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H Inano

Publications and source records attributed to H Inano.

At least 73 records · Page 4Linked to original sources
PubMed1986

Purification and characterization of rat adrenal 3 beta-hydroxysteroid dehydrogenase with steroid 5-ene-4-ene-isomerase.

After solubilization of rat adrenal microsomes with sodium cholate, 3 beta-hydroxysteroid dehydrogenase with steroid 5-ene-4-ene isomerase (abbreviated as steroid isomerase) activity was purified to a homogeneous state. The following characteristics of the enzyme were obtained: 3 beta-Hydroxysteroid dehydrogenase together with steroid isomerase was detected as a single protein band in SDS-polyacrylamide gel electrophoresis, where its mol. wt was estimated as 46,500. Either NAD+ or NADH was required for demonstration of steroid isomerase activity. Treatment of the enzyme with 5'-p-fluorosulfonylbenzoyladenosine, an affinity labeling reagent for NAD+-dependent enzyme, diminished both the enzyme activities.

3-Hydroxysteroid Dehydrogenases↗
PubMed1985

Affinity labeling of the cofactor-binding site of estradiol 17 beta-dehydrogenase of human placenta by 5'-p-fluorosulfonylbenzoyl adenosine.

An analogue of adenosine nucleotide, 5'-p-fluorosulfonylbenzoyl adenosine (5'-FSB-Ado), appears to interact irreversibly with the cofactor-binding site of estradiol 17 beta-dehydrogenase of human placenta. This conclusion is based on the following observations: (1) The estradiol 17 beta-dehydrogenase is inhibited by 5'-FSB-Ado. When NAD+ is the variable component in the presence of saturated amount of steroid, the type of the inhibition is competitive in nature. When the steroid is the variable component, mode of the inhibition becomes non-competitive. The results suggest reversible binding of 5'-FSB-Ado to the cofactor-binding site of the dehydrogenase. (2) 5'-FSB-Ado inactivates irreversibly the estradiol 17 beta-dehydrogenase in time- and concentration-dependent manners, following pseudo-first-order kinetics. But, no inactivation is observed in the presence of p-fluorosulfonylbenzoic acid, suggesting that adenosine moiety of 5'-FSB-Ado is essential for the affinity labeling of estradiol 17 beta-dehydrogenase. (3) NADP+ protects completely estradiol 17 beta-dehydrogenase from the inactivation of 5'-FSB-Ado, whereas NAD(H) is partially protective against the inactivation, suggesting that phosphate moiety at 2'-position of NADP+ disturbs the covalent binding of 5'-FSB-Ado at or near the cofactor-binding site of the enzyme. (4) 2',5'-ADP shows the significant protection against the inactivation by 5'-FSB-Ado, but less effect is observed in the presence of nicotinamide mononucleotides. These results suggest that 5'-FSB-Ado is an affinity ligand for binding-site of adenosine nucleotide moiety of the cofactor.

17-Hydroxysteroid Dehydrogenases↗
PubMed1985

The presence of essential carboxyl group for binding of cytochrome c in rat hepatic NADPH-cytochrome P-450 reductase by the reaction with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

NADPH-cytochrome P-450 reductase (EC 1.6.2.4) purified from rat hepatic microsomal fraction was inactivated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), a specific agent for modification of carboxyl groups in a protein. The inactivation exhibited pseudo-first order kinetics with a reaction order approximately one and a second-order-rate constant of 0.60 M-1 min-1 in a high ionic strength buffer and 0.08 M-1 min-1 in a low ionic strength buffer. By treatment of NADPH-cytochrome P-450 reductase with EDC, the pI value changed to 6.5 from 5.0 for the native enzyme, and the reductase activity for cytochrome c, proteinic substrate, was strongly inactivated. When an inorganic substrate, K3Fe(CN)6, was used for assay of the enzyme activity, however, no significant inactivation by EDC was observed. The rate of inactivation by EDC was markedly but not completely decreased by NADPH. Also, the inactivation was completely prevented by cytochrome c, but not by K3Fe(CN)6 or NADH. The sulfhydryl-blocked enzyme prepared by treatment with 5,5'-dithio-bis(2-nitrobenzoic acid), which had no activity, completely recovered its activity in the presence of dithiothreitol. When the sulfhydryl-blocked enzyme was modified by EDC, the enzyme in which the carboxyl group alone was modified was isolated, and its activity was 35% of the control after treatment with dithiothreitol. In addition, another carboxyl reagent, N-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward reagent K), decreased cytochrome c reductase activity of NADPH-cytochrome P-450 reductase. These results suggest that the carboxyl group of NADPH-cytochrome P-450 reductase from rat liver is located at or near active-site and plays a role in binding of cytochrome c.

Animals↗
PubMed1984

The reaction of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide with an essential carboxy group of human placental estradiol 17 beta-dehydrogenase.

Estradiol 17 beta-dehydrogenase from human placenta was inactivated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), a specific reagent for modification of carboxyl group in a protein. Inactivation of the dehydrogenase by EDC progressed in time-dependent, concentration-dependent and ionic strength-dependent manners. In the presence of NAD+ or NADP+, the inactivation rate by EDC was markedly accelerated. NMN+ and acetylpyridine-DPN+ also appeared to increase the rate of modification of the dehydrogenase by EDC, but no acceleration of the inactivation was observed in the presence of the reduced forms of those nucleotides. Inactivation by EDC in the presence of NAD+ was accompanied by incorporation of 2 mol of NAD+/mol of dimer (Mr = 68,000) of the enzyme. When the dehydrogenase was incubated with EDC together with estriol, the activity was lost slower than that of control without the steroid. Similarly, the inactivation was prevented by addition of estrone and estradiol as well as aminoestrogens. From those results, we suggest that the carboxyl group essential for the function of estradiol 17 beta-dehydrogenase is buried in the enzyme molecule, and the positive charge of nicotinamide N1 of the oxidized form of the cofactor plays an important role for enhancement of the inactivation by EDC.

17-Hydroxysteroid Dehydrogenases↗
PubMed1984

Contribution of cytochrome b5 to androgen synthesis in rat testicular microsomes.

Cytochrome b5 was purified from porcine testicular microsomes, and its amino acid composition was determined. Rabbit antibody against the purified cytochrome b5 was prepared in order to study the contribution of cytochrome b5 to testicular microsomal oxygenases related to androgen production. In the presence of NADPH alone as the electron donor, the antibody against cytochrome b5 inhibited the activities of steroid 17 alpha-hydroxylase and C-17-C-20 lyase of rat testicular microsomal fraction. Addition of NADH to the NADPH-supported oxygenase assay system enhanced both steroid oxygenase activities, and addition of the antibody against cytochrome b5 decreased the NADH-caused stimulation of steroid 17 alpha-hydroxylase and C-17-C-20 lyase activities. When dehydroepiandrosterone and NAD+ were added as substrates for 3 beta-hydroxy-delta 5-steroid dehydrogenase in order to synthesize NADH by enzymatic reaction, the NADPH-supported activities of steroid 17 alpha-hydroxylase and C-17-C-20 lyase were further stimulated as compared with the addition of NADH, and this stimulation was suppressed by the antibody against cytochrome b5. These results suggest that cytochrome b5, together with 3 beta-hydroxy-delta 5-steroid dehydrogenase, contributes to the activities of steroid 17 alpha-hydroxylase and C-17-C-20 lyase in the testicular microsomal fraction.

3-Hydroxysteroid Dehydrogenases↗
PubMed1984

Oxygenation of 18-hydroxycorticosterone as the final reaction for aldosterone biosynthesis.

Incubation of 18-hydroxycorticosterone with the sonicated mitochondrial preparation of bovine adrenal glomerulosa tissue leads to the production of aldosterone, as measured by radioimmunoassay. The in vitro production of aldosterone from 18-hydroxycorticosterone requires both molecular oxygen and NADPH, and is inhibited by carbon monoxide. Cytochrome P-450 inhibitors such as metyrapone, SU 8000, SU 10603, SKF 525A, amphenone B and spironolactone decrease the biosynthesis of aldosterone from 18-hydroxycorticosterone. These results support the conclusion that the final reaction in aldosterone synthesis from 18-hydroxycorticosterone is catalyzed by an oxygenase, but not by 18-hydroxysteroid dehydrogenase. By the same preparation, the production of [3H]aldosterone but not [3H]18-hydroxycorticosterone from [1,2-3H]corticosterone is decreased in a dose-dependent manner by addition of non-radioactive 18-hydroxycorticosterone.

18-Hydroxycorticosterone↗
PubMed1984

Chemical modification of tyrosine residues in active-site of human placental estradiol 17 beta-dehydrogenase by tetranitromethane.

The native estradiol 17 beta-dehydrogenase purified from human placenta was rapidly inactivated by tetranitromethane, which is a reagent for chemical modification of tyrosine and cysteine residues. The inactivation followed pseudo-first-order reaction kinetics, and the activity was partially recovered by addition of dithiothreitol. On the other hand, the enzyme sulfhydryl-blocked by 5,5'-dithiobis(2-nitrobenzoic acid) was treated with tetranitromethane, and the activity was assayed in the presence of dithiothreitol. Tetranitromethane inactivated the enzyme in a time-dependent manner, following pseudo-first-order kinetics. The rate of inactivation was significantly decreased by addition of NADP(H), 2'-AMP, 5'-ATP, 2',5'-ADP, 3-pyridine aldehyde-DPN and 3-acetylpyridine-DPN(reduced form). These results suggest that the tyrosyl residues are located at or near the cofactor-binding site of the estradiol 17 beta-dehydrogenase and play an essential role in the catalytic function of the enzyme.

17-Hydroxysteroid Dehydrogenases↗
PubMed1983

Affinity labeling of arginyl residues at the catalytic region of estradiol 17 beta-dehydrogenase from human placenta by 16-oxoestrone.

16-Oxoestrone inhibited competitively the activity of estradiol 17 beta-dehydrogenase from human placenta against estradiol in phosphate buffer (pH 7.2), suggesting reversible binding of 16-oxoestrone to the substrate-binding site. 16-Oxoestrone irreversible inactivated the estradiol 17 beta-dehydrogenase in borate buffer (pH 8.5) in a time-dependent manner, following pseudo-first-order kinetics. The rate constant (k3) obtained for the inactivation by 16-oxoestrone was 8.3 x 10(-4) s-1. The rate of inactivation was significantly decreased by addition of estrone, estradiol, estriol, NAD(H) and NADP+. Also, the rate was reduced markedly by 2'AMP, 5'ATP and 2',5' ADP, but not by NMN(H) and 3-pyridinealdehyde adeninediphospho nucleotide. The inactivation by 16-oxoestrone was neither prevented by sodium azide nor influenced by light. From these data, 16-oxoestrone, an alpha-dicarbonyl steroid, was suggested to inactive estradiol 17 beta-dehydrogenase by modification of arginyl residues located around the substrate-binding site of the enzyme. Biphasic inactivation of the enzyme by 16-oxoestrone was observed with an increase of modified arginyl residues. The first phase of the inactivation was regarded as an affinity labeling of the arginyl residues at or near the substrate-binding site of the enzyme. Stoichiometry of the inactivation indicated that two arginyl residues were essential for maintenance of the enzyme activity. The second phase was considered as chemical modification of the arginyl residues outside of the catalytic region of the enzyme.

17-Hydroxysteroid Dehydrogenases↗
PubMed1983

Photochemical inactivation of human placental estradiol 17 beta-dehydrogenase in the presence of 2,3-butanedione.

Estradiol 17 beta-dehydrogenase of human placenta was rapidly inactivated by 2,3-butanedione under u.v. light, and no protection against the inactivation was observed in the presence of sodium azide. Under ordinary laboratory illumination, the inactivation was biphasically progressed in time-dependent and concentration-dependent manners, while a partial protection from the inactivation was indicated by sodium azide. These results suggest that the inactivation mechanism of the dehydrogenase by 2,3-butanedione under laboratory illumination is different from that under u.v. light. Therefore, the inactivation under laboratory illumination proceeded by a reaction with excited singlet molecular oxygen (1 delta g or 1 sigma +g states), and that under u.v. light was caused by a reaction of substrate with triplet sensitizer. In the presence of NADP+, the inactivation of the enzyme by 2,3-butanedione was markedly reduced. The maximum protection by NADP+ was about 80% of the initial enzyme activity. Amino acid analysis of the enzyme treated with 2,3-butanedione under laboratory illumination showed that the modified enzyme contained considerably less of the following amino acids than the native enzyme: histidine, arginine, threonine, methionine, tyrosine and leucine. In addition, other dicarbonyl reagents, 1,4-dibromo-2,3-butanedione, 1-phenyl-1,2-propanedione, phenylglyoxal, 16-oxoestrone, 1,2-cyclohexanedione, 2,4-pentanedione and glyoxal were found to decrease the dehydrogenase activity in various degree.

17-Hydroxysteroid Dehydrogenases↗
PubMed1983

Functional site of human placental estradiol 17 beta-dehydrogenase involves tyrosine residues, as evidenced by reaction to p-nitrobenzenesulfonyl fluoride.

Native estradiol 17 beta-dehydrogenase (EC 1.1.1.62) from human placenta was inactivated in time dependent manner by p-nitrobenzenesulfonyl fluoride (NBSF), which is a reagent for chemical modification of tyrosine. The sulfhydryl-blocked enzyme by 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) was also reacted with NBSF more slowly in pseudo-first-order kinetics. After the sequential treatments with DTNB, NBSF and dithiothreitol (DTT), the enzyme in which tyrosine residues alone were modified was isolated, and its activity was decreased. These results suggest that tyrosyl residues of the estradiol 17 beta-dehydrogenase from human placenta are located at or near its catalytic site, and play a functional role in the enzyme reaction.

17-Hydroxysteroid Dehydrogenases↗
PubMed1982

Effects of age and 1,4,6-androstatriene-3,17-dione on activities of hCG-induced 19-hydroxylase and aromatase of rat testes.

The testicular aromatase activity was significantly increased by administration of hCG to 18-day old rats, but not increased in 29-day old rats. 1,4,6-Androstatriene-3,17-dione did not inhibit the in vitro conversion of testosterone to 19-hydroxytestosterone by the testicular cell-free homogenates of the hCG-treated 18-day old rats, but strongly suppressed production of estrogen from 19-hydroxyandrostenedione. On the other hand, the 19-hydroxylase activity of 18-day old rats stimulated by hCG was reduced to about 50% of the control value by SKF-525A, SU-4,885, SU-8,000 and SU-9,055 at their concentration of 10(-4) M. From our results, it is postulated that there are two distinct steps in the process of aromatization of testosterone, the one, the primary 19-hydroxylation which is inhibited by SKF-525A and SU-compounds, but not by 1,4,6-androstatriene-3,17-dione, and the other, aromatization of 19-hydroxylated androgen which is inhibited by both 1,4,6-androstatriene-3,17-dione and non-steroidal inhibitors.

Aging↗
PubMed1982

Kinetic mechanism of porcine testicular 17 beta-hydroxysteroid dehydrogenase.

A study on product inhibition of 17 beta-hydroxysteroid dehydrogenase from porcine testes was carried out by measuring the initial velocities of NADPH formation using testosterone as the substrate steroid. Type of inhibition by NADPH against NADP+ was competitive in both saturated and unsaturated concentrations of testosterone. In the saturated concentration of NADP+, activity of the enzyme was not inhibited by NADPH against testosterone. In the unsaturated concentrations of NADP+, however, NADPH brought mixed type inhibition against testosterone. The similar modes of inhibition by the product steroid, androstenedione were observed in the saturated and unsaturated concentrations of NADP+ and testosterone. The fluorescence of NADPH was increased in the presence of the enzyme, and fluorometric titration indicated that 1 mol of NADPH was bound to 1 mol of the 17 beta-hydroxysteroid dehydrogenase. Addition of testosterone to enzyme-NADPH complex reduced the intensity of fluorescence of NADPH, suggesting formation of testosterone-enzyme-NADPH complex as a ternary dead end complex. From the analyses of product inhibition and spectral changes of NADPH, the kinetic mechanism of the enzyme was revealed as rapid equilibrium random system with two dead end complexes which consisted of the two reduced reactants bound to the enzyme and the two oxidized ones bound to it.

17-Hydroxysteroid Dehydrogenases↗