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PubMed1979

Multiple molecular forms of catechol-O-methyltransferase. Evidence for two distinct forms, and their purification and physical characterization.

Catechol-O-methyltransferase (COMT: EC 2.1.1.6) has been shown to exist in the soluble fraction of rat liver as two distinct molecular forms, designated COMT I and COMT II, which are separable by gel filtration, ion exchange chromatography, and sedimentation. The predominant form, COMT I, has a smaller Mr of about 24,000, as determined by gel filtration and sedimentation, and less negative charge, whereas the minor form, COMT II, has a larger Mr of about 47,500 and more negative charge. The COMT I and COMT II have been purified 450- and 205-fold, respectively, from rat liver by a newly developed procedure which gives homogeneous enzyme preparations with respect to catechol-methylating activities. The molecular properties of the predominant form, COMT I, were: s20,w, 2.7; D20,W, 10.5; Stokes radius, 20.1 A; f/fo, 1.08; and pI, 4.9. For the minor form, COMT II, the values were s20,w, 3.8; D20,w, 7.3; Stokes radius, 28.7 A; f/fo, 1.23; and pI, 4.8. Catechol-O-methyltransferase was found to exhibit tissue-specific isozymic patterns in the distribution of its two variant forms. In the rat tissues, the liver and kidney exhibited the presence of the two physically separable forms. Catechol-O-methyltransferase was also found as two distinct molecular forms in human tissues, including liver, brain, and placenta. The two forms of human catechol-O-methyltransferase were not distinguishable by the criteria of gel filtration from their counterparts in rat liver, indicating that the two molecular forms of human and rat liver catechol-O-methyltransferase are homologous. No interconversion of one molecular form of catechol-O-methyltransferase into the other was observed under experimental conditions employed. Available evidence indicates that the two molecular forms of catechol-O-methyltransferase are genetically dissimilar proteins.

Animals↗

PubMed2026

Coordinated use of three homocysteine methyltransferases supports l-methionine biosynthesis and environmental adaptation among plant-associated bacteria.

Plant pathogens colonize multiple plant-associated habitats throughout their life cycle, encountering distinct nutrient conditions and microbial communities. l-methionine is required for bacterial growth and environmental adaptation. However, how plant pathogens coordinate l-methionine biosynthetic pathways to adapt to different plant-associated environments remains poorly understood. Here, using the plant pathogen Xanthomonas campestris pv. campestris strain XC1 as a model, we show that three homocysteine methyltransferase pathways allow XC1 to catalyze the final step of l-methionine biosynthesis using different methyl donors and cofactors under different environmental conditions. Bioinformatic and transcriptional analyses identified three homocysteine methyltransferase-associated operons in XC1, mesMXD, mmuPM, and metHRHaHb, corresponding to the MesD-, MmuM-, and MetHaHb-dependent pathways, respectively. MesD uses an endogenously synthesized methyl donor and functions as the dominant homocysteine methyltransferase under l-methionine-limiting conditions, supporting bacterial growth, intracellular l-methionine accumulation, and full virulence. Furthermore, MmuM enables XC1 to use plant-derived S-methylmethionine for l-methionine biosynthesis, whereas MetHaHb enables XC1 to use vitamin B12 supplied by a neighboring bacterium for l-methionine biosynthesis in co-culture. Expression analyses showed that mesMXD was the only homocysteine methyltransferase-associated operon that responded to l-methionine availability, and its expression also decreased when S-methylmethionine- or vitamin B12-dependent pathways supported l-methionine biosynthesis. Comparative genomic analysis further showed that the three-homocysteine methyltransferase configuration is conserved in Xanthomonas and is also present in other plant-associated bacteria. Together, these findings show that a plant pathogen can coordinate endogenous, plant-derived, and microbially supported homocysteine methyltransferase pathways to maintain l-methionine biosynthesis, providing a metabolic strategy for adaptation to plant-associated environments.

Methionine↗

PubMed1977

Synthesis of mRNA guanylyltransferase and mRNA methyltransferases in cells infected with vaccinia virus.

Guanylyltransferase and methyltransferases that modify the 5'-terminals of viral mRNA's to form the structures m7G(5')pppAm- and m7G(5')pppGm- appear to be synthesized afte- vaccinia virus infection of HeLa cells. Elevations in these enzyme activities were detected within 1 h after virus inoculation and increased 15- to 30-fold by 4 to 10 h. Increases in the guanylyl- and methyltransferase activities were prevented by cycloheximide, an inhibitor of protein synthesis, but not by cytosine arabinoside, an inhibitor of DNA synthesis. The latter results suggest that the mRNA guanylyl- and methyltransferases are "early" or prereplicative viral gene products. The guanylyltransferase and two methyltransferases, a guanine-7-methyltransferase and nucleoside-2'-methyltransferase, were isolated by column chromatography from infected cell extracts and found to have properties similar or identical to those of the corresponding enzyme previously isolated from vaccinia virus cores. In contrast, enzymes with these properties could not be isolated from uninfected cells.

Cycloheximide↗

PubMed1979

Guanidoacetate methyltransferase. Purification and molecular properties.

Guanidoacetate methyltransferase has been purified about 140-fold from pig liver. Polyacrylamide gel electrophoresis of the purified enzyme showed four protein bands, each of which is associated with guanidoacetate methyltransferase activity. During gel electrophoresis at pH 3 in 8 M urea, guanidoacetate methyltransferase migrated as a single component. The molecular weight of the purified guanidoacetate methyltransferase was estimated to be 31,000 by sodium dodecyl sulfate-gel electrophoresis, which also showed only one protein component with guanidoacetate methyltransferase activity. This molecular weight is in agreement with that estimated by Sephadex G-75 chromatography. Guanidoacetate methyltransferase is inhibited by adenosylhomocysteine, 3-deazaadenosylhomocysteine, and sinefungin with Ki values of 16 microM, 39 microM, and 18 microM, respectively.

Amino Acids↗

PubMed1977

5-demethylubiquinone-9-methyltransferase from rat liver mitochondria. Characterization, localization, and solubilization.

The methyltransferase responsible for the conversion of 5-demethylubiquinone-9 to ubiquinone-9 in rat liver mitochondria has been shown to be localized in the inner membrane of rat liver mitochondria. NADH was required to generate the hydroquinone, which was the immediate substrate for methylation. The Km for 5-demethylubiquinone-9 was estimated to be in the range of 60 to 80 nM and the Km for S-adenosylmethionine was found to be 22 micronM. The methyl-transferase was solubilized by Triton X-100, a procedure which inactivated the 5-demethylubiquinone-9 reductase. Dithionite was found to partially substitute for NADH in both membranous and soluble systems. Inhibitors of catechol-O-methyltransferase were not effective inhibitors of 5-demethylubiquinone-9-methyltransferase. In addition, catechol-O-methyltransferase and 5-demethylubiquinone-9-methyltransferase were found to have reciprocal subcellular localizations. It is likely that the hydrophobic side chain of ubiquinone, added to p-hydroxybenzoate in the first biosynthetic step, is required for attachment to the lipid bilayer. This permits subsequent metabolism of the ring system by membrane-bound enzymes, including the final methylation to form ubiquinone-9.

Animals↗

PubMed1978

Potential inhibitors of S-adenosylmethionine-dependent methyltransferases. 7. Role of the ribosyl moiety in enzymatic binding of S-adenosyl-L-homocysteine and S-adenosyl-L-methionine.

A series of 2',3'-acyclic analogues of S-adenosyl-L-homocysteine were synthesized and evaluated as inhibitors of S-adenosyl-L-methionine-dependent methyltransferases. The 2',3'-acyclic analogues were prepared by periodate oxidation of the corresponding ribonucleosides, followed by reduction of the intermediate dialdehydes with sodium borohydride. These 2',3'-acyclic ribonucleosides were inactive as inhibitors of histamine N-methyltransferase, catechol O-methyltransferase, phenylethanolamine N-methyltransferase, and hydroxyindole O-methyltransferase. These results suggest that the rigidity of the ribosyl ring of S-adenosyl-L-homocysteine is crucial to its enzymatic bindings.

Animals↗

PubMed1976

Subcellular localization of S-adenosyl-L-methionine:tRNA methyltransferases with aminoacyl-tRNA synthetases in human and mouse: normal and leukemic leukocytes.

The subcellular distributions of S-adenosyl-L-methionine:tRNA methyltransferases and aminoacyl-tRNA synthetases were investigated with the use of human and mouse normal and leukemic leukocyte cell lines. Differential centrifugation of homogenized cell suspensions produced three pelleted subcellular fractions (nuclear and membrane, microsomal, and postribosomal) and a supernatant fraction. Each fraction was assayed for both methyltransferase activity and synthetase activity. The largest amounts, 40-50%, of total methyltransferase and synthetase activities were localized in either the microsomal or the postribosomal fractions, depending on cell type. In addition, the highest specific activities of these two enzyme systems were found to be present in the microsomal and postribosomal fractions. The psotribosomal fraction from leukemic leukocytes had a methyltransferase specific activity higher than that of the microsomal fraction, while the same two fractions of normal leukocytes had approximately equal activities. Specific activities of aminoacyl-tRNA synthetases were found to be approximately equal for these two fractions, whether they were from normal or leukemic leukocytes. The activity of tRNA methyltransferases and synthetases within the postribosomal fraction of the cytoplasm suggests the existence of high-molecular-weight enzyme complexes for the modification as well as the aminoacylation of tRNA.

Amino Acyl-tRNA Synthetases↗

PubMed1977

Stringent regulation of the synthesis of a transfer ribonucleic acid biosynthetic enzyme: transfer ribonucleic acid(m5U)methyltransferase from Escherichia coli.

This paper describes the regulation of a transfer ribonucleic acid (tRNA) biosynthetic enzyme, the tRNA(m5U)methyltransferase (EC 2.1.1.35). This enzyme catalyzes the formation of 5-methyluridine (m5U, ribothymidine) in all tRNA chains of Escherichia coli. Partial deprivation of charged tRNAVal can be imposed by shifting strains carrying a temperature-sensitive valyl-tRNA ligase from a permissive to a semipermissive temperature. By using two such strains differing only in the allelic state of the relA gene, it was possible to show the tRNA(m5U)methyltransferase to be stringently regulated. Upon partial deprivation of charged tRNAVal, the differential rate of tRNA(m5U)methyltransferase synthesis was found to decrease in a strain with stringent RNA control (relA+), whereas it increased in the strain carrying the relA allele. This increase of accumulation of tRNA(m5U)methyltransferase activity required protein synthesis. Thus, when tRNA is partially uncharged in the cell, the relA gene product influences the expression of tRNA(m5U)methyltransferase gene.

Bacterial Proteins↗

PubMedDate not supplied

Separation of the S-adenosylmethionine: 5- and 8-hydroxyfuranocoumarin O-methyltransferases of Ruta graveolens L. by general ligand affinity chromatography.

Two S-adenosyl-L-methionine:furanocoumarin O-methyltransferases of R. graveolens, acting at the 5- and 8-hydroxyl of the psoralen nucleus, were completely resolved by adsorption on a general affinity ligand, 5-(3-carboxypropanamido) xanthotoxin, followed by specific desorption by bergaptol and xanthotoxol, respectively. The 5-O-methyltransferase was purified 450-fold by this procedure, the 8-O-methyltransferase 112-fold, and both enzyme fractions were electrophoretically homogeneous. No resolution could be achieved of the activity against two 5-hydroxypsoralens or of the activity against two 8-hydroxypsoralens, and conclusive evidence is presented for the existence of only one 5-O-methyltransferase and only one 8-O-methyltransferase acting on linear furanocoumarins.

Chromatography, Affinity↗

PubMed1975

Isolation and characterization of tryptophan transaminase and indolepyruvate C-methyltransferase. Enzymes involved in indolmycin biosynthesis in Streptomyces griseus.

Two enzymes, tryptophan transaminase and indolepyruvate C-methyltransferase, which are active in the initial steps of the biosynthetic pathway of the antibiotic indolmycin, have been detected and partially purified from cell-free extracts of Streptomyces griseus. The transaminase has been purified 3-fold by ammonium sulfate fractionation. At this stage of purification, it catalyzes the alpha-ketoglutarate and pyridoxal phosphate-dependent transamination of L-tryptophan, 3-methyltryptophan, L-pphenylalanine, and L-tyrosine. The C-methyltransferase catalyzes the transfer of a methyl group from S-adenosylmethionine to position 3 of the aliphatic side chain of indolepyruvate. No cofactors are required. The C-methyltransferase has been purified 110-fold by ammonium sulfate fractionation, Sephadex G-150 gel filtration, DEAE-Sephadex column chromotography, and Bio-Gel A-5m gel filtration. The enzyme has a broad pH optimum of 7.5 to 8.5. A molecular weight of 55,000 +/- 5,000 has been determined by Sephadex G-200 gel filtration with reference proteins and a molecular weight of 58,000 +/- 8,000 has been determined by sucrose density gradient centrifugation. The enzyme is relatively stable at temperatures of 0-5 degrees but is destroyed by freezing or by heating. The C-methyltransferase is inhibited strongly by the thiol reagents p-chloromercuribenzoate and N-ethylmaleimide. The Zn2+ and Fe2+ chelators 1,10-phenanthroline and 2,2'-bipyridine also inhibit the enzyme activity but EDTA does not. Michaelis-Menten constants have been determined for the 110-fold purified enzyme as 1.2 X 10(-5) M for S-adenosylmethionine and 4.8 X 10(-6) M for indolepyruvate. The enzyme activity in the crude extract is inhibited competitively by indolmycin (Ki equals 2.3 mM) and L-tryptophan (Ki equals 0.17 mM), but these effects are not observed after the enzyme has been passed through the Sephades G-150 column during purification. The crude extract is capable of methylating phenylpyruvate and p-hydroxyphenylpyruvate but this capability is lost upon purification of the indolepyruvate C-methyltransferase activity. No methylation of L-tryptophan occurs under the conditions used.

Anti-Bacterial Agents↗

PubMed1979

Pineal N-acetyltransferase and hydroxyindole-O-methyltransferase: control by the retinohypothalamic tract and the suprachiasmatic nucleus.

The visual pathway and central neural structures involved in the photic and endogenous regulation of the activity of pineal N-acetyltransferase and hydroxyindole-O-methyltransferase were investigated. The results indicate that the visual pathway regulating both enzymes is the retinohypothalamic tract, and that the inferior accessory optic tract is clearly not involved in the regulation of hydroxyindole-O-methyltransferase activity, as has been previously thought. In addition, the suprachiasmatic nucleus was found to be necessary for the generation of a rhythm in N-acetyltransferase activity in blinded animals, and to be responsible for the tonic elevation of hydroxyindole-O-methyltransferase activity in blinded animals. Finally, it was concluded that the rapid and large daily changes in N-acetyltransferase activity seen in a normal lighting cycle and the much slower and smaller changes in hydroxyindole-O-methyltransferase activity seen only after weeks in constant lighting conditions are mediated by the same neural tract; the different time courses of the effects of environmental lighting may be explained on the basis of different intracellular regulatory mechanisms.

Acetylserotonin O-Methyltransferase↗

PubMed1975

Properties of catechol O-methyltransferases from brain and liver of rat and human.

Kinetic and electrophoretic properties of catechol O-methyltransferases (EC 2.1.1.6) from brain and liver were studied. The enzyme of either rat or human tissues exhibited a single molecular form when subjected to electrophoresis at pH7.9. At pH9 a second, apparently oxidized, form was detected. Isoelectric-focusing experiments also indicated only one enzyme form, which was identical from extracts of brain and liver of each species (pI = 5.2 for rat, 5.5 for human). Similarities between brain and liver catechol O-methyltransferase of a given species were also demonstrated by kinetic parameters, meta/para ratios of products, and inhibitor potencies. Human catechol O-methyltransferase exhibited lower Km values than did the rat enzyme for S-adenosyl-L-methionine, dopamine and dihydroxybenzoic acid. Adrenochrome inhibited both rat and human enzyme. It was concluded (1) that only a single enzyme form could be demonstrated in the physiological pH region; (2) that catechol O-methyltransferase of brain could not be distinguished from the liver enzyme of the same species; and (3) that species differences exist between the enzymes of rat and human tissues.

Adrenochrome↗

PubMed1975

Alteration of glycine N-methyltransferase activity in fetal, adult, and tumor tissues.

Glycine N-methyltransferase activity has been examined in a number of fetal and adult organs, as well as in several rodent hepatomas, using both enzymatic and immunological techniques. In fetal rabbit liver, the activity first appears at a low level at about 20 days postfertilization and rises to high levels after birth, reaching maximum in the adult liver. In fast-growing hepatomas, the activity could not be detected by either enzymatic or immunological assay. It could be detected in the slower-growing hepatomas, but in considerably diminished levels compared with that of normal adult rat liver. Immunoassays gave no evidence for inactive forms of the enzyme in the tissues that had no enzymatic activity. Transfer RNA methyltransferase assays carried out simultaneously showed an inverse relationship to the glycine N-methyltransferase activity. The levels of transfer RNA methyltransferase activity were high in fetal and tumor tissues and lower in normal adult tissues.

Animals↗

PubMed1979

Age-dependent changes in the specificity of tRNA methyltransferases in the cerebellum of the icteric and nonicteric Gunn rat.

The activity of tRNA methyltransferases present in the cerebellum of 6- and 21-day-old nonicteric and icteric Gunn rats was compared using purified E. coli tRNAs as substrates. At 6 days the tRNA methyltransferases of the icteric animals were significantly more effective in methylating tRNAGlu2 and tRNAPhe than were those of their nonicteric counterparts. This relationship reversed itself at 21 days. The action of the tRNA methyltransferases from the 6-day-old icteric animals led to higher proportions of 1-methyladenine in tRNAGlu2 and tRNAPhe than were obtained using the corresponding enzymes of the nonicteric animals. The proportion of N2-methylguanine was also higher, yet only in tRNAfMet and not in tRNAPhe. The study reveals much more extensive fluctuations in the activity and in the substrate recognition specificity among the cerebellar tRNA methyltransferases of the icteric than among those of the nonicteric controls during the crucial 6--21 day period of cerebellar development.

Aging↗

PubMed1976

Effect of ethanol ingestion on choline phosphotransferase and phosphatidyl ethanolamine methyltransferase activities in liver microsomes.

The effect of ethanol ingestion on choline phosphotransferase and phosphatidyl ethanolamine methyltransferase activities, the two enzymes involved in phosphatidyl choline biosynthesis in liver microsomes, has been investigated. Female rats were fed a 5% ethanol-liquid diet containing amino acids, minerals, vitamins, with and without choline, for 2, 6 and 10 weeks. Control animals were pair-fed the same isocaloric diet with 5% sucrose with and without choline. Ethanol administration with or without dietary choline stimulated significantly (P less than 0.001) the specific activities of phosphatidyl ethanolamine methyltransferase in liver microsomes in the animals fed 5% ethanol for 2, 6, and 10 weeks, when compared to those control animals pair-fed the isocaloric diet with or without choline. Ethanol administration with or without dietary choline for 2 weeks stimulated significantly (P less than 0.02) the specific activities of choline phosphotransferase. The specific activities of phosphatidyl ethanolamine methyltransferase continued to increase in the liver microsomes from the animals in which dietary choline was omitted for 2, 6, and 10 weeks in the sucrose controls and alcohol-fed animals. Ethanol administration stimulates significantly (P less than 0.001) the phosphatidyl ethanolamine methyltransferase specific activities in liver microsomes of animals fed the liquid diet with dietary omission of choline and methionine for 2 weeks.

Alcohol Drinking↗

PubMed1977

The effect of vitamin A deficiency on testicular transfer RNA methyltransferase activity.

Testicular transfer RNA methyltransferase activity was examined in normal and vitamin A-deficient rats. The specific activity was reduced by 50% in the vitamin A-deficient rats. In addition, a 5-fold decrease in the extent of tRNA methylation was observed with enzyme preparations from deficient testes. Both the rate and extent of tRNA methylation returned to control levels in vitamin A-repleted rats. In contrast, retinoic acid repletion did not reverse the effect of vitamin A deficiency on testicular tRNA methyltransferase activity. The methylated nucleoside composition of tRNA methylated by extracts of vitamin A-deficient testes was altered dramatically compared to that of tRNA methylated by control testicular enzymes. Decreased testicular tRNA methyltransferase activity was noted in midly deficient rats before the onset of testicular degeneration suggesting that the decreased tRNA methyltransferase activity in the testes is primarily the result of vitamin A deficiency.

Animals↗

PubMed1979

An assay for human erythrocyte catechol-O-methyltransferase activity using a catechol estrogen as the substrate.

A radiometric assay for catechol-O-methyltransferase (COMT) activity in human erythrocytes is described that employs 2-hydroxy[3H]estrone, and non-radiolabeled S-adenosylmethionine (SAM) as the cosubstrates. The ease of separation of the product of the reaction, 2-methoxy[3H]estrone from 2-hydroxy[3H]estrone makes it possible to achieve low reaction blanks. The assay is very sensitive, and only 200 microliter of whole blood are used per determination. The assay is highly reproducible. The interassay variability (coefficient of variation) was 6.5% for 24 assays of COMT activity in red blood cells in blood obtained daily for 24 days from one person. In incubations conducted at 37 degrees C for 30 min, the catechol-O-methyltransferase activity was a linear function of enzyme concentration (equivalent to 11 to 180 microliter of packed red blood cells). Employing this assay, we evaluated the catalytic conversion of 2-hydroxyestrone to 2-methoxyestrone by catechol-O-methyltransferase from human red blood cells and found that the apparent Michaelis constant and the apparent maximal rate of reaction were 3 x 10(-7) M and 6.7 x 10(-9) mol . ml-1 erythrocytes . h-1, respectively. The catechol-O-methyltransferase activity measured in erythrocytes obtained from 100 healthy subjects (men and nonpregnant women) was 8.2 +/- 0.17 (mean +/- S.E.) nmol 2-methoxyestrone . ml-1 erythrocytes . h-1.

Carbon Radioisotopes↗

PubMed1976

Membrane-associated reactions in ubiquinone biosynthesis. 2-Octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone methyltransferase.

The O-methylation of 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone, which has been previously postulated to be the final reaction in the biosynthesis of ubiquinone was demonstrated in vitro using cell extracts of Escherichia coli. S-Adenosyl-L-methionine was active as the methyl donor for the reaction. The enzyme concerned, S-adenosyl-L-methionine: 2-octaprenyl-3-methyl-5-hydroxy-6-methoxy-1,4-benzoquinone-O-methyltransferase, was partially purified and shown to have a molecular weight of about 50 000 and to require a divalent metal and dithiothreitol for optimal activity in vitro. The methyltransferase was absent from extracts from ubiG- mutants suggesting that the ubiG gene is the structural gene coding for the methyltransferase. The enzyme, although not firmly membrane-bound, showed some affinity for the cell membrane in broken cell preparations and could utilize the benzoquinone substrate when the latter was free or bound to the cell membrane, with about equal efficiency. It is concluded that in vivo, the methyltransferase reaction probably occurs at the internal surface of the cytoplasmic membrane.

Animals↗