Purification and characterization of sulfatases from Haliotis rufescens: evidence for changes in synthesis and heterogeneity during development.
Purification and characterization of sulfatases from Haliotis rufescens: evidence for changes in synthesis and heterogeneity during development.
复制标题
红鲍硫酸酯酶的纯化和表征：发育过程中合成和异质性变化的证据。
DOI：
10.1007/bf00257905
复制
发表时间：
1991
期刊：
影响因子：
--
通讯作者：
Morse,DE
中科院分区：
文献类型：
--
作者：
Spaulding,DC;Morse,DE
许多海洋软体动物的消化腺中含有丰富的芳基硫酸酯酶，这些酶可能在草食性动物饲料中的硫酸多糖的降解中起作用。芳基硫酸酯酶，部分纯化的红鲍，Haliotis rufescens肝胰腺，进行了调查方面的异质性，催化的要求，并在发展过程中的诱导时间。采用疏水相互作用和阴离子交换层析相结合的方法从成年动物中纯化了四种肝胰腺酶。在非变性条件下通过电泳产生的四种部分纯化的酶的酶谱揭示了第五种，相对更碱性的同工酶。所有四种部分纯化的酶似乎是单体的，分子量约为43 000 Da，每一个，通过凝胶过滤测量。的亲和力为p-硝基儿茶酚硫酸盐，pH值的最佳值，和这些酶显示的阴离子的抑制强度是类似的其他软体动物芳基硫酸酯酶的值。四种酶中有三种对对硝基儿茶酚硫酸酯的Km值在0.8和2.0 mM之间;剩余的酶（A2）的Km为6.7 mM。所有四种酶的最适pH和最适温度分别为5.5和45°C。四种酶中有三种的-t1/2（50 ℃）值为3.5 min;酶A4的t1/2（50 ℃）为8.5分钟。当通过蛋白质印迹分析时，针对A1 b型的单克隆抗体不与任何其它肝胰腺芳基硫酸酯酶交叉反应，证实了成体酶的结构异质性。总芳基硫酸酯酶活性在鲍鱼发育早期以双相方式增加，第一次增加发生在幼虫成熟的早期。酶表达的secoad阶段是依赖于诱导的解决和变态的主管面盘幼虫，强烈表明芳基硫酸酯酶的合成（和成熟的消化腺，肝胰腺）的表达是控制的遗传事件发生的结果变态。有能力的面盘幼虫只表达两种芳基硫酸酯酶的形式，这与成人肝胰腺酶的许多物理化学和动力学特征。然而，无论是幼虫芳基硫酸酯酶是公认的单克隆抗体形成A1 b从成人肝胰腺。内源性酶抑制剂水平在整个芳基硫酸酯酶诱导期内保持恒定，因此在发育过程中对芳基硫酸酯酶活性水平的控制没有贡献，这些结果是第一次记录了鲍鱼变态过程中特定蛋白质的发育诱导。芳基硫酸酯酶表达的时间的意义进行了讨论，在有关潜在的生理底物和饮食开关发生在变态。也被认为是与观察到的这些酶的表达模式一致的可能的遗传事件。
The digestive glands of many marine molluscs are rich sources of arylsufatase enzymes which may function in the catabolism of sulfated polysaccharides in the diets of herbivorous species. Arylsulfatases, partially purified from the hepatopancreas of the red abalone,Haliotis rufescens, were investigated with respect to heterogeneity, catalytic requirements, and timing of induction during development. Four hepatopancreatic enzymes were purified from adult animals using a combination of hydrophobic interaction and anion-exchange chromatography. Zymograms of the four partially-purified enzymes produced by electrophoresis under nondenaturing conditions revealed a fifth, relatively more basic isozyme. All four partially-purified enzymes appear to be monomeric, with molecular weights of approximately 43 000 Da each, as measured by gel filtration. The affinities forp-nitrocatechol sulfate, pH optima, and strengths of inhibition by anions displayed by these enzymes are similar to the values reported for other molluscan arylsulfatases. Three of the four enzymes haveKmvalues between 0.8 and 2.0 mMforp-nitrocatechol sulfate; the remaining enzyme (A2) has aKmof 6.7 mM. All four enzymes have pH and temperature optima of 5.5 and 45°C, respectively. Three of the four enzymes have-t1/2(50°C) values of 3.5 min; the enzyme A4has a t1/2has a t1/2(50°C) of 8.5 min. A monoclonal antibody directed against form A1bdoes not cross react with any of the other hepatopancreatic arylsulfatases when assayed by Western blot, confirming the structural heterogeneity of the adult enzymes.Total arylsulfatase activity increases in a biphasic manner during early abalone development, with the first increase occurring early in larval maturation. The secoad phase of enzyme expression is dependent upon the induction of settlement and metamorphosis of the competent veliger larvae, strongly suggesting that the expression of arylsulfatase synthesis (and the maturation of the digestive gland, the hepatopancreas) is controlled by genetic events which occur as a result of metamorphosis. Competent veliger larvae express only two arylsulfatase forms, which share many physicochemical and kinetic characteristics with the adult hepatopancreatic enzymes. However, neither of the larval arylsulfatases is recognized by the monoclonal antibody to form A1bfrom adult hepatopancreas. Endogenous enzyme inhibitor levels in larvae remain constant throughout the period of arylsulfatase induction, and therefore do not contribute to the control of arylsulfatase activity levels during development.These results are the first documentation of the developmental induction of a specific protein(s) in abalone as a result of metamorphosis. The significance of the timing of arylsulfatase expression is discussed in relation to potential physiological substrates and the dietary switching which occurs at metamorphosis. Possible genetic events which are consistent with the observed patterns of expression of these enzymes also are considered.