Origin of the methyl carbon of methyl coniine in Conium maculatum

Studies with [methyl-¹⁴C]-l-methionine have established that the methyl carbon of l-methionine can act as a precursor of the N-methyl group of methyl coniine in Conium maculatum.     

A new alkaloid from south african Conium species

Recent screening of South African Conium species for alkaloids as part of taxonomic studies has yielded γ-coniceine, coniine, methylconiine, conhydrine and a new alkaloid N-methylpseudoconhydrine. The relative stereochemistry of N-methylpseudoconhydrine was ascertained by ¹H NMR decoupling experiments. This latter alkaloid was found in significant amounts in the leaf and stem of some plants investigated and was the major alkaloid along with conhydrine in the leaf and stem of one group of high altitude plants. These plants also contained significant amounts of volatile oil, the major monoterpene being myrcene.     

Product inhibition studies and the reaction sequence of rabbit adrenal norepinephrine N-methyl transferase isozymes

The effects of reaction products on the steady-state kinetic properties of the five charge isozymes of rabbit adrenal norepinephrine N-methyl transferase have been investigated. Qualitative and quantitative differences were observed for the isozymes. The only characteristic that was common to all isozymes was the competition between S-adenosylmethionine and S-adenosylhomocysteine for the binding site. In most instances, the product inhibition constants were sufficiently low to suggest that product inhibition may be an important factor in regulating the activities of the isozymes. A reaction model is proposed for rabbit adrenal norepinephrine N-methyl transferase which is consistent with results observed in investigations of the steady-state kinetic properties of the five charge isozymes. The proposed model is that of an ordered sequential reaction sequence in which the active center contains a binding site for S-adenosylmethionine and S-adenosylhomocysteine, and a binding site for norepinephrine and epinephrine. The proposed model includes the formation of a number of abortive complexes between enzyme and substrate and product, but not all of the abortive complexes are significant kinetically in the case of some of the isozymes. The differences in the steady-state kinetic characteristics of the isozymes are attributed to differences in the magnitudes of the rate constants of some of the individual steps.     

Identification of N5,N10-methylene tetrahydrofolate reductase as the enzyme involved in the 5-methyl tetrahydrofolate-dependent formation of a beta-carboline derivative of 5-hydroxytryptamine in human platelets.

An enzyme in human platelets or rat brain incubated with 5-methyl tetrahydrofolate (5MeH₄folate) yields formaldehyde (4, 13), which will combine with biogenic amines to form β-carbolines (5) or tetrahydroisoquinolines. This activity was purified 500-fold from human platelets which are the main storage site for 5-hydroxytryptamine in man. This enzyme was identical to N⁵, N¹⁰-methylene tetrahydrofolate (N⁵,N¹⁰-methylene H₄folate) reductase by the following criteria: (i) co-purification, (ii) heat denaturation, (iii) pH response, (iv) molecular weight, (5) cofactor requirements. A mechanism involving the enzymatic generation of formaldehyde followed by adduct formation with a biogenic amine is proposed.     

Crystal structure of the PHF8 Jumonji domain, an N-methyl lysine demethylase

Crystallographic analysis of the catalytic domain of PHD finger protein 8 (PHF8), an N^ε-methyl lysine histone demethylase associated with mental retardation and cleft lip/palate, reveals a double-stranded β-helix fold with conserved Fe(II) and cosubstrate binding sites typical of the 2-oxoglutarate dependent oxygenases. The PHF8 active site is highly conserved with those of the FBXL10/11demethylases, which are also selective for the di-/mono-methylated lysine states, but differs from that of the JMJD2 demethylases which are selective for tri-/di-methylated states. The results rationalize the lack of activity for the clinically observed F279S PHF8 variant and they will help to identify inhibitors selective for specific N^ε-methyl lysine demethylase subfamilies.     

7β-Methyl substituent is a structural locus associated with activity cliff for nepenthone analogues

With the purpose of identifying novel selective κ opioid receptor (KOR) antagonists as potential antidepressants from nepenthone analogues, starting from N-nor-N-cyclopropylmethyl-nepenthone (SLL-020ACP), a highly selective and potent KOR agonist, a series of 7β-methyl-nepenthone analogues was conceived, synthesized and assayed on opioid receptors based on the concept of hybridization. According to the pharmacological results, the functional reversal observed in orvinol analogues by introduction of 7β-methyl substituent could not be reproduced in nepenthone analogues. Alternatively, introduction of 7β-methyl substituent was associated with substantial loss of both subtype selectivity and potency but not efficacy for nepenthone analogues, which was not found in 7β-methyl orvinol analogues. Surprisingly, SLL-603, a 7β-methyl analogue of SLL-020ACP, was identified to be a KOR full agonist. The possible molecular mechanism for the heterogeneity in activity cliff was also investigated. In conclusion, 7β-methyl substituent was a structural locus associated with activity cliff and demonstrated as a pharmacological heterogeneity between nepenthone and orvinol analogues that warrants further investigations.     

Structural insights into cholinesterases inhibition by harmane β-carbolinium derivatives: A kinetics – molecular modeling approach

The natural indole alkaloids, the β-carbolines, are often associated with cholinesterase inhibition, especially their quaternary salts, which frequently have higher activity than the free bases. Due to lack of information explaining this fact in the literature, the cholinesterase inhibition by the natural product harmane and its two β-carbolinium synthetic derivative salts (N-methyl and N-ethyl) was explored, together with a combination of kinetics and a molecular modeling approach. The results, mainly for the β-carbolinium salts, demonstrated a noncompetitive inhibition profile, ruling out previous findings which associated cholinesterase inhibition by β-carbolinium salts to a possible mimicking of the choline moiety of the natural substrate, acetylcholine. Molecular modeling studies corroborate this kind of inhibition through analyses of inhibitor/enzyme and inhibitor/substrate/enzyme complexes of both enzymes.     

Determination of putrescine N-methyltransferase by high performance liquid chromatography

A novel procedure is described for the chemical synthesis of N-methylputrescine, the product of the title enzyme. This is obtained from putrescine by formylation followed by the reduction of the monoformylputrescine intermediate with LiA1H₄. An assay method for putrescine N-methyltransferase was developed which depends on the determination of N-methylputrescine in the presence of an excess of putrescine. This method, which makes use of a radiolabeled substrate unnecessary, is based on dansylation of the product followed by HPLC separation on a reversed-phase column. The enzyme activity of the protein peak extracted from plant material was measured after treatment by gel filtration on prepacked disposable PD 10 columns. The specific enzyme activities determined in the extract from the roots of Nicotiana tabacum and Datura stramonium plants, and from a root culture of D. stramonium, are reported. With an enzyme preparation from the last root culture, K_m values for putrescine and S-adenosylmethionine (SAM) were determined as 0.88 mM and 0.15 mM, respectively.     

Two new mononuclear gold(I)-nitrogen [N,N′-di(2,6-methyl)phenylformamidine] complexes: Syntheses and crystal structures

Two gold(I) mononuclear complexes have been prepared by reacting gold(I) tetrahydrothiophene with N,N′-di(2,6-methyl)phenylformamidine. The neutral complex [N,N′-di(2,6-methyl)phenylformamidine)-gold(I) chloride (C₁₇H₂₀AuClN₂) (1), crystallizes in the triclinic group while the cationic [N,N′-di(2,6-methyl)phenylformamidine](tetrahydrothiophene)-gold(I) (C₂₁H₂₈AuN₂S) (2) crystallizes with a nitrate anion in the monoclinic group P2(1)/n. Both compounds are good starting materials for synthetic gold chemistry.     

Induction of delta-Aminolevulinic Acid Synthase Activity and Inhibition of Heme Synthesis in Euglena gracilis by N-Methyl Mesoporphyrin IX

N-Methyl mesoporphyrin IX, an inhibitor of heme synthesis, increases extractable δ-aminolevulinic acid (ALA) synthase activity when administered to growing cultures of Euglena gracilis Klebs strain Z Pringsheim in micromolar concentrations. Wild-type light-grown green cells and white aplastidic cells exhibited 2.8-fold and 1.8-fold increases, respectively, in ALA synthase activity within five to six hours after incubation with 4 × 10⁻⁶ molar N-methyl mesoporphyrin IX. Protoheme levels were decreased and ⁵⁹Fe incorporation into heme was inhibited by N-methyl mesoporphyrin IX, indicating that, as in animal cells, N-methyl mesoporphyrin IX acts specifically to block iron insertion into protoporphyrin IX. Chlorophyll synthesis in wild-type cells was not affected within the first 6 hours after administration of N-methyl mesoporphyrin IX.     

Effect of adenine nucleotides on the oxidation of N-methyl groups in liver mitochondria

In phosphorylating mitochondria, isolated in 0.25 M sucrose and suspended in a glycylglycine-KC1 medium at pH 7.4, the N-methyl group of sarcosine is oxidized to formaldehyde, formate, and CO₂. The initial rate of O₂ uptake in this system is only about half as great as with phosphate-washed mitochondria, in which the N-methyl carbon is oxidized only to the level of “active formaldehyde” and can be recovered as serine-β-carbon and/or formaldehyde. In the glycylglycine-KC1 medium, the O₂ uptake with sarcosine occurs in a biphasic manner and the initial slower rate can be extended by the addition of Mg²⁺, and ADP, AMP, or ATP. O₂ uptake is similarly restrained by ADP in mitochondria buffered with imidazole or pyrophosphate. The ADP effect is not observed in the presence of dinitrophenol. The patterns of O₂ uptake obtained with ADP in these various media are not altered when the oxidation of the formaldehyde, derived from the N-methyl group, is suppressed by the addition of either semicarbazide or rotenone. With dimethylglycine, another component of the “1-C cycle”, the initial rate of oxidation in glycylglycine or imidazole is enhanced by ADP rather than being decreased. These results together with appropriate coenzyme analyses suggest that reactions of “one carbon compounds” can provide sensitive markers for assessing compartition of cofactors such as the pyridine nucleotides, flavins, and folates in the mitochondrial matrix.     

Ion binding by repeat hexapeptide of elastin

Synthetic elastin hexapeptides, N-formyl-Val-Ala-Pro-Gly-Val-Gly-O-methyl, n-formyl-Ala-Pro-Gly-Val-Gly-Val-O-methyl, and N-formyl-Pro-Gly-Val-Gly-Val-Ala-O-methyl were studied in order to determine their affinity and selectivity for calcium, magnesium, strontium, sodium, and potassium ions. Of the three permutations N-forrnyl-Val-Ala-Pro-Gly-Val-Gly-O-methyl had the highest calcium affinity, k_a ～- 0.5 × 10⁶. All three hexamers displayed a selectivity toward calcium, although strontium did bind well also. The peptides could not compete effectively with water for magnesium ions and had little affinity for sodium and potassium ions. The significance of these results lies in the possible role of this hexameric sequence in calcium binding in natural elastin.     

Biosynthesis of cyclosporin A

Short term feeding of the mould Tolypocladium inflatum with ¹⁴C-labelled amino acids revealed a selective incorporation of l-leucine, l-valine, glycine and d, l-alanine into cyclosporins A and C. Feeding of l-[Me-¹⁴C]methionine exclusively labelled the N-methyl moieties of the cyclosporins. The distribution of radioactivity from this substrate was directly proportional to the number of the relevant N-methyl amino acids in cyclosporin A, indicating a simultaneous methylation of these residues.     

Oxidation of methyl groups by grass grubs and vertebrate liver enzymes

1. Oxidation rates of p-nitrotoluene, p-acetamidotoluene and p-toluidine by intact grass grubs and vertebrate liver preparations were measured. 2. The effect of p-substitution in increasing the rate of conversion of the methyl into a carboxy group was in the order acetamido> nitro[unk] amino in mice and grass grubs. 3. Rates of oxidation of the N-methyl group in some alkylaryl N-methylcarbamates was measured and the effect of ring substituents in increasing the rate was in the order hydrogen> o-methyl or o-isopropyl> p-methyl or p-isopropyl> m-methyl or m-isopropyl. 4. Rates of oxidation of the N-methyl groups were similar to those of the p-substituted toluenes.     

Alcaloïdes d'Alstonia quaternata

Eleven alkaloids have been isolated from Alstonia quaternata. Three of them, namely 11-methoxy-epi-3α-yohimbine, 10,11-dimethoxy-picrinine designated quaternine, and 19,20-epoxy-N_(a)-methyl, desacetyl, desformo, 2βH-dihydroakuammiline designated quaternoxine, are new alkaloids.     

A rat kidney neutral peptidase that degrades B chain of insulin, glucagon, and ACTH: purification by affinity chromatography and some properties.

A metallo-endopeptidase that catalyzes at near neutral pH the hydrolysis of certain polypeptides was purified from rat kidney microsomes by a simplified procedure using affinity chromatography on Sepharose 4B coupled with insulin B chain. The purified enzyme showed a single component by chromatography on diethylaminoethyl cellulose and by gel filtration on a Sephadex G-200 column. The native enzyme has a molecular weight of approximately 213,000. Studies on its substrate specificity showed that the purified enzyme rapidly degrades insulin B chain, glucagon, adrenocorticotropin, and, at a significantly lower rate, insulin A chain. The enzyme has a very weak or no activity toward ribonuclease and vasopressin. In contrast, the enzyme does not degrade denatured hemoglobin, bovine serum albumin, insulin (nano- or micromolar), oxytocin, furylacryloylglycyl-leucine amide (FAGLA), synthetic substrates of cathepsin C (β-napthalamides of glycine-l-arginine and l-histidine-l-serine), or synthetic substrates of aminopeptidases (l-arginine- or l-glutamic acid-β-napthylamide). The enzyme degrades reduced or oxidized B chain at about the same rate, but S-sulfonated B chain is degraded at a markedly lower rate. The effect of several potential activators and inhibitors on the enzyme's activity was investigated. Activity of the enzyme is markedly inhibited by chelating agents (EDTA and o-phenanthroline) and, modestly, by high concentrations of citrate and histidine. Activity of the enzyme is also markedly inhibited by simple thiol compounds (dithiothreitol, glutathione, and mercaptoethanol), but not by sulfhydryl reagents (N-ethylmaleimide or iodoacetate). The inactive apoenzyme, prepared by treatment of the enzyme with EDTA followed by dialysis, was reactivated by Zn²⁺ > Ca²⁺, minimally by Cu²⁺, but not by Hg²⁺. Some anions (phosphate, borate, and bicarbonate) were strongly inhibitory, but chloride had no effect. The following agents were found to have no effect: soybean and lima bean trypsin inhibitors, N^?-tosyl-l-phenylalanine chloromethyl ketone (TPCK), N^α,?-tosyl-l-lysine chloromethyl ketone (TLCK), aprotinin (Trasylol), phenylmethylsulfonyl fluoride (a serine protease inhibitor), 1-methyl histidine, 3-methyl histidine, histamine, imidazole, and heparin.     

Synthetic peptide K+ carrier with Ca2+ inhibition

Based on a proposed solution conformation of the Ca²⁺ ion complex of the repeat hexapeptide of elastin, l-Val-l-Ala-l-Pro-Gly-l-Val-Gly, it is possible to modify the molecule making it more lipophilic for lipid bilayer permeation while retaining its complexation features. Therefore the two peptides, For-MeVal-Ala-Pro-Sar-Pro-Sar-OMe and For-MeVal-Ala-Pro-Sar-Pro-Sar-OH, were synthesized and evaluated for lipid bilayer activity and cation binding (For, N-formyl; Me, N-methyl; Sar, N-methyl glycine). Both peptides bound Ca²⁺ preferentially but did not exhibit the properties of a Ca²⁺ carrier. They were however active as K⁺ carriers although K⁺ ion titration curves showed a much lower affinity for K⁺ than for Ca²⁺. The addition of Ca²⁺ or Mg²⁺ to the bilayer system inhibited the peptide K⁺ carrier activity. Three possible explanations of this interesting Ca²⁺ inhibition of carrier activity are irreversible complexation of Ca²⁺, mixed ligand complex formation involving Ca²⁺, lipid and peptide, and impermeability of the lipid layer when peptide is complexed with a divalent cation.     

Phenazine ethosulfate as a preferred electron acceptor to phenazine methosulfate in dye-linked enzyme assays.

Rapid nonenzymatic reduction of 2,6-dichlorophenolindophenol by N-methyl phenazonium methosulfate has been observed in aqueous solution and has been found to increase with increasing pH and ionic strength. The instability of N-methyl phenazonium methosulfate in aqueous solution has been explored in terms of change of absorption spectrum and formation of free radicals as evidenced by EPR spectroscopy. N-Ethyl phenazonium ethosulfate has been found to be much more stable than the methyl analog and did not reduce dichlorophenolindophenol nonenzymatically. The implications of these findings with respect to use of these dyes as artificial electron acceptors are discussed and the recommendation made that, wherever possible, use of N-methyl phenazonium methosulfate be discontinued in favor of use of the N-ethyl analog.     

Structural studies on the cytochrome c oxidase proton pump using a spin-label probe

We report studies in which we have used N-(2,2,6,6-tetramethylpiperidyl-1-oxyl)-N′-cyclohexylcarbodiimide, a spinlabel analogue of N,N′-dicyclohexylcarbodiimide, to investigate the structural aspects of the cytochrome c oxidase proton pump. We establish that the spin label binds to the reconstituted enzyme at the same site as does N,N′-dicyclohexylcarbodiimide, i.e., within subunit III. ESR studies of the bound spin label indicate that its binding site is situated in an apolar region of the enzyme, though close to its surface. The binding of the spin label to the free oxidase is different from that with the reconsituted enzyme, leading to spin-spin exchange between the bound probe molecules. From this and the fact that N,N′-dicyclohexylcarbodiimide binds to subunits III and IV in the free oxidase, we conclude that these two subunits are at the most 20 Å apart.     

Protein immobilization on poly-(N-substituted) acrylamides

A series of water-soluble polymers containing side chains derived from N-acryloyl-β-alanine, N-ethylacrylamide and N-[3-(N′,N′,N′-trimethylammonio)propyl] acylamide chloride has been prepared and characterized. A related series of insoluble gels was also prepared. Protein may be attached to these materials by means of amide bond formation between carboxyl groups on the polymers and amino groups of the protein; the preparation and characterization of conjugates formed with α-chymotrypsin are described. Polymers bearing negatively or positively charged side chains are attached to this enzyme at only a single amino acid and the integrity of the active site is largely preserved in these systems. The corresponding gels are not able to bind as much enzyme as are the soluble polymers and bound enzyme is less active in these cases.