TYROSINE-HYDROXYLASE - PURIFICATION FROM PC-12 CELLS, CHARACTERIZATION AND PRODUCTION OF ANTIBODIES
TYROSINE-HYDROXYLASE - PURIFICATION FROM PC-12 CELLS, CHARACTERIZATION AND PRODUCTION OF ANTIBODIES
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DOI：
10.1016/0197-0186(87)90036-2
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发表时间：
1987-01-01
影响因子：
4.2
通讯作者：
BILLINGSLEY, ML
中科院分区：
文献类型：
--
作者：
KUHN, DM;BILLINGSLEY, ML
酪氨酸羟化酶已从培养的PC-12细胞中纯化至均一。该蛋白在十二烷基硫酸钠聚丙烯酰胺电泳上以分子量为60，000的单一条带迁移。纯酶的双向电泳分离出三个点（每个点的分子量为60，000），等电点为5.4、5.8和5.9。这种电荷异质性不能解释的存在下，糖或脂质部分的酶。氨基酸分析表明，相对较高的疏水氨基酸含量和较低的丝氨酸含量比其他制剂的酪氨酸羟化酶。该酶以其酪氨酸羟基化速率的约1%羟基化色氨酸，但不催化苯丙氨酸的羟基化。在兔中产生的多克隆抗体对纯酪氨酸羟化酶，并通过Western印迹分析判断为单特异性的。从血清中分离出IgG组分，当偶联到溴化氰活化的琼脂糖凝胶上时，可用于在一个步骤中从粗提物中纯化酪氨酸羟化酶。该抗血清在酪氨酸羟化酶的免疫沉淀和免疫细胞化学实验中被证明是非常有用的。
Tyrosine hydroxylase has been purified to homogeneity from cultured PC-12 cells. The protein migrates as a single band with a molecular weight of 60,000 on sodium dodecyl sulfate polyacrylamide electrophoresis. Two-dimensional electrophoresis of the pure enzyme resolves three spots (each with molecular weights of 60,000) with isoelectric points of 5.4, 5.8 and 5.9. This charge heterogeneity cannot be explained by the presence of sugar or lipid moieties on the enzyme. Amino acid analysis indicated a relatively high content of hydrophobic amino acids and a lower serine content than other preparations of tyrosine hydroxylase. The enzyme hydroxylates tryptophan at approximately 1% of its rate of tyrosine hydroxylation but will not catalyze the hydroxylation of phenylalanine. Polyclonal antibodies were produced in rabbits against pure tyrosine hydroxylase and were judged to be monospecific by Western blot analysis. The IgG fraction was isolated from serum, and when coupled to cyanogen bromide activated Sepharose, could be used to purify tyrosine hydroxylase from crude extracts in a single step. The antiserum proved to be very useful in immunoprecipitation and immunocytochemical experiments with tyrosine hydroxylase.