Da Silva M E F, Silva J A, Marangoni S, Novello J C, Meirelles N C
Laboratory of Biomembranes, Department of Biochemistry, Institute of Biology, State University of Campinas, CP 6109, Cidade Universitária Zeferino Vaz, Barão Geraldo, 13083-970 Campinas, SP, Brazil.
Comp Biochem Physiol C Toxicol Pharmacol. 2004 May;138(1):67-74. doi: 10.1016/j.cca.2004.05.004.
Cytochromes P450 constitute a superfamily of the phase I enzymes whose primary task is the detoxification of both endogenous and xenobiotic compounds. Fish, among non-mammalian species, have received great interest because they are a direct food source for humans as well as conveyors of toxic chemicals to human beings. The aim of the present study was the purification of the hepatic isoform of CYP1A in Prochilodus scrofa (Prochilodontidae), a Brazilian fish, using only one chromatographic step. The purification of CYP1A was done by Reverse Phase HPLC on a C18 column. Purified CYP1A was characterized with respect to electrophoretic, immunochemical and biocatalyst properties. CYP1A fractions produced a single uniform band on SDS-PAGE with an apparent molecular mass of 58 kDa. Purified CYP1A of P. scrofa showed strong cross-reactivity with antibodies directed against CYP1A from trout. The fraction was also encapsulated in two different reconstituted systems; one composed of neutral lipids and another of negatively charged lipids. In both of them, we could detect EROD activity but not PROD activity, which confirms that the CYP1A was purified with all its enzyme activity. There was an increase of activity when CYP1A and NADPH cytochrome P450 (CYP) reductase were encapsulated in negatively charged lipids, which confirms that the charge of lipid is essential to CYP1A activity. All these characteristics strongly suggest that this new procedure is efficient for purifying hepatic CYP1A from P. scrofa, showing that the CYP1A isoform of this fish has a highly conserved protein region.
细胞色素P450构成了I相酶的一个超家族，其主要任务是对内源性和外源性化合物进行解毒。在非哺乳动物物种中，鱼类备受关注，因为它们是人类的直接食物来源，也是有毒化学物质的传播者。本研究的目的是仅通过一步色谱法纯化巴西鱼类苏氏无须魮（脂鲤科）肝脏中的CYP1A同工型。通过在C18柱上进行反相高效液相色谱法来纯化CYP1A。对纯化的CYP1A进行了电泳、免疫化学和生物催化剂特性方面的表征。CYP1A组分在SDS-PAGE上产生了一条单一的均匀条带，表观分子量为58 kDa。苏氏无须魮纯化的CYP1A与针对鳟鱼CYP1A的抗体表现出强烈的交叉反应。该组分还被包裹在两种不同的重构体系中；一种由中性脂质组成，另一种由带负电荷的脂质组成。在这两种体系中，我们都能检测到EROD活性，但检测不到PROD活性，这证实了纯化的CYP1A具有其所有的酶活性。当CYP1A和NADPH细胞色素P450（CYP）还原酶被包裹在带负电荷的脂质中时，活性有所增加，这证实了脂质的电荷对CYP1A活性至关重要。所有这些特性都强烈表明，这种新方法对于从苏氏无须魮中纯化肝脏CYP1A是有效的，表明这种鱼类的CYP1A同工型具有高度保守的蛋白质区域。
