CATALYTIC PROPERTIES OF A PURIFIED PHOSPHATIDYLINOSITOL-4-PHOSPHATE KINASE FROM RAT-BRAIN
CATALYTIC PROPERTIES OF A PURIFIED PHOSPHATIDYLINOSITOL-4-PHOSPHATE KINASE FROM RAT-BRAIN
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DOI：
10.1042/bj2370025
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发表时间：
1986-07-01
影响因子：
4.1
通讯作者：
CHAMBAZ, EM
中科院分区：
文献类型：
--
作者：
COCHET, C;CHAMBAZ, EM
从大鼠脑提取液中通过几个层析步骤纯化了磷脂酰肌醇-4-磷酸(PIP)激酶活性，得到了一种活性制剂(磷脂酰肌醇4，5-二磷酸/分钟掺入~(32)P的比活性为1微克/分钟/毫克蛋白质)，其天然形式的表观分子尺寸为100-110 kDa。分离的PIP激酶活性需要镁离子(最好为20-30 mM)，不受钙离子影响。该酶以ATP(Km25µm)和GTP(Km133mum)为磷源，以PIP(Km3.3mug/ml)为脂底物。PIP-磷酸化反应被微摩尔浓度的肝素[ID50(Conn.给予50%抑制)2微克/毫升]和黄酮类Qercetin(ID50 0.2微米)。肝素对PIP具有竞争性抑制作用，而栎素则对三磷酸腺苷(或GTP)具有竞争性。用高活性的纯化蛋白激酶C对该制剂进行磷酸化处理并不能明显改变PIP激酶的活性。12-O-十四酰佛波酯和各种磷脂对PIP活性无影响，而磷脂酰丝氨酸则呈剂量依赖性地激活PIP活性。这表明磷脂酰丝氨酸-PIP激酶的相互作用可能被认为是细胞-膜水平的一种调节过程。
A phosphatidylinositol-4-phosphate (PIP) kinase activity was purified from rat brain extract through several chromatographic steps to yield an active preparation (specific activity 1 .mu.mol of 32P incorporated into phosphatidylinositol 4,5-bisphosphate/min per mg of protein) with an apparent molecular size of 100-110 kDa in the native form. The isolated PIP kinase required Mg2+ (optimally 20-30 mM) for its activity and was not influenced by Ca2+. The enzyme used ATP (Km 25 .mu.M) and GTP (Km 133 .mu.M) as phosphate sources and appeared specific for PIP (Km 3.3 .mu.g/ml) as the lipid substrate. The PIP-phosphorylation reaction was inhibited by micromolar concentrations of heparin [ID50 (concn. giving 50% inhibition) 2 .mu.g/ml] and the flavonoid quercetin (ID50 0.2 .mu.M). Whereas heparin behaves as a competitive inhibitor to PIP, quercetin was competitive towards ATP (or GTP). Phosphorylation of the preparation by a highly active purified protein kinase C did not detectably alter PIP kinase activity. Whereas 12-O-tetradecanoylphorbol acetate and various phospholipids had no effect, phosphatidylserine elicited a dose-dependent activation of PIP activity. This suggests that a phosphatidylserine-PIP kinase interaction may be considered as a possible regulatory process at the cell-membrane level.