Structural requirements of phospholipase C delta1 for regulation by spermine, sphingosine and sphingomyelin.
Structural requirements of phospholipase C delta1 for regulation by spermine, sphingosine and sphingomyelin.
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精胺、鞘氨醇和鞘磷脂调节磷脂酶 C delta1 的结构要求。
DOI：
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
A. Matecki
中科院分区：
文献类型：
--
作者：
Tadeusz Pawelczyk;A. Matecki
我们研究了鞘磷脂、钙、精胺和鞘氨醇在磷脂酶C（PLC）δ 1活性调节中的关系。抑制PLC δ 1鞘磷脂促进精胺和钙离子，并部分取消鞘氨醇。鞘氨醇和精胺的作用完全依赖于Ca ~（2+）。在不存在Ca 2+的情况下，没有观察到这些物质对PLC δ 1活性的影响。使用缺失突变体和活性片段的PLC delta 1有限的蛋白水解产生的，我们研究了这些化合物的调节酶的结构要求。缺失突变体的PLC delta 1缺乏的前58个氨基酸和缺乏整个pleckstrin同源性（PH）域的突变体是完全活跃的洗涤剂测定，和他们的活动的影响精胺，鞘氨醇，Ca 2+和鞘磷脂的天然酶的相同程度。PLC delta 1的有限的蛋白水解产生两个片段的40 kDa和30 kDa，形成一个稳定的活性复合物。Ca 2+浓度和酶活性之间的关系是几乎相同的原生PLC delta 1和蛋白水解复合物。由40 kDa和30 kDa肽形成的蛋白水解复合物的活性不受精胺和鞘氨醇的影响。鞘磷脂抑制复合物略低于本地PLC delta 1，这种抑制并没有促进精胺。这些观察结果表明，为激活PLC delta 1的精胺和鞘氨醇，该地区跨越域的高度保守，命名为X和Y，必须是完整的。相比之下，PH结构域和完整的跨越区域的X和Y结构域是不是必不可少的抑制PLC delta 1鞘磷脂。
We studied the relationship between sphingomyelin, calcium, spermine and sphingosine in regulation of phospholipase C (PLC) delta1 activity. Inhibition of PLC delta1 by sphingomyelin was promoted by spermine and Ca2+ and was partially abolished by sphingosine. The effect of sphingosine and spermine entirely depended on Ca2+. In the absence of Ca2+, no effect of these substances on PLC delta1 activity was observed. Using deletion mutants and active fragments of PLC delta1 generated by limited proteolysis, we have studied the structural requirements of the enzyme for regulation by these compounds. The deletion mutant of PLC delta1 lacking the first 58 amino acids and the mutant lacking the entire pleckstrin homology (PH) domain were fully active in the detergent assay, and their activities were affected by spermine, sphingosine, Ca2+ and sphingomyelin to the same extent as the native enzyme. The limited proteolysis of PLC delta1 generated two fragments of 40 kDa and 30 kDa, which formed a stable active complex. The relationship between Ca2+ concentration and enzymatic activity was almost identical for the native PLC delta1 and the proteolytic complex. The activity of the proteolytic complex formed by the 40 kDa and 30 kDa peptides was not affected by spermine and sphingosine. Sphingomyelin inhibited the complex slightly less than the native PLC delta1, and this inhibition was not promoted by spermine. These observations suggest that for activation of PLC delta1 by spermine and sphingosine, the region spanning domains of high conservation, named X and Y, must be intact. In contrast, the PH domain and the intact spanning region of the X and Y domains are not essential for inhibition of PLC delta1 by sphingomyelin.
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DOI：
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期刊：
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