Bolwell G P, Bell J N, Cramer C L, Schuch W, Lamb C J, Dixon R A
Eur J Biochem. 1985 Jun 3;149(2):411-9. doi: 10.1111/j.1432-1033.1985.tb08941.x.
L-Phenylalanine ammonia-lyase (EC 4.3.1.5) has been purified over 200-fold from cell cultures of bean (phaseolus vulgaris L.) exposed to elicitor heat-released from the cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum. Four forms of the enzyme, with identical Mr but differing apparent pI values of 5.4, 5.2, 5.05 and 4.85, were observed following the final chromatofocussing stage of the purification. A preparation (purified 43-fold by ammonium sulphate precipitation, gel-filtration and ion-exchange chromatography) containing all four forms exhibited apparent negative rate cooperativity with respect to substrates. However, the individual forms displayed normal Michaelis-Menten kinetics, with Km values of 0.077 mM, 0.122 mM, 0.256 mM and 0.302 mM in order of decreasing apparent pI value. A preparation purified 200-fold and containing all four forms was used to immunise rabbits for the production of anti-(phenylalanine ammonia-lyase) serum. The antiserum was characterised by: immunotitration experiments; solid phase enzyme-linked immunosorbent assays; comparison of immunoprecipitates of 35S-labelled phenylalanine ammonia-lyase subunits (synthesized both in vivo and in vitro) on both one-dimensional and two-dimensional polyacrylamide gels after immunoprecipitation with the bean antiserum or antisera raised against pea and parsley phenylalanine ammonia-lyase preparations and immune blotting. SDS/polyacrylamide gels and SDS/polyacrylamide gel electrophoresis followed by immune blotting, indicated that the Mr of newly synthesized (in vivo and in vitro) bean phenylalanine ammonia-lyase subunits is 77000; a 70000-Mr form is readily generated as a partial degradation product during purification. Immunoprecipitates of bean phenylalanine ammonia-lyase synthesized both in vivo and in vitro showed the presence of multiple subunit types of identical Mr but differing in pI. Furthermore, treatment of bean cultures with Colletotrichum elicitor resulted in a 10-fold increase in phenylalanine ammonia-lyase extractable activity within 8 h, and chromatofocussing analysis indicated that this was associated with differential increased appearance of the high-pI, low-Km forms as compared to the two higher Km forms. This differential induction was further confirmed by immune blotting of crude extracts subjected to isoelectric focussing.
从暴露于致病真菌菜豆炭疽菌细胞壁释放的激发子的菜豆（Phaseolus vulgaris L.）细胞培养物中，L-苯丙氨酸解氨酶（EC 4.3.1.5）已被纯化了200多倍。在纯化的最后阶段聚焦层析后，观察到该酶有四种形式，其相对分子质量相同，但表观等电点值不同，分别为5.4、5.2、5.05和4.85。一种含有所有四种形式的制剂（通过硫酸铵沉淀、凝胶过滤和离子交换层析纯化了43倍）对底物表现出明显的负协同速率。然而，各个形式表现出正常的米氏动力学，按照表观等电点值降低的顺序，其米氏常数分别为0.077 mM、0.122 mM、0.256 mM和0.302 mM。一种纯化了200倍且含有所有四种形式的制剂被用来免疫兔子以制备抗（苯丙氨酸解氨酶）血清。该抗血清通过以下方法进行表征：免疫滴定实验；固相酶联免疫吸附测定；在用菜豆抗血清或针对豌豆和欧芹苯丙氨酸解氨酶制剂产生的抗血清进行免疫沉淀后，对一维和二维聚丙烯酰胺凝胶上35S标记的苯丙氨酸解氨酶亚基（体内和体外合成）的免疫沉淀物进行比较以及免疫印迹。SDS/聚丙烯酰胺凝胶和SDS/聚丙烯酰胺凝胶电泳后进行免疫印迹表明，新合成的（体内和体外）菜豆苯丙氨酸解氨酶亚基的相对分子质量为77000；在纯化过程中，一种70000相对分子质量的形式很容易作为部分降解产物产生。体内和体外合成的菜豆苯丙氨酸解氨酶的免疫沉淀物显示存在相对分子质量相同但等电点不同的多种亚基类型。此外，用菜豆炭疽菌激发子处理菜豆培养物，在8小时内可使可提取的苯丙氨酸解氨酶活性增加10倍，聚焦层析分析表明，这与高表观等电点、低米氏常数形式相对于两种高米氏常数形式的差异增加出现有关。通过对经等电聚焦的粗提物进行免疫印迹，进一步证实了这种差异诱导。
