Assay of N-acetylheparosan deacetylase with a capsular polysaccharide from Escherichia coli K5 as substrate.
Assay of N-acetylheparosan deacetylase with a capsular polysaccharide from Escherichia coli K5 as substrate.
复制标题
以大肠杆菌 K5 荚膜多糖为底物测定 N-乙酰乙酰肝素脱乙酰酶。
DOI：
10.1016/0003-2697(83)90741-8
复制
发表时间：
1983
影响因子：
2.9
通讯作者：
Rodén,L
中科院分区：
文献类型：
--
作者：
Navia,JL;Riesenfeld,J;Vann,WF;Lindahl,U;Rodén,L
制备了一种新的脱乙酰基酶底物，该酶催化肝素生物合成过程中N-乙酰肝素原中N-乙酰基的脱除。从大肠杆菌010：K5：H4，这是结构相同的N-乙酰肝素原的荚膜多糖，部分N-脱乙酰肼解，然后用[3 H]乙酸酐N-乙酰化放射性标记。在孵育标记的多糖与微粒体从Furth肥大细胞瘤，[3 H]乙酰基被释放，表明细菌多糖是一个底物的N-脱乙酰基酶。建立了定量测定N-脱乙酰酶活性的反应条件;测定的Km为74 mg多糖/升，相当于2.1 × 10− 4 m，以糖醛酸浓度表示; Vmax为3.4 nmol/mg蛋白/升。为了证实以前的结果，观察到（a）反应被3′-磷酸腺苷酰硫酸盐刺激（在0.5 mM浓度下最高达45%），表明发生了N-硫酸化，这促进了N-脱乙酰酶的持续作用，和（B）NaCl和KCl抑制该酶，在25 mM的浓度下，活性降低50%。在这项工作的过程中，使用了一种简单的单瓶测定方法。用含10%异戊醇的甲苯或二甲苯基闪烁液从酸化反应混合物中提取释放的[3 H]乙酸盐，并通过闪烁光谱法直接测量。
A new substrate for the deacetylase which catalyzes the removal of the N-acetyl groups from N-acetylheparosan in the course of heparin biosynthesis has been prepared. The capsular polysaccharide from Escherichia coli 010:K5:H4, which is structurally identical to N-acetylheparosan, was partially N-deacetylated by hydrazinolysis and was then radioactively labeled by N-acetylation with [3H]acetic anhydride. Upon incubation of the labeled polysaccharide with microsomes from the Furth mastocytoma, [3H]acetyl groups were released, demonstrating that the bacterial polysaccharide was a substrate for the N-deacetylase. Reaction conditions were established which permitted the quantitative assay of N-deacetylase activity; a Kmof 74 mg polysaccharide/liter was determined, which correponds to 2.1 × 10−4m, expressed as concentration of uronic acid; Vmaxwas 3.4 nmol/mg protein/liter. In confirmation of previous results, it was observed (a) that the reaction was stimulated by 3′-phosphoadenylylsulfate (up to a maximum of 45% at a concentration of 0.5 mm), suggesting that N-sulfation occurred which facilitated continued action of the N-deacetylase, and (b) that NaCl and KCl inhibited the enzyme, with 50% reduction of activity at a concentration of 25 mm. In the course of this work, a simple, single-vial assay procedure was used. Released [3H]acetate was extracted from the acidified reaction mixture with a toluene- or xylene-based scintillation fluid containing 10% isoamyl alcohol and measured directly by scintillation spectrometry.