Raunio H, Valtonen J, Honkakoski P, Lang M A, Ståhlberg M, Kairaluoma M A, Rautio A, Pasanen M, Pelkonen O
Department of Pharmacology and Toxicology, University of Oulu, Finland.
Biochem Pharmacol. 1990 Dec 1;40(11):2503-9. doi: 10.1016/0006-2952(90)90092-y.
Polyclonal antibodies generated to four distinct mouse liver phenobarbital-inducible cytochrome P450 isoforms were used to analyse related forms in human liver. N-terminal sequence analysis and biochemical properties of the P450s used as antigens suggest that they belong to P450 subfamilies IIB (P450PBI), IA (P450PBII), IIC (P450PBIII) and IIA (P450Coh). In immunoblot analysis, anti-P450PBII detected a single protein presumed to be P450IA2 in all the human livers tested. No proteins corresponding with P450IA1 could be detected. Anti-PBIII and anti-P450Coh antibodies each detected one band (54 and 48 kDa, respectively) in the liver samples. No bands were revealed by anti-P450PBI antibody. Protein dot-immunobinding analysis showed that P450s immunodetectable by anti-P450PBII, anti-P450PBIII and anti-P450Coh antibodies are expressed in human liver (range 9 to 69 pmol P450/mg protein). In immunoinhibition experiments the activity of 7-ethoxyresorutin O-deethylase (EROD) was blocked up to 90% by the anti-P450PBII antibody. Aryl hydrocarbon hydroxylase (AHH) was inhibited only by anti-P450PBIII, and coumarin 7-hydroxylase (COH) only by anti-P450Coh antibody. Testosterone hydroxylations in positions 6 beta, 7 alpha, 15 alpha and 16 alpha were not affected significantly by any of the antibodies. These data suggest that the human liver P450IA2 is responsible for most of the elevated EROD activity, P450s in the IIC subfamily for constitutive AHH and P450s in the IIA subfamily for all of COH activity.
针对四种不同的小鼠肝脏苯巴比妥诱导型细胞色素P450同工型产生的多克隆抗体被用于分析人类肝脏中的相关形式。用作抗原的P450的N端序列分析和生化特性表明它们属于P450亚家族IIB（P450PBI）、IA（P450PBII）、IIC（P450PBIII）和IIA（P450Coh）。在免疫印迹分析中，抗P450PBII在所有测试的人类肝脏中检测到一种单一蛋白质，推测为P450IA2。未检测到与P450IA1对应的蛋白质。抗PBIII和抗P450Coh抗体在肝脏样品中各自检测到一条带（分别为54和48 kDa）。抗P450PBI抗体未显示出条带。蛋白质斑点免疫结合分析表明，抗P450PBII、抗P450PBIII和抗P450Coh抗体可免疫检测到的P450在人类肝脏中表达（范围为9至69 pmol P450/毫克蛋白质）。在免疫抑制实验中，抗P450PBII抗体可将7-乙氧基间苯二酚O-脱乙基酶（EROD）的活性阻断高达90%。芳烃羟化酶（AHH）仅被抗P450PBIII抑制，香豆素7-羟化酶（COH）仅被抗P450Coh抗体抑制。6β、7α、15α和16α位的睾酮羟化不受任何一种抗体的显著影响。这些数据表明，人类肝脏P450IA2负责大部分升高的EROD活性，IIC亚家族中的P450负责组成型AHH，IIA亚家族中的P450负责所有的COH活性。
