The effect of vitamin B12 inhibition in vivo: Impaired folate polyglutamate biosynthesis indicating that 5-methyltetrahydropteroylglutamate is not its usual substrate

Using 5-[¹⁴C] methyltetrahydropteroylglutamate and [³H] pteroylglutamate it was found that vitamin B₁₂ dependent methyltransferase can be inhibited $\text{in vivo}$ in mice by nitrous oxide. Examination of the liver [³H] folates showed as expected that both controls and nitrous oxide treated mice had synthesised the reduced 5-methyl form. However, the controls had converted [³H] almost exclusively into a polyglutamate with virtually no monoglutamate present, while nitrous oxide treated mice had failed to add glutamates to over half of the incorporated folate. Thus $\text{in vivo}$ demethylation may be necessary prior to polyglutamate biosynthesis and act as a method of controlling folate accumulation, or be used to create a concentration gradient with the circulating folate.     

Proton NMR study of iron(II)-bleomycin: Assignment of resonances by saturation transfer experiments

The assignment of the paramagnetically shifted resonances of the Fe(II)-bleomycin complex in D₂O has been accomplished using the transfer of saturation method. A number of additional resonances arising from labile N$\text{H}$ protons which are shifted by the metal ion are observed in the ¹H spectrum of the complex in H₂O. The temperature dependence of the chemical shifts is consistent with the formation of an isolated 1:1 complex, but does not obey either the Curie Law or the Curie-Weiss Law. The magnitude of the shifts suggests that the valeric acid hydroxyl (or carbonyl) group, the α-amino group, the imidazole N^π, the carbamoyl oxygen, the pyrimidine N₁ and/or the secondary amino group may be coordinated to the iron(II).     

Proton and carbon magnetic resonance studies of the synthetic polypentapeptide of elastin.

Proton and ¹³C magnetic resonance studies are reported on the synthetic polypentapeptide of elastin, HCO-(Val₍₁₎-Pro₍₂₎-Gly₍₃₎-Val₍₄₎-Gly₍₅₎)_n-Val-OMe, where n ∼- 18. Temperature and solvent dependence of peptide N$\text{H}$ chemical shift and solvent dependence of peptide carbonyl chemical shift were used to delineate these moieties preliminary to identification of secondary structure.Based on these studies it is proposed, for the organic solvents of dimethyl sulfoxide, methanol, and low-temperature trifluoroethanol, that dynamic hydrogen bonds form in order of decreasing frequency of occurrence between the Val₍₁₎$\text{C}$O and the Val₍₄₎ N$\text{H}$ (a β-turn), between the Gly₍₃₎ N$\text{H}$ and the Gly₍₅₎$\text{C}$O (an 11-atom, hydrogen-bonded ring), and a more limited interaction between the Gly₍₃₎$\text{C}$O and the Gly₍₅₎ N$\text{H}$ (a γ-turn).Arguments are presented that relate the conformational features proposed above to the coacervate, which is a filamentous state.     

Respiration-driven proton translocation with nitrite and nitrous oxide in Paracoccus denitrificans

(1) $\text{H}$⁺/electron acceptor ratios have been determined with the oxidant pulse method for cells of denitrifying Paracoccus denitrificans oxidizing endogenous substrates during reduction of O₂, NO^?₂ or N₂O. Under optimal H⁺-translocation conditions, the ratios $\text{H}{}^{\mathrm{+}}\text{O}$, $\text{H}{}^{\mathrm{+}}\text{N}{}_2\text{O}$, $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂ and $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂O were 6.0–6.3, 4.02, 5.79 and 3.37, respectively. (2) With ascorbate/N,N,N′,N′-tetramethyl-p-phenylenediamine as exogenous substrate, addition of NO^?₂ or N₂O to an anaerobic cell suspension resulted in rapid alkalinization of the outer bulk medium. $\text{H}{}^{\mathrm{+}}\text{N}{}_2\text{O}$, $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂ and $\text{H}{}^{\mathrm{+}}\text{NO}{}^{\mathrm{?}}{}_2$ for reduction to N₂O were ?0.84, ?2.33 and ?1.90, respectively. (3) The $\text{H}{}^{\mathrm{+}}\text{oxidant}$ ratios, mentioned in item 2, were not altered in the presence of $\text{valinomycin}\text{K}{}^{\mathrm{+}}$ and the triphenylmethylphosphonium cation. (4) A simplified scheme of electron transport to O₂, NO^?₂ and N₂O is presented which shows a periplasmic orientation of the nitrite reductase as well as the nitrous oxide reductase. Electrons destined for NO^?₂, N₂O or O₂ pass two H⁺-translocating sites. The $\text{H}{}^{\mathrm{+}}\text{electron}$ acceptor ratios predicted by this scheme are in good agreement with the experimental values.     

Ruthenium(II) complexes derived from 2-(methylamino)pyridine

2-(Methylamino)pyridine reacts with RuCl₂(CO)₃ to give a carbamoyl complex, [$\text{Ru(C(O)N(CH}{}_3\text{)(C}{}_5\text{H}{}_4\text{N}$)Cl(CO)₂], which yields with pyridine (py) and acetylacetone (Hacac), respectively, [$\text{Ru(C(O)N(CH}{}_3\text{)C}{}_5\text{H}{}_4\text{N}$)Cl(CO)₂(py)] and [$\text{Ru(C(O)N(CH}{}_3\text{)C}{}_5\text{H}{}_4\text{N}$)(CO)₂(acac)]. These complexes are characterized spectroscopically. The amino group of the ligand is carbonylated and the resulted carbamoyl ligand is chelating through a pyridine ring-N and a carbamoyl-C atom. 2-Aminopyridine and 2-aminopyrimidine react similarly with RuCl₂(CO)₃ to give the corresponding carbamoyl complexes.     

Dialkyl- and alkylenedithiophosphates of gallium(III)

Gallium(III) tris-dialkyldithiophosphates, Ga[S₂P(OR)₂]₃ (R = C₂H₅, n-C₃H₇, i-C₃H₇, n-C₄H₉ and i-C₄H₉) and gallium(III) tris-alkylenedithiophosphates, Ga(S₂$\text{POGO}$)₃ [G = -CH₂C(C₂H₅)₂CH₂-, -C(CH₃)₂C(CH₃)₂ and -C(CH₃)₂CH₂CH(CH₃)] have been synthesized for the first time by the reactions of gallium(III) chloride with the alkali metal salt of the corresponding ligand in anhydrous benzene in 1:3 molar ratio respectively.These compounds are crystalline solids or viscous liquids and are soluble in common organic solvents, in which they show monomeric behaviour. Based on elemental analyses, molecular weight determinations, IR and NMR (¹H and ³¹P) spectral data, chelate octahedral structures have been proposed for these derivatives.     

Mass spectrometry and 13C nuclear magnetic resonance spectroscopy of compounds modeling the glycopeptide linkage of glycoproteins

The properties of several compounds useful as models for three-dimensional conformational studies and the investigation of the chemical degradation of glycopeptide linkages both of the N- and O-glycosidic type are described. Using the method of differential chemical shift in H₂O and D₂O as solvents, the carbon NMR spectrum of N-acetylglucosaminylasparagine, 1-N-acetyl-β-d-glucopyranosylamine, and 1-N-acetyl-2-acetamido-β-d-glucopyranosylamine has been assigned. Electron impact mass spectra of the peracetylated derivatives of the latter two compounds show a peak apparently unique to glycopyranosylamides at $\text{m}\text{e}\text{= 269}$, no analog of which is observed in the mass spectra of other peracetylated sugars. As models of the α-O-glycosidic linkage, fully assigned carbon NMR spectra of α-methyl-N-acetylgalactosamine (GalNAc), α-methyl-3-O-methyl GalNAc,and -GlcNAc as well as the disaccharide Glc-β-1 → 3 GalNAc are reported. Because certain anomalies in the chemical shifts and ¹J_CH observed in the disaccharide and in O-glycosylated glycoproteins are not observed in the simple model compounds, they may result from conformational interactions in the glycopeptides.     

Kinetics of the reduction of C-9 substituted acridinium cations by 1,4-dihydronicotinamides

Second-order rate constants (k₂) are reported for the reduction of 9-R-10-methylacridinium cations (5:R = H, CH₃, CH₃CH₂, C₆H₅CH₂, (CH₃)₂CH, C₆H₅, 4-(CH₃)₂NC₆H₄) by 1-benzyl-1,4-dihydronicotinamide (2:R = C₆H₅CH₂) in 20% CH₃CN-80% H₂O at 25°C. All 5:R ≠ H are reduced in the range 20- to 140-fold more slowly than 5:R = H. However, there is no simple relationship between k₂ and the nature of R, nor between k₂ and the second-order rate constant for hydroxide ion attack at C-9 of these cations in pseudobase formation. Rates of reduction of 5 by 1-benzyl-4,4-dideuterio-1,4-dihydronicotinamide allow the calculation of the following kinetic isotope effects in this solvent medium: 5:R, $\text{k}{}^{\mathrm{<mtext>H</mtext>}}\text{k}{}^{\mathrm{<mtext>D</mtext>}}\text{:}\text{H}$, 1.56; C₆H₅CH₂, 2.7; C₆H₅, 5.4. Substituent effects upon k₂ were evaluated for the reduction of 5 by 1-(X-benzyl)-1,4-dihydronicotinamides, and lead to the following Hammett ? parameters: 5:R, ?: H, ?0.68; C₆H₅CH₂, ?0.92; C₆H₅, ?0.96. The latter two values require essentially complete unit positive charge generation on the nicotinamide moiety in the rate-determining transition state. It is shown that these Hammett ? values and the above isotope effects can only be rationalized by a two-step e^? + H^? mechanism for hydride transfer from 2 to 5 in this solvent system. This result contrasts with our earlier conclusion of direct, one-step hydride transfer in the reduction of isoquinolinium cations by 2, but is consistent with our observation that acridinium cations are reduced 37500-fold faster by 2 than predicted on the basis of the relative rates of nucleophilic attack (hydroxide ion) on acridinium and isoquinolinium cations. It is suggested that the availability of both Hammett ? values and primary kinetic isotope effects will generally allow the establishment of the mechanism of hydride transfer in these systems. Application of these ideas to literature data suggests that 5:R = H is reduced by direct hydride transfer in acetonitrile solution, in contrast to the above result in predominantly aqueous solution. The ready formation of acridanyl radicals by electron transfer to acridinium cations is demonstrated by the formation of Wurster's Blue radical cation upon mixing solutions of acridinium cations with N,N,N′,N′-tetramethyl-p-phenylenediamine.     

Deuterium magnetic resonance study of the interaction between chondroitin sulfate and human plasma low density lipoproteins

[²H]Chondroitin sulfate was prepared by partial $\text{N}$-deacetylation of chondroitin sulfate ($\text{via}$ hydrazinolysis) followed by treatment with [²H₆]acetic anhydride. ²H NMR spectra of [²H]chondroitin sulfate in the presence of human plasma low density lipoprotein provide evidence for a soluble complex stoichiometry of 3 (and possibly 2) lipoproteins per polysaccharide molecule, and allow a rough estimation of the dissociation constant K_d.     

Extracts of interferon-treated cells can inhibit reticulocyte lysate protein synthesis.

Rumen bacteria retained methanogenic activity when stored at ?60° under H₂. This activity, which resides in $\text{Methanobacterium ruminantium}$ and $\text{Methanobacterium mobilis}$, is not lost when the cells are broken, as has been suggested. Unlike in $\text{Methanosarcina barkerii}$ and $\text{Methanobacterium M.o.H.}$, in rumen bacteria methanogenic enzymes are not soluble but readily precipitated at 15,000 g. Methane was synthesized from tetrahydrofolate derivatives but at slower rates than from CO₂. From the data, it was not possible to determine if methyl- and methylene tetrahydrofolate were oxidized to CO₂ prior to reduction to CH₄. In room light, CH₃-B₁₂ was reduced to CH₄ non-enzymatically in the presence of protein. When the reactions were carried out in the dark, very little CH₄ was formed from CH₃-B₁₂ by rumen bacterial enzymes. The cell-free particulate fraction did not require added ATP for methanogenesis but showed an absolute requirement for H₂.     

Effect of pH on H-2H exchange,H2 production and H2 uptake,catalysed by the membrane-bound hydrogenase of Paracoccus denitrificans

(1) The kinetics of isotope exchange catalysed by the membrane-bound hydrogenase of Paracoccus denitrificans have been studied by measuring H²H, H₂ or ²H₂ produced when the enzyme catalyses the exchange between ²H₂ and H₂O or H₂ and ²H₂O. (2) In the ²H₂-H₂O system the measured rate of H₂ production was always higher than that of H²H. The $\text{H}{}_2\text{H}{}^2\text{H}$ ratio remained constant (about 1.70) in the protein concentration range 0.08–1.32 mg. The very rapid formation of H₂ with respect to H²H is consistent with the hypothesis of a heterolytic cleavage of ²H₂ into a deuteron and an enzyme hydride that can exchange with the solvent. (3) In the H₂-²H₂O system, the exchange rate was much lower than in the ²H₂-H₂O system, indicating a marked isotopic effect of ²H₂O. (4) The H-²H exchange activity, determined from the initial velocity of H²H formation, is optimal at pH 4.5. A second maximum of activity is observed at pH 8.3. The pH value of 4.5 is also the pH optimum for H₂ production while at pH 8.3–8.5 there is a maximum of H₂ oxidation activity. (5) In ordinary H₂O the K_m for hydrogen uptake estimated either from H₂ consumption or from benzyl viologen reduction was 0.06–0.07 μM for both H₂ and ²H₂ indicating a strong affinity of the enzyme for hydrogen at pH 8.3–8.5. Shifting from H₂O to ²H₂O does not affect the K_m of the enzyme for H₂ but lowers the V_max value about 10-fold. The K_m for benzyl viologen and methyl viologen was 0.08 and 2 mM, respectively.     

Specific platinum chelation by the guanines of the deoxyhexanucleotide d(T-G-G-C-C-A) upon reaction with cis-[Pt(NH3)2(H2O)2](NO3)2

The stoichiometric reaction between d-TpGpGpCpCpA (d(T-G-G-C-C-A)) and $\text{cis}$-[Pt(NH₃)₂(H₂O)₂](NO₃)₂ (8.4 × 10^?6 to 1.3 × 10^?4M in water at pH 5.5–6) gives a single complex. High pressure gel permeation chromatography and pH-dependent ¹H NMR analyses of the nonexchangeable base protons, show that it is a platinum chelate with the $\text{cis}$-Pt^II(NH₃)₂ moiety bound to the two N7 atoms of the adjacent guanines. A 3 × 10^?3M reaction gives the same platinum chelate, via the formation of intermediate complexes, together with unsoluble adducts.     

High-performance liquid chromatographic determination of the distribution of naturally occurring folic acid derivatives in rat liver

Procedures which allow extraction and quantitation of labile, reduced folic acid derivatives in rat liver have been developed. These procedures entail extraction of hepatic folates at 100°C in 2% ($\text{w}\text{v}$) sodium ascorbate, 0.2 m 2-mercaptoethanol, pH 7.85. The extract was treated with conjugase to hydrolyze folate polyglutamates and reverse-phase, ion-pair high-performance liquid chromatography was used to separate the resulting monoglutamates which were measured by microbiological assay using Lactobacillus casei. Experiments with HPLC-purified standard derivatives, so treated, showed excellent stability of tetrahydropteroylglutamic acid (H₄PteGlu), 10-formyl-H₄PteGlu, 5-formyl-H₄PteGlu, 5-methyl-H₄PteGlu, and pteroylglutamic acid (PteGlu). Under these conditions, approximately 56% of H₂PteGlu was recovered unchanged while about 27% was converted to PteGlu; 5,10-methylene-H₄PteGlu was quantitatively recovered as H₄PteGlu. These procedures were applied to the task of measuring the distribution of naturally occurring folate cofactors in rat liver. These results indicated that rat liver folates have the following compositions: 5-methyl-H₄PteGlu, 37.2%; H₄PteGlu, 32.7%; 10-formyl-H₄PteGlu, 22.6%; and 5-formyl-H₄PteGlu, 7.7%. Experiments with [³H]PteGlu injection showed that all hepatic folates had the same specific radioactivity as determined by radioassay and L. casei assay, indicating that L. casei exhibited the same growth response to all the folates detected in rat liver.     

Effect of N-alkyl-N,N,N-trimethylammonium ions on phosphatidylcholine model membrane structure as studied by 31P-NMR

The interaction of |C_nH_2n+1N⁺(CH₃)₃| · I^? (n = 3, 6, 9, 12, 14, 16 or 18) with egg-yolk phosphatidylcholine-water dispersions has been studied by ³¹P-NMR spectroscopy. It is shown that the effective anisotropy of ³¹P chemical shift (?Δσ_eff) of the lamellar phospholipid liquid-crystalline phase L_α increases with increasing concentration and alkyl chain length of the drug. Addition of $\text{|}\text{C}{}_6\text{H}{}_{13}\text{N}{}^{\mathrm{+}}\text{(CH}{}_3\text{)}{}_3\text{| ·I}{}^{\mathrm{?}}$ or |C₉H₁₉N⁺(CH₃)₃|·I^? to the phospholipid-water dispersion at a molar ratio ammonium salt:phospholipid > 0.8 induces in the dispersion a structure with an effective isotropic phospholipid motion. This structure is unstable and slowly transforms into the hexagonal phase. These effects have not been observed in phospholipid-water dispersions mixed with the ammonium derivatives with the longer alkyl chains n  12, 14, 16 or 18. It is proposed that these results might explain the effects of the investigated drugs on the nerve, muscle and bacterial cells.     

Conversion of thyroxine to 3,3',5'-triiodothyronine (reverse-T3) by a soluble enzyme system of rat liver

Infrared spectra of N₂O in a variety of solvents and in the brain of a dog under typical conditions of halothane-N₂O anesthesia have been determined. The appearance or disappearance of N₂O in the brain was readily followed as N₂O was administered or withdrawn. The sites in brain were of two major types; one, with ν₃ = 2229.8 ± 0.4 cm^?1 and $\text{Δν}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 13.0 ± 0.6}\text{cm}{}^{\mathrm{?1}}$, is rather like the polar site in water and the other, with ν₃ = 2216.8 ± 0.8 cm^?1 and $\text{Δν}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 9.6 ± 1.0}\text{cm}{}^{\mathrm{?1}}$, is non-polar and is probably associated with membrane lipid. The significant variations in the antisymmetric stretch (ν₃) of N₂O as the polarity and other properties of the medium (solvent) vary make possible the characterization of $\text{in tissue}$ sites occupied by this anesthetic.     

High-pressure liquid chromatography separation and determination of rat liver folates

The endogenous levels of the various folate compounds in rat liver were determined using high-pressure liquid chromatography for the rapid separation of folate monoglutamate forms with specific quantitation of the folates by microbiological analysis of eluted fractions. The eight folate derivatives that were assayable were tetrahydrofolic acid (H₄PteGlu), 5-methyl-H₄PteGlu, 10-formyl-H₄PteGlu, 5-formyl-H₄PteGlu, 5,10-methenyl-H₄PteGlu, 5,10-methylene-H₄PteGlu, H₂PteGlu, and PteGlu. New techniques for the preparation of tissues were developed in order to reduce the degradation of the folates. Tissue folates were converted to the monoglutamate form by a partially purified hog kidney polyglutamate hydrolase preparation and incubations were carried out at pH 6.0. This minimized folate degradation but still allowed for maximal polyglutamate hydrolase activity. Rapid removal of tissues was compared with freeze-clamping techniques. The major folates in rat liver were H₄PteGlu and 5-methyl-H₄PteGlu, comprising 42 and 39%, respectively, of the total liver folate pool of 27.30 nmol/g liver (about 13 μg/g liver). In addition, 10-formyl-H₄PteGlu and 5-formyl-H₄PteGlu each comprised 10% of the total folate pool. No endogenous PteGlu, H₂PteGlu, or 5,10-methylene-H₄PteGlu was detected in rat liver samples under our conditions. Distribution of ¹⁴C derived from a previous [¹⁴C]folic acid injection paralleled the distribution of folate as determined microbiologically after high-pressure liquid chromatography separation. The importance of these methods for the direct determination and estimation of flux of H₄PteGlu, 5-methyl-H₄PteGlu, and 10-formyl-H₄PteGlu in studies dealing with the folate system was emphasized.     

Folylpolyglutamate synthetase activities of Neurospora

The folylpolyglutamate synthetase (FPGS) activities of Neurospora crassa, wild type (FGSC 853) and two polyglutamate-deficient mutants, met-6,35809 (FGSC 1330) and mac, 65108 (FGSC 3609), were examined after growth in defined media. Extracts of the wild type produced H₄PteGlu₆ (60 %), H₄PteGlu₃ (35 %) and H₄PteGlu₂ (15 %). Met-6 extracts formed H₄PteGlu₂ but lacked the ability to utilize H₄PteGlu₄ or H₄PteGlu₅. The mac mutant failed to catalyse glutamate addition to H₄PteGlu but H₄PteGlu₂ was an effective substrate for tri- and hexaglutamate synthesis. These polyglutamates were also formed by reaction systems containing mixtures of met-6 and mac protein or heterokaryon protein derived from mycelial fusions of met-6 and mac. Extract fractionations and heat treatments provided evidence for more than one FPGS activity in the wild type. A mitochondrial FPGS catalysed the H₄PteGlu₂ → H₄PteGlu₃ reaction but a cytosolic fraction synthesized di-, tri- and hexaglutamates when incubated with H₄PteGlu and glutamate. The latter system contained a temperature-sensitive diglutamate-forming activity and a relatively stable H₄PteGlu₂ → H₄PteGlu₆ activity. Polyglutamate synthesis in N. crassa appears to involve more than one step, H₄PteGlu → H₄PteGlu₂ followed by H₄PteGlu₂ → H₄PteGlu₆, in addition to the mitochondrial activity. These partial activities are lacking in mac and met-6 respectively. Consequently, these mutants are unable to form the folylhexaglutamates that predominate the folate pool of the wild type.     

Acylated des-(Ala1-Gly2)-somatostatin analogs: prolonged inhibition of growth hormone secretion

The following eight analogs of somatostatin were synthesized by solid phase: des-[Ala¹-Gly²]-somatostatin (I); des-[Ala¹-Gly²]-H₂somatostatin (II); $\text{N}$-acetyl-Cys³-somatostatin (III); $\text{N}$-acetyl-Cys³-H₂somatostatin (IV); $\text{N}$-pyvalyl-Cys³-H₂somatostatin (V); $\text{N}$-acrylyl-Cys³-H₂somatostatin (VI); $\text{N}$-benzoyl-Cys³-H₂somatostatin (VII); $\text{N}$-hexanoyl-Cys³-H₂somatostatin (VIII). Deletion of the N-terminal dipeptide Ala¹-Gly² is compatible with high biological activity. A single s.c. injection of these analogs as a microsuspension in saline inhibits for 24–72 hours (depending on the compound) the secretion of growth hormone normally stimulated in rats by pentobarbital.