1
40
4
-
Text
A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text.
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n/a
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Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
Pages
399-405
Issue
4
Volume
25
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Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Title
A name given to the resource
Coexpression of cytochrome P4502A6 and human NADPH-P450 oxidoreductase in the baculovirus system
Publisher
An entity responsible for making the resource available
Drug Metabolism and Disposition
Date
A point or period of time associated with an event in the lifecycle of the resource
1997
1997-04
Subject
The topic of the resource
human; Pharmacology & Pharmacy; protein; enzymes; reductase; liver-microsomes; catalytic properties; cdna-directed expression; insect cells; nuclear polyhedrosis-virus; promoters; vector system
Creator
An entity primarily responsible for making the resource
Chen L P; Buters J T M; Hardwick J P; Tamura S; Penman B W; Gonzalez F J; Crespi C L
Description
An account of the resource
Heterologous expression using baculovirus vectors has become a popular method for the production of catalytically active cytochrome P450s (CYPs). We have systematically optimized the multiplicity of infection (MOI) for a coinfection approach for the coexpression of CYP2A6 (viral vector designated v2A6) and NADPH-P450 oxidoreductase (OR; viral vector designated vOR) using Sf9 insect cells. A 3000-fold range of MOI was examined in stationary culture and stirred suspension culture. Surprisingly, our results Indicate that the best CYP2A6 catalytic activity (850-1300 pmol/ min/mg total lysate protein as measured by coumarin 7-hydroxylase activity) was obtained only when using a low MOI of v2A6 (1.5-3 x 10(-2)) and a vOR of 10- to 20-fold less, This activity was similar to 7- to 11-fold higher than the best activity obtained when infecting cells with v2A6 alone. At this level of coinfection, the P450 content ranged from 180 to 250 pmol/mg total lysate protein, and the NADPH cytochrome c reductase activity ranged from 350 to 520 nmol/min/mg total lysate protein. Increasing the MOI of both viruses to 50-fold higher resulted in lower overall activity with the optimum (250 pmol/min/mg total lysate protein) being seen earlier postinfection (60 vs. 72 hr). Increasing the MOI of vOR to levels comparable with those of v2A6, decreased coumarin 7-hydroxylase activity 14-fold. These results suggest that the best CYP2A6 catalytic activity depends on properly posttranslationally modified proteins accumulating in a right ratio as a result of primary, secondary, and possibly tertiary infection of both viruses. These results also suggest that high OR expression results in degradation of P450.
Identifier
An unambiguous reference to the resource within a given context
n/a
Format
The file format, physical medium, or dimensions of the resource
Journal Article or Conference Abstract Publication
1997
Buters J T M
catalytic properties
cdna-directed expression
Chen L P
Crespi C L
Drug Metabolism and Disposition
Enzymes
Gonzalez F J
Hardwick J P
Human
insect cells
Journal Article or Conference Abstract Publication
liver-microsomes
nuclear polyhedrosis-virus
Penman B W
Pharmacology & Pharmacy
promoters
Protein
reductase
Tamura S
vector system
-
Text
A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text.
URL Address
n/a
Rights
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
Pages
696-701
Issue
7
Volume
23
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Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Title
A name given to the resource
CDNA-DIRECTED EXPRESSION OF HUMAN CYTOCHROME-P450 CYP1A1 USING BACULOVIRUS - PURIFICATION, DEPENDENCY ON NADPH-P450 OXIDOREDUCTASE, AND RECONSTITUTION OF CATALYTIC PROPERTIES WITHOUT PURIFICATION
Publisher
An entity responsible for making the resource available
Drug Metabolism and Disposition
Date
A point or period of time associated with an event in the lifecycle of the resource
1995
1995-07
Subject
The topic of the resource
rat; liver; cancer; Pharmacology & Pharmacy; metabolism; activation; enzymes; acid; oxidation; assay; yeast saccharomyces-cerevisiae
Creator
An entity primarily responsible for making the resource
Buters J T M; Shou M G; Hardwick J P; Korzekwa K R; Gonzalez F J
Description
An account of the resource
A recombinant baculovirus containing the human cytochrome P450 (CYP) 1A1 cDNA was constructed and used to express CYP1A1 in Spodoptera frugiperda (SF9) insect cells (0.14+/-0.04 nmol/mg protein, 53+/-14 nmol/liter, N=30). The enzyme represented approximate to 1% of total cellular protein and was partially purified by a three-column procedure to specific content of 5.0 nmol/mg protein. Catalytic activity was reconstituted with both the purified enzyme using lipid and NADPH-P450 oxidoreductase, and the SF9 insect cell membrane fraction without purification using NADPH-P450 oxidoreductase and small amounts of detergent. Catalytic activity of the enzyme after reconstitution was optimum using molar ratios of CYP1A1 to NADPH-P450 oxidoreductase of 1:8. Cytochrome b(5) had no additional stimulating effect. The enzyme metabolized substrates characteristic for CYP1A1: benzo a pyrene (4.0+/-0.3 nmol/min/nmol CYP), 7-ethoxy-4-trifluoromethylcoumarin (36+/-2), ethoxyresorufin (37+/-1), but not pentoxyresorufin (0.77+/-0.02). Recombinant baculovirus expresses the highest amounts of all expression systems published to date of catalytically active CYP1A1. Because human CYP1A1 has never been isolated in a catalytically active state from human tissue, nor has recombinant unmodified human CYP1A1, this system is an excellent alternative for the isolation and characterization of this CYP.
Identifier
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n/a
Format
The file format, physical medium, or dimensions of the resource
Journal Article or Conference Abstract Publication
1995
acid
activation
assay
Buters J T M
Cancer
Drug Metabolism and Disposition
Enzymes
Gonzalez F J
Hardwick J P
Journal Article or Conference Abstract Publication
Korzekwa K R
Liver
Metabolism
oxidation
Pharmacology & Pharmacy
rat
Shou M G
yeast saccharomyces-cerevisiae
-
Text
A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text.
URL Address
n/a
Rights
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
Pages
688-692
Issue
5
Volume
22
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Dublin Core
The Dublin Core metadata element set is common to all Omeka records, including items, files, and collections. For more information see, http://dublincore.org/documents/dces/.
Title
A name given to the resource
CDNA-DIRECTED EXPRESSION OF HUMAN CYTOCHROME-P450 CYP3A4 USING BACULOVIRUS
Publisher
An entity responsible for making the resource available
Drug Metabolism and Disposition
Date
A point or period of time associated with an event in the lifecycle of the resource
1994
1994-09
Subject
The topic of the resource
pharmacokinetics; Pharmacology & Pharmacy; metabolism; polymorphism; identification; reductase; oxidation; vaccinia virus; human-liver; catalytic activities; hydroxylation
Creator
An entity primarily responsible for making the resource
Buters J T M; Korzekwa K R; Kunze K L; Omata Y; Hardwick J P; Gonzalez F J
Description
An account of the resource
A recombinant baculovirus containing the human CYP3A4 cDNA was constructed and used to express CYP3A4 in SF9 insect cells (0.46 +/- 0.13 nmol/mg protein, 103 +/- 29 nmol/liter, N = 15). The enzyme represented similar to 2-3% of total cellular protein and could be purified by a two column procedure to a specific content of 12.7 nmol/mg protein. Catalytic activity of the purified enzyme after reconstitution was optimum using molar ratios of CYP3A4 to cytochrome b(5) to NADPH-P450 oxidoreductase of 1:3:20, respectively. The enzyme metabolized cortisol, erythromycin, testosterone, and (R)-warfarin. Recombinant baculovirus expresses the highest amounts of all expression systems published to date of catalytically intact CYP3A4. This system is an excellent alternative for the isolation and characterization of P450 forms from human liver.
Identifier
An unambiguous reference to the resource within a given context
n/a
Format
The file format, physical medium, or dimensions of the resource
Journal Article or Conference Abstract Publication
1994
Buters J T M
catalytic activities
Drug Metabolism and Disposition
Gonzalez F J
Hardwick J P
human-liver
Hydroxylation
identification
Journal Article or Conference Abstract Publication
Korzekwa K R
Kunze K L
Metabolism
Omata Y
oxidation
pharmacokinetics
Pharmacology & Pharmacy
Polymorphism
reductase
vaccinia virus
-
Text
A resource consisting primarily of words for reading. Examples include books, letters, dissertations, poems, newspapers, articles, archives of mailing lists. Note that facsimiles or images of texts are still of the genre Text.
URL Address
<a href="http://doi.org/10.1002/(sici)1098-2744(199612)17:4%3C241::aid-mc8%3E3.0.co;2-g" target="_blank" rel="noreferrer noopener">http://doi.org/10.1002/(sici)1098-2744(199612)17:4%3C241::aid-mc8%3E3.0.co;2-g</a>
Rights
Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
Pages
241-249
Issue
4
Volume
17
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Title
A name given to the resource
Specificity of cDNA-expressed human and rodent cytochrome P450s in the oxidative metabolism of the potent carcinogen 7,12-dimethylbenz a anthracene
Publisher
An entity responsible for making the resource available
Molecular Carcinogenesis
Date
A point or period of time associated with an event in the lifecycle of the resource
1996
1996-12
Subject
The topic of the resource
12-dimethylbenz(a)anthracene; 12-dimethylbenz(a)anthracene; 12-dimethylbenzanthracene metabolites; 7; binding; Biochemistry & Molecular Biology; cDNA expression; chromatography; cytochrome P450; high-performance liquid; human-tissues; hydrocarbons; liver; lung-cancer; metabolism of polycyclic aromatic; microsomes; mouse skin; Oncology; purification; rat; vaccinia virus
Creator
An entity primarily responsible for making the resource
Shou M G; Korzekwa K R; Krausz K W; Buters J T M; Grogan J; Goldfarb I; Hardwick J P; Gonzalez F J; Gelboin H V
Description
An account of the resource
7,12-Dimethylbenz[a]anthracene (DMBA), a potent carcinogen, requires metabolic activation by cytochrome P450s (P450s) to electrophilic metabolites that result in DNA modification, mutagenicity, and carcinogenicity. In this study, we used eight human forms, four rodent forms, and one rabbit form of P450 expressed from recombinant vaccinia or baculovirus vectors to define their specificity for metabolizing DMBA. Of the eight human P450s, 1A1 was the most active (specific activity = 14.7 nmol/min/nmol of P450) in total metabolism of DMBA and showed approximately 6- to 33-fold more activity than other P450s. 2B6, 2C9, and 1A2 were also capable of metabolizing DMBA (2.0-2.5 nmol/min/nmol of P450), whereas 2C8, 2E1, 3A4, and 3A5 exhibited relatively low activities. Among animal P450s, mouse 1A1 exhibited activity similar to that of human 1A1 and had 5.0- to 37-fold more activity than other rodent and rabbit P450s. In regard to enzyme regioselectivity, most human and rodent P450s predominantly formed the 8,9-diol, but human 2B6 and rat 2B1 preferentially formed the 5,6-diol. In the production of monohydroxymethyl metabolites, all the enzymes yielded more 7-hydroxymethyl-12-methylbenz[a]anthracene (7HOM12MBA) than 12-hydroxymethyl-7-methylbenz[a]anthracene (7M12HOMBA), except for human 1A1, which presented the reverse selectivity. Human liver microsomes from 10 organ donors were shown to metabolize DM BA and in most circumstances generated the meta belie profile DM BA trans-8,9-d dihydrodiol > 7HOM12MBA greater than or equal to DMBA trans-5,6-dihydrodiol greater than or equal to 7,12-dihydroxymethylbenz[a]anthracene > 7M12HOMBA > DMBA trans-3,4-dihydrodiol. Thus, the combined activity of hepatic microsomal 2C9, 1A2, and 2B6 may contribute to the metabolic activation and the metabolism of DMBA in normal human liver. (C) 1996 Wiley-Liss, Inc.
Identifier
An unambiguous reference to the resource within a given context
<a href="http://doi.org/10.1002/(sici)1098-2744(199612)17:4%3C241::aid-mc8%3E3.0.co;2-g" target="_blank" rel="noreferrer noopener">10.1002/(sici)1098-2744(199612)17:4%3C241::aid-mc8%3E3.0.co;2-g</a>
Format
The file format, physical medium, or dimensions of the resource
Journal Article
12-dimethylbenz(a)anthracene
12-dimethylbenzanthracene metabolites
1996
7
Binding
Biochemistry & Molecular Biology
Buters J T M
cDNA expression
Chromatography
cytochrome P450
Gelboin H V
Goldfarb I
Gonzalez F J
Grogan J
Hardwick J P
high-performance liquid
human-tissues
Hydrocarbons
Journal Article
Korzekwa K R
Krausz K W
Liver
lung-cancer
metabolism of polycyclic aromatic
Microsomes
Molecular carcinogenesis
mouse skin
oncology
purification
rat
Shou M G
vaccinia virus