IH764—3对胶原生物合成中脯氨酰羟化的作用

从13天龄鸡胚胎提取原胶原脯氨酰羟化酶,在FeSO_4、2-酮戊二酸、抗坏血酸、过氧化氢酶等辅助因子存在下,将底物(Pro-Pro-Gly)10·9H_2O中的Pro羟化成羟脯氨酰(HOPro)的羟化反应体系,观察IH764-3对胶原生物合成中脯氨酰羟化作用的影响。结果表明,脯氨酰羟化酶催化的原胶原脯氨酰底物(Pro-Pro-Gly)10·9H_2O的羟基化作用可被IH764-3所抑制。在酶促反应标准条件下,IH764-3为0.24mmol/L时,对脯氨酰羟化的抑制率为50.78±16.03％。本实验还证明,IH764-3不能与脯氨酰羟化酶结合,提示IH764-3对酶促脯氨酰羟化的抑制是由于其同酶反应所需的Fe~(2 )形成络合物所致。平衡移动法表明,IH764-3与Fe~(2 )形成的稳定络合物的Fe(2 ):IH764-3摩尔比为1:3,表现稳定常数Ks=1.04×10~(12)。

Effect of IH764-3 on Prolyl Hydroxylation in Collagen Biosynthesis

Previous study showed that IH764-3 selectively inhibited the synthesis and secretion of collagen, suggesting that the effect of the drug might be due to inhibition of prolyl hydroxylation. In this papsr, we report the effect of IH764-3 on prolyl hydroxylation in collagen biosynthesis.Our results showed that the protocollagen substrate (Pro-Pro-Gly) 10 9H2O of prolyl hydroxylation by proline hydroxylase isolated from 13-day-old chick embryos in the presence of a-ketoglutarate, ascorbic acid and Fe2 was inhibited by IH764-3. The inhibition rate was about 50% at a concentration of 0.24 mmol/L.Further investigation demonstrated that IH764-3 did not bind to prolyl hydroxylase, and the inhibition of enzymatic prolyl hydroxylation was found to be due to chelation of the Fe2 required for the enzymatic reaction. The molar ratio of Fe2 to IH764-3 (1:3) was determined by the equilibrium movement method, and the chelate was postulated to be a octahedral complex.