Biosynthesis and proteolytic activation of cystathionine beta-synthase in rat liver.
Biosynthesis and proteolytic activation of cystathionine beta-synthase in rat liver.
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DOI：
10.1016/s0021-9258(17)43702-1
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发表时间：
1984-01
期刊：
影响因子：
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通讯作者：
F. Skovby;J. Kraus;L. Rosenberg
中科院分区：
文献类型：
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作者：
F. Skovby;J. Kraus;L. Rosenberg
我们研究了半胱硫醚-β-合酶(EC 4.2.1.22)在大鼠肝组织和肝组织中的生物合成。用免疫沉淀法回收酶，用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法进行分析。当用无细胞翻译方法检测大鼠肝脏mRNA时，只检测到一个编码63,000道尔顿多肽的单一mRNA物种。另一方面，通过免疫沉淀从新鲜的肝脏提取液中回收了两种多肽：主要的MR=63,000多肽和次要的MR=48,000的多肽。当这些提取物在4℃下孵育7天时，合酶活性增加2-3倍，伴随而来的是MR=63,000多肽的消失和MR=48,000多肽的一些增加。此外，现在发现含有较小亚基的合成酶的比活力大约是含有较大亚基的合成酶的60倍。至少在一定程度上，这种比活性的增加反映了对同型半胱氨酸的亲和力增加了30倍。在体外，Nα-对甲苯磺酰-L-赖氨酸氯甲基酮、止痛剂和亮肽素等酶抑制剂可以阻止亚基大小和活性的改变，但不能被其他几种酶抑制剂阻止。对大鼠肝脏切片的脉冲追逐实验表明，MR=63,000的多肽在翻译后缓慢地转化为MR=48,000的多肽。综上所述，我们的发现与以下可能性是一致的，即大亚基形式的合成酶在生理条件下基本上是不活跃的，合成酶的活性受到有限的蛋白质分解的调节。
We investigated the biosynthesis of cystathionine beta-synthase (EC 4.2.1.22) in a cell-free translation system programmed with rat liver mRNA and in slices of rat liver. The enzyme was recovered by immunoprecipitation and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Only a single mRNA species, coding for a 63,000-dalton polypeptide, was detected when rat liver mRNA was assayed by cell-free translation. On the other hand, two polypeptides were recovered by immunoprecipitation from fresh liver extracts: a predominant Mr = 63,000 polypeptide and a minor Mr = 48,000 polypeptide. When such extracts were incubated at 4 degrees C for 7 days, the synthase activity increased 2-3-fold with a concomitant disappearance of the Mr = 63,000 polypeptide and some increase of the Mr = 48,000 polypeptide. Moreover, the specific activity of synthase containing the smaller subunits was now found to be approximately 60-fold higher than that containing the larger ones. At least in part, this increased specific activity reflected a 30-fold greater affinity for homocysteine. The changes in subunit size and activity could be prevented in vitro by protease inhibitors such as N alpha-p-tosyl-L-lysine chloromethyl ketone, antipain, and leupeptin, but not by several other protease inhibitors. Pulse-chase experiments with slices of rat liver suggested a slow, post-translational conversion of the Mr = 63,000 polypeptide to the Mr = 48,000 polypeptide. Taken together, our findings are consistent with the possibility that the large subunit form of synthase is essentially inactive under physiologic conditions, and that synthase activity is regulated by limited proteolysis.