Purification and characterization of human erythrocyte phosphatidylinositol 4-kinase. Phosphatidylinositol 4-kinase and phosphatidylinositol 3-monophosphate 4-kinase are distinct enzymes.
Purification and characterization of human erythrocyte phosphatidylinositol 4-kinase. Phosphatidylinositol 4-kinase and phosphatidylinositol 3-monophosphate 4-kinase are distinct enzymes.
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人红细胞磷脂酰肌醇 4-激酶的纯化和表征。
DOI：
10.1042/bj2840039
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发表时间：
1992
期刊：
影响因子：
--
通讯作者：
Cantley,LC
中科院分区：
文献类型：
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作者：
Graziani,A;Ling,LE;Endemann,G;Carpenter,CL;Cantley,LC
PtdIns 4-激酶已从人红细胞膜中纯化了83，000倍。通过SDS/PAGE检测的主要蛋白质的分子量为56 kDa，并且酶活性可以从凝胶的该条带复性。这种酶的特性与先前描述的其他II型PtdIns激酶相似：Triton X-100胶束中存在的PtdIns比PtdIns囊泡更优选作为底物，该酶对ATP具有相对低的Km（20 μ M），并且腺苷是有效的抑制剂。牛脑II型PtdIns 4-激酶的单克隆抗体是纯化酶的有效抑制剂。PtdIns（4，5）P2抑制约当与PtdIns等摩尔量添加时，50%; PtdIns 4P对活性几乎没有影响。在红细胞裂解物中也检测到PtdIns 3 P 4-激酶活性。大约三分之二的这种活性是在胞质部分和三分之一的膜部分。在纯化的II型PtdIns 4-激酶制剂中没有检测到PtdIns 3 P 4-激酶活性，在牛脑III型PtdIns 4-激酶制剂中也没有检测到这种活性。抑制II型PtdIns 4-激酶的单克隆抗体不影响膜组分中PtdIns 3 P 4-激酶的活性。胞质PtdIns 3 P 4-激酶可以有效地从60%饱和的细胞中回收。（NH 4）2SO 4沉淀物，其几乎不含PtdIns 4-激酶活性。我们得出结论，PtdIns 3 P 4-激酶是一种新的酶不同于以前的特点PtdIns 4-激酶，这种酶更喜欢PtdIns 3 P超过PtdIns作为底物。
PtdIns 4-kinase has been purified 83,000-fold from human erythrocyte membranes. The major protein detected by SDS/PAGE is of molecular mass 56 kDa, and enzymic activity can be renatured from this band of the gel. The characteristics of this enzyme are similar to other type II PtdIns kinases previously described: PtdIns presented in Triton X-100 micelles is preferred as a substrate over PtdIns vesicles, the enzyme possesses a relatively low Km for ATP (20 microM), and adenosine is an effective inhibitor. A monoclonal antibody raised against bovine brain type II PtdIns 4-kinase is an effective inhibitor of the purified enzyme. PtdIns(4,5)P2 inhibits by approx. 50% when added in equimolar amounts with PtdIns; PtdIns4P has little effect on activity. A PtdIns3P 4-kinase activity has also been detected in erythrocyte lysates. Approximately two-thirds of this activity is in the cytosolic fraction and one-third in the membrane fraction. No PtdIns3P 4-kinase activity could be detected in the purified type II PtdIns 4-kinase preparation, nor could this activity be detected in a bovine brain type III PtdIns 4-kinase preparation. The monoclonal antibody that inhibits the type II PtdIns 4-kinase does not affect the PtdIns3P 4-kinase activity in the membrane fraction. The cytosolic PtdIns3P 4-kinase can be efficiently recovered from a 60%-satd.-(NH4)2SO4 precipitate that is virtually free of PtdIns 4-kinase activity. We conclude that PtdIns3P 4-kinase is a new enzyme distinct from previously characterized PtdIns 4-kinases, and that this enzyme prefers PtdIns3P over PtdIns as a substrate.