Ando-Yamamoto M, Hayashi H, Sugiyama T, Fukui H, Watanabe T, Wada H
J Biochem. 1987 Feb;101(2):405-14. doi: 10.1093/oxfordjournals.jbchem.a121925.
L-DOPA decarboxylase [DDC, aromatic-L-amino acid carboxyl-lyase, EC 4.1.1.28] was purified 800-fold from rat liver by several column chromatographic steps. The enzyme (specific activity, about 6 mumol/min X mg protein) had a molecular weight of 100,000 and gave a single band with a molecular weight of 50,000 on SDS-polyacrylamide gel electrophoresis. Its isoelectric point was pH 5.7. The absorption spectrum in the visible region of the purified DDC showed maxima at 330 and 420 nm. Polyclonal and monoclonal antibodies against DDC were produced by using this purified protein as an antigen. Polyclonal anti-DDC serum immunoprecipitated the DDC activities of rat, guinea-pig and rabbit livers (about 1, 10, and more than 100 microliter of antiserum, respectively, were required for 50% precipitation of 2 nmol/min of activity of these enzymes). The monoclonal antibody, named MA-1, belonged to the IgG1 subclass and immunoprecipitated the DDC activities of rat and guinea-pig livers to the same extent (about 0.5 micrograms of IgG was required to immunoprecipitate 2 nmol/min activity of each enzyme), but it did not affect the rabbit enzyme. The antibody MA-1 detected DDC molecules of both the purified enzyme and crude homogenate of rat liver blotted onto a nitrocellulose sheet. Immunohistochemically this antibody also stained specific neurons in the substantia nigra, raphe nucleus and locus coeruleus of rat brain.
通过几步柱色谱法从大鼠肝脏中纯化出800倍的L-多巴脱羧酶[DDC，芳香族L-氨基酸羧基裂解酶，EC 4.1.1.28]。该酶（比活性约为6 μmol/分钟×毫克蛋白）分子量为100,000，在SDS-聚丙烯酰胺凝胶电泳上呈现一条分子量为50,000的条带。其等电点为pH 5.7。纯化的DDC在可见光区域的吸收光谱在330和420 nm处有最大值。以这种纯化的蛋白质为抗原制备了针对DDC的多克隆抗体和单克隆抗体。多克隆抗DDC血清免疫沉淀大鼠、豚鼠和兔肝脏的DDC活性（分别需要约1、10和超过100微升抗血清才能使这些酶2 nmol/分钟的活性沉淀50%）。名为MA-1的单克隆抗体属于IgG1亚类，对大鼠和豚鼠肝脏的DDC活性免疫沉淀程度相同（每种酶2 nmol/分钟的活性进行免疫沉淀大约需要0.5微克IgG），但对兔酶没有影响。抗体MA-1能检测到印迹在硝酸纤维素膜上的纯化酶和大鼠肝脏粗匀浆中的DDC分子。免疫组织化学研究表明，该抗体还能对大鼠脑黑质、中缝核和蓝斑中的特定神经元进行染色。
