Determination of “active” cytochrome P-450 from relaxation kinetics of product formation

We estimate the active part of cytochrome P-450, which is involved in a special substrate transformation, by measuring the initial change of the production rate as a function of the relaxation transitions between two different steady states of the reaction cycle of cytochrome P-450 using the light-reversibility of the carbon monoxide inhibition. The kinetic data of such relaxations are interpreted within a model cycle, which reduces the reaction cycle to three steps. The estimation of the rate constant of the first reduction step, derived from model simulation of the production rate, is confirmed by independent experimental study of the reduction kinetics.An application of our model to the O-deethylation of 7-ethoxycoumarin reveals that — in a time average — 10%–15% of the spectroscopically detectable cytochrome P-450 is involved in that transformation.Abbreviations Cyt. P-450 microsomal cytochrome P-450 - 7-EC 7-ethoxycoumarin     

Induction of cytochrome P-450 and P-448 in the outer membrane of mouse liver nuclei by phenobarbital and benzo [α-]pyrene

This investigation confirms the presence of the inducible mixed function hydroxylase enzyme system in nuclear membranes. The cytochrome P-450 spectrum and demethylase activity, markers of the enzyme system, were used to define its localization to the outer membrane envelope. Intact BALB/c mouse liver nuclei isolated and purified in Mg⁺⁺ sucrose media of low ionic strength gave CO-dithionite reduced difference spectra of cytochrome P-450 and P-448. Phenobarbital induced P-450 by 40% while the carcinogenic hydrocarbon, benzo [α] pyrene, induced P-448 twofold. A corresponding increase was also observed in the microsomes of the same tissue preparations. No microsomal contamination of nuclear preparations was found. Intact nuclei stripped of their outer membrane by 0.5% Triton X-100 treatment resulted in a striking absence of the P-450 which, however, was found to be present in isolated outer nuclear membranes.     

Two modes of binding of adrenal NADPH–cytochrome P-450 reductase to liposomal membranes

NADPH-cytochrome P-450 reductase, purified from bovine adrenocortical microsomes, was shown to bind in two different modes to liposomal membranes composed of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine at a molar ratio of 5:3:1. As demonstrated by Ficoll density gradient centrifugation and HPLC gel filtration, the cholate dialysis method made the reductase bind tightly to the liposomal membranes, while the incubation with the preformed vesicles made the reductase bind loosely to the membranes. From the experiments of electron transfer to P-450_C21 residing at the other vesicles, the loosely bound reductase was found to be transferable between the vesicles, whereas the tightly bound reductase was not readily transferred. The rates of the binding and the release of the loosely bound reductase to and from the membranes were measured with the stopped-flow method by observing the reduction of P-450_C21 embedded in the vesicles. These kinetic studies showed that the rate-limiting step of the reductase transfer between the vesicles was the release of the reductase from the membranes. The reductase in both binding modes well supported the steroid 21-hydroxylase activity.     

Asymmetric distribution of cytochrome P-450 and NADPH–cytochrome P-450 (cytochrome c) reductase in vesicles from smooth endoplasmic reticulum of rat liver

1. The topography of cytochrome P-450 in vesicles from smooth endoplasmic reticulum of rat liver has been examined. Approx. 50% of the cytochrome is directly accessible to the action of trypsin in intact vesicles whereas the remainder is inaccessible and partitioned between luminal-facing or phospholipid-embedded loci. Analysis by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis reveals three major species of the cytochrome. Of these, the variant with a mol.wt. of 52000 is induced by phenobarbitone and this species is susceptible to trypsin. 2. After trypsin treatment of smooth membrane, some NADPH–cytochrome P-450 (cytochrome c) reductase activity remains and this remaining activity is enhanced by treatment with 0.05% deoxycholate, which renders the membranes permeable to macromolecules. In non-trypsin-treated control membranes the reductase activity is increased to a similar extent. These observations suggest an asymmetric distribution of NADPH–cytochrome P-450 (cytochrome c) reductase in the membrane. 3. As compared with dithionite, NADPH reduces only 44% of the cytochrome P-450 present in intact membranes. After tryptic digestion, none of the remaining cytochrome P-450 is reducible by NADPH. 4. In the presence of both a superoxide-generating system (xanthine plus xanthine oxidase) and NADPH, all the cytochrome P-450 in intact membrane (as judged by dithionite reducibility) is reduced. The cytochrome P-450 remaining after trypsin treatment of smooth vesicles cannot be reduced by this method. 5. The superoxide-dependent reduction of cytochrome P-450 is prevented by treatment of the membranes with mersalyl, which inhibits NADPH–cytochrome P-450 (cytochrome c) reductase. Thus the effect of superoxide may involve NADPH–cytochrome P-450 reductase and cytosolically orientated membrane factor(s).     

A form of rabbit liver cytochrome P-450 that catalyzes the 7α-hydroxylation of cholesterol

A form of cytochrome P-450 which comigrates with cytochrome P-450_LM4 (molecular weight, 55000) on SDS-polyacrylamide gel was purified from liver microsomes of cholestyramine-treated rabbits. This form of cytochrome P-450 catalyzed the 7α-hydroxylation of cholesterol with an activity of 37.5 pmol/min per nmol cytochrome P-450 in the reconstituted enzyme system containing cytochrome P-450 and NADPH-cytochrome P-450 reductase. The substrate specificity of this form of cytochrome P-450 was compared with cytochrome P-450_LM4 isolated from phenobarbital- and β-naphthoflavone-treated rabbit liver microsomes. The latter two isoenzymes do not catalyze 7α-hydroxylation of cholesterol, but are more active in O-deethylation of 7-ethoxycoumarin and p-nitrophenetole. Ouchterlony double diffusion revealed cross-reactivity between anti-P-450_LM4 (phenobarbital) IgG and cytochrome P-450 isolated from cholestyramine- or β-naphthoflavone-treated rabbit liver microsomes. A two-dimensional iodinated tryptic peptide fingerprint indicated only minor structural differences among these three cytochrome P-450_LM4 preparations.     

A Novel Method for the Preparation of Substrate-Free Cytochrome P-45011β

A new method for the removal of the stabilizing substrate, deoxycorticosterone, from adrenal cytochrome P-450_11β, has been developed. Dextran coated charcoal is used for the adsorption of the steroid and the adsorbed steroid is separated from the cytochrome P-450-preparation by low speed centrifugation. The substrate-free enzyme, obtained in this manner, has all the characteristic spectral properties of low-spin cytochrome P-450_11β, and may be converted to the high-spin form by the addition of deoxycorticosterone.

The dextran coated charcoal method has the following advantages over the previously used method of substrate removal. It does not require the addition of the cofactors for cytochrome P-450-dependant hydroxyla-tion of deoxycorticosterone, small amounts of enzyme may be prepared in a short time and the enzyme preparation is not diluted to any great extent during the process.     

P-糖蛋白的研究进展

P-糖蛋白是一类能量依赖性的转运蛋白,能将许多结构不同的化合物逆向转运出细胞.P-糖蛋白的过表达与肿瘤细胞的多药耐药性(Multidrug Resistance,MDR)密切相关,是导致肿瘤化疗失败的主要原因.随着对MDR机制认识的深入,已针对P-糖蛋白的结构设计出多种形式的MDR逆转药物.近年研究发现,P-糖蛋白广泛存在于正常的组织和器官,参与药物和内、外源毒素的吸收、分布和排泄,行使解毒和防御保护的功能.因此,通过转植P-糖蛋白基因可有效地降低经济鱼类、虾等水产品和经济作物中有毒污染物的积累,对保护人类健康将有着积极意义.     

Studies on cytochrome P-450 (P-45017α,lyase)from guinea pig adrenal microsomes. Dual function of a single enzyme and effect of cytochrome b5

We have reported (Kominami S., Shinzawa K. and Takemori S. (1982) Biochem. Biophys. Res. Commun. 109, 916–921) that a cytochrome P-450 purified from guinea pig adrenal microsomes shows 17α-hydroxylase and C-17,20-lyase activities in a reconstituted system with NADPH-cytochrome P-450 reductase. The homogeneity of the purified cytochrome P-450 was examined with the following methods: isoelectric focusing, immunoelectrophoresis and affinity chromatography on cytochrome b₅-immobilized Sepharose. It was found that progesterone competitively inhibited C-17,20-lyase reaction and that progesterone was converted into androstenedione by 17α-hydroxylation followed by the lyase reaction. These results indicate that the dual activities are carried out by a single enzyme (P-450_17α,lyase). P-450_17α,lyase had the maximum activity at pH 6.1 both for 17α-hydroxylation (6.0 nmol/min per nmol of P-450) and the lyase reaction (11.0 nmol/min per nmol of P-450). Upon addition of cytochrome b₅ to the reconstituted system, the optimal pH for 17α-hydroxylation was shifted to 7.0 and that of the lyase reaction to 6.6. The maximum activities at these optimal pH values were almost the same in the presence or absence of cytochrome b₅. With the addition of cytochrome b₅, both the activities were stimulated above pH 6.3–6.5 and were suppressed below pH 6.3–6.5. These results indicate that cytochrome b₅ plays some important role in controlling the dual activities of P-450_17α,lyase.     

家蝇P-450基因研究进展

较细致地介绍了近年来家蝇P-450基因的研究进展,总结了目前已克隆的8个家蝇P-450基因在同源性、遗传多态性、基因结构、基因的表达与调控等方面的特点。     

Biotransformation of Steroids by a Recombinant Yeast Strain Expressing Bovine Cytochrome P-45017α

The cDNA encoding cytochrome P-45017 from bovine adrenal cortex was expressed in Saccharomyces cerevisiae under the control of the galactose-inducible GAL10 promoter. Carbon monoxide difference spectra of the galactoseinduced yeast cells showed expression of about 240 nmol of P-45017 per liter of the culture. Binding of progesterone to the cytochrome P-45017 was clearly detectable already with intact yeast cells as judged by the formation of type I substrate difference spectra. Yeast cells grown on minimal medium containing galactose actively converted progesterone to 17-hydroxyprogesterone, this indicating the functional integrity of the heterologously expressed P-45017 and its efficient coupling with the constitutive NADPH-cytochrome P-450 reductase. More than 80% of the metabolite produced was secreted into the culture medium. Cultivation in a rich non-selective medium resulted in the formation of an additional product, which was identified by mass spectrometry as 17-hydroxy-20-dihydroprogesterone. Kinetic analysis revealed that its production followed the cytochrome P-45017-dependent hydroxylation reaction. The reduction of the 20-keto group of 17-hydroxyprogesterone was also observed in the non-induced yeast culture, this suggesting the involvement of the constitutive enzyme. Among several substrates tested, progesterone was hydroxylated by the cytochrome P-45017 expressed with the highest activity. The activity towards other substrates decreased in the sequence: 11- > 11- > 19-hydroxyprogesterone. In conclusion, the present results show that the host–vector system used is suitable for high-level functional expression of P-45017 and further application of enzymatic properties of this protein to perform specific steroid biotransformations.     

Differential down-regulation and induction responses of testicular steroidogenic cytochromes P-450(cscc) and P-450(C17α) to human choriogonadotropin

Evidence is presented that the regulation of the cytochrome P-450(C17) of the steroid-17-monooxygenase and of the cytochrome P-450(cscc) of the cholesterolmonooxygenase by human choriogonadotropin (hCG)in vivo is mediated by differential mechanisms in the adult rat testis. An initial down-regulation of the cytochrome P-450(C17) but not of the P-450(cscc) can be demonstrated. Furthermore, induction of the cytochrome P-450(cscc) requires exposure to higher hCG doses (3270 of the maximal induction rate of 43.7 pmol/(testis x d) are achieved with 4 IU hCG/single dose) than induction of the P-450(C17) (59% of the maximal induction rate of 48.4 pmol/(testis x d) with 4 IU hCG/single dose), Finally, induction ofcytochrome P-450(cscc) starts faster after initiation of hCG treatment than induction of P-450(C 17).     

Purification of Cytochrome P-450«/A from n-Alkane-grown Cells of Candida maltosa,and Cloning and Nucleotide Sequencing of the Encoding Gene

When the cells of an n-alkane-assimilating yeast, Candida maltosa I AM12247, were transferred from a glucose medium to an n-alkane medium, various enzymes are induced in the endoplasmic reticulum and peroxisome. Cytochrome P-450alk, one of these enzymes in the endoplasmic reticulum, was purified after mild solubilization of the membrane, followed by a few steps of chromatography. The enzyme was characterized spectrophotometrically and its N-terminal amino acid sequence (12 residues) was determined.

Using oligonucleotide probes prepared to match parts of the N-terminal amino acid sequence and of the partial cDNA sequence of cytochrome P-450alk of C. maltosa EH 15, we isolated from a gene library of C. maltosa I AM 12247 a clone which had a gene encoding cytochrome P-450a/Ar. By nucleotide sequencing of this gene, the amino acid sequence of this enzyme was deduced. It consisted of 523 amino acids (59,838 daltons), with a non-cleavable signal sequence in the N-terminal region. The structure of this enzyme was compared with some other members of the cytochrome P-450 superfamily.     

P-(Alkyl)-Nucleoside 5′-Hydrogenphosphonates as Depot Forms of Antiviral Nucleotide Analogues

Abstract

P-(Alkyl)esters of AZT 5′-hydrogenphosphonate were synthesized and their stabilities in the phosphate buffer and human serum were evaluated. The esters bearing residues of primary and secondary alcohols were degraded to give AZT, whereas those containing tertiary alkyl groups yielded AZT 5′-hydrogenphosphonate. The corresponding derivatives of d2A and d4T showed the same properties.     

Immobilization of digitoxin 12β-hydroxylase,a cytochrome P-450-dependent enzyme from cell cultures of Digitalis lanata EHRH

Attempts were made to immobilize digitoxin 12-hydroxylase, a membrane-bound, cytochrome P-450-dependent monooxygenase from cell cultures of Digitalis lanata. The optimum procedure was the entrapment of microsomes in 2% alginate by crosslinking the polysaccharide chains with CaCl₂. After the immobilization of the enzyme about 70% of its activity was retained. The kinetic data such as the pH optimum and the optimum substrate concentrations were identical for the immobilized enzyme and freely suspended microsomes. Using -methyldigitoxin as a substrate enzyme activity could be observed for more than 20 h. A continuous flow system for immobilized digitoxin 12-hydroxylase is described.Abbreviations -mdg -methyldigoxin - -mdt -methyldigitoxin     

31P-15N One-Bond Couplings of Diastereoisomeric Adenosine Cyclic 3′,5′-Phosphoramidates

Abstract

¹J(³¹P¹⁵N) coupling constants of R _p and S _p adeno-sine cyclic 3′,5′-phosphoramidates (1), -N-methylphosphor-amidates (2) and -N,N-dimethylphosphoramidates (3) increase in the order of 1<2<3 and obey the Stec rule (J(R _p)< J(S _p)). A possible interpretation of coupling constant differences based on differences in substituent electronegativities and variation in hybridization at nitrogen atom, is suggested.     

Cytochrome P-450 heme and the regulation of δ-aminolevulinic acid synthetase in the liver

Hepatic δ-aminolevulinic acid synthetase was induced in rats injected with allylisopropylacetamide. The induction process was studied in relation to experimental perturbation of cytochrome P-450 in the liver. Animals were treated with either administered endotoxin or exogenous heme, both of which accelerate degradation of cytochrome P-450 heme. These manipulations were effective in blocking induction of δ-aminolevulinic acid synthetase, and the effect of each compound was proportional to its ability to stimulate degradation of cytochrome P-450 heme. The findings suggest that the heme moiety of cytochrome P-450 dissociates reversibly from its apoprotein and, prior to its degradation, mixes with endogenously synthesized heme to form a pool that regulates δ-aminolevulinic acid synthetase activity. A similar or identical heme fraction appears to mediate stimulation of heme oxygenase, which suggests that the regulation of δ-aminolevulinic acid synthetase and of heme oxygenase in the liver are closely interrelated.     

P-糖蛋白结构及作用机制

ABC (ATP-binding cassette) 转运蛋白广泛存在于各种生物体细胞中,例如细菌的内层细胞浆膜和真核生物的细胞膜和细胞器膜.其利用与ATP的结合和水解供能进行底物的跨膜转运,其中一部分ABC转运蛋白能转运多种疏水性分子.P-糖蛋白隶属于ABC转运蛋白超家族,是研究最为透彻的一员,主要功能是防止机体对外来有害物质的摄入.P-糖蛋白(P-glycoprotein)由4 个基本结构域组成,2 个跨膜区和2 个位于细胞浆内的核苷酸结合区.核苷酸结合区参与ATP的结合和水解,而各由6 个α 跨膜螺旋组成的2个跨膜区联合构成了底物跨膜转运的通道.P 糖蛋白能转运多种不同结构的底物,包括脂类、胆汁酸、多肽和外源性化学物质,这对机体的生存至关重要,但同时也存在不利的一面,包括干扰了药物的运输,从而导致了多药耐药现象的产生.本文就P-糖蛋白的分子结构和作用机制的最新研究进展进行综述.     

细胞色素P-450研究概况

肝细胞色素P-450是人及动物体内,代谢内源性及外源性化合物的一族酶,其氧化药物的机制已被进一步阐明,并发现P-450虽可使大多数外源性化合物代谢解毒,但也与某些药物的毒性作用以及化学致癌有关。现已证明,肝内有多种形式的P-450,分别为不同的诱导剂所诱导。它们在光谱特性、底物特异性以及催化反应等方面有明显的区别,并证明酶的诱导是由特定基因调节的。此外,近年来多种P-450的分离分析方法也有了一定的提高。     

细胞色素P-450研究概况

肝细胞色素P-450是人及动物体内,代谢内源性及外源性化合物的一族酶,其氧化药物的机制已被进一步阐明,并发现P-450虽可使大多数外源性化合物代谢解毒,但也与某些药物的毒性作用以及化学致癌有关。现已证明,肝内有多种形式的P-450,分别为不同的诱导剂所诱导。它们在光谱特性、底物特异性以及催化反应等方面有明显的区别,并证明酶的诱导是由特定基因调节的。此外,近年来多种P-450的分离分析方法也有了一定的提高。