Comparison between avian and human prolyl 4-hydroxylases: studies on the holomeric enzymes and their constituent subunits.
Comparison between avian and human prolyl 4-hydroxylases: studies on the holomeric enzymes and their constituent subunits.
复制标题
禽类和人类脯氨酰 4-羟化酶的比较：全聚酶及其组成亚基的研究。
DOI：
10.1002/jcb.240480209
复制
发表时间：
1992
影响因子：
4
通讯作者：
Desnick,RJ
中科院分区：
文献类型：
--
作者：
Guzman,NA;Ascari,WQ;Cutroneo,KR;Desnick,RJ
4-羟基脯氨酸酶是胶原蛋白生物合成的关键酶，它催化前胶原蛋白中新生的或完整的前-α链中特定的脯氨基残基转化为反式-羟脯氨酸。该酶是由两个不同亚基组成的四聚体，分别命名为α和β。为了比较不同来源的酶及其亚基的异同，对鸡胚胎和人胎盘组织中的4-羟基脯氨酸酶进行了纯化，并测定了它们的理化和免疫学性质。这两种酶都是表观分子量在400 kDa到600 kDa之间的糖蛋白。氨基酸和碳水化合物分析表明，这两种全聚酶之间略有差异，与它们各自的cDNA推导的氨基酸序列一致。人胎盘组织中的Pro-4-羟基酶比鸡胚酶含有更紧密结合的铁。人胎盘酶与纯化的鸡胚脯氨酰4-羟基酶抗体的免疫扩散显示部分相同，表明它们的三级结构中存在不同的抗原决定因素。用高分辨毛细管电泳法对这两种酶进行了分离，表明天然鸡胚胎和人胎盘蛋白的电荷密度不同。电泳学研究表明，人的Pro-4-羟基酶是一个四聚体酶，含有两个不同的亚基，约为和62 kDa，比例约为1：2，分别命名为α和β。相比之下，鸡胚α和β亚基的比例为1：1。值得注意的是，在变性条件下进行电泳时，人α亚基部分降解。类似地，当鸡胚酶受到有限的蛋白降解时，观察到α亚基的选择性降解。最后，只有α亚基与刀豆蛋白A结合，表明两种动物的4-羟基脯氨酸酶的α亚基都是糖基化的。利用生化技术，这些结果表明，人胎盘胶原中的4-反式-羟基-L-脯氨酸残基是由一种酶合成的，该酶的一级结构和免疫学性质与以前描述的鸡胚酶的一级结构和免疫学性质不同，与最近从cDNA序列推导出的一级结构一致。
Prolyl 4‐hydroxylase, a key enzyme in collagen biosynthesis, catalyzes the conversion of selected prolyl residues totrans‐hydroxyproline in nascent or completed pro‐α chains of procollagen. The enzyme is a tetramer composed of two nonidentical subunits, designated α and β. To compare the enzyme and its subunits from different sources, the chick embryo and human placental prolyl 4‐hydroxylases were purified to homogeneity and their physicochemical and immunological properties were determined. Both enzymes were glycoproteins with estimated apparent molecular weights ranging between 400 and 600 kDa. Amino acid and carbohydrate analyses showed slight differences between the two holomeric enzymes, consistent with their deduced amino acid sequences from their respective cDNAs. Human placental prolyl 4‐hydroxylase contained more tightly bound iron than the chick embryo enzyme. Immunodiffusion of the human placental enzyme with antibodies raised against the purified chick embryo prolyl 4‐hydroxylase demonstrated partial identity, indicating different antigenic determinants in their tertiary structures. The enzymes could be separated by high‐resolution capillary electrophoresis, indicating differential charge densities for the native chick embryo and human placental proteins.Electrophoretic studies revealed that the human prolyl 4‐hydroxylase is a tetrameric enzyme containing two nonidentical subunits of about 64 and 62 kDa, in a ratio of approximately 1 to 2, designated α and β, respectively. In contrast, the chick embryo α and β subunit ratio was 1 to 1. Notably, the human α subunit was partially degraded when subjected to electrophoresis under denaturing conditions. Analogously, when the chick embryo enzyme was subjected to limited proteolysis, selective degradation of the α subunit was observed. Finally, only the α subunit was bound to Concanavalin A demonstrating that the α subunits of prolyl 4‐hydroxylase in both species were glycosylated. Using biochemical techniques, these results demonstrated that the 4‐trans‐hydroxy‐L‐proline residues in human placental collagens are synthesized by an enzyme whose primary structure and immunological properties differ from those of the previously well‐characterized chick embryo enzyme, consistent with their recently deduced primary structures from cDNA sequences.