Characterization of the phosphatidylinositol-specific phospholipase C-released form of rat osseous plate alkaline phosphatase and its possible significance on endochondral ossification
Characterization of the phosphatidylinositol-specific phospholipase C-released form of rat osseous plate alkaline phosphatase and its possible significance on endochondral ossification
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DOI：
10.1007/bf01076074
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发表时间：
1995-11-22
影响因子：
4.3
通讯作者：
Leone, FA
中科院分区：
文献类型：
--
作者：
Pizauro, JM;Ciancaglini, P;Leone, FA
通过将大鼠骨板膜结合酶与磷脂酰肌醇特异性磷脂酶C孵育，碱性磷酸酶活性从膜上释放至100%。释放的酶的分子量在Sephacryl S-300凝胶过滤上为145，000，在PAGE-SDS上为66，000，表明二聚体结构。用磷脂酶C溶解膜结合酶并不破坏其水解PNPP、ATP和焦磷酸的能力。ATP和PNPP的水解磷脂酰肌醇特异性磷脂酶C-释放酶表现出“迈克尔”动力学与K-0.5=70和979 μ M，分别。对于焦磷酸盐，K-0.5为128 μ M，并观察到位点间的相互作用（n=1.4）。镁离子对磷脂酰肌醇特异性磷脂酶C释放酶有刺激作用（K-0.5=1.5 mM），锌离子对磷脂酰肌醇特异性磷脂酶C释放酶有强的非竞争性抑制作用（K-1 =6.2 μ M）。然而，随着温度从40-60 ℃升高，该酶迅速失活，遵循一级动力学，热失活常数从5.08 × 10（-4）min（-1）变化到0.684 min（-1）。镁（K-0.5=29.5 μ M）、锰（K-0.5=5 μ M）和钴离子（K-0.5=10.1 μ M）恢复了Chelex处理的酶的活性，证明了其金属酶性质。由钙离子（K-0.5=653 μ M）的Chelex处理的酶的刺激是不太有效的（只有26%），并发生与站点的相互作用（n=0.7）。锌离子没有刺激作用。的可能性，可溶性形式的酶，检测endochondriossification过程中，将出现由骨板碱性磷酸酶的PI锚定形式的水解进行了讨论。
Alkaline phosphatase activity was released up to 100% from the membrane by incubating the rat osseous plate membrane-bound enzyme with phosphatidylinositol-specific phospholipase C. The molecular weight of the released enzyme was 145,000 on Sephacryl S-300 gel filtration and 66,000 on PAGE-SDS, suggesting a dimeric structure. Solubilization of the membrane-bound enzyme with phospholipase C did not destroy its ability to hydrolyse PNPP, ATP and pyrophosphate. The hydrolysis of ATP and PNPP by phosphatidylinositol-specific phospholipase C-released enzyme exhibited 'Michaelian' kinetics with K-0.5=70 and 979 mu M, respectively. For pyrophosphate, K-0.5 was 128 mu M and site-site interactions were observed (n=1.4). Magnesium ions were stimulatory (K-0.5=1.5 mM) and zinc ions were a powerful noncompetitive inhibitor (K-i=6.2 mu M) of phosphatidylinositol-specific phospholipase C-released enzyme.Phosphatidylinositol-specific phospholipase C-released alkaline phosphatase was relatively stable at 40 degrees C. However, with increasing temperature from 40-60 degrees C, the enzyme was inactivated rapidly following first order kinetics and thermal inactivation constants varied from 5.08 x 10(-4) min(-1) to 0.684 min(-1).Treatment of phosphatydilinositol-specific phospholipase C-released alkaline phosphatase with Chellex 100 depleted to 5% its original PNPPase activity. Magnesium (K-0.5=29.5 mu M), manganese (K-0.5=5 mu M) and cobalt ions (K-0.5=10.1 mu M) restored the activity of Chelex-treated enzyme, demonstrating its metalloenzyme nature. The stimulation of Chelex-treated enzyme by calcium ions (K-0.5=653 mu M) was less effective (only 26%) and occurred with site-site interactions (n=0.7). Zinc ions had no stimulatory effects.The possibility that the soluble form of the enzyme, detected during endochondral ossification, would arise by the hydrolysis of the Pl-anchored form of osseous plate alkaline phosphatase is discussed.