Abstract:
Carboxylative enzymes are involved in many pathways and their regulation plays a crucial role in many of these pathways. In particular, γ-glutamylcarboxylase (GGCX) converts glutamate residues (Glu) into γ-carboxyglutamate (Gla) of the vitamin K-dependent proteins (VKDPs) activating them. VKDPs include at least 17 proteins involved in processes such as blood coagulation, blood vessels calcification, and bone mineralization. VKDPs are activated by the reduced form of vitamin K, naturally occurring as vitamin K1 (phylloquinone) and K2 (menaquinones, MKs). Among these, MK7 is the most efficient in terms of bioavailability and biological effect. Similarly to other trans isomers, it is produced by natural fermentation or chemically in both trans and cis. However, the efficacy of the biological effect of the different isomers and the impact on humans are unknown. Our study assessed carboxylative efficacy of trans and cis MK7 and compared it with other vitamin K isomers, evaluating both the expression of residues of carboxylated Gla-protein by western blot analysis and using a cell-free system to determine the GGCX activity by HPLC. Trans MK7H2 showed a higher ability to carboxylate the 70 KDa GLA-protein, previously inhibited in vitro by warfarin treatment. However, cis MK7 also induced a carboxylation activity albeit of a small extent. The data were confirmed chromatographically, in which a slight carboxylative activity of cis MK7H2 was demonstrated, comparable with both K1H2 and oxidized trans MK7 but less than trans MK7H2 . For the first time, a difference of biological activity between cis and trans configuration of menaquinone-7 has been reported.
摘要:
羧基酶参与许多途径,它们的调节在许多途径中起着至关重要的作用。特别是,γ-谷氨酰羧化酶(GGCX)将谷氨酸残基(Glu)转化为维生素K依赖性蛋白(VKDP)的γ-羧基谷氨酸(Gla),从而激活它们。VKDP包括至少17种参与血液凝固等过程的蛋白质,血管钙化,和骨矿化。VKDP被还原形式的维生素K激活,天然存在的维生素K1(苯醌)和K2(甲基萘醌,MKs)。其中,MK7在生物利用度和生物效应方面是最有效的。与其他反式异构体类似,它是通过自然发酵或以反式和顺式化学方式产生的。然而,不同异构体的生物效应的功效和对人类的影响尚不清楚。我们的研究评估了反式和顺式MK7的羧基功效,并将其与其他维生素K异构体进行了比较,通过蛋白质印迹分析评估羧化Gla蛋白残基的表达,并使用无细胞系统通过HPLC测定GGCX活性。TransMK7H2显示出更高的羧酸70KDaGLA蛋白的能力,先前通过华法林治疗在体外抑制。然而,顺式MK7也诱导羧化活性,尽管程度很小。数据经色谱确认,其中证明了顺式MK7H2的轻微羧基活性,与K1H2和氧化反式MK7相当,但低于反式MK7H2。第一次,据报道,甲基萘醌-7的顺式和反式构型的生物活性存在差异。