found: 8.4 μM for adrenaline, 9.4 μM for noradrenaline and 6.8 μM for dopamine with 2-hydroxyoestradiol-17β as inhibitor; the corresponding values with 2-hydroxy-17α-ethinyloestradiol-17β as inhibitor were 4.5 μM for adrenaline, 6.8 μM for noradrenaline and 3.8 μM for dopamine. As for the inhibitory effect of 2-hydroxylated oestrogens, no significant differences were observed in the thalamic, hypothalamic, medullar and hypophysial areas, respectively.

Complementation in Vitro by Mutant Restriction Enzymes from Escherichia coli K

Abstract Two mutant strains of Escherichia coli K, K-18 lacking restriction activity (endonucleolytic cleavage of foreign DNA), and the other, K-19 lacking both restriction and modification activities (specific DNA methylation that protects against the homologous restriction activity), complement in vivo to yield a wild type phenotype. Although neither mutant extract alone binds unmodified DNA, the wild type extract and the mixture of mutant extracts do. Retention of the DNA-enzyme complex on membrane filters was used as an assay to purify the two mutant restriction enzymes. Both of these sediment like the wild type enzyme. Complementation in vitro by the two mutant enzymes could be demonstrated by specific DNA binding, cleavage of the unmodified DNA, and restriction-dependent ATP hydrolysis. All of these activities are absent in the individual mutant endonucleases but present in the wild type restriction endonuclease from E. coli K. However, both mutant enzymes show an activity that hydrolyzes ATP which is different from that of the wild type enzyme since it does not require unmodified DNA, but is dependent on the presence of S-adenosylmethionine.

Coproporphyrinogenase activities in extracts of Rhodopseudomonas spheroides and Chromatium strain D.

1. The anaerobic coproporphyrinogenase activity in an extract of Rhodopseudomonas spheroides is inhibited by 1,10-phenanthroline, alphaalpha'-bipyridyl, flavins, 2,4-dinitrophenol and 1,4-naphthaquinone. These compounds have no effect on the aerobic coproporphyrinogenase activity. 2. On removal of small-molecular-weight material from a crude extract, the anaerobic system becomes very unstable; it can be stabilized by adding succinate. Now nicotinamide nucleotides, in addition to Mg(2+), ATP and methionine, are required for protoporphyrin to be formed. 3. A mechanism for the anaerobic reaction is proposed, based on the cofactor requirements and the effect of inhibitors. 4. The enzyme responsible for aerobic activity has been partially purified and some of its properties are reported. 5. A crude extract of Chromatium strain D also exhibits coproporphyrinogenase activity under anaerobic conditions in the presence of S-adenosylmethionine or ATP plus methionine. The requirement for other cofactors is variable.

Methionine synthetase. Existence and interconversion of two enzyme species.

A purification procedure is described which permits the isolation of the vitamin B12‐de‐pendent methionine synthetase (5‐methyl‐tetrahydrofolate‐homocysteine‐transmethylase) from Escherichia coli largely in its native, i. e. methylated form. The enzymatic activity of this species does not depend on the presence of S‐adenosylmethionine. The native enzyme can be demethylated and thus made dependent on S‐adenosylmethionine; this reaction is reversible. The inter‐conversions of the enzyme can be recognized by the spectra. The demethylation is effected by a macromolecular factor present in E. coli cells. By gel filtration, both methylated and demethylated enzyme have been found to possess a molecular weight of about 255000.

Metabolism of S-adenosylmethionine in germinating pea seeds: Turnover and possible relationships between recycling of sulfur and transmethylation reactions

Tissue slices of pea cotyledons incubated with methionine labeled in the methyl group with 3H and 14C and with methionine 35S, rapidly incorporated radioactivity into S-adenosylmethionine. Such slices transferred to unlabeled methionine solutions showed a rapid turnover of labeled S-adenosylmethionine. Kinetic studies showed that sulfur-labeled and methyl-labeled S-adenosylmethionine had quite different rates of turnover. Such differences were related to the possible recycling of sulfur. Attempts to demonstrate direct methylation of S-adenosylhomocysteine in vitro were not successful. Experiments with S-adenosyl-8- 14C- l-homocysteine demonstrated conversion to adenosine- 14C and cell-free extracts catalyzed the synthesis of methionine from homocysteine, in the presence of S-adenosylmethionine, S-methylmethionine, or 5-methyltetrahydrofolate. It is concluded that in the metabolic turnover of S-adenosylmethionine the resulting S-adenosylhomocysteine is rapidly cleaved to give rise to adenosine and homocysteine. Homocysteine is then methylated by 5-methyltetrahydrofolate and the resulting methionine is utilized for regeneration of S-adenosylmethionine.

On the Role of S-Adenosyl-L-methionine in the Biosynthesis of Spermidine by Rat Prostate

Abstract The isolation from the rat ventral prostate of a soluble enzyme preparation producing spermidine, CO2, and methylthioadenosine from putrescine and S-adenosyl-l-methionine is described. This preparation catalyzes limited decarboxylation of S-adenosylmethionine in the absence of any additional reactants but maximal rates of CO2 release from S-adenosylmethionine require the presence of putrescine. When putrescine is present, spermidine and methylthioadenosine are produced in amounts stoichiometric with the CO2 released. The decarboxylation of S-adenosylmethionine in the presence of putrescine is inhibited by isonicotinic acid hydrazide and by 4-bromo-3-hydroxybenzyloxyamine. This finding suggests that pyridoxal phosphate is a cofactor of the reaction. The prostatic enzyme preparation also catalyzes the formation of spermidine from putrescine and exogenous decarboxylated S-adenosylmethionine (prepared by the action of Escherichia coli S-adenosylmethionine decarboxylase). The affinities for S-adenosylmethionine, decarboxylated S-adenosylmethionine, and putrescine were measured. The prostatic preparation could not be separated into two enzymic fractions, one catalyzing the decarboxylation of S-adenosylmethionine, and the other catalyzing spermidine synthesis, as is the case with the spermidine-synthesizing system of E. coli. The differences between the prostatic and the bacterial spermidine-synthesizing systems are discussed. Spermine is synthesized by the preparation from the rat ventral prostate in the presence of S-adenosylmethionine and spermidine, but at a considerably slower rate than spermidine synthesis from putrescine and S-adenosylmethionine.