Biosynthesis of securinine,the main alkaloid of Securinega suffruticosa

Biosynthesis of securinine was studied by incorporation experiments in Securinega suffruticosa. Among presumed precursors tested, lysine, cadaverine, and tyrosine showed the highest incorporation into securinine. Degradation experiments revealed that cadaverine-[1,5-¹⁴C] labelled specifically the piperidine ring of securinine and the radioactivity from dl-tyrosine-[2-¹⁴C] was introduced into the C-11 lactone carbonyl. Experiments with L-tyrosine-[U-¹⁴C] and L-tyrosine-[3′,5′-³H; U-¹⁴C] prove that the remaining C₆Sz.sbnd;C₂ moiety is derived from the aromatic ring and the C-2 and C-3 or tyrosine.     

The stereochemistry of biosynthesis of decaprenoxanthin in a cell-free system

Intact cells of Flavobacterium dehydrogenans grown on glucose or acetate did not incorporate mevalonic acid-[¹⁴C]. After treatment with lysozyme the protoplasts were lysed by sonication in a dilute medium containing mevalonic acid-[¹⁴C] and the cell-free system produced incorporated label into uncyclized C₄₀, monocyclic C₄₅ and bicyclic C₅₀ carotenoids of which decaprenoxanthin was the most abundant.With mevalonate-[2-¹⁴C,4R-4-³H₁] the ¹⁴C:³H ratios of the carotenoids showed that the hydrogen atoms at C-2 and C-6 of the ring and that at C-3 of the 1-hydroxy, 2-methyl but-2-ene-4-yl residues of decaprenoxanthin were derived from the 4-pro-R hydrogen atom of mevalonic acid.Mevalonate-[2-¹⁴C,2R-2-³H₁] and mevalonate-[2-¹⁴C,2S-2-³H₁] gave ratios which showed that the C-4 hydrogen atoms of decaprenoxanthin were derived from the 2-pro-S hydrogen atom of mevalonic acid.     

Comparisons of affinity constants of PGEs-receptor interaction with concentrations of PGEs needed for half maximal stimulation of adenylate cyclase

The specific binding of ³H-prostaglandin E₁ (³H-PGE₁) to bovine corpus luteum cell membranes was not affected by cholesterol or various progestins at concentrations of up to 9.0 × 10⁻⁶M. At concentrations above 2.5 × 10⁻⁶M; estrone, 17β-estradiol (but not 17α-estradiol or 17β-estradiol glucuronide), estriol, equilin, D-equilenin, 17-ethynyl estradiol, diethylstilbestrol, cortisol, corticosterone, deoxycorticosterone and aldosterone inhibited specific binding of ³H-PGE₁. On the other hand, testosterone and dihydrotestosterone (DHT) (but not androstenedione) significantly enhanced ³H-PGE₁ binding. These findings permitted the following correlations between steroid structure and modulation of ³H-PGE₁ binding: steroids with a free phenolic ring and a 17β-hydroxyl or 17-keto group or C-21 steroids with a C-20 ketone and a C-21 hydroxy group decrease, whereas C-19 steroids with a C-17 hydroxy group enhance specific binding of ³H-PGE₁. PGE receptors are heterogeneous with respect to affinity for ³H-PGE₁. The steroids that decreased ³H-PGE₁ binding caused a lowering to a complete loss of low affinity PGE receptors. Steroids that increased ³H-PGE₁ binding caused appearance of new low affinity PGE receptors. Association rate constants for ³H-PGE₁ binding were decreased by 17β-estradiol (61%) and increased by DHT (59%).     

Studies in phytosterol biosynthesis. Mechanism of biosynthesis of cycloartenol

1. The mechanism of cycloartenol biosynthesis in leaves of Solanum tuberosum was investigated with the use of [2-¹⁴C,(4R)-4-³H₁]mevalonic acid. 2. The ³H/¹⁴C atomic ratio in cycloartenol was 6:6, the same as that in squalene; this eliminates lanosterol as a possible biosynthetic precursor of cycloartenol, and indicates that a hydrogen migration from C-9 to C-8 occurs. 3. Chemical isomerization of the cycloartenol to lanosterol (³H/¹⁴C ratio 5:6) and parkeol (³H/¹⁴C ratio 6:6) confirms the hydrogen migration from C-9 to C-8. 4. Possible mechanisms for the biosynthesis of cycloartenol and parkeol are discussed. 5. The ³H/¹⁴C ratio for 24-methylenecycloartanol was 6:6, demonstrating that the hydrogen atom at C-24 is retained during alkylation of the cycloartenol side chain.     

Mechanism of C4 photosynthesis in Chloris gayana: pool sizes and kinetics of 14CO2 incorporation into 4-carbon and 3-carbon intermediates.

In one group of C₄ species, including Chloris gayana, C₄ acids are decarboxylated via phosphoenolpyruvate carboxykinase to give phosphoenolpyruvate as the initial C₃ product. This paper presents an analysis of the kinetics of labeling of various photosynthetic intermediates in Chloris gayana leaves exposed to ¹⁴CO₂, and the pool sizes of these intermediates, primarily to provide information about the subsequent metabolism of phosphoenolpyruvate. Saturation labeling of the C-4 of aspartate and malate, and the C-1 of 3-phosphoglycerate, indicated photosynthetically active pools of 0.45, 0.22, and 0.95 μol/mg chlorophyll, respectively. For aspartate and 3-phosphoglycerate, the total leaf pools and the photosynthetic pools were of similar size, but the total pool of malate was about 100 times larger than the photosynthetically active pool. From the relative rates of labeling of phosphoenolpyruvate, pyruvate, alanine, and C-1, C-2 plus C-3 of aspartate, during steady-state ¹⁴CO₂ assimilation, relative pool sizes were calculated to be about 10:11:78:100, respectively. Pulse/chase labeling of leaves provided estimates of relative photosynthetic pool sizes in the ratio of about 6:15:90:100, respectively, where aspartate is arbitrarily assigned a value of 100 in both cases. Notably, labeling of alanine was consistent with its derivation from the C-1, C-2 plus C-3 carbons of aspartate, and the alanine pool was at least eight times larger than the phosphoenolpyruvate pool that showed similar labeling kinetics. Results were consistent with the view that at least most of the phosphoenolpyruvate produced by C₄ acid decarboxylation is metabolized via alanine.     

Formation of 3H2O from [2-3H]- and [4-3H]estradiol by rat uteri in vitro: Possible role of peroxidase

The mitochondrial fraction of diethylstilbestrol-treated rat uteri, known to contain an estrogen-induced peroxidase, was able to catalyze the release of ³H₂O from either [2-³H]- or [4-³H]estradiol. Hydrogen peroxide added to this system increased the yield of ³H₂O but had no effect on mitochondrial preparations from ovariectomized rat uteri having only very low peroxidase activity. The reaction was inhibited by catalase and also occurred with lactoperoxidase in the presence of H₂O₂ but 2-hydroxyestradiol was not detected in any of these experiments. Under similar conditions, tyrosinase catalyzed the formation of the catechol estrogen with loss of ³H from [2-³H]- or [2,4,6,7-³H]- but not [4-³H]- or [6,7-³H]estradiol. It is proposed that the formation of ³H₂O from ³H-labeled estradiol in the estrogen-treated rat uterus may occur by a peroxidative mechanism which does not necessarily result in hydroxylation of the steroid.     

Properties of peroxidases from tobacco cell suspension culture

An enzyme preparation from suspension cultured tobacco cells oxidized IAA only in the presence of added cofactors, Mn²⁺ and 2,4-dichlorophenol, and showed two pH optima for the oxidation at pH 4·5 and 5·5. Effects of various phenolic compounds and metal ions on IAA oxidase activity were examined. The properties of seven peroxidase fractions separated by column chromatography on DEAE-cellulose and CM-Sephadex, were compared. The peroxidases were different in relative activity toward o-dianisidine and guaiacol. All the peroxidases catalysed IAA oxidation in the presence of added cofactors. The pH optima for guaiacol peroxidation were very similar among the seven isozymes, but the optima for IAA oxidation were different. The anionic and neutral fractions showed pH optima near pH 5·5, but the cationic isozymes showed optima near pH 4·5. With guaiacol as hydrogen donor, an anionic peroxidase (A-1) and a cationic peroxidase (C-4) were very different in H₂O₂ concentration requirements for their activity. Peroxidase A-1 was active at a wide range of H₂O₂ concentrations, while peroxidase C-4 showed a more restricted H₂O₂ requirement. Gel filtration and polyacrylamide gel studies indicated that the three cationic peroxidases have the same molecular weight.     

Evidence for a C-2→C-1 intramolecular hydrogen-transfer during the acid-catalyzed isomerization of D-glucose to D-fructose ag]

In mechanistic studies by isotope-exchange tecniques of the conversion of D-fructose and D-glucose into 2-(hydroxyacetyl)furan, it was shown that both sugars are converted in acidified, tritiated water into the furan containing essentially no carbon-bound tritium. As the hydroxymethyl carbon atom of the furan corresponds to C-1 of the hexose, this result suggests that one of the hydrogen atoms in this group, when it is produced from D-glucose, must arise intramolecularly. This hypothesis was verified by synthesizing D-glucose-2-³H and converting it into the furan in acidified water. The 2-(hydroxyacetyl)furan obtained was labeled exclusively on the hydroxymethyl carbon atom, thus showing that intramolecular hydrogen-transfer occurs, during the conversion, from C-2 of D-glucose to the carbon atom corresponding to C-1. The specific activities of the product and reactant permitted calculation of the tritium isotope-effect (k_h/k_t4.4) for the reaction. The precise step for the transfer from C-2 of the aldose to the carbon atom corresponding to C-1 was found to be during the isomerization of D-glucose to D-fructose, as evidenced by the conversion of D-glucose-2-³H into D-fructose-1-³H in acidified water.     

Methyl β-lactoside: 600-MHz H- and 75-MHz C-n.m.r. studies of H- and C-enriched compounds

Using UDP-d-galactose : 2-acetamido-2-deoxy-d-glucose 4-β-d-galactosyltransferase (EC 2.4.1.22), several methyl β-lactosides have been prepared with ²H- and/or ¹³C-enrichment at specific sites to facilitate study by ¹³C (75 MHz) and ¹H (600 MHz) n.m.r. spectroscopy. ¹³C-Chemical shift assignments were verified and the ¹H-spectrum of β-lactoside was fully assigned. Sites of enrichment were selected to permit all of the potential three-bond C-C and C-H couplings through the glycosidic bond to be obtained. Replacement of H-3 of the d-glucose residue of methyl β-lactoside with ²H allowed resolution of C-1–H-4′ coupling in the 600-MHz ¹H-spectrum. Single or multiple ¹³C-enrichment at C-1, C-2, C-3, C-1′, C-3′, and/or C-4′ in the disaccharide allowed observation of intra- and inter-residue couplings. ¹³C-Spin-lattice relaxation-times (T₁) are interpreted in terms of molecular motion in solution. The data suggest that methyl β-lactoside has an extended conformation with little rotation about the glycosidic bond. Inter-residue couplings are best explained by tortion angles of φ ~ 40° and ψ ~ 15°, indicating that the conformations of β-lactoside in solution and in the crystal are similar.     

Steroid hormone modulation of 3H-prostaglandin E1 binding to bovine corpus luteum cell membranes

The specific binding of ³H-prostaglandin E₁ (³H-PGE₁) to bovine corpus luteum cell membranes was not affected by cholesterol or various progestins at concentrations of up to 9.0 × 10⁻⁶M. At concentrations above 2.5 × 10⁻⁶M; estrone, 17β-estradiol (but not 17α-estradiol or 17β-estradiol glucuronide), estriol, equilin, D-equilenin, 17-ethynyl estradiol, diethylstilbestrol, cortisol, corticosterone, deoxycorticosterone and aldosterone inhibited specific binding of ³H-PGE₁. On the other hand, testosterone and dihydrotestosterone (DHT) (but not androstenedione) significantly enhanced ³H-PGE₁ binding. These findings permitted the following correlations between steroid structure and modulation of ³H-PGE₁ binding: steroids with a free phenolic ring and a 17β-hydroxyl or 17-keto group or C-21 steroids with a C-20 ketone and a C-21 hydroxy group decrease, whereas C-19 steroids with a C-17 hydroxy group enhance specific binding of ³H-PGE₁. PGE receptors are heterogeneous with respect to affinity for ³H-PGE₁. The steroids that decreased ³H-PGE₁ binding caused a lowering to a complete loss of low affinity PGE receptors. Steroids that increased ³H-PGE₁ binding caused appearance of new low affinity PGE receptors. Association rate constants for ³H-PGE₁ binding were decreased by 17β-estradiol (61%) and increased by DHT (59%).     

Properties of metal-ion coordinated imidazoles: nmr and C-2 H Exchange in Co(III) Complexes

The ¹H and ¹³C nmr spectra of Co(NH₃)₅ImH³⁺ and the ¹H nmr spectra of αCotrien(ImH)₂³⁺ and βCotrien(ImH)₂³⁺ are reported. The pK_a values determined from the dependence of the chemical shift on pH are 10.0, 9.6, and 10.1, respectively. The range of the chemical shift between the acid and base forms is unusually small in the ¹H nmr, 0.5–0.7 ppm for the C-2 H and about 0.25 ppm for the C-4 H and C-5 H. In the ¹³C nmr, C-2 and C-4 have large shifts to low field and C-5 a small shift to high field on deprotonation. The C-2 proton is not exchanged with solvent ²H under acidic or basic conditions, in marked contrast to the corresponding proton in both imidazole and 1-methylimidazole. These spectroscopic and chemical properties should be useful for the direct identification of metal-ion coordinated histidines in proteins.     

X-Ray crystal and molecular structure of 1,2,3,5-tetra-O-acetyl-4-deoxy-4-C-[(S)-ethylphosphinyl]-α-d-ribofuranose,and conformations of aldopentofuranose analogs having phosphorus in the hemiacetal ring

X-Ray crystallographic analysis was performed on the compound to which had been assigned the structure 1,2,3,5-tetra-O-acetyl-4-deoxy-4-C-[(S)-ethylphosphinyl]-α-d-ribofuranose. The results showed that the compound has the proposed configuration, the five-membered ring is in the ³T₂ conformation with a tendency towards the E₂ form, the substituents on C-1, C-4, and P-5 are linked bisectionally, and the acetoxyl groups on C-2 and C-3 are respectively attached axially and equatorially. Based on the X-ray crystallographic and ¹H-n.m.r.-spectral data, favored conformations of P-in-ring analogs of aldopentofuranose peracetates in solution are discussed.     

Triaryl (Z)-olefins suitable for radiolabeling with carbon-11 or fluorine-18 radionuclides for positron emission tomography imaging of cyclooxygenase-2 expression in pathological disease

A group of (Z)-1,1-diphenyl-2-(4-methylsulfonylphenyl)alk-1-enes were synthesized using methodologies that will allow incorporation of a [¹¹C]OCH₃ substituent at the para-position of the C-1 phenyl ring, a [¹¹C]SO₂CH₃ substituent at the para-position of the C-2 phenyl ring, a [¹⁸F]OCH₂CH₂F substituent at the para-position of the C-1 phenyl ring, and a [¹⁸F]CH₂CH₂F substituent at the C-2 position of the olefinic bond. The [¹¹C] and [¹⁸F] radiotracers are designed as potential radiopharmaceuticals to image cyclooxygenase-2 (COX-2) expression in any organ where COX-2 is upregulated. The COX-1/COX-2 inhibition data acquired suggest that compounds having a [¹¹C]OMe or [¹⁸F]OCH₂CH₂F substituent at the para-position of the C-1 phenyl ring may be more suitable for imaging COX-2 expression in view of their ability to exclusively inhibit the COX-2 isozyme.     

Kristall- und molekülstruktur des mono-O-isopropylidenderivatives der dimeren 1,6-anhydro-β-D-arabino-hexopyranos-3-ulose

Acetonation of dimeric 1,6-anhydro-β-D-arabino-hexopyranos-3-ulose yields, besides a monomeric di-O-isopropylidene compound, the dimer 2, which crystallizes in space group P2₁2₁2₁ with a  1.3680 (9), b  1.0686 (7), and c  1.0319 (7) nm, Z  4. The crystal and molecular structure of 2 have been determined by X-ray analysis with direct methods and was refined to a final R_w of 5.55% for 2468 reflections. Compound 2 has not the same dimeric structure as the parent compound with a central 1,4-dioxane ring, but contains instead a central 1,3-dioxolane ring. The pyranose ring bearing the isopropylidene group adopts an almost ideal sofa conformation, with a nearly planar arrangement of C-1, C-2, C-3, C-4, and C-5. By analogy, it was concluded that the dimeric mono-O-isopropylidene derivative 7 of 1,6-anhydro-β-D-xylo-hexopyranos-3-ulose has the same asymmetric structure. The 360-MHz ¹H-n.m.r. spectra of both compounds are in full agreement with the proposed structures.     

Glucose Respiration in the Intact Chloroplast of Chlamydomonas reinhardtii

Chloroplastic respiration was monitored by measuring ¹⁴CO₂ from ¹⁴C glucose in the darkened Chlamydomonas reinhardtii F-60 chloroplast. The patterns of ¹⁴CO₂ evolution from labeled glucose in the absence and presence of the inhibitors iodoacetamide, glycolate-2-phosphate, and phosphoenolpyruvate were those expected from the oxidative pentose phosphate cycle and glycolysis. The K_m for glucose was 56 micromolar and for MgATP was 200 micromolar. Release of ¹⁴CO₂ was inhibited by phloretin and inorganic phosphate. Comparing the inhibition of CO₂ evolution generated by pH 7.5 with respect to pH 8.2 (optimum) in chloroplasts given C-1, C-2, and C-6 labeled glucose indicated that a suboptimum pH affects the recycling of the pentose phosphate intermediates to a greater extent than CO₂ evolution from C-1 of glucose. Respiratory inhibition by pH 7.5 in the darkened chloroplast was alleviated by NH₄Cl and KCl (stromal alkalating agents), iodoacetamide (an inhibitor of glyceraldehyde 3-phosphate dehydrogenase), or phosphoenolpyruvate (an inhibitor of phosphofructokinase). It is concluded that the site which primarily mediates respiration in the darkened Chlamydomonas chloroplast is the fructose-1,6-bisphosphatase/phosphofructokinase junction. The respiratory pathways described here can account for the total oxidation of a hexose to CO₂ and for interactions between carbohydrate metabolism and the oxyhydrogen reaction in algal cells adapted to a hydrogen metabolism.     

Mechanism of alkylation at C-24 during ergosterol biosynthesis in Phycomyces blakesleeanus

Ergosterol isolated from Phycomyces blakesleeanus grown in the presence of methionine-[methyl-²H₃] contained two ²H atoms showing that one ²H atom is lost during transmethylation. Ergosterol isolated from P. blakesleeanus grown in the presence of mevalonic acid-[2-¹⁴C,(4R)-4-³H₁] had a ¹⁴C:³H atomic ratio of 5:3. Chemical degradation of 2,3-dimethylbutanal obtained by ozonolysis of the doubly-labelled ergosterol showed that the ³H atom originally at C-24 of lanosterol is transferred to C-25 of ergosterol during transmethylation. The mechanism of formation of the ergosterol side chain in P. blakesleeanus is presented.     

6-Ethyl-5-hydroxy-2,7-dimethoxy-1,4-naphthoquinone from Hendersonula toruloidea: A biosynthetic study using 13C labels detected by nuclear magnetic resonance and 14C tracers

Production of 6-ethyl-5-hydroxy-2,7-dimethoxy-1,4-naphthoquinone was obtained by growth of Hendersonula toruloidea on Czapek-Dox broth supplemented with malt extract. Stationary cultures were grown at 28°C for 21–22 days yielding about 6 mg of metabolite per 700 ml of culture fluid. The best incorporations of isotopic tracers were obtained by addition at the 20th day of growth, followed by harvest 24–48 hr later. With [2-¹⁴C]acetate, incorporation values were in the range of 0.1–0.3% with dilution values from 2000 to 5900. With [1-¹⁴C]propionate, incorporations were much lower (0.04%) and dilutions much higher (120,000). Activity from [${}^{14}\text{CH}{}_3$]methionine was incorporated only into the OCH₃ groups (incorporation values, 0.5–0.7%). Nuclear magnetic resonance studies confirmed that propionate was not a precursor. Using [1,2-¹³C]acetate, substantial enrichments were obtained at all carbon atoms except those of the OCH₃ groups. The following pairs of carbon atoms were shown to be derived from acetate units: C-1 + 2, C-3 + 4, C-5 + 10, C-6 + 7, C-8 + 9, C-11 + 12. The biosynthetic pathway is clearly that of acetate plus polymalonate. Experiments with [2-¹³C²H₃]acetate suggested that the “starter” acetate unit was located at positions C-12 + 11.     

Autophagy induction by tetrahydrobiopterin deficiency

Tetrahydrobiopterin (BH₄) deficiency is a genetic disorder associated with a variety of metabolic syndromes such as phenylketonuria (PKU). In this article, the signaling pathway by which BH₄ deficiency inactivates mTORC1 leading to the activation of the autophagic pathway was studied utilizing BH₄-deficient Spr^-/- mice generated by the knockout of the gene encoding sepiapterin reductase (SR) catalyzing BH₄ synthesis. We found that mTORC1 signaling was inactivated and autophagic pathway was activated in tissues from Spr^-/- mice. This study demonstrates that tyrosine deficiency causes mTORC1 inactivation and subsequent activation of autophagic pathway in Spr^-/- mice. Therapeutic tyrosine diet completely rescued dwarfism and mTORC1 inhibition but inactivated autophagic pathway in Spr^-/- mice. Tyrosine-dependent inactivation of mTORC1 was further supported by mTORC1 inactivation in Pah^enu2 mouse model lacking phenylalanine hydroxylase (Pah). NIH3T3 cells grown under the condition of tyrosine restriction exhibited autophagy induction. However, mTORC1 activation by RhebQ64L, a positive regulator of mTORC1, inactivated autophagic pathway in NIH3T3 cells under tyrosine-deficient conditions. In addition, this study first documents mTORC1 inactivation and autophagy induction in PKU patients with BH₄ deficiency.Key words: tetrahydrobiopterin, autophagy, mTORC1, tyrosine, phenylalanine, phenylketonuria, Akt, AMPK     

The stereochemistry of hexahydroprenol, ubiquinone and ergosterol biosynthesis in the mycelium of Aspergillus fumigatus Fresenius

1. The mycelium of Aspergillus fumigatus has been shown to incorporate mevalonate into squalene, ubiquinone, ergosterol and hexahydroprenol. 2. The ³H/¹⁴C ratio in ubiquinone, biosynthesized from [2-¹⁴C-(4R)-4-³H₁]mevalonate, is the same as in the squalene; essentially no ³H was incorporated from [2-¹⁴C-(4S)-4-³H₁]mevalonate, indicating the biosynthesis of biogenetically trans-isoprene units. 3. The ³H/¹⁴C ratio for ergosterol (from `4R-mevalonate') was 3:5, showing that the proton at C-24 is not lost during alkylation of the side chain; it probably migrates to C-25. 4. As ³H from both mevalonates was incorporated into the hexahydroprenols the biosynthesis of both cis- and trans-isoprene units must occur. 5. The saturated ω- and ψ-isoprene units are shown to be biogenetically trans, as are two of the unsaturated residues. 6. The saturated α- and unsaturated β-isoprene residues are both biogenetically cis. 7. An inexplicable loss of approximately half of the olefinic protons from the cis-portion of hexahydroprenol occurs; possible reasons for this loss are discussed. 8. Increase in chain length of the hexahydroprenols is by a cis addition. 9. A biosynthesis of hexahydroprenols by addition of cis-isoprene units to all-trans-geranylgeranyl pyrophosphate, or a dihydro or tetrahydro derivative thereof, is suggested.     

Trichothecene Biosynthesis in Fusarium sporotrichioides: Origin of the Oxygen Atoms of T-2 Toxin

Mass spectral analysis of T-2 toxin formed during the growth of Fusarium sporotrichioides (ATCC 24043) in the presence of H₂¹⁸O showed incorporation of up to three ¹⁸O atoms per toxin molecule. The carbonyl oxygens of the acetates at C-4 and C-15 and of the isovalerate at C-8 were derived from H₂O. Toxin formed in the presence of ¹⁸O molecular oxygen incorporated up to six ¹⁸O atoms per toxin molecule. The overall incorporation was 78 and 92% of toxin molecules labeled for H₂¹⁸O and ¹⁸O₂ labeled samples, respectively. The oxygens of position 1, the 12,13-epoxide, and the hydroxyl groups at C-3, C-4, C-8, and C-15 were all derived from molecular oxygen.