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Joørn Hejgaard - One of the best experts on this subject based on the ideXlab platform.
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Differential gene expression for Suicide‐Substrate serine proteinase inhibitors (serpins) in vegetative and grain tissues of barley
Journal of experimental botany, 2003Co-Authors: Thomas H. Roberts, Salla Marttila, Soøren K. Rasmussen, Joørn HejgaardAbstract:Proteins of the serpin superfamily (∼43 kDa) from mature cereal grains are in vitro Suicide-Substrate Inhibitors of specific mammalian serine proteinases of the chymotrypsin family. However, unlike the 'standard-mechanism' serine proteinase inhibitors (
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differential gene expression for Suicide Substrate serine proteinase inhibitors serpins in vegetative and grain tissues of barley
Journal of Experimental Botany, 2003Co-Authors: Thomas H. Roberts, Salla Marttila, Soøren K. Rasmussen, Joørn HejgaardAbstract:Proteins of the serpin superfamily (∼43 kDa) from mature cereal grains are in vitro Suicide-Substrate Inhibitors of specific mammalian serine proteinases of the chymotrypsin family. However, unlike the 'standard-mechanism' serine proteinase inhibitors (<25 kDa), the biological functions of plant serpins are unknown. Expression studies of genes encoding members of three subfamilies of serpins (BSZx, BSZ4 and BSZ7) in developing grain and vegetative tissues of barley (Hordeum vulgare L.) showed that transcripts encoding BSZx, which inhibits distinct proteinases at overlapping reactive centres in vitro, were ubiquitous at low levels, but the protein could not be detected. EST analysis showed that expression of genes for serpins with BSZx-type reactive centres in vegetative tissues is widespread in the plant kingdom, suggesting a common regulatory function. For BSZ4 and BSZ7, expression at the protein level was highest in the maturing grain (≥15 d post-anthesis), where these serpins were localized by immunomicroscopy to the central and peripheral starchy endosperm, subaleurone, and (at lower levels) to the aleurone. Serpins were also localized to the meristem and vascular tissues of roots, and to the phloem of coleoptiles and leaves. The identification of BSZ4 in vegetative tissues by western blotting was confirmed for the roots by purification and amino acid sequencing, and for the leaves by in vitro reactive-centre loop cleavage studies. Plant serpins are likely to use their irreversible inhibitory mechanism in the inhibition of exogenous proteinases capable of breaking down seed storage proteins, and in the defence of specific cell types in vegetative tissues.
Thomas H. Roberts - One of the best experts on this subject based on the ideXlab platform.
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Differential gene expression for Suicide‐Substrate serine proteinase inhibitors (serpins) in vegetative and grain tissues of barley
Journal of experimental botany, 2003Co-Authors: Thomas H. Roberts, Salla Marttila, Soøren K. Rasmussen, Joørn HejgaardAbstract:Proteins of the serpin superfamily (∼43 kDa) from mature cereal grains are in vitro Suicide-Substrate Inhibitors of specific mammalian serine proteinases of the chymotrypsin family. However, unlike the 'standard-mechanism' serine proteinase inhibitors (
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differential gene expression for Suicide Substrate serine proteinase inhibitors serpins in vegetative and grain tissues of barley
Journal of Experimental Botany, 2003Co-Authors: Thomas H. Roberts, Salla Marttila, Soøren K. Rasmussen, Joørn HejgaardAbstract:Proteins of the serpin superfamily (∼43 kDa) from mature cereal grains are in vitro Suicide-Substrate Inhibitors of specific mammalian serine proteinases of the chymotrypsin family. However, unlike the 'standard-mechanism' serine proteinase inhibitors (<25 kDa), the biological functions of plant serpins are unknown. Expression studies of genes encoding members of three subfamilies of serpins (BSZx, BSZ4 and BSZ7) in developing grain and vegetative tissues of barley (Hordeum vulgare L.) showed that transcripts encoding BSZx, which inhibits distinct proteinases at overlapping reactive centres in vitro, were ubiquitous at low levels, but the protein could not be detected. EST analysis showed that expression of genes for serpins with BSZx-type reactive centres in vegetative tissues is widespread in the plant kingdom, suggesting a common regulatory function. For BSZ4 and BSZ7, expression at the protein level was highest in the maturing grain (≥15 d post-anthesis), where these serpins were localized by immunomicroscopy to the central and peripheral starchy endosperm, subaleurone, and (at lower levels) to the aleurone. Serpins were also localized to the meristem and vascular tissues of roots, and to the phloem of coleoptiles and leaves. The identification of BSZ4 in vegetative tissues by western blotting was confirmed for the roots by purification and amino acid sequencing, and for the leaves by in vitro reactive-centre loop cleavage studies. Plant serpins are likely to use their irreversible inhibitory mechanism in the inhibition of exogenous proteinases capable of breaking down seed storage proteins, and in the defence of specific cell types in vegetative tissues.
Soøren K. Rasmussen - One of the best experts on this subject based on the ideXlab platform.
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Differential gene expression for Suicide‐Substrate serine proteinase inhibitors (serpins) in vegetative and grain tissues of barley
Journal of experimental botany, 2003Co-Authors: Thomas H. Roberts, Salla Marttila, Soøren K. Rasmussen, Joørn HejgaardAbstract:Proteins of the serpin superfamily (∼43 kDa) from mature cereal grains are in vitro Suicide-Substrate Inhibitors of specific mammalian serine proteinases of the chymotrypsin family. However, unlike the 'standard-mechanism' serine proteinase inhibitors (
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differential gene expression for Suicide Substrate serine proteinase inhibitors serpins in vegetative and grain tissues of barley
Journal of Experimental Botany, 2003Co-Authors: Thomas H. Roberts, Salla Marttila, Soøren K. Rasmussen, Joørn HejgaardAbstract:Proteins of the serpin superfamily (∼43 kDa) from mature cereal grains are in vitro Suicide-Substrate Inhibitors of specific mammalian serine proteinases of the chymotrypsin family. However, unlike the 'standard-mechanism' serine proteinase inhibitors (<25 kDa), the biological functions of plant serpins are unknown. Expression studies of genes encoding members of three subfamilies of serpins (BSZx, BSZ4 and BSZ7) in developing grain and vegetative tissues of barley (Hordeum vulgare L.) showed that transcripts encoding BSZx, which inhibits distinct proteinases at overlapping reactive centres in vitro, were ubiquitous at low levels, but the protein could not be detected. EST analysis showed that expression of genes for serpins with BSZx-type reactive centres in vegetative tissues is widespread in the plant kingdom, suggesting a common regulatory function. For BSZ4 and BSZ7, expression at the protein level was highest in the maturing grain (≥15 d post-anthesis), where these serpins were localized by immunomicroscopy to the central and peripheral starchy endosperm, subaleurone, and (at lower levels) to the aleurone. Serpins were also localized to the meristem and vascular tissues of roots, and to the phloem of coleoptiles and leaves. The identification of BSZ4 in vegetative tissues by western blotting was confirmed for the roots by purification and amino acid sequencing, and for the leaves by in vitro reactive-centre loop cleavage studies. Plant serpins are likely to use their irreversible inhibitory mechanism in the inhibition of exogenous proteinases capable of breaking down seed storage proteins, and in the defence of specific cell types in vegetative tissues.
Salla Marttila - One of the best experts on this subject based on the ideXlab platform.
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Differential gene expression for Suicide‐Substrate serine proteinase inhibitors (serpins) in vegetative and grain tissues of barley
Journal of experimental botany, 2003Co-Authors: Thomas H. Roberts, Salla Marttila, Soøren K. Rasmussen, Joørn HejgaardAbstract:Proteins of the serpin superfamily (∼43 kDa) from mature cereal grains are in vitro Suicide-Substrate Inhibitors of specific mammalian serine proteinases of the chymotrypsin family. However, unlike the 'standard-mechanism' serine proteinase inhibitors (
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differential gene expression for Suicide Substrate serine proteinase inhibitors serpins in vegetative and grain tissues of barley
Journal of Experimental Botany, 2003Co-Authors: Thomas H. Roberts, Salla Marttila, Soøren K. Rasmussen, Joørn HejgaardAbstract:Proteins of the serpin superfamily (∼43 kDa) from mature cereal grains are in vitro Suicide-Substrate Inhibitors of specific mammalian serine proteinases of the chymotrypsin family. However, unlike the 'standard-mechanism' serine proteinase inhibitors (<25 kDa), the biological functions of plant serpins are unknown. Expression studies of genes encoding members of three subfamilies of serpins (BSZx, BSZ4 and BSZ7) in developing grain and vegetative tissues of barley (Hordeum vulgare L.) showed that transcripts encoding BSZx, which inhibits distinct proteinases at overlapping reactive centres in vitro, were ubiquitous at low levels, but the protein could not be detected. EST analysis showed that expression of genes for serpins with BSZx-type reactive centres in vegetative tissues is widespread in the plant kingdom, suggesting a common regulatory function. For BSZ4 and BSZ7, expression at the protein level was highest in the maturing grain (≥15 d post-anthesis), where these serpins were localized by immunomicroscopy to the central and peripheral starchy endosperm, subaleurone, and (at lower levels) to the aleurone. Serpins were also localized to the meristem and vascular tissues of roots, and to the phloem of coleoptiles and leaves. The identification of BSZ4 in vegetative tissues by western blotting was confirmed for the roots by purification and amino acid sequencing, and for the leaves by in vitro reactive-centre loop cleavage studies. Plant serpins are likely to use their irreversible inhibitory mechanism in the inhibition of exogenous proteinases capable of breaking down seed storage proteins, and in the defence of specific cell types in vegetative tissues.
Mitsuteru Numazawa - One of the best experts on this subject based on the ideXlab platform.
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aromatase inactivation by 2 substituted derivatives of the Suicide Substrate androsta 1 4 diene 3 17 dione
The Journal of Steroid Biochemistry and Molecular Biology, 2009Co-Authors: Madoka Takahashi, Wakako Handa, Hiromi Umeta, Saki Ishikawa, Kouwa Yamashita, Mitsuteru NumazawaAbstract:To gain the structure-activity relationship of Delta(1)-androstenediones (Delta(1)-ADs) as mechanism-based inactivator of aromatase, series of 2-alkyl- and 2-alkoxy-substituted Delta(1)-ADs (6 and 9) as well as 2-bromo-Delta(1)-AD (14) were synthesized and tested. All of the inhibitors examined blocked aromatase in human placental microsomes in a competitive manner. In a series of 2-alkyl-Delta(1)-ADs (6), n-hexyl compound 6f was the most powerful inhibitor with an apparent K(i) value of 31 nM. The inhibitory activities of 2-alkoxy steroids 9 decreased in relation to length of the alkyl chain up to n-hexyloxy group (K(i): 95 nM for methoxy 9a). All of the alkyl steroids 6 along with the alkoxy steroid 9, except for the ethyl and n-propyl compounds 6b and 6c, caused a time-dependent inactivation of aromatase. The inactivation rates (k(inact): 0.020-0.084 min(-1)) were comparable to that of the parent compound Delta(1)-AD. The inactivation was prevented by the Substrate AD, and no significant effect of l-cysteine on the inactivation was observed in each case. The results indicate that the 2-hexyl compound 6f act as the most powerful mechanism-based inactivator of aromatase among Delta(1)-AD analogs and may be submitted to the preclinical study in estrogen-dependent breast cancer.
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Aromatase Inactivation by a Suicide Substrate, Androst-5-ene-4, 7, 17-trione : The 5β, 6β-Epoxy-19-oxo Derivative, as a Possible Reactive Electrophile Irreversibly Binding to the Active Site
Biological & pharmaceutical bulletin, 1997Co-Authors: Mitsuteru Numazawa, Mii TachibanaAbstract:In order to understand the mechanism involved in the aromatase inactivation by androst-5-ene, 4, 7, 17-trione (4), a Suicide Substrate of aromatase, 5β, 6β-epoxyandrosta-4, 7, 17, 19-tetraone (6) was synthesized as a candidate for a reactive electrophile involved in irreversible binding to the active site of aromatase upon treatment of 19-oxo-5-ene steroid 5 with hydrogen peroxide in the presence of NaHCO3. The epoxide 6 was a competitive inhibitor of human placental aromatase (K1=34 μM); moreover, it inactivated the enzyme in an active-site-directed manner in the absence of NADPH (K1=36 μM, a rate constant for inactivation (kinact)=0.027 min-1). NADPH stimulated the inactivation rate, but the Substrate androst-4-ene-3, 17-dione blocked the inactivation. A nucleophile, L-cysteine, did not cause a significant change in the inactivation. When both the epoxide 6 and its 19-methyl analog 7 were subjected separately to a reaction with N-acetyl-L-cycteine in the presence of NaHCO3, the 19-oxo compound 6 disappeared from the reaction mixture more rapidly (t/1/2=6.0 min) than the 19-methyl analog 7 (t1/2=16 min). On the basis of these results, it is suggested that the 5β, 6β-epoxy-19-oxo steroid 6 may be the reactive electrophile that alkylates a nucleophilic residue of the amino acid of the active site.
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Mechanism for aromatase inactivation by a Suicide Substrate, androst-4-ene-3,6,17-trione: The 4β,5β-epoxy-19-oxo derivative as a reactive electrophile irreversibly binding to the active site
Biochemical pharmacology, 1996Co-Authors: Mitsuteru Numazawa, Ayako Mutsumi, Mii TachibanaAbstract:Abstract Aromatase is a cytochrome P450 enzyme complex that catalyzes the conversion of androst-4-ene-3,17-dione to estrone through three sequential oxygenations of the 19-methyl group. Androst-4-ene-3,6,17-trione (1) is a Suicide Substrate of aromatase. The inactivation mechanism for steroid 1 has been studied to show that the inactivation reaction proceeds through the 19-oxo intermediate 3. To further clarify the mechanism, 4β,5β-epoxyandrosta-3,6,17,19-tetraone (6) was synthesized as a candidate for a reactive electrophile involved in irreversible binding to the active site of aromatase, upon treatment of compound 3 with hydrogen peroxide in the presence of NaHCO3. The epoxide 6 inhibited human placental aromatase in a competitive manner (Ki = 30 μM); moreover, it inactivated the enzyme in an active-site-directed manner in the absence of NADPH (KI = 88 μM, fkinact = 0.071 min−1). NADPH and BSA both stimulated the inactivation rate without a significant change of the K, in either case (kinact: 0.133 or 0.091 min−1), in the presence of NADPH or BSA, respectively). The Substrate androst-4-ene-3,17-dione protected the inactivation, but a nucleophile, l -cysteine, did not. When both the epoxide 6 and its 19-methyl analog 4 were subjected separately to reaction with N-acetyl- l -cysteine in the presence of NaHCO3, the 19-oxo steroid 6 disappeared from the reaction mixture more rapidly ( T 1 2 = 40 sec ) than the 19-methyl analog 4 ( T 1 2 = 3.0 min ). The results clearly indicate that the 4β,5β-epoxy-19-oxo compound 6, which is possibly produced from 19-oxo-4-ene steroid 3 through the 19-hydroxy-19-hydroperoxide intermediate, is a reactive electrophile that irreversibly binds to the active site of aromatase. BIOCHEM PHARMACOL 52;8:1253–1259, 1996.
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Androst-5-ene-7,17-dione: a novel class of Suicide Substrate of aromatase.
Biochemical and biophysical research communications, 1992Co-Authors: Mitsuteru Numazawa, Ayako Mutsumi, Kumiko Hoshi, Yuko TanakaAbstract:Summary 5-En-7-one steroid 1 was found to be a potent inhibitor of aromatase. This along with its 19-hydroxy derivative 7 was characterized as Suicide Substrate of human placental aromatase (k inact 's of 0.069 and 0.058 min −1 and K i 's of 143 nM and 11.1 μM, respectively, or steroids 1 and 7 ). The results suggest that the 19-oxygenation would be involved in the irreversible inactivation of aromatase by the 5-en-7-one steroids.
