The isolation and comparison of multiple forms of CYP2B from untreated and phenobarbital-treated rabbit liver microsomes.
The isolation and comparison of multiple forms of CYP2B from untreated and phenobarbital-treated rabbit liver microsomes.
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从未经处理和苯巴比妥处理的兔肝微粒体中分离和比较多种形式的 CYP2B。
DOI：
10.1006/abbi.1995.1038
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发表时间：
1995
期刊：
影响因子：
--
通讯作者：
Schenkman,JB
中科院分区：
文献类型：
--
作者：
Jansson,I;Mole,JE;Schenkman,JB
在低水平的苯巴比妥诱导的两种形式的同工酶2（LM 2; CYP 2B 4）的纯化过程中获得的细胞色素P450从兔肝微粒体。在高水平诱导下，仅存在一种形式（LM 2A）。尽管两种纯化形式（LM 2 A和LM 2 B）非常相似，但它们的不同之处在于：（a）CM-琼脂糖凝胶上的峰洗脱，（B）还原P450-CO光谱的最大波长，和（c）几种底物的代谢，其中LM 2 B的活性范围为LM 2 A的0.6至2.65倍。从未处理的兔肝脏中分离出第三种LM 2组分（2C），尽管在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳上是均一的，但似乎是LM 2B和除LM 2A以外的P450 LM 2形式的混合物。在未处理的兔肝微粒体中未发现LM 2A。在CM-Sepharose上，级分2C的洗脱与LM 2B的洗脱重叠。级分2C的表观分子量和对抗LM 2A IgG的免疫应答与LM 2A和LM 2B相同。使用胰蛋白酶的肽图谱显示LM 2A和LM 2B之间没有差异，但一致地显示级分2C至少有一条额外条带。在溴化氰裂解和LM 2A和LM 2B片段的高压液相色谱分离后，LM 2A的以Pro（347）开始的肽（峰4A）比LM 2B的以Pro（347）开始的肽（峰4 B）晚1 - 2分钟洗脱，表明片段中的差异，尽管部分NH 2-末端氨基酸序列和分子量相同。级分2C的相应CNBr片段分裂成两个峰（4C：1和4C：2），保留时间分别对应于4 B和4A。4C的质量：1。4C：2的质量与4A、4 B有显著差异。两个片段具有与4A和4 B相同的部分NH 2-末端氨基酸序列。在比较这些分离和纯化的LM 2形式与cDNA表达形式2B-B 0、2B-B1、2B-B2和2B-Bx的物理化学性质以及催化活性之后[参见R. Ryan等人（1993）Arch.Biochem.Biophys. 304，454-463]，数据表明LM 2A是指定为2B-B 0（LM 2）的形式，LM 2B是2B-Bx，并且级分2C是含有2B-B1和2B-Bx的混合物。这是首次从家兔肝脏中分离鉴定出三种LM 2同工酶蛋白。
At low levels of phenobarbital induction two forms of isoenzyme 2 (LM2; CYP2B4) were obtained during purification of cytochrome P450 from rabbit liver microsomes. At high levels of induction only one form (LM2A) was present. Although the two purified forms (LM2A and LM2B) were very similar they differed in: (a) peak elution on CM-Sepharose, (b) wavelength maximum of the reduced P450-CO spectrum, and (c) metabolism of several substrates, where the activities of LM2B ranged from 0.6 to 2.65 times that of LM2A. A third LM2 fraction (2C) was isolated from untreated rabbit liver and, although homogenous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, appeared to be a mixture of LM2B and a form of P450 LM2 other than LM2A. LM2A was not found in the untreated rabbit liver microsomes. On CM-Sepharose the elution of fraction 2C overlapped that of LM2B. The apparent molecular weight and immunoresponse to anti-LM2A IgG were the same for fraction 2C as for LM2A and LM2B. Peptide mapping using trypsin showed no difference between LM2A and LM2B, but consistently revealed at least one extra band with fraction 2C. After CNBr cleavage and high-pressure liquid chromatography separation of the LM2A and LM2B fragments the peptide beginning with Pro(347) of LM2A (peak 4A) eluted 1 2 min later than that of LM2B (peak 4B) indicating a difference in the fragments, although partial NH2-terminal amino acid sequences and molecular masses were the same. The corresponding CNBr fragment of fraction 2C splits into two peaks (4C:1 and 4C:2) with retention times corresponding to 4B and 4A, respectively. The mass of 4C:1. was the same as that of 4B, while the mass of 4C:2 markedly differed from that of 4A and 4B. Both fragments had the same partial NH2-terminal amino acid sequence as 4A and 4B. After comparing the physicochemical properties as well as catalytic activities of these isolated and purified LM2 forms with the cDNA-expressed forms 2B-B0, 2B-B1, 2B-B2, and 2B-Bx [see R. Ryan et al. (1993) Arch. Biochem. Biophys. 304, 454-463], the data suggest that LM2A is the form designated as 2B-B0 (LM2), LM2B is 2B-Bx, and fraction 2C is a mixture containing 2B-B1 and 2B-Bx. This is the first isolation and identification of the three isozymic LM2 proteins from rabbit liver.