High production of beta-thujaplicin glycosides by immobilized plant cells of Nicotiana tabacum.

beta-Thujaplicin (hinokitiol) is a tropolone derivative present in the heartwood of cupressaceous plants and is used as a medicine, a food additive, and a preservative, and in cosmetics as hair tonic. The cultured plant cells of Nicotiana tabacum glycosylated beta-thujaplicin to two glucosides, 4-isopropyltropolone 2-O-beta-D-glucoside (6%) and 6-isopropyltropolone 2-O-beta-D-glucoside (12%), and two gentiobiosides, 4-isopropyltropolone 2-O-beta-D-gentiobioside (2%) and 6-isopropyltropolone 2-O-beta-D-gentiobioside (5%) after 48 h incubation. The use of immobilized cells of N. tabacum in sodium alginate gel much improved the yield of the products; the glycosylation of beta-thujaplicin with immobilized N. tabacum gave the glycoside products, 4-isopropyltropolone 2-O-beta-D-glucoside (11%), 4-isopropyltropolone 2-O-beta-D-gentiobioside (6%), 6-isopropyltropolone 2-O-beta-D-glucoside (20%), and 6-isopropyltropolone 2-O-beta-D-gentiobioside (10%). On the other hand, 4-isopropyltropolone 2-O-beta-D-glucoside (14%), 4-isopropyltropolone 2-O-beta-D-gentiobioside (7%), 6-isopropyltropolone 2-O-beta-D-glucoside (33%), and 6-isopropyltropolone 2-O-beta-D-gentiobioside (13%) were obtained through the biotransformation with immobilized cells in the medium without iron ions. In comparison with the case of bioconversion in the normal medium containing iron ions, removal of iron ions improved the yields of products.

Formation of gamma-glutamylpropargylglycylglycine from propargylglycine in human blood and erythrocytes.

Gamma-Glutamylpropargylglycylglycine (gamma-Glu-PPG-Gly) was isolated as a metabolite of propargylglycine (2-amino-4-pentynoic acid, a natural and synthetic inhibitor of cystathionine gamma-lyase) from human blood incubated with D,L-propargylglycine in the presence of L-glutamate and glycine, and identified by fast-atom-bombardment mass spectrometry, indicating that human blood can metabolize propargylglycine to gamma-Glu-PPG-Gly. When whole blood was incubated with 2 mM D,L-propargylglycine in the presence of 10 mM L-glutamate and 10 mM glycine at 37 degrees C for 16h, 0.094+/-0.013 micromol of gamma-Glu-PPG-Gly was formed per ml of whole blood. When erythrocytes were incubated under the same conditions for 16h, 0.323+/-0.060 micromol of gamma-Glu-PPG-Gly was formed per ml of erythrocytes, suggesting a large contribution of erythrocytes to gamma-Glu-PPG-Gly formation in whole blood. The apparent Km value of gamma-Glu-PPG-Gly formation in human erythrocytes for D,L-propargylglycine was 0.32 mM. The observed rate of gamma-Glu-PPG-Gly formation and the Km value for D,L-propargylglycine suggest that metabolism of propargylglycine to gamma-Glu-PPG-Gly can play a definite biological role in human subjects who are loaded with propargylglycine.

Different effect of diastereoisomers of L-cystathionine sulfoxide on the stimulus coupled responses of human neutrophils.

The priming effect of L-cystathionine sulfoxide, which is one of the unusual cystathionine metabolites found in the urine of patients with cystathioninuria, on the stimulus-induced superoxide generation by human neutrophils was examined. The synthetic L-cystathionine sulfoxide significantly enhanced the superoxide generations induced by N-formyl-methionyl-leucyl-phenylalanine [fMLP], opsonized zymosan [OZ], arachidonic acid [AA], and phorbol 12-myristate 13-acetate [PMA]. Then the synthetic L-cystathionine sulfoxide was separated into two diastereoisomers, CS-I and CS-II, which showed a peak at 76 and 83 min on chromatogram by amino acid analyzer, respectively. CS-I enhanced the superoxide generations induced by AA and PMA but not those induced by fMLP and OZ. On the contrary, CS-II enhanced the superoxide generations induced by fMLP and OZ but not those induced by AA and PMA. The superoxide generation induced by PMA with CS-I was suppressed by H-7 and was enhanced by genistein, while that by fMLP with CS-II was suppressed by genistein and was enhanced by H-7.

Priming effect of N-acetyl-S-(3-oxo-3-carboxy-n-propyl)cysteine in human neutrophils and tyrosyl phosphorylation of 45 kDa protein.

Human peripheral blood polymorphonuclear leukocytes were preincubated with N-acetylcystathionine and N-acetyl-S-(3-oxo-3-carboxy-n-propyl)cysteine (NAc-OCPC) found in the urine of a patient with cystathioninuria. NAc-OCPC significantly enhanced the N-formyl-methionyl-leucyl-phenylalanine-induced superoxide generation, whereas N-acetylcystathionine did not enhance the superoxide generation. When the cells were incubated with NAc-OCPC, the tyrosyl phosphorylation of 45 kDa protein of the cell was markedly increased with time. The phosphorylation process was dependent on the concentration of NAc-OCPC. Both the superoxide generation and the tyrosyl phosphorylation of 45 kDa protein increased by NAc-OCPC were inhibited by genistein and herbimycin A, the inhibitors of protein tyrosine kinase, but were rather enhanced by staurosporine, an inhibitor of protein kinase C.

Identification of cyclic cystathionine sulfoxide and N-acetylcyclic cystathionine in the urine of a patient with cystathioninuria using liquid chromatography-mass spectrometry with an atmospheric pressure chemical ionization interface system.

Perhydro-1,4-thiazepine-4,5-dicarboxylic acid sulfoxide (cyclic cystathionine sulfoxide [cyclic cystaSO]) and N-acetylperhydro-1,4-thiazepine-3,5-dicarboxylic acid (NAc-cyclic cysta) have been identified in the urine of a patient with cystathioninuria as new metabolites of cystathionine for the first time using liquid chromatography-mass spectrometry with an atmospheric pressure chemical ionization interface system (LC/APCI-MS). The concentrations of cyclic cystaSO and NAc-cyclic cysta in the urine of a patient with cystathioninuria have also been determined for the first time using this method: 18.24 +/- 0.79 and 25.23 +/- 0.83 mg/g creatinine, respectively.

Identification of perhydro-1,4-thiazepine-3,5-dicarboxylic acid, cystathionine mono-oxo acids, cystathionine ketimines, cystathionine sulfoxide and N-acetylcystathionine sulfoxide in the urine sample of D,L-propargylglycine treated rats.

Novel cystathionine metabolites, perhydro-1,4-thiazepine-3,5-dicarboxylic acid (PHTZDC), cystathionine mono-oxo acids [S-(3-oxo-3-carboxy-n-propyl)cysteine and S-(2-oxo-2-carboxyethyl)homocysteine], cystathionine ketimines, cystathionine sulfoxide and N-acetylcystathionine sulfoxide were identified previously in the urine of patients with cystathioninuria. We have identified these compounds for the first time in the urine of D,L-propargylglycine-treated rats using LC/APCl-MS (liquid chromatography-mass spectrometry with an atmospheric pressure chemical ionization interface system) and an amino acid analyzer. Cystathionine mono-oxo acids and cystathionine ketimines were easily interconvertible depending on the pH of the solution. The excretion of PHTZDC, total cystathionine ketimine (cystathionine mono-oxo acids plus cystathionine ketimines), cystathionine sulfoxide and Nac-cystathionine sulfoxide in the rat urine increased in proportion to that of cystathionine content after D,L-propargylglycine administration.

Identification of N-acetyl-S-(3-oxo-3-carboxy-n-propyl)cysteine in the urine of a patient with cystathioninuria using LC/APCI-MS.

A new cystathionine metabolite has been identified in the urine of a patient with cystathioninuria using liquid chromatography-mass spectrometry with an atmospheric pressure chemical ionization interface system (LC/APCI-MS). By this method a very intense quasi-molecular ion was observed as a base peak of synthetic N-acetyl-S-(3-oxo-3-carboxy-n-propyl)cysteine (NAc-OCPC). The quasimolecular ion [M + H]+ of NAc-OCPC observed in the urine of a patient with cystathioninuria was the same as that of the authentic compound (m/z 264). The retention time and Rf value on paper chromatography of the synthetic compound were the same as those of the urinary compound from the patient with cystathioninuria. From these results, this new cystathionine metabolite was identified as N-acetyl-S-(3-oxo-3-carboxy-n-propyl)cysteine.

Increase in cystathionine content in rat liver mitochondria after D,L-propargylglycine administration.

Intraperitoneal administration of D,L-propargylglycine to rats resulted in an increase in the cystathionine content of whole liver and liver mitochondria. Cystathionine in mitochondria was identified by amino acid analysis, thin layer chromatography, high-voltage paper electrophoresis and liquid chromatography-mass spectrometry. The cystathionine content of whole liver was 5.37 ± 1.59µmol per g of fresh liver at 14 h after the administration of 50 mg of D,L-propargylglycine per kg of body weight, while 0.07 ± 0.02µmol of cystathionine per g of fresh liver was detected in the control rats. The cystathionine content of liver mitochondria from both groups of rats was 9.40 ± 1.20 and 0.19 ± 0.04 nmol of cystathionine per mg of protein, respectively. The mitochondrial cystathionine increased dose-dependently with the increase of D,L-propargylglycine administered. The increase was proportional to the time after the administration up to 12 h, and then decreased. The increase of cystathionine in the liver mitochondria was linearly proportional to that in the whole liver. These results suggest that cystathionine in liver mitochondria is in an equilibrium with that in the cytosol.

Increase in tissue cysteine level and excretion of sulfate and taurine after intragastric administration ofL-2-oxothiazolidine-4-carboxylate in rats.

Five mmol ofL-2-oxothiazolidine-4-carboxylate (OTC)/kg of body weight was administered into the stomach of rats, and cysteine levels in tissues and sulfate and taurine excreted in the urine were determined. The cysteine (plus cystine expressed as cysteine) concentration in the liver increased to 170-200% of the original level at 30 min and that in the blood to 160% at 60 min after the OTC administration. These high levels were maintained until 8 h after the administration and decreased gradually thereafter. Excretion of sulfate and taurine increased after the OTC administration and the increase corresponded to 26% and 15%, respectively, of the OTC administered. These findings suggest that at least about 40% of the OTC administered into the stomach was taken up and converted to cysteine, which was metabolized to sulfate and taurine.

Isolation and characterization of 3-[(carboxymethyl)thio]-3-(1H-imidazol-4-yl)propanoic acid from human urine and preparation of its proposed precursor, S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]cysteine.

3-[(Carboxymethyl)thio]-3-(1H-imidazol-4-yl)propanoic acid (I) was isolated from healthy human urine by using ion-exchange column chromatography, and characterized by physicochemical analyses involving i.r., m.s. and n.m.r. spectrometries as well as chemical synthesis. The urinary content was 0.04-0.07 mumol/l. Compound (I) was synthesized by the addition of mercaptoacetic acid to urocanic acid. In order to establish the origin of the compound. S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]cysteine (II) and S-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]glutathione (III) were produced by similar reactions of urocanic acid with cysteine and GSH respectively. The yield of compound (II) was markedly increased by sunlight irradiation of the reaction mixture or by the use of cis-urocanic acid rather than the trans isomer. Incubation of compound (II) with rat liver homogenate in a phosphate buffer, pH 7.40, formed a major and some minor products of enzymic degradation, one of which was identified with compound (I). Exposure of rats to the sunlight for 2 days resulted in increase of the epidermal content of trans-urocanic acid from the normal value of 0.38 to 1.70 micrograms/mg wet wt. of skin, accompanied by formation de novo of the epidermal cis isomer. After sunlight irradiation, the content of the trans isomer decreased at a constant rate of 0.03 micrograms/mg wet wt. of skin per day, whereas the cis isomer was eliminated more quickly, having a phase of rapid decrease in the early period. From these results we suggest that compound (I) may participate in the metabolism of urocanic acid and natural thiol compounds such as cysteine and GSH.

S-[2-Carboxy-1-(1H-imidazol-4-yl)ethyl]cysteine in normal human urine.

A compound, which had the same mobility on a high-voltage paper electrophoretogram and the sameR F value on a thin-layer chromatogram as those ofS-[2-carboxy-1-(1H-imidazol-4-yl)ethyl]cysteine (I), was partially purified from human urine by ion-exchange column chromatography. The compound gave a signal at m/z 260 on its FAB mass spectrum, which was assigned as MH(+) of compound I. These results suggest that the urinary compound is compound I and it is a physiological precursor of 3-[(carboxymethyl)thio]-3-(1H-imidazol-4-yl)propanoic acid [Kinuta et al., (1991) Biochem J 275: 617-621].

Preparation and analysis of a volatile derivative of cysteic acid.

We have reported preparations and gas chromatographic analyses of volatile derivatives of sulfuric acid and taurine (Masuoka et al., 1988; 1989). By extending these studies, we have developed a method for the gas chromatographic determination of cysteic acid. Cysteic acid was converted to the N-isobutoxycarbonyl derivative by the reaction with isobutyl chloroformate in the presence of sodium hydroxide. After desalting with a cation-exchange column, the derivative was converted to the silver salt by reacting with silver oxide. The resulting silver salt was quantitatively esterified with methyl iodide in the presence of dimethyl sulfate and silver oxide. Dimethyl N-isobutoxy-carbonylcysteate [methyl 2-(N-isobutoxycarbonylamino)-3-(methoxysulfonyl) propanoate] formed was analyzed by gas chromatography. The calibration curve was linear up to 5.0µmol per ml of cysteic acid and the recovery was more than 95%.

Excretion of 3-mercaptolactate-cysteine mixed disulfide, sulfate and taurine in human urine before and after oral administration of sulfur-containing amino acids.

The excretion of 3-mercaptolactate-cysteine mixed disulfide [S-(2-hydroxy-2-carboxyethylthio)-L-cysteine, HCETC], sulfate and taurine in the urine of normal adults was investigated before and after oral administration of L-cysteine and related sulfur-containing amino acids. Before the loading of amino acids, the excretion (mean +/- SD) per kg of body weight per day of HCETC, free sulfate and taurine was 0.096 +/- 0.042, 305.7 +/- 66.1 and 31.9 +/- 8.7 mumols, respectively. After the loading of L-cysteine (800 mumols/kg of body weight), the average excretion in the 24-h urine of HCETC increased 2-fold and that of taurine increased 1.6-fold. The average excretion of free sulfate after the L-cysteine loading was 989.4 +/- 145.1 and 388.8 +/- 51.6 mumols/kg per day in the first and second 24-h urine, respectively, indicating that the sulfur corresponding to 85% of the L-cysteine loaded was excreted as free sulfate in 24 h. Administration of L-cystine (400 mumols/kg) resulted in similar results. The increase in HCETC after L-cysteine or L-cystine administration indicates that L-cysteine is metabolized in part through the transamination pathway (3-mercaptopyruvate pathway) and that an equilibrium exists between the intake and excretion of sulfur in humans.

Formation of sulfate from L-cysteine in rat liver mitochondria.

Formation of sulfate in rat liver mitochondria was studied. About 0.1 mumol of sulfate was formed in mitochondria from 1 g of liver in 60 min when 10 mM L-cysteine was used as the substrate. Addition of either 10 mM 2-oxoglutarate or 10 mM glutathione to this system increased sulfate formation 3 to 4 times. The addition of both 2-oxoglutarate and glutathione resulted in a 20-fold increase in sulfate formation. Sulfate formation in the presence of 5 mM L-cysteine was 58% of that with 10 mM L-cysteine. L-Cysteine-glutathione mixed disulfide was not a good substrate, indicating that this mixed disulfide was not an intermediate of sulfate formation in the present system. Incubation of 3-mercaptopyruvate with rat liver mitochondria also resulted in sulfate formation, and the addition of glutathione accelerated it. Formation of sulfite and thiosulfate was also detected. These results indicate that sulfate is produced in mitochondria, at least in part, from L-cysteine through the transamination pathway (3-mercaptopyruvate pathway).

Visualization of sialoglycoproteins in polyacrylamide gels by acidic ninhydrin reaction.

A new method for staining sialoglycoproteins in polyacrylamide gel after disc electrophoresis is described. The method utilizes the reaction of sialic acids with an acidic ninhydrin reagent which yields a stable color with an absorption maximum at 470 nm. After electrophoresis, the polyacrylamide gel is placed in a test tube and heated with 5 ml of the acidic ninhydrin reagent for 10 min in a boiling water bath. Sialoglycoproteins are detected as brown bands. No additional procedure such as destaining is necessary. When 20 micrograms fetuin, a sialoglycoprotein, per gel is applied, the band remains visible for at least 2 h. Stained gel can be scanned with a gel scanner at 470 nm. When the stained gel was dried on a sheet of polypropylene filter, the color was stable for at least one month. The present method is superior to the method using Stains-all (3,3'-diethyl-9-methyl-4,5,4',5'-dibenzothiacarbocyanine) in specificity and simplicity for the detection of sialoglycoproteins.

Preparation of a volatile derivative of taurine and application to gas chromatographic determination of urinary taurine.

A new volatile derivative of taurine, N-isobutoxycarbonyltaurine methyl ester (methyl 2-(N-isobutoxycarbonylamino)ethanesulfonate), was prepared by a three-step procedure for the gas chromatographic determination of taurine in urine. First, taurine was converted to its silver salt by reaction with silver oxide; next the silver salt was reacted with isobutyl chloroformate to form the N-isobutoxycarbonyl derivative, and finally the derivative was reacted with methyl iodide to form N-isobutoxycarbonyltaurine methyl ester. The volatile derivative was analyzed by gas chromatography using a column of 3% OV-101 on Chromosorb W. When methyl 3-(N-isobutoxycarbonylamino) propanesulfonate was used as an internal standard, the calibration curve was linear between 0.5 and 5.0 mumol of taurine/ml and showed a good reproducibility. This method was applied to the determination of taurine in human urine. Recovery was 98.6 +/- 5.2%, when 1.25 to 5.0 mumol/ml of taurine was added to human urine.

Direct determination of bound sialic acids in sialoglycoproteins by acidic ninhydrin reaction.

A simple and rapid method for sialic acid determination in sialoglycoproteins by acidic ninhydrin reaction is described. The method is based on the reaction of sialic acids with an acidic ninhydrin reagent (K. Yao and T. Ubuka (1987) Acta Med. Okayama 41, 237-241). By heating a sample solution containing sialoglycoprotein with the reagent at 100 degrees C for 10 min, a stable color with an absorption maximum at 470 nm was produced. The standard curve was linear in the range of 20 micrograms to 3 mg of fetuin, a sialoglycoprotein, per 3.0 ml of the reaction mixture. The reaction is specific only for sialoglycoproteins among various proteins examined. The acidic ninhydrin method was applied to the determination of sialic acids in sialoglycoproteins in ascites fluids of Ehrlich ascites tumor-bearing mice.

Gas chromatographic determination of sulfuric acid and application to urinary sulfate.

A new gas chromatographic method for the determination of sulfate was developed. In this method, sulfate was quantitatively converted to a volatile derivative, dimethyl sulfate, by a two-step procedure. First, sulfate was converted to silver sulfate by reaction with silver oxide, and then to dimethyl sulfate by reaction with methyl iodide. The derivative was analyzed by gas chromatography. Methyl methanesulfonate was used as an internal standard. The method was applied to the determination of total urinary sulfate. Phosphate and chloride ions, which interfered with the present method, were eliminated with the use of basic magnesium carbonate and an excess of silver oxide, respectively. Recovery was over 96% when 5 to 40 mumol/ml of sulfate was added to human urine samples.

Determination of isoelectric point value of 3-mercaptopyruvate sulfurtransferase by isoelectric focusing using ribonuclease A-glutathione mixed disulfides as standards.

The pI value of rat erythrocyte 3-mercaptopyruvate sulfurtransferase (EC 2.8.1.2) was determined to be 5.9 at 10 degrees C by isoelectric focusing in a horizontal slab polyacrylamide gel containing 2% carrier ampholyte (pH 3-10). In this study, ribonuclease A-glutathione mixed disulfides (RNase-SG's) (T. Ubuka et al. (1986) J. Chromatogr., 363, 431-437) were used as pI standards. A mixture of RNase-SG was prepared by reducing bovine pancreatic ribonuclease A (RNase) with dithiothreitol and then treating the reduced RNase with oxidized glutathione. The mixture was composed of eight species which contained 1 (RNase-SG1) to 8 (RNase-SG8) mol of glutathione per mole of RNase, and the pI values of these species were determined under conditions minimizing the effect of carbon dioxide. The newly determined pI values of RNase-SG1 through RNase-SG8 were 8.8, 8.2, 7.7, 7.3, 6.9, 6.4, 5.8, and 5.3, respectively. The average change in pI values of these disulfides was 0.50 pH unit per mole of the bound glutathione per mole of RNase. The RNase-SG mixture was stable in acidic solutions and could be stored at 4 degrees C as well as at -20 degrees C with little change for at least 1 year. Thus, the mixture is shown to be an excellent standard for the determination of pI values of proteins by isoelectric focusing in the wide range of pI value.

Reaction of S-(2-amino-2-carboxyethylsulfonyl)-L-cysteine with thiosulfate: synthesis of L-alanine sulfodisulfane and application to the determination of thiosulfate.

A new reaction of S-(2-amino-2-carboxyethylsulfonyl)-L-cysteine (ACESC) with thiosulfate is described. The reaction proceeded quantitatively in formic or acetic acid solutions, yielding equimolar amounts of L-alanine sulfodisulfane (2-amino-2-carboxyethyl sulfodisulfane) and L-alanine 3-sulfinic acid. L-Alanine sulfodisulfane was obtained as pure monosodium salt; the yield was 92% of the theoretical. A new method is described for the determination of thiosulfate. The method is based on the quantitative reaction between ACESC and thiosulfate, and L-alanine sulfodisulfane, one of the reaction products, was determined using acid ninhydrin reagent 2 of M. K. Gaitonde (1967, Biochem. J. 104, 627-633). The recovery was over 95%. When samples contained sulfite in addition to thiosulfate, S-sulfo-L-cysteine (T. Ubuka et al., 1982, Anal. Biochem. 126, 273-277) was produced in addition to L-alanine sulfodisulfane by the treatment with ACESC. Both products were separated by a small Dowex 1 column and determined with the acid ninhydrin reagent 2. The recoveries were over 95%. The new method was applied to the thiosulfate sulfurtransferase reaction, in which thiosulfate, a substrate, and sulfite, a product, were determined separately.