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The methylation sites and the number of methyl groups of 26-S and 17-S ribosomal RNA (rRNA) of Saccharomyces carlsbergensis were investigated. 26-S and 17-S rRNA contain 43 and 24 methyl groups per molecule, respectively, of which in both molecules 6 are attached to the heterocyclic bases. In 26-S rRNA the base-methyl groups are present as two copies of m³U, m5C as well as m¹A, whereas the methylated bases in 17-SrRNA are one mTG, one methylated cytidine and two of m26A. In 17-S rRNA there are 11 different alkali-stable (i.e. ribose-methylated) dinucleotides, while 26-S rRNA contains 15 different alkali-stable dinucleotides, and in addition 4 alkali-stable trinucleotides (Am-Cm-Gp, Am-Gm-Cp, Am-Am-Up and Um-Gm-ψp). Most of the methylated oligonucleotides found in a ribonuclease T1 digest of 26-S or 17-S rRNA occur in approximately one-molar amounts. The combined data suggest that yeast ribosomal RNA is essentially homogeneous in its methylated sequences and in addition that the methylation is a highly specific process. [ABSTRACT FROM AUTHOR]

An S-adenosylmethionine: catechol-O-inethyltransferase occurs in the 105000 ×g supernatant of human liver As compared with the homogenate, the enzyme was purified 380-fold by adjusting the 24000 × g supernatant to pH 5, fractionation with (NH4)2SO4, gel filtration on Sephadex G-200 and G-100 columns and ion-exchange chromatography on CM-Sephadex and DEAE-cellulose columns. The purified enzyme preparation was homogeneous when subjected to further chromatography on Sephadex G-100 and DEAE-cellulose, disc electrophoresis on polyacrylamide gel at pH 9.5 and analytical ultracentrifugation The enzyme exhibited a s20, w of 3.6 S with a molecular weight of 29000 The final preparation of catechol-O-methyltransferase was very labile, but could be stabilised by the addition of EDTA and MgCl2 in equimolar concentrations The activity of the enzyme was assayed in the presence of cysteine and MgCI2 with 2-hydroxyoestradiol- 17β and radioactive S-adenosylmethionine as substrates The metabolites of 2-hydroxyoestradiol-17β formed during meubation were identical with 2-methoxyoestradiol-17β and 2-hydroxyoestradiol-17β 3-methyl ether. Throughout the purification procedure, the ratio of the 2- and 3-methyl ethers, respectively, remained constant at 2.1. [ABSTRACT FROM AUTHOR]

The role of 1-methyladenosine and 7-methylguanosine in the structure and function of (yeast)tRNAPhe was investigated by studying the NaBH4-reduction of this RNA, of fragments containing 1-methyladenosine and 7-methylguanosine and of the nucleosides themselves. 1. Treatment of 1-methyladenosine with NaB³H4 yielded 1-methyl-6-[³H]hydroadenosine which was fairly stable when kept under a nitrogen atmosphere in a weakly acidic solution. 1-Methyladenosine was converted to the reduction production when it was part of an octanucleotide or of a fragment comprising the CCA-half of (yeast)tRNAPhe, but it was not reduced when it was part of intact (yeast)tRNAPhe. 2. The reduction product of 7-methylguanosine, 7-methyl-8-hydroguanosine, as too unstable to be isolated under the conditions of oligonucleotide anaylsis. The reduction of 7-methyl-guanosine and the reoxidation could be followed, however, by measuring the absorption of the reduction product at 310 nm. It turned out that also 7-methylguanosine was reduced when part of an oligonucleotide or a larger fragment of the tRNA while it was protected in the intact (yeast)tRNAPhe. 3. The finding that in intact tRNA 1-methyladenosine and 7-methylguanosine are inaccessible to NaBH4 demands a revision of parts of the current three-dimensional models of tRNA. Our results support the hypothesis that it is the function of the positively charged odd nucleotides, as 1-methyladenosine and 7-methylguanosine to stabilize certain regions of the tRNA structure. 4. The amino acid acceptance of mixtures of CCA- and pG-halves of (yeast)tRNAPhe was not altered when 1-methyladenosine was reduced. 1-Methyladenosine is therefore probably not an essential part of the synthetase recognition site of this tRNA. [ABSTRACT FROM AUTHOR]

The Indian plant Glycosmic arborea (Roxb.) DC. (Rutaceae) contains alkaloids of different types. The main alkaloid of the Glycosmis leaves is the 4-quinazolone derivative arborine (VI). Previously it has been shown that labelled anthranilic acid (X) and phenylalanine (XI) are specifically incorporated into this alkaloid. This study was performed in order to elucidate the possible pathway of arborine formation in viva. During these investigations an improved procedure for the isolation of arborine was developed. The following results were obtained: After feeding of [14CH3]methionine the N-methyl group of VI was specifically labelled. The same result was obtained after the application of [14CH]3N- methylanthranilic acid. These results seem to indicate that anthranilic acid gets methylated prior to the condensation with phenylalanine which leads to VI. But on the other hand after feeding of [2-14C]glycosminine (V) labelled arborine was also isolated. However in this case the incorporation rate was much lower compared with those of the two other mentioned precursors. It should be mentioned that V is only sparingly soluble in solvents suitable for feeding experiments. The N-1 atom of VI is provided by the nitrogen of anthranilic acid. The experiment with [3- 14C,15N]phenylalanine shows clearly that with the exception of the carboxyl group the aliphatic side chain of XI is incorporated into arborine, Therefore phenylacetic acid can be excluded as an immediate precursor of VI. [U-³H]anthranoylphenylalanine (XIV) was a very poor precursor of arborine. N-Methyl-N-phenyl- [14CO-CH2] acetylanthranilamid (XV) was transformed with high efficiency in Glycosmis into arborine. However it is doubtful whether the N-phenylacetyl derivative of N-methylanthranilamide is a true precursor in arborine biosynthesis. Surprisingly it could be shown that the transformation of XV →VI takes also place in pea plants. Apparently the ring closure of XV is catalyzed by an enzym or enzymatic system which is unspecific and widely distributed. [ABSTRACT FROM AUTHOR]

1. By chemical synthesis starting from phenylacetylene and utilizing stereospecific reactions, two specimens of acetic acid were prepared in which the following conditions were closely approached: (a) some of the methyl groups coated of one hydrogen, one deuterium, and one tritium atom attached to carbon; (b) all such methyl groups in one specimen had the B configuration, and all such methyl groups in the other specimen had the S configuation, and the correct absolute configuration was assignable to each specimen from the method of synthesis used; (e) all methyl groups containing a tritium atom also contained a deuterium atom. 2. Each specimen of acetic acid was converted enzymically into acetyl-coenzyme A and (in the same incubation) this product was condensed with glyoxylic acid on malate synthase to form two specimens of S-malic acid. 3. Each specimen of malic acid was incubated with fumarase until the tritium content of the organic acids (fumarate + malate) had ceased to fall. 4. Malic acid synthesized from R-acetic acid retained 69 % of its carbon-bound tritium (in fumarate + malate) on incubation with fumarase. Malic acid synthesised from S-acetic acid retained 31 % of its carbon-bound tritium. 5. The enzymic assay described in paragraphs 2-4 constitutes a method for determining the absolute configuration of asymmetric methyl groups, provided that a substantial proportion of the tritium in the specimen examined is present in such groups. 6. The method thus established can be used to study the mechanism of enzymic reactions in which a methylene group is converted stereospecifically into a methyl group. With one logical proviso-that it is necessary to known whether protium or deuterium is preferentially removed by the enzsyme from methyl groups also containing tritium — the method is also applicable to enzymic reactions in which a methyl group is converted stereospecifically into a methylene group. 7. Subject to the above proviso, it is indicated that on malate synthase the removal of hydrogen and the attachment of the glyoxylate residue occurs with inversion of configuration. [ABSTRACT FROM AUTHOR]

High speed supernatant enzyme fractions derived from each of three tissues (livers and spleens of normal rats, and spleens of rats with the Dunning leukemia) were compared in their ability to transfer methyl groups from S-adenosyl-methionine to Escherichia coli transfer-RNA (tRNA). In comparison to the supernatant fractions from the normal tissues, that from leukemic spleen had a six-fold higher tRNA methylase activity and methylated E. coli tRNA to a much higher extent. A tRNA methylase which methylated adenine in the 1-position was isolated from each high speed supernatant fraction by DEAE-cellulose and Sephadex G-200 chromatography. Although the purified leukemic spleen enzyme contained six-fold higher methylase activity, it. methylated E. coli, yeast, and mammalian tRNA to the same extent as did the corresponding enzyme from normal liver or spleen. The normal and leukemic tissue enzyme preparations appeared to methylate the same sequences in E. coli tRNA, and could methylate this tRNA to an extent of about one methyl group per molecule. The results suggest that in comparison to that of normal tissue, 1-adenine methylase in the leukemic spleen tissue is increased in activity but possesses identical substrate specificity. [ABSTRACT FROM AUTHOR]

1. The replacement of the phenolic proton of tyrosine by alkyl, aryl or acyl groups completely abolishes the chymotryptic hydrolysis of tyrosine esters. Similarly, chymotrypsin fails to hydrolyze the tyrosyl bonds of dinitrophenyladrenocorticotropin. 2. Several model substrates have been synthesized and characterized. The action of chymotrypsin was followed potentiometrically in buffer containing some alcohol. The hydrolysis of the analogous compounds unsubstituted at the phenolic group was normal. 3. We conclude that inhibition is due, not to an inductive effect, but to a blocking effect which is markedly affected by replacement of the phenolic proton, even with a group as small as a methyl group. [ABSTRACT FROM AUTHOR]

When 2-hydroxyoestradiol-17β was incubated in the presence of S-adenosylmethionine with a 380-fold purified cateehol O-methyltransferase from human liver, 2-methoxyoestradiol-17β and 2-hydroxyoestradiol-17β 3-methyl ether were identified as the only metabolites. No dimethylation, transmethylation or demethylation was observed. The purified enzyme was active only in the presence of cysteine and divalent cations; magnesium was found to be the most effective cation. The temperature optimum for the methylation of 2-hydroxyoestradiol-17β was 42 °C; the activation energy amounted to 20.9 kcal/mol. The enzyme activity had two pH optima; the pH optimum between 6.8 and 8.4 was due to maximal formation of 2-methoxyoestradiol- 17β, whereas the pH optimum at 9.2 was due to the formation of both the 2- and 3-monomethyl ether. The formation of the two isomeric monomethyl ethers increased to a maximum at a substrate concentration of 50 μM (Km value 14 μM) and then decreased with an inflection point between 75 and 100 μM (Ki value under standard assay conditions 95 μM). Whereas both monomethyl ethers were formed at the same rate up to 50 μM substrate concentration, the decrease of formation of 2-hydroxyoestradiol- 17β 3-methyl ether was more pronounced than that of 2-methoxy-oestradiol-17β. Both substrate inhibition and the ratio of methylation depended on the concentrations of S-adenosylmethionine and MgCl2. Product inhibition was also demonstrated with Ki values of 24 μM for 2-methoxyoestradiol- 17β, 80 μM for 2-hydroxyoestradiol- 17β 3-methyl ether and 39 μM for S-adenosylhomocysteine. In contrast, increasing amounts of S-adenosylmethionine did not produce inhibition or changes of the ratio of methylation (Km value 8.5 μM). [ABSTRACT FROM AUTHOR]

The conformations of 1-anilinonaphthalene-8-sulphonate in 2H2O and C2H3O2H have been determined by proton magnetic resonance spectroscopy, and its fluorescence spectra as a solid, m a methyl-cellulose matrix, and in solution in water, methanol and aqueous MgCl2 have been studied. The aromatic rings of anilinonapbthalene-sulphonate are more nearly coplanar in C2H3O2H than in 2H2O. The NH proton fails to exchange with 2H from the solvent in C2H3O2H, and its chemical shift indicates that there is a strong interaction with the sulphonate group. The solvents in which rapid exchange of the NH proton occurs are those in which the quantum yield of fluorescence of anilinonaphthalene-sulphonate is quenched. The fluorescence emission is enhanced and blue-shifted in strong aqueous MgCl2 solutions. Under some conditions the overall emission is composed of contributions from two separable spectra. It is argued that an efficient mechanism for quenching the first excited singlet of anilinonaphthalene-sulphonate is through vibrations of bonds to the nitrogen, and that the shifts of emission maxima in non-aqueous solvents may be partly a result of the molecule displaying different spectra m different conformations (rather than simply a solvent-polarity-induced change). There is no convincing reason for the assumption that when a fluorescence enhancement follows the binding of anilinonaphthalene-sulphonate to a biological structure the site of interaction must be hydrophobic. [ABSTRACT FROM AUTHOR]

Fifty normal (sham operated) and 48 castrated male Swiss mice were painted with methylcholanthrene solution for 16 weeks resulting in warty papules and squamous cell carcinomas. A comparison was made between the two groups as to the median neoplastic surface area involved, the number of mice developing papules of at least 2 mm in diameter, and the mean number of tumors per mouse. The castrated group had lower values for all three comparisons, although only the comparison of numbers of mice developing papules was statistically significant (P < 5% at 15 weeks, and P < 20% at 16 weeks). [ABSTRACT FROM AUTHOR]

The role of 1-methyladenosine and 7-methylguanosine in the structure and function of (yeast)tRNAPhe was investigated by studying the NaBH4-reduction of this RNA, of fragments containing 1-methyladenosine and 7-methylguanosine and of the nucleosides themselves. 1. Treatment of 1-methyladenosine with NaB³H4 yielded 1-methyl-6-[³H]hydroadenosine which was fairly stable when kept under a nitrogen atmosphere in a weakly acidic solution. 1-Methyladenosine was converted to the reduction production when it was part of an octanucleotide or of a fragment comprising the CCA-half of (yeast)tRNAPhe, but it was not reduced when it was part of intact (yeast)tRNAPhe. 2. The reduction product of 7-methylguanosine, 7-methyl-8-hydroguanosine, as too unstable to be isolated under the conditions of oligonucleotide anaylsis. The reduction of 7-methyl-guanosine and the reoxidation could be followed, however, by measuring the absorption of the reduction product at 310 nm. It turned out that also 7-methylguanosine was reduced when part of an oligonucleotide or a larger fragment of the tRNA while it was protected in the intact (yeast)tRNAPhe. 3. The finding that in intact tRNA 1-methyladenosine and 7-methylguanosine are inaccessible to NaBH4 demands a revision of parts of the current three-dimensional models of tRNA. Our results support the hypothesis that it is the function of the positively charged odd nucleotides, as 1-methyladenosine and 7-methylguanosine to stabilize certain regions of the tRNA structure. 4. The amino acid acceptance of mixtures of CCA- and pG-halves of (yeast)tRNAPhe was not altered when 1-methyladenosine was reduced. 1-Methyladenosine is therefore probably not an essential part of the synthetase recognition site of this tRNA. [ABSTRACT FROM AUTHOR]

This article presents information on the research paper "The Effect of Structural Alterations of the Erythemal Activity of Furocoumarins (Psoralens)," by M.A. Pathek, J.H. Fellman and K.D. Kaufman. Thirty-six furocoumarin derivatives and forty-two coumarin derivatives were tested for photosensitizing action to ultra-violet light. None of the compounds was more active than psoralen itself. Methyl substitution in several positions reduced the activity, as did substitution with methoxy, amino, nitro, acetyl, acetamino, bromo groupings in the 5 or 8 positions.

The article reports on a study that investigated the optimal pH effect for N-methyl-N-nitrosourea-induced odontogenic neoplasms in rats. The conclusion is that with a pH of four the gingival epithelium has the characteristics of dental lamina and appears to be the source of keratocyts, ghost cells, dentinoid masses, and denticles. Radiographs show a neoplasm or expansile lesion in the mandible and dentinoid tissue in the periodontal ligament. Research methods include 26 Wistar female rats that were separated into three groups.