Solubilization of hyaluronic acid synthetic activity from streptococci and its activation with phospholipids.
Solubilization of hyaluronic acid synthetic activity from streptococci and its activation with phospholipids.
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DOI：
10.1016/s0021-9258(17)38485-5
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发表时间：
1986-05
期刊：
影响因子：
--
通讯作者：
M. Triscott;I. van de Rijn
中科院分区：
文献类型：
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作者：
M. Triscott;I. van de Rijn
迄今为止，所有的玻尿酸合成系统都是颗粒性质的，并且在增溶方面的尝试都不成功。这阻碍了阐明透明质酸产生机制的努力。本研究证明，C群链球菌膜透明质酸的合成活性用2%地黄芩苷溶解，并以心磷脂重组为最佳的磷脂/蛋白比（微克/微克）为5:1。此外，对溶解的合成酶的色谱分析表明，它在Sepharose CL-6B柱的空隙体积后被洗脱。CHAPSO、辛基吡喃葡萄糖苷、胆酸钠、Triton X-100和两性增溶剂314均抑制或不能增溶合成活性。心磷脂以外的磷脂，特别是磷脂酰乙醇胺和磷脂酰丝氨酸，也能重建洋地黄苷提取物的活性。在我们的系统中，透明质酸合成酶的比活性比完整膜的系统提高了63倍。此外，重组体系的总活性是完整膜的4.9倍。可溶性酶系统与膜结合合成酶在生成三氯乙酸溶性和不溶性透明质酸的动力学上表现出相似之处，所产生的透明质酸分子量相当。
To date all hyaluronic acid synthetic systems have been of a particulate nature, and attempts at solubilization have been unsuccessful. This has hampered attempts to elucidate the mechanism by which hyaluronic acid is produced. In this paper we demonstrate that the hyaluronic acid synthetic activity from group C streptococcal membranes was solubilized using 2% digitonin and that the activity was optimized by reconstitution with cardiolipin at an optimum phospholipid/protein ratio (microgram/microgram) of 5:1. Furthermore, chromatography of the solubilized synthetase demonstrated that it eluted after the void volume of a Sepharose CL-6B column. CHAPSO, octyl glucopyranoside, sodium cholate, Triton X-100, and zwittergent 314 either inhibited or failed to solubilize the synthetic activity. Phospholipids other than cardiolipin also reconstituted the activity from the digitonin extract, particularly phosphatidylethanolamine and phosphatidylserine. In our system, the specific activity of hyaluronic acid synthetase was increased up to 63 times that of the system of the intact membrane. Furthermore, the total activity of the reconstituted system was 4.9 times greater than that of intact membranes. The soluble enzyme system showed similarities to the membrane-bound synthetase in the kinetics of production of trichloroacetic acid-soluble and -insoluble hyaluronic acid, and the hyaluronic acid produced was of comparable molecular weight.