Volonté C, Merlo D, Ciotti M T, Calissano P
Institute of Neurobiology, CNR, Rome, Italy.
J Neurochem. 1994 Dec;63(6):2028-37. doi: 10.1046/j.1471-4159.1994.63062028.x.
Primary neuronal cultures from 8-day-old rat cerebellum were incubated in the presence of exogenously added 16 nM [gamma-32P]ATP. Phosphorylation of a 45-kDa endogenous protein was detected within 1 min and increased linearly for approximately 20 min. Unlike what was seen with [gamma-32P]ATP, in the presence of [32P]orthophosphate no visible phosphorylation of protein was detected after 10 min, but a different pattern of phosphorylation was obtained in 30 min. The phosphorylation of the 45-kDa protein was reduced by 80-90% in the presence of 1 microM unlabeled ATP, 5 U/ml of apyrase, or 0.01% trypsin but not 1 mM PO4(3-). Phosphorylation was inversely proportional to cell density and was unaffected by addition to the cells of 56 mM KCl or 100 microM glutamate for 3 min. The presence of exogenously added cellular protein extracts or pretreatment of the cells for up to 20 min in phosphorylation buffer also did not affect the observed phosphorylation of the 45-kDa protein. The phosphorylation was found to be insensitive to MgCl2 but inhibited in the presence of MnCl2 or NaF and in the absence of CaCl2. Analogues of ATP suppressed phosphorylation of the 45-kDa protein by 80-90%. A similar inhibition was obtained in the presence of ADP or AMP. In this study, we establish via several different means that the phosphorylation of the 45-kDa protein in primary neuronal granule cultures occurs extracellularly through an ectokinase activity, which is furthermore distinguishable from a series of other presently characterized ecto-protein enzymes and intracellular kinases.
将来自8日龄大鼠小脑的原代神经元培养物在外源添加16 nM [γ-32P]ATP的情况下进行孵育。在1分钟内检测到一种45 kDa内源性蛋白质的磷酸化，并且线性增加约20分钟。与[γ-32P]ATP的情况不同，在存在[32P]正磷酸盐的情况下，10分钟后未检测到可见的蛋白质磷酸化，但在30分钟时获得了不同的磷酸化模式。在存在1 μM未标记的ATP、5 U/ml的腺苷三磷酸双磷酸酶或0.01%胰蛋白酶但不存在1 mM PO4(3-)的情况下，45 kDa蛋白质的磷酸化减少了80 - 90%。磷酸化与细胞密度成反比，并且在向细胞中添加56 mM KCl或100 μM谷氨酸3分钟后不受影响。外源添加细胞蛋白提取物或在磷酸化缓冲液中对细胞进行长达20分钟的预处理也不影响观察到的45 kDa蛋白质的磷酸化。发现该磷酸化对MgCl2不敏感，但在存在MnCl2或NaF以及不存在CaCl2的情况下受到抑制。ATP类似物使45 kDa蛋白质的磷酸化抑制了80 - 90%。在存在ADP或AMP的情况下也获得了类似的抑制作用。在本研究中，我们通过几种不同的方法确定，原代神经元颗粒培养物中45 kDa蛋白质的磷酸化是通过一种胞外激酶活性在细胞外发生的，而且它与目前已鉴定的一系列其他胞外蛋白酶和细胞内激酶不同。
