Kuhn D M, Billingsley M L
Section on Biochemical Pharmacology, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD 20205, U.S.A.
Neurochem Int. 1987;11(4):463-75. doi: 10.1016/0197-0186(87)90036-2.
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 indicates 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.
酪氨酸羟化酶已从培养的PC - 12细胞中纯化至同质。该蛋白质在十二烷基硫酸钠聚丙烯酰胺电泳上以单一分子量为60,000的条带迁移。纯酶的二维电泳分离出三个斑点（每个分子量为60,000），其等电点分别为5.4、5.8和5.9。这种电荷异质性不能用酶上糖或脂质部分的存在来解释。氨基酸分析表明，与其他酪氨酸羟化酶制剂相比，其疏水氨基酸含量相对较高，丝氨酸含量较低。该酶将色氨酸羟化的速率约为其酪氨酸羟化速率的1%，但不会催化苯丙氨酸的羟化。用纯酪氨酸羟化酶在兔体内产生了多克隆抗体，并通过蛋白质印迹分析判断其为单特异性。从血清中分离出IgG组分，当与溴化氰活化的琼脂糖偶联时，可用于一步从粗提物中纯化酪氨酸羟化酶。事实证明，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非常有用。
