Azhar S, Menon K M
Eur J Biochem. 1975 Oct 1;58(1):105-15. doi: 10.1111/j.1432-1033.1975.tb02354.x.
The solubilization of plasma membrane fractions FI and FII associated protein kinases has been attempted using monovalent salts of high ionic strength and various detergent treatments. Extraction of FI and FII plasma membranes with high ionic strength salt solutions did not release more than 20% of the protein kinase activity. Similarly, monovalent salts released little adenosine 3':5'-monophosphate (cyclic AMP) binding activity, but after extraction binding capacity of cyclic [3H]AMP to plasma membranes was increased about 150-200%. Triton X-100 was a better solubilizing agent that Lubrol WX or deoxycholate. In addition to solubilization, 0.1% Triton X-100 also stimulated the protein kinase activity 150-200%. The properties of Triton X-100 solubilized FI and FII and purified cytosol KII were characterized with respect to protein substrate specificity, effect of cyclic AMP, cyclic nucleotide specificity, effects of divalent metal ion and gonadotropins. Upon sucrose density gradient centrifugation, FI solubilized protein kinase and cyclic AMP binding activities co-sedimented with a sedimentation coefficient of 6.3 S. The FII solubilized protein kinase sedimented as two components with sedimentation coefficients of 7.7 S and 5.5 S. The cyclic AMP binding activity also sedimented as two components with sedimentation coefficient 6.7 S and 5.5 S. Cyclic AMP caused dissociation of solubilized protein kinase from FI into a single catalytic (4.8 S) and two cyclic AMP binding subunits (8.1 S and 6.7 S). FII solubilized enzyme was dissociated into one catalytic (4.8 S) and one cyclic AMP binding subunit (6.3 S). Fractionation of FI and FII solubilized enzymes on DEAE-cellulose column chromatography resolved them each into two peaks Ia, Ib and IIa, IIb, respectively. Peaks Ib and IIb were more sensitive to cyclic AMP STIMULATION THAN Ia and IIa peaks. From these studies it is concluded that the plasma-membrane associated and cytosol protein kinases have similar catalytic properties but differ in some of their physical properties.
已尝试使用高离子强度的单价盐和各种去污剂处理来溶解与质膜组分FI和FII相关的蛋白激酶。用高离子强度盐溶液提取FI和FII质膜时，释放的蛋白激酶活性不超过20%。同样，单价盐释放的腺苷3':5'-单磷酸（环磷酸腺苷）结合活性很少，但提取后，环[3H]AMP与质膜的结合能力增加了约150 - 200%。Triton X - 100是比Lubrol WX或脱氧胆酸盐更好的增溶剂。除了增溶作用外，0.1%的Triton X - 100还能使蛋白激酶活性提高150 - 200%。就蛋白底物特异性、环磷酸腺苷的作用、环核苷酸特异性、二价金属离子和促性腺激素的作用而言，对Triton X - 100溶解的FI和FII以及纯化的胞质KII的特性进行了表征。在蔗糖密度梯度离心中，FI溶解的蛋白激酶和环磷酸腺苷结合活性以沉降系数6.3 S共同沉降。FII溶解的蛋白激酶沉降为两个组分，沉降系数分别为7.7 S和5.5 S。环磷酸腺苷结合活性也沉降为两个组分，沉降系数为6.7 S和5.5 S。环磷酸腺苷导致溶解的蛋白激酶从FI解离为一个单一的催化亚基（4.8 S）和两个环磷酸腺苷结合亚基（8.1 S和6.7 S）。FII溶解的酶解离为一个催化亚基（4.8 S）和一个环磷酸腺苷结合亚基（6.3 S）。在DEAE - 纤维素柱色谱上对FI和FII溶解的酶进行分级分离，分别将它们分离为两个峰Ia、Ib和IIa、IIb。峰Ib和IIb比峰Ia和IIa对环磷酸腺苷刺激更敏感。从这些研究得出结论，质膜相关的和胞质蛋白激酶具有相似的催化特性，但在一些物理特性上有所不同。
