Gardner J M, Kado C I
J Bacteriol. 1976 Jul;127(1):451-60. doi: 10.1128/jb.127.1.451-460.1976.
An endopolygalacturonic acid trans-eliminase (EC 4.2.2.2), released by osmotic shock of Erwinia rubrifaciens cells, has been purified to near homogeneity (3, 100-fold) by column chromatography on diethylaminoethyl-cellulose, phosphocellulose, and hydroxyapatite-cellulose followed by isoelectric focusing. It has a molecular weight of 41,000, s20,w of 3.09S, an isoelectric point of pH 6.25, pH optimum of 9.5, and a temperature optimum of 37 C and requires Ca2+ with an optimum concentration of 0.5 to 1.0 mM. Mg2+ could not substitute for Ca2+. Tyrosinyl residues seem essential for enzyme catalysis based on rapid inactivation by tetranitromethane. The enzyme prefers unmethylated polygalacturonic acid as the substrate, cleaving alpha-1,4-glycosidic linkages randomly to form unsaturated galacturonides at a Vmax of 1,166 mumol of product/min per mg of protein and a Km of 5 mg of polygalacturonic acid per ml. Over 90% of the enzyme activity is released from osmotically shocked E. rubrifaciens cells. Unlike E. rubrifaciens, trans-eliminase is not released from Erwinia carotovora cells by osmotic shock treatment, but enzyme activity is detected in the culture medium. The release of the enzyme is reduced fivefold by the addition of dibutyryl cyclic adenosine 5'-monophosphate. The hypersensitive reaction in tobacco leaves was induced within 60 min after injection of less than 1 mug of purified E. rubrifaciens trans-eliminase. Single cells of tobacco in suspension culture are readily killed by the enzyme, whereas tobacco protoplasts remain unaffected when treated in the same manner. These results indicate that endopolygalacturonic acid trans-eliminase is a constitutive enzyme possibly located in the periplasmic space of the E. rubrifaciens cell and releases enzyme into the culture medium in the presence of substrate. The release of the enzyme in tobacco tissue and the trans-eliminative cleavage of plant cell wall components may be steps leading to hypersensitivity of the tobacco tissue.
通过对红斑欧文氏菌细胞进行渗透压休克释放出的一种内聚半乳糖醛酸反式消除酶（EC 4.2.2.2），先后经二乙氨基乙基纤维素柱色谱、磷酸纤维素柱色谱和羟磷灰石纤维素柱色谱，然后进行等电聚焦，已被纯化至接近均一状态（纯化了3100倍）。该酶的分子量为41,000，沉降系数s20,w为3.09S，等电点为pH 6.25，最适pH为9.5，最适温度为37℃，且需要Ca2+，最适浓度为0.5至1.0 mM。Mg2+不能替代Ca2+。基于四硝基甲烷能使其快速失活，酪氨酸残基似乎对酶催化至关重要。该酶更倾向于以未甲基化的聚半乳糖醛酸为底物，随机切割α-1,4-糖苷键，以1166 μmol产物/（min·mg蛋白质）的最大反应速度和5 mg聚半乳糖醛酸/ml的米氏常数形成不饱和半乳糖醛酸苷。超过90%的酶活性是从经渗透压休克处理的红斑欧文氏菌细胞中释放出来的。与红斑欧文氏菌不同，胡萝卜软腐欧文氏菌细胞经渗透压休克处理后不会释放反式消除酶，但在培养基中可检测到酶活性。添加二丁酰环腺苷5'-单磷酸可使该酶的释放减少五倍。注射不到1 μg纯化的红斑欧文氏菌反式消除酶后60分钟内，烟草叶片即可诱导出过敏反应。悬浮培养的烟草单细胞很容易被该酶杀死，而烟草原生质体以相同方式处理时则不受影响。这些结果表明，内聚半乳糖醛酸反式消除酶是一种组成型酶，可能位于红斑欧文氏菌细胞的周质空间，并在有底物存在时将酶释放到培养基中。该酶在烟草组织中的释放以及对植物细胞壁成分的反式消除切割可能是导致烟草组织过敏的步骤。
