Simantov R, Sachs L
Eur J Biochem. 1975 Nov 1;59(1):89-95. doi: 10.1111/j.1432-1033.1975.tb02428.x.
A clone of neuroblastoma cells has been selected for its ability to survive and multiply at 40 degrees C. This temperature-resistant clone, like clones of neuroblastoma cells selected for resistance to dibutyryladenosine 3':5'-monophosphate (Bt2-Ado-3':5'-P) showed an increased tumorogenicity in animals and an increased saturation density at 37 degrees C. The Ado-3':5'-P-binding proteins and Ado-3':5'-P-dependent protein kinases from the temperature-resistant and non-resistant cells have been partially purified by chromatography on a DEAE-cellulose column. The Ado-3':5'-P-binding proteins from temperature-resistant cells were more sensitive to temperature than the binding proteins from non-resistant cells. After incubation of binding proteins from resistant cells at 37 degrees C, the specific activity of Ado-3':5'-P-binding to proteins was decreased about 50% and the apparent association constant (Ka) for Ado-3':5-p-binding was decreased from 7.4 X 10(7)M-1 to 4.4 x 10(7)M-1. There was no such decrease with binding proteins from non-resistant cells. A decrease in the activity of binding proteins from the temperature-resistant cells, but not of those from non-resistant cells, was also found when the proteins were stored at 2 degrees C. Treatment with 2-mercaptoethanol made binding proteins from the resistant cells less temperature-sensitive. In the absence of added Ado-3:5-P the protein kinase activity from the temperature-resistant cells was about 50% of the activity from non-resistant cells. Kinase activity was increased by addition of Ado-3:5-P and there was a greater increase with kinases from resistant cells. The maximum protein kinase activity was found in the presence of 10muM Ado-3':5'-P for the temperature-resistant cells and 0.1 muM Ado-3':5'-P for the non-resistant cells. The results indicate that the temperature sensitivity of Ado-3':5'-P-binding proteins, and the activity of protein kinase from cells selected for resistance to high temperature, are similar to those of cells selected for resistance to Bt2-Ado-3':5'-P. It is suggested that the temperature sensitivity of Ado-3':5'-P-binding proteins and the activity of Ado-3':5'-P-dependent protein kinases are involved in the regulation of malignancy and of cell growth at different temperatures.
已筛选出一个神经母细胞瘤细胞克隆，其具有在40摄氏度下存活和增殖的能力。这个耐温克隆，与筛选出的对二丁酰腺苷3':5'-单磷酸（Bt2-Ado-3':5'-P）具有抗性的神经母细胞瘤细胞克隆一样，在动物体内显示出更高的致瘤性，并且在37摄氏度时具有更高的饱和密度。通过在DEAE-纤维素柱上进行色谱分离，已部分纯化了来自耐温和非耐温细胞的腺苷3':5'-磷酸结合蛋白和腺苷3':5'-磷酸依赖性蛋白激酶。耐温细胞的腺苷3':5'-磷酸结合蛋白比非耐温细胞的结合蛋白对温度更敏感。将耐温细胞的结合蛋白在37摄氏度下孵育后，与蛋白结合的腺苷3':5'-磷酸的比活性降低了约50%，腺苷3':5'-磷酸结合的表观缔合常数（Ka）从7.4×10⁷M⁻¹降至4.4×10⁷M⁻¹。非耐温细胞的结合蛋白没有这种降低。当蛋白保存在2摄氏度时，也发现耐温细胞的结合蛋白活性降低，但非耐温细胞的结合蛋白活性未降低。用2-巯基乙醇处理使耐温细胞的结合蛋白对温度的敏感性降低。在没有添加腺苷3':5'-磷酸的情况下，耐温细胞的蛋白激酶活性约为非耐温细胞活性的50%。添加腺苷3':5'-磷酸可增加激酶活性，并且耐温细胞的激酶增加幅度更大。对于耐温细胞，在存在10μM腺苷3':5'-磷酸时发现最大蛋白激酶活性，对于非耐温细胞，在存在0.1μM腺苷3':5'-磷酸时发现最大蛋白激酶活性。结果表明，腺苷3':5'-磷酸结合蛋白的温度敏感性以及从筛选出的耐高温细胞中获得的蛋白激酶活性，与筛选出的对Bt2-Ado-3':5'-P具有抗性的细胞相似。有人提出，腺苷3':5'-磷酸结合蛋白的温度敏感性和腺苷3':5'-磷酸依赖性蛋白激酶的活性参与了不同温度下恶性肿瘤和细胞生长的调节。
