Irvine R F, Letcher A J, Dawson R M
Biochem J. 1984 Feb 15;218(1):177-85. doi: 10.1042/bj2180177.
The phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] [and to a lesser extent, the phosphatidylinositol-4-phosphate (PtdIns4P)] phosphodiesterase and monoesterase activities of a rat brain supernatant have been studied by using 32P-labelled substrates prepared from human red blood cells. PtdIns(4,5)P2 monoesterase is maximally stimulated by Mg2+, though some activity is detectable in Ca2+/EDTA (Mg2+-free) buffers. The phosphodiesterase, however, is Ca2+-dependent, and in Ca2+/EDTA buffers with the pure lipid as substrate, shows maximal activity at 100 nM-Ca2+. If PtdIns(4,5)P2 is presented as a component of a lipid mixture of similar composition to that of the inner half of the lipid bilayer of a rat liver plasma membrane, the phosphodiesterase shows considerable activity at 1 microM-Ca2+, and is maximal at 100 microM-Ca2+. However, if it is assayed against the same substrate in Ca2+/EGTA buffers with 3mM-Mg2+ and 80 mM-KCl present (as an approximate parallel with the ionic environment in vivo), it shows no detectable activity below 100 microM-Ca2+, and is maximal at 1 mM-Ca2+. The monoesterase can hydrolyse PtdIns(4,5)P2 in such a lipid mixture at all Ca2+ concentrations with 1 or 3 mM-Mg2+ present. PtdIns(4,5)P2 phosphodiesterase can be induced to attack its substrate under ionic conditions similar to those in vivo (0.1-1 microM-Ca2+; 1 mM-Mg2+; 80 mM-KCl) by the conversion of its substrate into a non-bilayer configuration. If given such a substrate [by mixing PtdIns(4,5)P2 with an excess of phosphatidylethanolamine (PtdEtn)] it shows a shallow Ca2+-dependency curve from 0.1 to 100 microM and then a steep rise to 1 mM-Ca2+. Together these observations lead us to the suggestion that a perturbation in a membrane in vivo equivalent to a non-bilayer configuration would be sufficient to induce phosphodiesterase-catalysed PtdIns(4,5)P2 breakdown. When given substrates mixed with excess PtdEtn at pH 7.25 (or 5.5), 1 microM-Ca2+, 1 mM-Mg2+ and 80 mM-KCl, the rat brain supernatant phosphodiesterase activity hydrolysed PtdIns(4,5)P 50-100-fold faster than it hydrolysed phosphatidylinositol (PtdIns). If the supernatant was presented with such a non-bilayer mixture containing a ten-fold excess of PtdIns over PtdIns(4,5)P2, the latter phospholipid was still hydrolysed by phosphodiesterasic cleavage at nearly ten times the rate of the former. Receptor-stimulated phosphodiesterase cleavage of polyphosphoinositides is an early event in cell activation by many agonists. The properties of PtdIns(4,5)P2 phosphodiesterase in vitro suggest that a change in the presentation of its substrate would be a sensitive and sufficient control on the enzyme's activity in vivo.
利用从人红细胞制备的32P标记底物，研究了大鼠脑匀浆上清液中磷脂酰肌醇-4,5-二磷酸[PtdIns(4,5)P2][以及程度较轻的磷脂酰肌醇-4-磷酸(PtdIns4P)]磷酸二酯酶和单酯酶的活性。PtdIns(4,5)P2单酯酶在Mg2+存在时受到最大程度的刺激，不过在Ca2+/EDTA(不含Mg2+)缓冲液中也可检测到一定活性。然而，磷酸二酯酶依赖Ca2+，在以纯脂质为底物的Ca2+/EDTA缓冲液中，在100 nM - Ca2+时表现出最大活性。如果将PtdIns(4,5)P2作为与大鼠肝质膜脂质双层内半部分组成相似的脂质混合物的一个成分，磷酸二酯酶在1 μM - Ca2+时表现出相当高的活性，在100 μM - Ca2+时达到最大活性。但是，如果在含有3 mM - Mg2+和80 mM - KCl的Ca2+/EGTA缓冲液中(近似模拟体内离子环境)以相同底物进行测定，在Ca2+浓度低于100 μM时未检测到活性，在1 mM - Ca2+时达到最大活性。单酯酶在有1或3 mM - Mg2+存在的情况下，在所有Ca2+浓度下均可水解这种脂质混合物中的PtdIns(4,5)P2。通过将底物转化为非双层结构，可诱导PtdIns(4,5)P2磷酸二酯酶在类似于体内的离子条件下(0.1 - 1 μM - Ca2+；1 mM - Mg2+；80 mM - KCl)攻击其底物。如果给予这样的底物[通过将PtdIns(4,5)P2与过量的磷脂酰乙醇胺(PtdEtn)混合]，它在0.1至100 μM范围内呈现出一条平缓的Ca2+依赖性曲线，然后在Ca2+浓度达到1 mM时急剧上升。这些观察结果共同使我们推测，体内膜中相当于非双层结构的扰动足以诱导磷酸二酯酶催化的PtdIns(4,5)P2分解。当在pH 7.25(或5.5)、1 μM - Ca2+、1 mM - Mg2+和80 mM - KCl条件下给予与过量PtdEtn混合的底物时，大鼠脑匀浆上清液中的磷酸二酯酶活性水解PtdIns(4,5)P的速度比水解磷脂酰肌醇(PtdIns)快50 - 100倍。如果向上清液提供这样一种非双层混合物，其中PtdIns的量比PtdIns(4,5)P2过量十倍，后者磷脂仍会通过磷酸二酯酶裂解，其水解速度几乎是前者的十倍。受体刺激的多磷酸肌醇磷酸二酯酶裂解是许多激动剂激活细胞的早期事件。体外PtdIns(4,5)P2磷酸二酯酶的特性表明，底物呈现方式的改变将是对该酶体内活性的一种敏感且充分的调控。
