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本文采用二亚酰胺酸二甲酯类双功能试剂的化学交联作用结合SDS聚丙烯酰胺凝胶电泳分析技术研究了高粱叶片PEP羧化酶四级结构的对称性。结果表明,此酶的四个亚基以同构缔合方式相联接,其四级结构具有D_2对称性。配基对PEP羧化酶与辛二亚酰胺酸二甲酯的交联作用有明显的影响。底物PEP,必须金属离子Mg~(2+),PEP-Mg~(2+)及抑制剂苹果酸均不同程度地提高了酶的交联程度,使酶分子内亚基相互作用力改变。激活剂G6P则防止酶与辛二亚酰胺酸二甲酯交联。不管G6P存在与否,PEP羧化酶都以活性的四聚体形式存在。因此推测G6P在此酶分子上的调节部位可能位于亚基接触区内。亚基间交联作用对PEP羧化酶催化功能影响不大,但却提高了酶对尿素、盐酸胍、酸及高温等不利条件的抗性。
In this paper, the chemical cross-linking of bis-amidite bifunctional reagents and SDS polyacrylamide gel electrophoresis were used to study the symmetry of the quaternary structure of PEP carboxylase in sorghum leaves. The results show that the four subunits of this enzyme are connected by isomorphism, and their quaternary structure has D_2 symmetry. Ligands have a significant effect on the cross-linking of PEP carboxylase and dimethyl sulfoximidate. Substrate PEP, metal ions Mg 2+, PEP-Mg 2+ and inhibitor malic acid all increased the degree of cross-linking of the enzyme to some extent and changed the subunit interaction force of the enzyme molecule. The activator G6P prevents the enzyme from cross-linking with octandiocidic acid dimethyl ester. Regardless of the presence or absence of G6P, PEP carboxylase exists as an active tetramer. Therefore, it is speculated that the regulatory region of G6P on this enzyme molecule may be located in the subunit contact region. Cross-linking between subunits has little effect on the catalytic function of PEP carboxylase, but increases the resistance of the enzyme to unfavorable conditions such as urea, guanidine hydrochloride, acid and high temperature.