2-(7,13-Dihydroxy-2-trans-octadecenoylamino)ethanesulfonic acid (lipotaurine) as an intermediate of taurolipids biosynthesis

A hydroxy fatty-acid-combined taurine (lipotaurine) was found in the taurolipids fraction of Tetrahymena thermophila. Lipotaurine accounted for about 1.4% of the total taurolipids of the cells, and was composed of taurine and 7,13-dihydroxy-2-trans-octadecenoic acid. By nuclear magnetic resonance, mass and infrared spectrometries, the chemical structure of lipotaurine was identified as 2-(7,13-dihydroxy-2-trans-octadecenoylamino)ethanesulfonic acid. When cells of T. thermophila were incubated with the double-labeled lipotaurine which was biosynthesized from [2(n)-3H]taurine and [1-14C]stearic acid, both the radioactivities were detected in taurolipid A, B and C. Furthermore, the ratio of the radioactivities of 3H and 14C in the lysotaurolipids were the same as that of the lipotaurine. From these results, it is suggested that lipotaurine is an intermediate of taurolipid biosynthesis.     

Effects of taurolipids on lysosomal enzymes in Tetrahymena

Effects of novel taurolipid A and B localized in Tetrahymena lysosomes on the activities of lysosomal enzymes purified from Tetrahymena were investigated. Both taurolipids activated acid phosphatase, while they did not affect α-glucosidase and β-hexosaminidase. The acid phosphatase activity was activated approximately 3-fold by both taurolipids A and B, with the half-maximum activations for taurolipid A and B being at approximately 1.03·10⁻⁴ and 0.72·10⁻⁴ M, respectively. When the purified acid phosphatase was incubated at 37°C in citrate-phosphate buffer (pH 5.0) its activity was rapidly inactivated, but the inactivation was prevented to a remarkable extent by the addition of taurolipids to the incubation medium. These results thus suggest that the taurolipids may be involved in activating and stabilizing acid phosphatase in Tetrahymena lysosomes.     

Tetrahydroxystearic acid-containing taurolipid isolated from Tetrahymena thermophila

The second taurine-containing lipid (taurolipid B) was found in cells of Tetrahymena thermophila. The lipid accounted for about 1.4% of the total lipids of the cells. The lipid was subjected to mild alkaline and methanolic hydrochloric acid hydrolyses, and the structures of the hydrolysis products were identified by mass and nuclear magnetic resonance spectrometry, as taurine, 2,3,7,13-tetrahydroxystearicacid and non-hydroxy fatty acids. By spin-decoupling analysis in nuclear magnetic resonance spectrometry, the structure of the taurolipid B was identified as 2-(3-acyloxy-2,7,13-trihydroxyoctadecanoyl)aminoethane-sulfonic acid. This structure shows that taurolipid B is a homologue of the first taurine-containing lipid (taurolipid A).     

Identification of pentahydroxystearic acid-containing taurolipid (taurolipid C) isolated from Tetrahymena thermophila

A pentahydroxystearic acid-containing taurolipid (taurolipid C) was found in cells of Tetrahymena thermophila. The lipid accounted for about 1.2% of the total taurolipids of the cells. The lipid was subjected to mild alkaline and methanolic hydrochloric acid hydrolyses, and the structures of the hydrolyses products were identified by mass and nuclear magnetic resonance spectrometries, as taurine, non-hydroxy fatty acid and 2,3,7,12,13-pentahydroxystearic acd, a novel fatty acid. The NMR spectra of the intact and acetylated lipid showed that the carboxyl group of the pentahydrxyostearic acid was combined with the amino group of taurine, and the hydroxy group at C-3 was esterified with non-hydroxy fatty acids. From these results, the pentahydroxystearic acid-containing taurolipid (taurolipid C) isolated from T. thermophila was identified as 2-(3-acyloxy-2,7,12,13-tetrahydroxyoctadecanoylamino)ethanesulf oni c acid.     

Definition of total biosynthesis pathway of taurolipids in Tetrahymena cells

Taurine-combined fatty acids were found in the lipotaurine fraction of cells of Tetrahymena thermophila. Taurine and fatty acid moieties of the compounds were identified by nuclear magnetic resonance and gas chromatography-mass spectrometry, respectively. The molar ratio of fatty acid methyl esters and taurine in the hydrolysate of the lipotaurine fraction by methanolic hydrochloric acid hydrolysis, was 1.06:1.00. From the results, the structures of six taurine-combined fatty acids including lipotaurine in the fraction were identified. These structures suggest that the compounds are precursors of lipotaurine as an intermediate of taurolipids biosynthesis, and lipotaurine is biosynthesized via 2-(octadecanoylamino)ethanesulfonic acid and 2-(7-hydroxy-13-octadecenoylamino)ethanesulfonic acid. From the results of the present study and our previous studies, the total biosynthesis pathway of taurolipids is defined.     

Conversion of 5-(1,2-epoxy-2,6,6-trimethylcyclohexyl) -3-methylpenta-cis-2-trans-4-dienoic acid into abscisic acid in plants

(±)-5-(1,2-Epoxy-2,6,6-trimethylcyclohexyl) -3-methyl[2-¹⁴C]penta-cis-2-trans-4-dienoic acid is converted into abscisic acid by tomato fruit in 1.8% yield (or 3.6% of one enantiomer if only one is utilized) and 15% of the abscisic acid is derived from the precursor. The 2-trans-isomer is not converted. The amounts of [2-³H]mevalonate incorporated into abscisic acid have shown that the 40-times higher concentration of (+)-abscisic acid in wilted wheat leaves in comparison with unwilted ones reported by Wright & Hiron (1969) arises by synthesis. The conversion of (±)-5-(1,2-epoxy-2,6,6-trimethylcyclohexyl) -3-methyl-[2-¹⁴C]penta-cis-2-trans-4-dienoic acid into abscisic acid by wheat leaves is also affected in the same way by wilting and it is concluded from this that the epoxide or a closely related compound derived from it is on the biosynthetic pathway leading to abscisic acid. The oxygen of the epoxy group was shown, by ¹⁸O-labelling, to become the oxygen of the tertiary hydroxyl group of abscisic acid.     

Tetrahydropyran ring-containing fatty acid-combined taurine (tetrathermoyltaurine) in the taurolipid fraction of Tetrahymena thermophila.

A tetrahydropyran ring-containing fatty acid-combined taurine (tetrathermoyltaurine) was found in the taurolipid fraction of Tetrahymena thermophila. Tetrathermoyltaurine accounted for about 0.6% of the total taurolipid of the cells. The compound was subjected to methanolic hydrochloric acid hydrolysis, and the structures of the hydrolysis products were identified by nuclear magnetic resonance and mass spectrometries, as taurine and 2,3-dihydroxy-9,13-oxy-7-trans-octadecenoic acid (tetrathermic acid). The chemical structure of tetrathermoyltaurine was identified as 2-(2,3-dihydroxy-9,13-oxy-7-trans-octadecenoylamino) ethanesulfonic acid. This structure suggests that tetrathermoyltaurine may be derived from taurolipid B as the major taurolipid of the cells. When cells of HL 60, as a human lymphoma, were cultured with tetrathermoyltaurine, 88% of the cell growth was inhibited at the concentration of 100 micrograms/ml.     

(2R)-Catalponone,a biosynthetic intermediate for prenylnaphthoquinone congeners of the wood of Catalpa ovata

In accord with the results of experiments using callus tissues of Catalpa ovata, the simultaneous administration of (2R)- [1-¹⁴C]catalponone and (2S)- [8-³H]catalponone to the wood of the same plant demonstrated that the main pathway for the biosynthesis of naphthoquinone congeners, including catalpalactone, catalponol and 4,9-dihydroxy-α-lapachone, proceeds through the 2R-enantiomer of catalponone.     

The unexpected reactions of [RuCl3(2mqn)NO] (H2mqn=2-methyl-8-quinolinol) with 2-chloro-8-quinolinol (H2cqn) and of [RuCl(2cqn)(2mqn)NO] on photoirradiation

The reaction of [RuCl₃(2mqn)NO]⁻ (H2mqn=2-methyl-8-quinolinol) with 2-chloro-8-quinolinol (H2cqn) afforded cis-1 [RuCl(2cqn)(2mqn)NO] (the oxygen of 2cqn is trans to the NO) (complex 1), cis-1 [RuCl(2cqn)(2mqn)NO] (the oxygen of 2mqn is trans to the NO) (complex 2) and a 1:1 mixture of cis-2 [RuCl(2cqn)(2mqn)NO] (the oxygen of 2mqn is trans to the NO) and cis-2 [RuCl(2cqn)(2mqn)NO] (the oxygen of 2cqn is trans to the NO) (complex 3). The reaction was compared with that of [RuCl₃(2mqn)NO]⁻ with 8-quinolinol (Hqn) or 5-chloro-8-quinolinol (H5cqn). Photoirradiation reaction of complex 1 at room temperature in deaerated CH₂Cl₂ in the presence of NO gave trans-[RuCl(2cqn)(2mqn)NO] (the Cl is trans to the NO) and complex 2 with recovery of complex 1. The reaction was contrasted with that of cis-1 [RuCl(qn)(2mqn)NO] or cis-1 [RuCl(5cqn)(2mqn)NO]. The crystal structure of complex 1 was determined by X-ray diffraction. The reactions were examined under consideration of atomic charge of the phenolato oxygen in 8-quinolinol and its derivatives calculated at the restricted Hartree-Fock/6-311G** level.     

Biosynthesis of p-hydroxybenzoic acid in poplar lignin

Biosynthetic pathways to p-hydroxybenzoic acid in polar lignin were examined by tracer experiments. High incorporation of radioactivity to the acid was observed when shikimic acid-[1-¹⁴C], phenylalanine-[3-¹⁴C], trans-cinnamic acid-[3-¹⁴C], p-coumaric acid-[3-¹⁴C] and p-hydroxybenzoic acid-[COOH-¹⁴C] were administered, while incorporation was low from shikimic acid-[COOH-¹⁴C], phenylalanine-[1-¹⁴C], phenylalanine-[2-¹⁴C], tyrosine-[3-¹⁴C], benzoic acid-[COOH-¹⁴C], sodium acetate-[1-¹⁴C] and d-glucose-[U-¹⁴C]. Thus p-hydroxybenzoic acid in poplar lignin is formed mainly via the pathway: shikimic acid → phenylalanine → trans-cinnamic acid → p-coumaric acid → p-hydroxybenzoic acid.     

The 2-hydroxylation of trans-cinnamic acid by chloroplasts from Melilotus alba Desr

Chloroplasts isolated from sweetclover leaves contain an enzyme which converts trans-[3-¹⁴C]cinnamic acid to 2-hydroxy-trans-[3-¹⁴C]cinnamic (o-coumaric) acid. The identity of the product has been verified by recrystallization with unlabeled o-coumaric acid to constant specific activity, and by gas-liquid cochromatography of unlabeled o-coumaric acid and the radioactive product.The enzyme has an optimum of pH 7.0 and its activity can be enhanced ~ 4-fold by adding 4 mm glucose-6-phosphate to the reaction mixture. Light can replace glucose-6-phosphate, presumably as a source of reducing power required for the hydroxylation system. It was found that approximately 50% of the hydroxylase activity is bound to the lamellar membranes, from which it can be released by sonication.     

Biosynthesis of azetidine-2-carboxylic acid in Convallaria majalis

Convallaria majalis plants were fed dl-methionine-[1-¹⁴C]. [1-¹⁴C, 4-³H], and [1-¹⁴C, 2-³H], S-adenosyl-l-methionine-[1-¹⁴C], and dl-homoserine-[1-¹⁴C], resulting in the formation of labeled azetidine-2-carboxylic acid (A-2-C). The complete retention of tritium relative to carbon-14 in the feeding experiment involving methionine-[1-¹⁴C, 4-³H] indicates that aspartic acid or aspartic-β-semialdehyde are not intermediates between methionine and A-2-C. However, since the A-2-C derived from methionine-[1-¹⁴C, 2-³H] had lost 95% of the tritium relative to the C-14, it is not considered that methionine or its S-adenosyl derivative are the immediate precursors of A-2-C. Our data and that of others is consistent with the intermediate formation of γ-amino-α-ketobutyric acid which on cyclization yields 1-azetine-2-carboxylic acid, A-2-C then being formed on reduction.     

Metabolism of [2-3H]- and [6-3H]-nicotinic acid in intact Nicotiana tabacum plants

Earlier observations of Dawson on the relative incorporation of [2-³H]- and [6-³H]-nicotinic acid into nicotine have been confirmed in intact Nicotiana tabacum plants. All the tritium in the nicotine derived from [2-³H]-nicotinic acid was located at C-2 of the pyridine ring. However the radioactive nicotine derived from [6-³H]-nicotinic acid was not labelled specifically at C-6 with tritium. By carrying out feeding experiments with [6-¹⁴-C, 2-³H]- and [6-¹⁴C, ³H]-nicotinic acids, it was established that there was very little loss of tritium from C-2 and C-6 of nicotinic acid during 5 days of metabolism in the tobacco plant.     

A criterion to determine whether cis-4-hydroxyproline is produced in animal tissues

Hydrolyzates of tissues that had been labeled with [¹⁴C]proline often contain significant amounts of cis-4-hydroxy[¹⁴C]proline. Since animal cells do not contain an enzyme which can effect formation of cis-4-hydroxyproline, there are only two possible explanations for its presence. Either it is formed during acid hydrolysis of trans-4-hydroxyproline (which is synthesized by cells and is a common constituent of connective tissues), or it is produced by a nonenzymatic mechanism such as attack by oxygen radicals. It is important to resolve this issue because if a nonenzymatic mechanism is active in connective tissues, then it will be necessary to reevaluate currently accepted ideas about production of hydroxyproline. This communication describes a method for distinguishing between the two alternate explanations. Tissues or cells are labeled with [¹⁴C]proline, and then a known amount of trans-4-hydroxy[³H]proline is added to each sample before hydrolysis; the relative amounts of [¹⁴C]- and [³H]-cis-4-hydroxyproline are compared after hydrolysis. It is known from a separate series of measurements with mixtures of [¹⁴C]- and [³H]-trans-4-hydroxyproline standards that there is a very high correlation (r = 0.998) between acid-induced formation of the [¹⁴C]- and [³H]-cis epimers. One can thus compare the amount of cis-4-hydroxy[¹⁴C]proline in a hydrolyzate from a biological system with the amount that would be expected if it were all formed during acid hydrolysis. This method was used to show that fibroblasts cultured under conditions commonly used to study collagen metabolism do not produce cis-4-hydroxyproline. This result strongly suggests that nonenzymatic hydroxylation does not normally occur in cell culture systems.     

Tetrapyrrole biosynthesis in Anacystis nidulans; incorporation of [1-13C]-, [2-13C]-, [1,2-13C]- and [2-13C, 2-2H3]acetate

Labelling experiments with [2-¹³C]- and [1,2-¹³C]acetate showed that both photopigments of Anacystis nidulans, chlorophyll a and phycocyanobilin, share a common biosynthetic pathway from glutamate. The fate of deuterium during these biosynthetic events was studied using [2-¹³C, 2-²H₃]acetate as a precursor and determining the labelling pattern by ¹³C NMR spectroscopy with simultaneous [¹H, ²H]-broadband decoupling. The loss of ²H (ca 20%) from the precursor occurred at an early stage during the tricarboxylic acid cycle. After formation of glutamate there was no further loss of ²H in the assembly of the cyclic tetrapyrrole intermediates or during decarboxylation and modification of the side-chains. Thus the labelling data support a divergence in the pathway to cyclic and linear tetrapyrroles after protoporphyrin IX.     

Studies on l-ascorbic acid biosynthesis and metabolism in Parthenocissus quinquefolia L. (vitaceae)

Parthenocissus quinquefolia (L.) Planch., commonly known as Virginia Creeper, is a vitaceous tartrate-accumulating vine that exhibits C-4/C-5 cleavage of l-ascorbic acid (AA) to produce l-tartaric acid (TA) from the C₄ fragment and carbohydrate pool material from the C₂ fragment. Experiments in which detached leaves were supplied d-[5-³H,1-¹⁴C]glucose or d-[5-³H,6-¹⁴C]glucose yielded AA devoid of ³H whereas the l-threonic acid (ThA) and TA recovered from the same tissues still retained some ³H. These comparative experiments also indicated that the ThA was derived from carbons 3 through 6 of d-glucose. ThA was shown to be a natural constituent of P. quinquefolia but apparently not an intermediate between AA and TA. Results are consistent with a biosynthetic pathway from d-glucose to AA that involves a hydrogen-exchanging epimerization at C-5 as reported earlier for the geraniaceous plant Pelargonium crispum, but differing from P.crispum in biosynthesis and metabolism of ThA.When l-[6-¹⁴C]idonate or its lactone was supplied to P. quinquefolia leaves, about 80% of the ¹⁴C appeared in the carbohydrates, an observation remarkably similar to previous observations with [6-¹⁴C]AA-labeled leaves. l-Idonate and its lactone appear to have an intermediate role in AA metabolism in vitaceous plants.     

Structure and Biosynthesis of Cuticular Lipids: Hydroxylation of Palmitic Acid and Decarboxylation of C(28), C(30), and C(32) Acids in Vicia faba Flowers

The structure and composition of the cutin monomers from the flower petals of Vicia faba were determined by hydrogenolysis (LiAlH₄) or deuterolysis (LiAlD₄) followed by thin layer chromatography and combined gas-liquid chromatography and mass spectrometry. The major components were 10, 16-dihydroxyhexadecanoic acid (79.8%), 9, 16-dihydroxyhexadecanoic acid (4.2%), 16-hydroxyhexadecanoic acid (4.2%), 18-hydroxyoctadecanoic acid (1.6%), and hexadecanoic acid (2.4%). These results show that flower petal cutin is very similar to leaf cutin of V. faba. Developing petals readily incorporated exogenous [1-¹⁴C]palmitic acid into cutin. Direct conversion of the exogeneous acid into 16-hydroxyhexadecanoic acid, 10, 16-dihydroxy-, and 9, 16-dihydroxyhexadecanoic acid was demonstrated by radio gas-liquid chromatography of their chemical degradation products. About 1% of the exogenous [1-¹⁴C]palmitic acid was incorporated into C₂₇, C₂₉, and C₃₁n-alkanes, which were identified by combined gas-liquid chromatography and mass spectrometry as the major components of the hydrocarbons of V. faba flowers. The radioactivity distribution among these three alkanes (C₂₇, 15%; C₂₉, 48%; C₃₁, 38%) was similar to the per cent composition of the alkanes (C₂₇, 12%; C₂₉, 43%; C₃₁, 44%). [1-¹⁴C]Stearic acid was also incorporated into C₂₇, C₂₉, and C₃₁n-alkanes in good yield (3%). Trichloroacetate, which has been postulated to be an inhibitor of fatty acid elongation, inhibited the conversion of [1-¹⁴C]stearic acid to alkanes, and the inhibition was greatest for the longer alkanes. Developing flower petals also incorporated exogenous C₂₈, C₃₀, and C₃₂ acids into alkanes in 0.5% to 5% yields. [G-³H]n-octacosanoic acid (C₂₈) was incorporated into C₂₇, C₂₉, and C₃₁n-alkanes. [G-³H]n-triacontanoic acid (C₃₀) was incorporated mainly into C₂₉ and C₃₁ alkanes, whereas [9, 10, 11-³H]n-dotriacontanoic acid (C₃₂) was converted mainly to C₃₁ alkane. Trichloroacetate inhibited the conversion of the exogenous acids into alkanes with carbon chains longer than the exogenous acid, and at the same time increased the amount of the direct decarboxylation product formed. These results clearly demonstrate direct decarboxylation as well as elongation and decarboxylation of exogenous fatty acids, and thus constitute the most direct evidence thus far obtained for an elongation-decarboxylation mechanism for the biosynthesis of alkanes.     

Time-course tracer studies on the metabolism of cinnamic acid in Cestrum poeppigii

Time-course tracer studies were performed on the metabolism of trans-cinnamic acid-[3-¹⁴C] and trans-p-coumaric acid-[2-¹⁴C] in the     

Accumulation and enzymatic synthesis of 2-O-acetyl-3-O-(p-coumaroyl)-meso-tartaric acid in spinach cotyledons

2-O-Acetyl-3-O-[(E)-p-coumaroyl]-meso-tartaric acid was isolated from cotyledons of Spinacia oleracea and its structure elucidated and characterized with the aid of TLC, HPLC, FAB MS and ¹H NMR. Accumulation and enzymatic synthesis of the diester are described, proceeding first via 1-O-(p-coumaroyl)-β-glucose in the formation of p-coumaroyltartaric acid and second via acetyl-CoA in the formation of 2-O-acetyl-3-O-[(E)-p-coumaroyl]-meso-tartaric acid. Some properties of the CoA-thioester-dependent acyltransferase activity were studied.     

Biosynthesis of pterocarpan,isoflavan and coumestan metabolites of Medicago sativa: chalcone,isoflavone and isoflavanone precursors

Comparative feeding experiments in CuCl₂,- and UV-treated lucerne (Medicago sativa) seedlings have shown that 2′,4,4′-trihydroxychalcone-[carbonyl-¹⁴C] and formononetin-[Me-¹⁴C] but not 2′,4′-dihydroxy-4-methoxychalcone-[carbonyl- ¹⁴C] or daidzein-[4-¹⁴C] were incorporated into the phytoalexins demethylhomopterocarpin, sativan and vestitol, and also into 9-O-methylcoumestrol. The synthesis of 9-O-methylcoumestrol is greatly stimulated by this abiotic treatment but coumestrol production is not noticeably affected. Daidzein and the trihydroxychalcone were precursors of coumestrol. The results are interpreted in favour of a mechanism in which methylation is an integral part of the aryl migration process associated with the biosynthesisof 4′-methoxyisoflavonoids. Formononetin, 2′,7-dihydroxy-4′-methoxyisoflavone-[Me-¹⁴C], 7-hydroxy-4′-methoxyisoflavanone-[Me-¹⁴C] and 2′,7-dihydroxy-4′-methoxyisoflavanone-[Me-¹⁴C] were all excellent precursors of demethylhomopterocarpin, sativan, vestitol and 9-O-methylcoumestrol, and thus a metabolic grid may be involved in their biosynthetic origin.