棉铃虫不同发育阶段微粒体P450酶系组成和活性的比较

比较了棉铃虫Helicoverpa armigera 6龄幼虫、蛹、成虫微粒体P450单加氧酶系组成及其活性。P450含量在6龄幼虫中肠>（脂肪体=蛹）>成虫,NADPH-细胞色素还原酶在幼虫中肠>幼虫脂肪体>蛹>成虫;6龄幼虫脂肪体微粒体与蛹脂肪体微粒体P450含量相近,但NADPH-细胞色素还原酶活性前者是后者的4.2倍;成虫微粒体的细胞色素P450和NADPH细胞色素P450还原酶含量很低,几乎未检测出。用对-硝基苯甲醚和艾氏剂为底物测定P450酶系活性表明,与6龄幼虫相比,蛹和成虫具有极低的单加氧酶活性,其O-脱甲基酶活性未检出,艾氏剂环氧化酶活性比幼虫低2～3个数量级。     

棉铃虫6龄幼虫中肠与脂肪体微粒体P450酶系的比较

报道了棉铃虫6龄幼虫中肠和脂肪体微粒体P450酶系的组成与加单氧酶活性。与脂肪体微粒体相比,中肠策粒体具有更高的细胞色素P450,细胞色素b5和NADPH－细胞色素P450还原酶含量,表现出较高的艾氏剂环氧化酶和对－硝基苯甲醚O－脱甲基酶活性。SDS－PAGE电泳显示,中肠与脂肪体微粒体介于P450分子量范围内（45－60kDa)的蛋白图谱有所不同,反映出中肠和脂肪体微粒体蛋白组成存在差异。通过对－硝基苯甲醚O－脱甲基酶的动力学分析发现脂肪体微粒体的对－硝基苯甲醚O－脱甲基酶对底物有更强的亲和性,表明不同组织来源的P450同功酶存在质的不同。     

棉铃虫中肠微粒体P450的分离纯化

为深入研究棉铃虫Helicoverpa armigera细胞色素P450的结构与功能,需要分离不同型的P450蛋白。作者建立了适用于棉铃虫中肠微粒体P450的纯化方法,包括聚乙二醇8000（PEG8000）沉淀、高效疏水作用色谱（HPHIC）和高效离子交换色谱（HPIEC）等连续分离步。SDS-PAGE（银染）显示,棉铃虫中肠微粒体经以上步骤分离纯化后,在含P450的馏分中检测出分子量分别为58 kD、47 kD、56 kD和45 kD的4条蛋白带。 P450的回收率为14.3%,比含量提高了39倍。     

2-十三烷酮对棉铃虫细胞色素P450的诱导作用

将2-十三烷酮按0.005%～0.01%（重量比）的浓度加到棉铃虫人工饲料中,连续诱导3代,测定棉铃虫中肠和脂肪体中细胞色素P450(cyt-P450)含量以及与标准配基（正丁醇、吡啶、苯胺、环己烷）形成的氧化型结合光谱。2-十三烷酮诱导品系的中肠cyt-P450与CO结合光谱的最大吸收峰在449 nm处,脂肪体cyt-P450与CO结合光谱的最大吸收峰在450.7 nm处。中肠cyt-P450除了在450 nm附近存在一个吸收峰外,在通入CO后依次在414、415、418 nm附近出现吸收峰,随后该峰消失,随着时间的推移（第31次扫描）在420 nm处又开始出现一个弱吸收峰。2-十三烷酮诱导品系的中肠、脂肪体cyt-P450与4种标准配基形成的差光谱与对照相比在峰型上存在着不同程度的差异。中肠cyt-P450与正丁醇形成双峰双谷的光谱;脂肪体cyt-P450与正丁醇形成的光谱最大吸收峰在416.61 nm处,波谷在424.91 nm处;中肠cyt-P450和脂肪体cyt-P450与吡啶形成的光谱为典型的Ⅱ型光谱,而与环己烷形成的光谱为不典型Ⅰ型光谱;中肠和脂肪体的cyt-P450与苯胺形成典型的Ⅱ型光谱,最大吸收峰分别在443.30和428.92 nm处,最小吸收分别在402.30和401.00 nm处。     

氰戊菊酯和苯巴比妥钠对敏感和抗性棉铃虫中肠多功能氧化酶系的诱导作用

用苯巴比妥钠（2mg/g）和氰戊菊酯（0.2mg/g）拌饲料处理,对敏感品系棉铃虫Helicoverpa armigera中肠的细胞色素P450和细胞色素c还原酶含量均具有明显的诱导作用（两者都使细胞色素P450含量提高了2.24倍,使细胞色素c还原酶的含量分别提高1.33和1.40倍）,但对细胞色素b5诱导作用不显著（仅为对照的1.23和1.15倍）;此外,苯巴比妥钠对敏感棉铃虫中肠的艾氏剂环氧化酶活性和甲氧试卤灵-O-脱甲基酶活性也有显著的诱导作用（分别提高了2.75和2.66倍）,但对7-乙氧香豆素-O-脱乙基酶活性没有诱导作用,而氰戊菊酯对敏感棉铃虫中肠的艾氏剂环氧化酶活性则有2.02倍的诱导作用。同一浓度的苯巴比妥钠和氰戊菊酯使抗性品系棉铃虫中肠的细胞色素P450含量分别提高1.21和1.15倍,使细胞色素c还原酶含量分别提高1.48和1.86倍（差异显著）,但是细胞色素b₅含量没有明显变化（分别为对照的1.15和0.98倍）;此外,氰戊菊酯能使抗性棉铃虫中肠的艾氏剂环氧化酶活性提高1.53倍,但苯巴比妥钠对该酶活性则有明显抑制作用。     

几种植物源化合物对棉铃虫海藻糖酶活性及相关碳水化合物含量的影响

碳水化合物对昆虫的能量代谢和物质合成具有重要的作用。本研究选用2种一般性生物碱（氢溴酸东莨菪碱和烟碱）以及2种β-葡萄糖苷类化合物（七叶灵和皂角苷）, 研究其在不同浓度下对棉铃虫Helicoverpa armigera (Hübner)幼虫体内海藻糖酶活性及相关碳水化合物代谢的影响。结果表明: 用饲喂法处理3龄幼虫96 h后, 皂角苷对棉铃虫幼虫的活体抑制效果明显, 且随添加物浓度增高, 棉铃虫死亡率上升, 10, 20, 40 g/L浓度下棉铃虫的均重分别是0.194, 0.089和0.034 g, 分别为对照的86.99%, 39.91%和15.24%。对海藻糖酶活性及其相关代谢酶的测定结果表明, 2种苷类化合物显著抑制中肠海藻糖酶活性, 饲喂40 g/L皂角苷的试虫中肠海藻糖酶比活力仅是对照组的54.21%; 饲喂30 g/L七叶灵的试虫中肠海藻糖酶比活力为对照组的83.73%。而2种生物碱类化合物显著抑制血淋巴和脂肪体中海藻糖酶活性, 20 g/L氢溴酸东莨菪碱对棉铃虫血淋巴和脂肪体组织的海藻糖酶活性抑制率分别为7.24%和71.43%; 而20 g/L烟碱对试虫血淋巴和脂肪体组织的海藻糖酶活性抑制率为26.29%和33.44%。用氢溴酸东莨菪碱、 烟碱和七叶灵处理试虫后, 血淋巴海藻糖含量都有所增高。4种化合物能够导致试虫糖原磷酸化酶活性变化, 其中, 皂角苷在中肠和脂肪体表现为显著抑制作用, 而随外源化合物浓度变化, 糖原含量和糖原磷酸化酶活性表现为此消彼长关系。饲喂4种植物源化合物的试虫血淋巴中葡萄糖浓度变化和其海藻糖变化一致。本研究证明β-葡萄糖苷类化合物是海藻糖酶抑制剂, 在作为先导化合物进行农药创制开发方面具有重要意义。     

棉酚和烟碱对棉铃虫的生长和细胞色素P—450单加氧酶活性的影响

以人工饲料添加测定了0．5％的棉酚和烟碱对棉铃虫的生长和细胞色素P－450单加氧酶（简称P－450酶系）活性的影响。研究结果显示,在测定浓度下,高龄棉铃虫短期取食含棉酚和烟碱的人工饲料后,对幼虫的生长没有显著影响,由此表明,棉铃虫对其主要寄主植物中的次生物质棉酚和烟碱具有很好的适应能力。与此同时,棉铃虫中肠微粒体P－450酶系的蛋白组成和酶活性发生了不同的变化,有升有降,有的没有变化。棉铃虫可能通过调整P－450酶系的各种蛋白含量和酶的活力水平,来适应对植物次生物质的代谢解毒的需要。另外,棉铃虫取食棉酚和烟碱后,细胞色素B5含量均显著提高,而细胞色素P－450含量均显著降低,细胞色素B5在棉铃虫对棉酚和烟碱的解毒代谢中可能发挥着更为重要的作用。     

棉铃虫蜕皮调节转录因子在大肠杆菌中的重组表达及其包涵体纯化

蜕皮调节转录因子(hormone receptor 3,HR3)在昆虫蜕皮过程中启动蜕皮相关早期基因簇表达,并抑制蜕皮相关晚期基因簇表达,对昆虫蜕皮级联反应起着关键的调控作用。利用合成的特异性引物通过RT-PCR扩增了棉铃虫 Helicoverpa armigera 蜕皮调节转录因子(HHR3),并与pGEX-4T-1载体连接,在大肠杆菌Escherichia coli DH5α内进行扩增,经过PCR筛选获得了HHR3-pGEX-4T-1重组质粒。 用该质粒转化大肠杆菌表达菌株BL21并进行诱导表达,获得了与谷胱甘肽-S转移酶（GST）融合表达的HHR3包涵体,分子量在94 kD左右,通过无离子去垢剂CAPS（3-［cyclohexylamino］-1-propanesulfonic acid）变性、复性后获得了可溶性GST-HHR3融合蛋白,经凝血酶裂解和SDS-PAGE分离得到纯化的HHR3,经蛋白质N-端测序确认表达正确。用重组表达的HHR3免疫家兔,制备了兔抗HHR3多克隆抗体,免疫印迹检测显示该抗体对HHR3有特异性识别能力, 可以用于HHR3功能与调控等下游研究。免疫印迹检测结果还表明,HHR3在5龄向6龄蜕皮的幼虫脂肪体中高表达,在进入6龄24 h 的幼虫脂肪体中含量明显下降,在6 龄72 h 的幼虫中肠中没有检测到HHR3表达;成虫卵巢中有HHR3表达。     

棉铃虫对氰戊菊酯-辛硫磷混剂的抗性演化及解毒酶活性变化

用氰戊菊酯-辛硫磷混剂（有效成分1∶10,简称氰-辛混剂）对棉铃虫Helicoverpa armigera室内品系(YS)进行16代的抗性选育,获得棉铃虫对氰-辛混剂的抗性品系（YS-FP）。YS-FP品系与YS品系相比,对氰-辛混剂的抗性为14.7倍,对其中的单剂氰戊菊酯和辛硫磷的抗性分别为2 170倍和3.1倍。随着筛选的进行,氰戊菊酯和辛硫磷之间的共毒系数在F₂代出现短暂的增加,然后逐渐降低,它们之间的互作由增效变为拮抗。交互抗性测定结果表明,YS-FP品系对氯氰菊酯、溴氰菊酯、三氟氯氰菊酯、三唑磷和灭多威产生了明显的交互抗性,对硫丹、多杀菌素和爱玛菌素没有产生交互抗性。YS-FP品系6龄幼虫中肠细胞色素P450氧化酶甲氧基香豆素O-脱甲基活性为YS品系的10倍,3龄幼虫谷胱甘肽S-转移酶和酯酶活性分别是YS品系的1.7倍（CDNB结合作用）和2.4倍（α-NA 酯酶水解作用）。氰-辛混剂的筛选导致了棉铃虫多种解毒酶活性的增加,特别是细胞色素P450氧化酶活性增强最为明显。本研究结果表明氰-辛混剂对棉铃虫的筛选导致了广谱的交互抗性和多种代谢抗性机理,并且两个单剂之间的互作由增效变为拮抗,因此氰 辛混剂在棉铃虫抗性治理中的作用是有限的和暂时的。     

微粒体多功能氧化酶系与棉铃虫对氰戊菊酯抗药性的关系

测定了棉铃虫Helicoverpa armigera抗氰戊菊酯种群及相对敏感种群不同组织微粒体的甲氧试卤灵-O-脱甲基酶、乙氧试卤灵-O-脱乙基酶、乙氧香豆素-O-脱乙基酶、芳烷基羟基化酶和艾氏剂环氧化酶的活性。结果表明：抗性种群棉铃虫中肠组织的这5种酶活性分别比敏感种群的活性提高了11.29、4.10、2.66、6.30和2.34倍,其脂肪体及体壁的相应酶活性则分别为敏感种群的1.46、6.80、1.36、4.05、1.48倍和12.32、2.2、1.33、0.80和0.51倍。两种群中,棉铃虫不同组织部位的各单加氧酶活性均不同,活性高低顺序在两种群间也不同。总体而言,均是中肠或脂肪体微粒体对不同底物的氧化代谢能力最强。     

Effect of different detergent systems on the molecular size of UDP glucuronosyltransferase and other microsomal drug-metabolizing enzymes

Mouse liver microsomes were solubilized in various detergent systems, and the resulting aggregate structures associated with cytochrome P-450, cytochrome c reductase, and UDP glucuronosyltransferase were sized by gel filtration chromatography. Cholate or its derivative, CHAPS, in combination with Emulgen 911 or Lubrol 12A9 were necessary to generate a particle of about 140 k daltons, the smallest structure associated with cytochrome P-450. Cholate or CHAPS alone was sufficient to generate a minimally sized aggregate of 200 k daltons associated with NADPH cytochrome c reductase activity. Cholate in combination with Emulgen 911 or Lubrol 12A9 generated particles of about 280 k daltons associated with UDP glucuronosyltransferase activity. CHAPS alone also generated similarly sized particles under conditions in which UDP glucuronosyltransferase activity toward 1-naphthol and morphine was two to about twenty times greater, respectively, than with the combination of detergents. This finding suggests that the zwitterionic CHAPS is superior to other detergent systems for studies concerned with the purification of transferase enzymes, a microsomal system in which investigation of the number of different forms has been hampered by the instability of the enzyme upon solubilization and subsequent manipulation.     

Purification of rat liver microsomal cytochrome P-450b without the use of nonionic detergent

Sodium cholate, Emulgen 911, and (3-[(-cholamidopropyl)-dimethyl- ammonio]-1-propanesulfonate) (CHAPS) were selected to examine the effects of ionic, nonionic, and zwitterionic detergents on testosterone hydroxylation catalyzed by four purified isozymes of rat liver microsomal cytochrome P-450, namely P-450a, P-450b, P-450c, and P-450h, in reconstituted systems containing optimal amounts of dilauroylphosphatidylcholine and saturating amounts of NADPH- cytochrome P-450 reductase (reductase). The major phenobarbital-inducible form of rat liver microsomal cytochrome P-450, designated P-450b, was extremely sensitive to the inhibitory effects of Emulgen 911, which is used in several procedures to purify this and other forms of cytochrome P-450. In contrast, sodium cholate and CHAPS had little effect on the catalytic activity of cytochrome P-450b, even at ten times the concentration of Emulgen 911 effecting 50% inhibition (IC-50). By substituting the zwitterionic detergent CHAPS for Emulgen 911, we purified cytochrome P-450b without the use of nonionic detergent. The protein is designated cytochrome P-450b^* to distinguish it from cytochrome P-450b purified with the use of Emulgen 911. NADPH-cytochrome P-450 reductase was also purified both with and without the use of nonionic detergent. The absolute spectra of cytochrome P-450b and P-450b^* were indistinguishable, as were the carbon monoxide (CO)- and metyrapone-difference spectra of the dithionite-reduced hemoproteins. When reconstituted with NADPH-cytochrome P-450 reductase and dilauroylphosphatidylcholine, cytochromes P-450b and P-450b^* catalyzed the N-demethylation of benzphetamine and aminopyrine, the 4-hydroxylation of aniline, the O-dealkylation of 7-ethoxycoumarin, the 3-hydroxylation of hexobarbital, and the 6-hydroxylation of zoxazolamine. Both hemo-proteins catalyzed the 16α- and 16β-hydroxylation of testosterone, as well as the 17-oxidation of testosterone to androstenedione. Both hemoproteins were poor catalysts of erythromycin demethylation and benzo[a]pyrene 3-/9-hydroxylation. The rate of biotransformation catalyzed by cytochrome P-450b^* was up to 50% greater than the rate catalyzed by cytochrome P-450b when reconstituted with either reductase or reductase^*. The activity of cytochrome P-450b and P-450b^* increased up to 50% when reconstituted with reductase^* instead of reductase. In addition to establishing the feasibility of purifying an isozyme of rat liver microsomal cytochrome P-450 without the use of nonionic detergent, these results indicate that the catalytic activity of cytochrome P-450 is not unduly compromised by residual contamination with the nonionic detergent Emulgen 911.     

Solubilization and partial purification of human placental cytochromes P-450

Human placental mitochondrial and microsomal cytochrome P-450 (P-450) was solubilized with sodium cholate in the presence of Emulgen 911 and the solubilized preparations purified by phenyl-Sepharose and DEAE-cellulose column chromatography. The final preparations exhibited specific activities between 3.0 and 7.0 nmol P-450/mg protein implying a 30–115-fold purification over the starting material. Androstenedione exhibited type I spectral interaction with the microsomal P-450 and pregnenolone a reverse type I spectrum with mitochondrial P-450. Hydrophobic column chromatography proved to be a rapid and efficient initial purification step for placental P-450s.     

Cytochrome P450 monooxygenases in the cotton bollworm (Lepidoptera: Noctuidae): tissue difference and induction

Differences in the microsomal P450 monooxygenase system and its inducibility by pentamethylbenzene (PMB) and naphathalene (NA) were investigated in midgut and fatbody tissues of the cotton bollworm, Helicoverpa armigera (Boddie), larvae. Orthogonal array design was used to establish the optimal conditions for measuring Aldrin epoxidation (AE). The optima for AE were similar for the midgut and the fatbody at a temperature of 30 degrees C, pH 7.4, and a time of 10 min. In comparison to fatbody, the midgut had higher levels of total cytochrome P450s, p-nitroanisole O-demethylation (ODM) and AE. In vivo administration of 0.2% PMB or 0.2% NA resulted in higher microsomal protein content and levels of total cytochrome P450 as well as the two examined monooxygenase activities. Total cytochrome P450 and ODM activity were induced to a greater degree in the fatbody. In the midgut, PMB was significantly more effective on ODM than NA. Differences existed in SDS-PAGE profiles between the midgut and the fatbody. The induction of the midgut with PMB and of the fatbody with NA and PMB resulted in marked intensification of the protein bands with molecular masses of 59,100, 53,400, 50,400 Da.     

Solubilization of human prostatic 5 alpha-reductase

A sensitive assay for 5 alpha-reductase was introduced which is capable of detecting at least 0.2 U of activity per sample. The assay was used in developing a method for the solubilization of human prostatic 5 alpha-reductase. Homogenisation conditions were devised under which 95% of the total prostatic 5 alpha-reductase was released into the microsomal fraction. A combination of 0.1 M sodium citrate, 0.1 M KCl, 20% (v/v) glycerol, 0.5 mM NADPH and 1 microM testosterone was found to stabilise 5 alpha-reductase in the presence of detergents. The effect of the presence of low concentrations of detergents in the assay on the activity of 5 alpha-reductase was studied. Triton X-100, Lubrol PX and Nonidet P-40, caused a concentration-dependent inhibition of activity. The ability of several detergents (Triton X-100 MEGA-9, Tween 20, Tween 80, digitonin, Lubrol PX and Nonidet P-40) to solubilise 5 alpha-reductase was studied. All detergents caused a concentration-dependent solubilization of 5 alpha-reductase. Significant amounts of active solubilized enzyme were recovered only with Lubrol PX at concentrations less than 1.1 mg/ml. Seventy percent of the 5 alpha-reductase was solubilized in an active form by extracting the membranes 3 times with 0.8 mg/ml Lubrol PX.     

Partial purification and characterization of phenobarbital-induced house fly cytochrome P-450

Two forms of phenobarbital-induced cytochrome P-450 were partially purified from the Rutgers diazinon-resistant strain of house fly using cholate solubilization, polyethylene glycol 6000 precipitation, and chromatography on DEAE cellulose. The preparation of highest purity had an absorbance maximum of 452 nm, a specific content of 10.0 nmol/mg protein, and an apparent molecular weight of 60,000 when examined by sodium dodecyl sulfate polyacrylamide electrophoresis. The yield of the highly purified cytochrome P-450 was 2–3%. This form contained proportionately less cytochrome P-420 than the original cholate solubilized microsomes, and is thus apparently more stable. A second form of cytochrome P-450 having a specific content of 0.50–0.89 nmol/mg protein was eluted from DEAE cellulose with a 0-0.25 M salt gradient. This is consistent with a previously reported elution pattern for Emulgen 913-solubilized house fly microsomes. Several methods of solubilizing house fly microsomes were examined. High salt, 2M KCI, in the absence of detergents effectively solubilized cytochrome P-450 (50–70% recovery) with little or no conversion to cytochrome P-(420).     

Solubilization of Myelin Membranes by Detergents

The major proteins of myelin have classically been extracted in organic solvents. Here we investigated some of the characteristics of brain myelin solubilization in aqueous detergent solutions. At comparable molar concentrations, two nonionic detergents, i.e., octyl glucoside and Lubrol PX, proved relatively better myelin solubilizers than the detergents related to the bile salts, i.e., cholate and CHAPS. The two former detergents solubilized more protein than lipid and the two latter ones more lipid than protein from myelin membranes. All four detergents solubilized the phospholipid more efficiently than the cholesterol component of myelin. The detergent concentrations required for myelin solubilization were reduced substantially if the temperature and the salt concentration of the media were increased. As much as 3 mg of lyophilized myelin (about 1 mg of protein) were solubilized readily per milliliter of a solution containing 30 mM octyl glucoside and 0.1 M sodium sulfate in 0.1 M sodium phosphate buffer, pH 6.7. Each of the detergents studied, including the above four, sodium dodecyl sulfate (SDS). Triton X-100, and Zwittergent 3-14, had its own advantages and drawbacks as myelin protein extractors. The nonionic amphiphiles and CHAPS left a small residue mainly composed of proteins of the Wolfgram fraction, as revealed by SDS-polyacrylamide gel electrophoresis. Octyl glucoside was preferred, given its versatility as solubilizer, ultraviolet transparency, and high critical micellar concentration. Observations on possible difficulties that may be encountered are also included.     

Reconstituted liver microsomal enzyme system that hydroxylates drugs, other foreign compounds and endogenous substrates. VII. Stimulation of benzphetamine N-demethylation by lipid and detergent

The resolved liver microsomal hydroxylation system required lipid for benzphetamine N-demethylation. Certain nonionic detergents, such as Emulgen 911, Triton N-101, and Triton X-100, at appropriate concentrations could substitute for lipid. These results suggest that lipid and detergent activate the cytochrome P-450-containing hydroxylation system by a similar mechanism, probably by enhancing the interaction between cytochrome P-450 and NADPH-cytochrome c reductase.     

Purification of cytochrome P-450, NADPH-cytochrome P-450 reductase,and epoxide hydratase from a single preparation of rat liver microsomes

A simplified procedure is presented for the simultaneous purification of the enzymes cytochrome P-450, epoxide hydratase (EC 3.3.2.3), and NADPH-cytochrome P-450 reductase (EC 1.6.2.4) from a single preparation of rat liver microsomes. All three enzymes can be recovered after chromatography of detergent-solubilized microsomes on a column of n-octylamino-Sepharose 4B. The major form of cytochrome P-450 (of phenobarbitaltreated rats) is purified by subsequent DEAE-cellulose chromatography, epoxide hydratase is purified by DEAE- and O-(carboxymethyl)-cellulose chromatography, and NADPH-cyto-chrome P-450 reductase is purified using 2′,5′-ADP agarose chromatography. The nonionic detergent Lubrol PX and the ionic detergents sodium cholate and deoxycholate are used in these procedures to permit utilization of uv-absorbance measurements in monitoring protein during purification. Overall yields of the three enzymes are approximately 20, 25, and 60%, respectively. All three enzymes are apparently homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and are functionally active. The same procedure can be used to obtain the major cytochrome P-450 present in liver microsomes isolated from β-naphthoflavone (5,6-benzoflavone)- or 3-methylcholanthrene-treated rats. Thus, the described procedures permit the rapid and reproducible purification of three major rat liver microsomal enzymes which can be coupled to study bioactivation and detoxification of a variety of xenobiotics in reconstituted systems.     

Purification of NADPH:cytochrome c (cytochrome P-450) reductase from hamster liver microsomes by detergent extraction and affinity chromatography

NADPH:cytochrome c (cytochrome P-450) reductase (Fp) from hamster liver microsomes has been purified to near homogeneity using a simple and rapid method. Microsomes were treated with the detergent Chaps (3-[(3-cholamidopropyl)dimethylammonio]propanesulfonic acid) in combination with 0.07% protamine sulfate and then centrifuged to pellet insoluble material. While over 60% of the total microsomal protein was solubilized, all Fp activity remained in the pellet. Fp was extracted from the Chaps-insoluble material using a combination of the detergents sodium cholate and Lubrol PX. This treatment resulted in a fivefold increase in Fp specific activity and allowed direct processing of the enriched Fp fraction by 2',5'-ADP agarose affinity chromatography. The purified Fp had a total flavin content of 23 nmol/mg protein (flavin adenine dinucleotide:flavin mononucleotide ratio = 1:1), a specific activity of 26,000 units/mg protein at 22 degrees C using cytochrome c as electron acceptor, and migrated as a single band on sodium-dodecyl sulfate-polyacrylamide gel electrophoresis with a relative molecular weight of 76,000. The purity, specific activity, and yield were nearly identical to results obtained when the flavoprotein was purified by conventional methods. This procedure eliminates the need for anion-exchange chromatography and allows for the rapid purification of large amounts of Fp suitable for use in studies concerning cytochrome P-450-mediated drug metabolism. Importantly, this method is equally effective when used to purify Fp from rat liver microsomes.