Search PubMed⌕ Search

Biomedical subjects

S Toki

Publications and source records attributed to S Toki.

At least 73 records · Page 4Linked to original sources
PubMed1990

Purification and characterization of hydroxypyruvate reductase from a serine-producing methylotroph, Hyphomicrobium methylovorum GM2.

Hydroxypyruvate reductase of a serine-producing methylotroph, Hyphomicrobium methylovorum GM2, was purified to complete homogeneity, crystallized and characterized, the first time for an enzyme from a methylotroph. The enzyme was found to be a dimer composed of identical subunits (38 kDa), the molecular mass of the enzyme being about 70 kDa. The enzyme was stable against heating at 25 degrees C for 10 min at pH values between 5 and 9. Optimal activity was observed at pH 6.8 and around 45 degrees C. The enzyme catalyzed the reduction of hydroxypyruvate with the oxidation of only NADH. Other than hydroxypyruvate, only glyoxylate served as a substrate. The Km values were found to be 0.175 mM for hydroxypyruvate and 10.8 mM for glyoxylate. Taking advantage of the high substrate specificity of this enzyme, a means of enzymatic determination of hydroxypyruvate was established.

Alcohol Oxidoreductases↗
PubMed1990

A new metabolic pathway of morphine: in vivo and in vitro formation of morphinone and morphine-glutathione adduct in guinea pig.

The biliary excretion of morphine and its metabolites by guinea pigs after s.c. injection of morphine (25 mg/kg) was determined by high-performance liquid chromatography procedures. The amounts of morphinone (MO), morphinone-glutathione adduct (MO-GSH), morphine and morphine-3-glucuronide excreted over a 4-hr period were 1.27, 9.35, 1.13 and 7.54% of the administeral dose, respectively. In addition, trace amounts of morphine-GSH adduct and morphinone-cysteine adduct, derived from MO-GSH, were detected. Although MO-GSH formation from MO proceeded nonenzymatically with a relatively high rate, GSH S-transferases participated in the reaction, especially at lower GSH concentrations. Pretreatment of the animals with diethyl maleate and lithocholic acid-3-sulfate decreased the total biliary excretion of MO. On the other hand, pretreatment with naloxone increased the excretion of total MO. In vitro experiments using liver cytosolic preparations showed that lithocholic acid-3-sulfate inhibited the enzymatic formation of MO from morphine and of MO-GSH from MO. The effects of naloxone on MO formation in vitro were more complex. A reaction was stimulated at low and suppressed at higher concentrations. The results indicated that MO-GSH formation from morphine is inhibited by lithocholic acid-3-sulfate through actions on both morphine 6-dehydrogenase and GSH S-transferases. Low levels of naloxone were found to stimulate the morphine 6-dehydrogenase-mediated dehydrogenation of morphine to MO.

Animals↗
PubMed1987

Purification and characterization of a serine hydroxymethyltransferase from an obligate methylotroph, Hyphomicrobium methylovorum GM2.

A serine hydroxymethyltransferase was purified to complete homogeneity from a serine-producing obligate methylotroph, Hyphomicrobium methylovorum GM2. The enzyme has a molecular mass of about 98 kDa and consists of two subunits of identical molecular mass. The holoenzyme exhibits absorption maxima at 280 nm, 340 nm and 415 nm in potassium phosphate buffer, pH 7.3, the last of which shifts with a change in pH (6.0-7.5) and contains 2 mol pyridoxal phosphate/mol enzyme. The holoenzyme is converted to the apoenzyme on incubation with phenylhydrazine and reconstituted on the addition of pyridoxal phosphate. The enzyme activity was inhibited on the addition of several sulfhydryl-modifying reagents and then recovered with 2-mercaptoethanol. One sulfhydryl group per subunit was found to be responsible for the activity. Isoelectric focusing showed that the enzyme has a pI of 5.6. The Km values for glycine, L-serine and DL-beta-phenylserine are 0.046 mM, 0.15 mM and 33 mM respectively.

Chromatography, High Pressure Liquid↗
PubMed1986

Guinea-pig liver morphine 6-dehydrogenase as a naloxone reductase.

Elution profiles of guinea-pig liver naloxone reductase and morphine 6-dehydrogenase on Matrex green A, Sephadex G-100 and DEAE-cellulose (DE32) column chromatography used sequentially in the purification procedure were identical. The ratios of the two enzyme activities were almost constant throughout all the purification steps. The two enzymes were similarly more stable at pH 6.0 than at pH 8.0 on storage at 4 degrees. The reversible inactivation of the two enzymes by the removal of 2-mercaptoethanol from the enzyme solution was the same. Inhibitory effects of lithocholic acid, CuSO4, quercitrin, phenylarsine oxide, and prostaglandin E1 on the two enzymes were almost the same. These results indicated that naloxone reductase is identical to morphine 6-dehydrogenase in the guinea-pig liver. For the reduction of naloxone, the enzyme utilized either NADPH or NADH as cofactor, and pH optima were 6.8 with NADPH and 6.2 with NADH. The Km values for NADPH and NADH were 6.5 and 2.2 microM respectively. The Vmax values for naloxone were 1.2 units/mg protein with NADPH and 0.5 unit/mg protein with NADH. The Km values for naloxone were 0.27 mM with NADPH and 0.44 mM with NADH. The reaction product formed by the enzyme was identified as 6 alpha-naloxol by thin-layer and gas-liquid chromatographic analyses. Accordingly, it is clear that the enzyme catalyzes the stereospecific reduction of naloxone to form the 6 alpha-hydroxyl congener.

Alcohol Oxidoreductases↗
PubMed1986

Crystalline serine hydroxymethyltransferase from an obligate methylotroph, Hyphomicrobium methylovorum.

Optimal culture conditions of a methylotrophic Hyphomicrobium methylovorum and improved purification of serine hydroxymethyltransferase from the bacterium were established for the large-scale preparation of the enzyme. The first crystalline serine hydroxymethyltransferase from the microbial source was obtained in the apo form and found to be homogeneous. Amino acid analysis revealed that the enzyme had higher value per subunit for acidic and neutral amino acids than that from rabbit liver. The carboxy-terminal amino acid analysis suggested the sequence -Ile-Ala-Tyr.

Amino Acids↗
PubMed1986

Effects of glutathione and phenobarbital on the toxicity of codeinone.

The ability of sulfhydryl compounds to provide protection against the acute toxicity of codeinone, a toxic metabolite of codeine, was investigated in mice. Subcutaneous administration of codeinone produced a slight reduction in hepatic glutathione concentration. Pretreatment of the mice with glutathione or cysteine significantly increased the survival rate for mice given a lethal dose of codeinone (10 mg/kg). The lethality of codeine was lowered by naloxone, whereas that of codeinone was not blocked by naloxone. The strychnine-like convulsant action of codeinone could be prevented by phenobarbital pretreatment. Glutathione pretreatment reduced the amounts of radioactivity in tissues of mice injected with [N-methyl-3-H]codeinone. A possible explanation for these observations is that glutathione reacts in vivo with codeinone and plays a role as a scavenger of this compound. This assumption is supported by the observation that codeinone reacts non-enzymatically with glutathione under physiological conditions.

Animals↗
PubMed1985

In vitro formation of codeinone from codeine by rat or guinea pig liver homogenate and its acute toxicity in mice.

In vitro metabolism of codeine was investigated by using a 9000 g supernatant fraction of rat or guinea pig liver homogenate. When a mixture of [N-14CH3] and [C-6-3H]codeine was incubated with the rat liver 9000 g supernatant fraction in the presence of NAD, formation of codeinone, morphine and norcodeine was detected. Replacement of NAD with NADP abolished only the formation of codeinone. On the other hand, when the guinea pig liver homogenate was used in the presence of NAD, codeinone was the main metabolite of codeine. NADP was also ineffective in forming codeinone with the guinea pig liver homogenate. The acute toxicity of codeinone was thirty times higher than that of codeine. The roles of codeinone as a metabolic intermediate and in the acute toxicity of codeine are discussed.

Animals↗
PubMed1985

Purification and characterization of guinea pig liver morphine 6-dehydrogenase.

Morphine 6-dehydrogenase, which catalyzes the dehydrogenation of morphine to morphinone, has been purified about 440-fold from the soluble fraction of guinea pig liver with a yield of 38%. The purified enzyme was a homogeneous protein on polyacrylamide gel disc electrophoresis and isoelectric focusing. The molecular weight and isoelectric point of the enzyme were 29,000 and 7.6, respectively. The enzyme utilizes both NAD and NADP as a cofactor, and the Km values were 0.12 mM for NAD and 0.42 mM for NADP. The Vmax values for morphine were 588 milliunits/mg of protein (with NAD) and 1600 milliunits/mg of protein (with NADP). The Km values for morphine were 0.12 mM (with NAD) and 0.49 mM (with NADP). The enzyme also exhibited activity for morphine-related compounds: nalorphine, normorphine, codeine, and ethylmorphine; however, 7,8-saturated congeners such as dihydromorphine and dihydrocodeine were poor substrates. The enzyme was inactivated by removal of 2-mercaptoethanol from the enzyme solution. The inactivated enzyme was rapidly recovered by the addition of 2-mercaptoethanol. Phenylarsine oxide and CdCl2 (dithiol modifiers) inhibited competitively toward cofactor binding and noncompetitively toward morphine binding. These results suggest that the enzyme possesses the essential thiol groups, probably vicinal dithiol, at or near the cofactor-binding site. Using the partially purified enzyme, 8-(2-hydroxyethylthio)dihydromorphinone was isolated as the product and identified by UV, mass, and NMR spectra. It was confirmed that morphinone proposed as the dehydrogenation product was nonenzymatically and covalently bound to 2-mercaptoethanol. Accordingly, the isolated morphinone-2-mercaptoethanol conjugate must be formed by two steps: enzymatic production of morphinone from morphine and then nonenzymatic binding of 2-mercaptoethanol to morphinone.

Alcohol Oxidoreductases↗
PubMed1985

Biotransformation of N-methylcyclobarbital in vivo in rabbit and rat.

Metabolism of N-methylcyclobarbital in the rabbit and rat has been studied in vivo for the purpose of comparison with the C5-methylated analogue, hexobarbital. In the rabbit, the main route of the metabolism of N-methylcyclobarbital is glucuronide formation after hydroxylation at the 3'-position of the parent compound. Dehydrogenation of the 3'-hydroxy product, a major pathway in the metabolism of hexobarbital, was a minor route in the case of N-methylcyclobarbital. In addition, a new type of metabolite, thought to be dihydroxylated products from spectral studies, was isolated. In the rat, there were almost no differences in the metabolic fates of N-methylcyclobarbital and hexobarbital. Profiles of metabolism of four analogous barbiturates (N-methylcyclobarbital, hexobarbital, cyclobarbital and norhexobarbital), which have a cyclohexene ring on the 5-carbon, reveal the contribution of alkyl substituents in the barbiturate ring on the bioavailability and metabolism of these compounds.

Animals↗
PubMed1982

Effect of morphinone on opiate receptor binding and morphine-elicited analgesia.

Specific binding of 3H-naloxone to opiate receptors was found to be irreversibly inactivated by morphine. This inactivation exhibited pseudo-first-order kinetics. The presence of sulfhydryl compounds or morphine during incubation with morphinone proved good protection. Morphinone-pretreated mice blocked the analgesic effect of morphine. The possible mechanism for these observations is proposed as follows: morphinone binds covalently to sulfhydryl group of opiate receptors, and inactivates irreversibly opiate binding sites, thus blocking the analgesic effect of morphine.

Analgesia↗
PubMed1982

Protective effect of sulfhydryl compounds on acute toxicity of morphinone.

The ability of sulfhydryl compounds to provide protection against the acute toxicity of morphinone was investigated in mice. Subcutaneous administration of morphinone produced a reduction of hepatic non-protein sulfhydryl concentration. Pretreatments of mice with glutathione or cysteine significantly increased the survival rate of mice given a lethal dose of morphinone, whereas morphinone lethality was markedly potentiated by diethyl maleate. On the other hand, the administration of morphine produce a dose dependent reduction of hepatic non-protein sulfhydryl contents. However, neither glutathione nor cysteine protected mice from the acute toxicity of morphine. A possible explanation for these observations was proposed as follows: morphine is oxidized by morphine 6-dehydrogenase to morphinone, and the morphinone thus produced decreases the sulfhydryl contents in the liver. This mechanism is supported by the fact that morphinone reacts easily with glutathione and cysteine in vitro.

Animals↗
PubMed1977

Purification and properties of a new testosterone 17beta-dehydrogenase (NADP+) from guinea-pig liver.

As a result of studies of guinea-pig live testosterone 17beta-dehydrogenase (NADP+) (EC 1.1.1.64), a new testosterone 17beta-dehydrogenase was discovered. The new enzyme was purified to a single homogeneous protein from the 105 000 g-supernatant fraction of guinea-pig liver by (NH4)2SO4 fractional precipitation and two gel-filtration stages, DEAE-cellulose column chromatography and hydroxyapatite column chromatography. It was characterized by many properties. The enzyme has almost the same properties as the classical testosterone 17beta-dehydrogenase (NADP+) (EC 1.1.1.64), with respect to cofactor requirement, pH optima for dehydrogenation, effect of phosphate ion on the NAD+-dependent reaction and molecular weight, but characteristic differences were observed in substrate-specificity between the two dehydrogenases. With various androstane derivatives, the configuration of the A/B-ring junction was closely connected with enzyme activity. 5alpha-Androstanes, such as 5alpha-androstane-3alpha,17beta-diol, 5alpha-androstane-3beta,17beta-diol and 17beta-hydroxy-5alpha-androstan-3-one, and 5beta-congeners, such as 5beta-androstane-3alpha,17beta-diol, 5beta-androstane-3beta,17beta-diol and 17beta-hydroxy-5beta-androstan-3-one, served as substrates for both the EC 1.1.1.64 enzyme and the new enzyme. The EC 1.1.1.64 enzyme oxidized testosterone more rapidly than did the new enzyme. These comparisons were based on the relative activities, apparent Km values and apparent Vmax values.

Androstanes↗
PubMed1977

Dehydrogenation of indanol by rabbit liver 3-hydroxyhexobarbital dehydrogenase.

1. Among the several enzyme activities in rabbit liver cytosol able to dehydrogenate 1-indanol, only the main activity was not separable from 3-hydroxyhexobarbital dehydrogenase during purification including polyacrylamide gel disc electrophoresis. 2. Results of mixed substrate method indicated that the same enzyme catalyses the dehydrogenation of 1-indanol and 3-hydroxyhexobarbital. The ratio between the two dehydrogenation activities was almost constant as the enzyme underwent thermal inactivation. The Ki values of p-chloromercuribenzoate, the Km values for NAD+, and the Km values for NADP+ were very similar for the two dehydrogenations. These results lead to the conclusion that the same enzyme catalyses the dehydrogenation of 3-hydroxyhexobarbital and 1-indanol. 3. 1-Tetralol, 1-acenaphthenol, 9-fluorenol, thiochroman-4-ol and 4-chromanol also served as substrate of the enzyme, but 2-indanol, 2-tetralol, and trans- and cis-indan-1,2-diol were not oxidized. 4. Reversibility of the reaction was also confirmed using 1-indanone as substrate.

Alcohol Oxidoreductases↗