Mitsuru Tateishi

Occurrence of cholesterol 7α- and 7β-hydroperoxides in rat skin as aging markers

Abstract Evidence for presence of cholesterol 7α- and 7β-hydroperoxides in rat skin was presented for the first time. The 7-hydroperoxides in rat skin were reduced with sodium borohydride and trimethylsilylated for identification with the authentic compounds by gas chromatography/mass spectrometry. A content of cholesterol 7-hydroperoxides in rat skin, determined by high performance liquid chromatography with a chemiluminescence detector, highly correlated with the age of rats ( r = 0.874; between 1 and 45 weeks old), indicating that cholesterol 7α- and 7β-hydroperoxides were good markers for aging.

Involvement of leukocytes in the oxygenation and chlorination reaction of phenylbutazone

A center carbon atom of 1,3-diketone moiety of phenylbutazone was oxidized to give three metabolites--4-hydroxyphenylbutazone (metabolite I), 4-hydroperoxyphenylbutazone (metabolite II) and 4-chlorophenylbutazone (metabolite III)--by the action of enzymes present in leukocyte extract obtained from peritoneal exudate of rats. Both metabolites II and III were produced by peroxidases, while metabolite I was produced by enzymes other than the peroxidases.

Involvement of Cystathionase in the Formation of Alkane-Thiols from Corresponding Cysteine Conjugates

1. Rat liver cystathionase [EC.4.4.1.1] mediated a cleavage reaction of the C-S bond of S-alkyl-L-cysteine conjugates to give equimolar amounts of corresponding thiols, ammonia, and pyruvic acid. Neither S-aryl- nor S-aralkyl-L-cysteine conjugates were acceptable substrates. 2. The Km value for S-(tert-butyl)-L-cysteine was 0.3 mM at pH 8.5 in Tris-HCl buffer. 3. The S-alkyl-L-cysteine lyase activity of cystathionase was inhibited with carbonyl reagents and dithiothreitol. 4. The present finding that cystathionase has activity towards cysteine conjugates may provide some insights into the role of this enzyme in the metabolism of xenobiotics.

Purification and properties of methyl sulfoxide reductases from rat kidney

Two kinds of enzymes (tentatively designated methyl sulfoxide reductases I and II) responsible for the reduction of the methyl sulfoxide group on various xenobiotics have been purified about 223- and 155-fold, respectively, from rat kidney cytosol. The molecular weight was determined to be 12,000 +/- 1000 for methyl sulfoxide reductase I and 24,000 +/- 1000 for methyl sulfoxide reductase II. Thioredoxin or dithiothreitol is essential in order for the reducing activity to occur. The respective Km values of p-bromophenylmethyl sulfoxide were 2.75 and 1.30 mM for methyl sulfoxide reductases I and II. Replacement of the methyl group on the sulfur atom with a longer alkyl group or phenyl group caused a markedly low or negligible substrate activity.

[31] Cysteine conjugate β-lyase

Publisher Summary This chapter presents a procedure for the preparation of cysteine conjugate β-lyase. The Pprinciple of assay method states that cysteine conjugates of aromatic compounds (R) serve as the substrate of β-lyase. Utilizing a substrate labeled with 35 S, the product of the reaction, R-[ 35 S]H, can be measured after converting to R-[ 35 S]CH 3 with methyl iodide. The derivatized product, thus, can be purified by thin-layer chromatography and its radioactivity can be determined. Purification procedure involves the extraction of supernatant fluid, followed by heat treatment and the addition of ammonium sulfate. After thisthe enzyme solution is concentrated to 20 ml with a Diaflo PM-10 membrane filter and is applied to a column of DEAE-cellulose equilibrated with the same buffer; further, the combined enzyme preparation is concentrated to approximately 15 ml with a Diaflo PM-10 membrane and is applied to a Sephadex G-200 column. Overall, 1100-fold purification is achieved through the Sephadex step.

Specific induction by glucocorticoids of steroid esterase in rat hepatic microsomes and its release into serum

Steroid esterase hydrolysing methylprednisolone 21-hemisuccinate was induced specifically and markedly in hepatic microsomes and serum of rats by various glucocorticoids. Among the glucocorticoids examined, dexamethasone and betamethasone showed the highest potency to induce the hepatic steroid esterase, the induction ratio being 32 and 33 times higher than the basal level (about 160 mU/g liver), respectively. Steroid esterase in the serum was induced greatly by fluocinolone acetonide and betamethasone to 92 and 79 times of the basal level of about 16 mU/mL, respectively, followed by dexamethasone and methylprednisolone. When dexamethasone was given to rats, the enzyme in other tissues except for duodenum and small intestine (of which activity was lowered to 50% of the basal level) was also elevated, but the induction ratio was much lower than that in the liver and serum. The induction of the steroid esterase is probably due to stimulation of de novo synthesis of the enzyme by glucocorticoids, because the elevation of esterase activity was inhibited by treatment with cycloheximide (a translation inhibitor) and actinomycin D (a transcription inhibitor), and about 4- and 10-hr lag time was observed before the elevation of esterase activity in liver and serum, respectively. Coupled with these observations the following results indicate that the steroid esterase in serum is probably synthesized in the liver and subsequently released into the blood via the Golgi apparatus: (1) when the liver of rats treated with dexamethasone was subjected to perfusion with a recycling system, significant amounts of steroid esterase were released into the perfusate; (2) anti-hepatic esterase antibody inhibited the steroid esterase activity not only in the liver but also in serum; and (3) monensin, which prevents the secretion of various kinds of secretory proteins by disrupting the function of the Golgi apparatus, inhibited the elevation of the steroid esterase activity in serum by dexamethasone but did not affect the induction in liver.

Purification and characterization of glucocorticoid-inducible steroid esterase in rat hepatic microsomes

A steroid esterase hydrolysing methylprednisolone 21-hemisuccinate was purified from the hepatic microsomes of rats treated with dexamethasone, a potent inducer of the esterase. The enzyme was solubilized by Lubrol WX and purified up to 30-fold over the microsomal fraction by ammonium sulfate fractionation and successive chromatographies with gel permeation, DEAE-cellulose and hydroxylapatite. The steroid esterase thus purified showed a single band and a molecular mass of 58 kDa on SDS-polyacrylamide gel electrophoregram. The enzyme appears likely to exist as two interconvertible forms, which can be distinguished by pI values, pI 4.9 and 5.1. The enzyme was completely inhibited by organic phosphates, indicating that it can be classed as a carboxylesterase (EC 3.1.1.1). Both negatively charged and uncharged esters of several steroids (methylprednisolone, hydrocortisone, deoxycorticosterone and dehydrotestosterone) as well as various non-steroidal esters including 4-nitrophenyl esters were hydrolysed by the enzyme, but none of the amides were substrates. The enzyme showed higher activity with increasing lipophilicity of the substrates. It is noticeable that the optimum pH for charged esters was 5.5, whereas the highest activity was observed around pH 7-8 for uncharged esters. When methylprednisolone 21-hemisuccinate (one of the charged esters) was used as substrate, the Km value was 2.8 mM and Vmax was 59.3 mumol/mg protein for 1 min at the optimum pH of 5.5. Regarding the methyl ester of methylprednisolone 21-hemisuccinate, Km and Vmax values were 1.8 mM and 193 mumol/mg protein/min, respectively, at the optimum pH of 7.0. On the basis of these results, the enzyme is most likely a carboxylesterase.

Purification and Characterization of Cysteine Conjugate Transaminases from Rat Liver

1. Soluble cysteine-conjugate alpha-ketoglutarate transaminase (CAT-I) was purified about 670-fold from rat liver cytosol using s-(p-bromophenyl)-L-cysteine as amino acid substrate. The enzyme preparation of the final step of purification showed a single band in polyacrylamide gel electrophoresis. CAT-I accounted for 64% of the transaminase activity in cytosol. 2. The mol. wt of the enzyme was about 64,000 as determined by gel filtration. Respective Km values for s-(p-bromophenyl)-L-cysteine and alpha-ketoglutaric acid were 1.0 and 1.3 mM in Tris-acetate buffer (pH 7.0). Aminooxyacetic acid, hydroxylamine, and KCN inhibited the enzyme activity. 3. In addition to CAT-I, two isozymes (CAT-IIA and CAT-IIB) were partially purified from rat liver cytosol. In respect of mol. wt, substrate specificity towards cysteine conjugates, and several other properties, CAT-IIA and CAT-IIB were very similar to CAT-I. However, differences were observed for these enzymes in the rate of reverse reaction (formation reaction of cysteine conjugates and alpha-ketoglutaric acid) and substrate specificity towards L-aspartic acid and L-cysteinesulphinic acid.

The Metabolism of Isopropylantipyrine in the Rat and Man

1. The major urinary metabolite of isopropylantipyrine in the rat and man was an enol glucuronide of N-desmethyl isopropylanipyrine, which comprised about 80% of the whole urinary metabolites. 2. Six other minor metabolites occurred in rat urine; all lacked the N-methyl group of the parent drug, and some had undergone further oxidation at the isopropyl group, the phenyl group or in the C-4 position.

DEVELOPMENT OF MICROPLATE-RADIOLUMINOGRAPHY FOR 32P

Basic research for designing a microplate which can be applied to determination of 32P by radioluminography was carried out. It was proven that 32P can be determined by using the microplate which has plural planchets placed at intervals on a thin plate and a brass collimator that fills the gaps between the planchets. The microplate is joined with the collimator, and exposed to an imaging plate for 24 h. The detection limit of the proposed method was estimated to be 6 m Bq. The cross-talk ratio was negligibly small (0.2%).