Abstract:
:In contrast to other P450 enzymes purified from rat liver microsomes, purified P450 IIIA1 (P450p) is catalytically inactive when reconstituted with NADPH-cytochrome P450 reductase and the synthetic lipid, dilauroylphosphatidylcholine. However, purified P450 IIIA1 catalyzes the oxidation of testosterone when reconstituted with NADPH-cytochrome P450 reductase, cytochrome b5, an extract of microsomal lipid, and detergent (Emulgen 911). The present study demonstrates that the microsomal lipid extract can be replaced with one of several naturally occurring phospholipids, but not with cholesterol, sphingosine, sphingomyelin, ceramide, cerebroside, or cardiolipin. The ratio of the testosterone metabolites formed by purified P450 IIIA1 (i.e., 2 beta-, 6 beta-, and 15 beta-hydroxytestosterone) was influenced by the type of phospholipid added to the reconstitution system. The ability to replace microsomal lipid extract with several different phospholipids suggests that the nature of the polar group (i.e., choline, serine, ethanolamine, or inositol) is not critical for P450 IIIA1 activity, which implies that P450 IIIA1 activity is highly dependent on the fatty acid component of these lipids. To test this possibility, P450 IIIA1 was reconstituted with a series of synthetic phosphatidylcholines. Those phosphatidylcholines containing saturated fatty acids were unable to support testosterone oxidation by purified P450 IIIA1, regardless of the acyl chain length (C6 to C18). In contrast, several unsaturated phosphatidylcholines supported testosterone oxidation by purified P450 IIIA1, and in this regard dioleoylphosphatidylcholine (PC(18:1)2) was as effective as microsomal lipid extract and naturally occurring phosphatidylcholine or phosphatidylserine. These results confirmed that P450 IIIA1 activity is highly dependent on the fatty acid component of phospholipids. A second series of experiments was undertaken to determine whether microsomal P450 IIIA1, like the purified enzyme, is dependent on cytochrome b5. A polyclonal antibody against purified cytochrome b5 was raised in rabbits and was purified by affinity chromatography. Anti-cytochrome b5 caused a approximately 60% inhibition of testosterone 2 beta-, 6 beta-, and 15 beta-hydroxylation by purified P450 IIIA1 and inhibited these same reactions by approximately 70% when added to liver microsomes from dexamethasone-induced female rats. Overall, these results suggest that testosterone oxidation by microsomal cytochrome P450 IIIA1 requires cytochrome b5 and phospholipid containing unsaturated fatty acids.
journal_name
Arch Biochem Biophysjournal_title
Archives of biochemistry and biophysicsauthors
Eberhart DC,Parkinson Adoi
10.1016/0003-9861(91)90128-6subject
Has Abstractpub_date
1991-12-01 00:00:00pages
231-40issue
2eissn
0003-9861issn
1096-0384pii
0003-9861(91)90128-6journal_volume
291pub_type
杂志文章abstract::The ionophore properties of cationomycin and monensin were studied on human erythrocytes by measuring Na+ influx by 23Na NMR and concomitant K+ efflux by potentiometry in the presence of increasing amounts of serum. Both ion currents (Na+ or K+) decreased linearly with the reciprocal of serum amount. The serum effects...
journal_title:Archives of biochemistry and biophysics
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abstract::Free and endoplasmic reticulum-bound polyribosomes from rat heart were examined for their ferritin mRNA content. A procedure for separation and purification of the two ribosome populations that produced good yields of homogeneous mono- and polyribosomes with no contaminating ultrastructures and gave distinctive sedime...
journal_title:Archives of biochemistry and biophysics
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journal_title:Archives of biochemistry and biophysics
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abstract::A protocol for dry weight determination of the concentration of protein, using 0.2-1.0 mg of protein per sample, has been presented and applied to nine proteins: bovine serum albumin, ovalbumin, bovine carbonic anhydrase B, galactoside binding protein (rabbit), lens calinaris lectin B, green pea lectin, soy bean agglu...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
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abstract::Forskolin, a diterpene that exerts several pharmacological effects, activates adenylate cyclase in brain and in some other mammalian tissues. Properties of forskolin activation of adenylate cyclase from central nervous system of the dipterous Ceratitis capitata are described. The interaction of forskolin with the inse...
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journal_title:Archives of biochemistry and biophysics
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journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
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pub_type: 杂志文章
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journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/0003-9861(92)90044-w
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journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/0003-9861(88)90148-8
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journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1006/abbi.1999.1142
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journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/0003-9861(84)90428-4
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abstract::Cytosolic extracts from the posterior adductor muscle of the bivalve mollusk Mytilus galloprovincialis contain significant amounts of both cGMP-binding and cGMP-stimulated protein kinase activities. However, photoaffinity labeling with 8-azido-[32P]cGMP revealed only a major cGMP-binding protein with an apparent molec...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1006/abbi.1998.0879
更新日期:1998-11-01 00:00:00
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journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/j.abb.2020.108562
更新日期:2020-10-30 00:00:00
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journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/0003-9861(92)90135-j
更新日期:1992-04-01 00:00:00
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journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/j.abb.2004.07.029
更新日期:2004-11-15 00:00:00
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journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1006/abbi.2001.2581
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abstract::We have cloned and overexpressed a variant of Arabidopsis thaliana beta-carbonic anhydrase (Q158A) that deletes the functional equivalent of the backbone amide NH of Thr-199 in human alpha-carbonic anhydrase II. The latter residue is hypothesized to be important in catalyzing the rate of CO(2)(-) HCO (3)(-) interconve...
journal_title:Archives of biochemistry and biophysics
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更新日期:2004-05-01 00:00:00
abstract::Diastolic dysfunction refers to an impaired relaxation and an abnormality in ventricular blood filling during diastole while systolic function is preserved. Cardiac myofibril hypersensitivity to Ca(2+) is a major factor that causes impaired relaxation of myocardial cells. The present study investigates the effect of t...
journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/j.abb.2015.03.015
更新日期:2015-05-01 00:00:00