Properties of adenosine 3',5'-monophosphate-dependent protein kinases isolated from bovine epididymal spermatozoa.
Properties of adenosine 3',5'-monophosphate-dependent protein kinases isolated from bovine epididymal spermatozoa.
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从牛附睾精子中分离出的腺苷 3,5-单磷酸依赖性蛋白激酶的特性。
DOI：
10.1016/s0021-9258(19)44348-2
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发表时间：
1973
期刊：
影响因子：
--
通讯作者：
H. Lardy
中科院分区：
文献类型：
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作者：
D. Garbers;N. First;H. Lardy
用DEAE-Sephadex离子交换层析和SephadexG-100凝胶过滤从牛附睾精子中分离到三种蛋白激酶。用SephadexG-100凝胶过滤法测得这三种激酶的分子量分别为120，000、78，000和56，000。3′，5 ′-环磷酸腺苷（cyclicadenosine 3′，5 ′-monophosphate，cAMP）可显著增强上述三种激酶的活性。在10 ~（-6）m环腺苷酸存在下，从三种蛋白激酶中分离出一个分子量为30，000 ~ 35，000的催化亚基。在这些相同的条件下，环AMP结合蛋白位于对应于分子量为78，000，35，000至40，000和17，000至18，000的组分中;后者可能是由蛋白水解引起的，因为在新鲜制剂中没有观察到。分子量为78，000的环腺苷酸结合蛋白与所有三种蛋白激酶的分离催化亚基结合，酪蛋白的表观Km值（约3 mg/ml）似乎对三种激酶没有不同，也不受加入环腺苷酸的影响。ATP的表观Km在3.5至8.7 µm之间变化，但所有三种蛋白激酶的表观Km似乎相同，并且似乎不受环AMP的影响。分离的分子量为120，000的激酶催化亚基对ATP的Km值为5.5 μm，与完整激酶的Km值一致，三种激酶以酪蛋白为底物时的最大激酶活性约为pH 6.5，以组蛋白为底物时的最大激酶活性约为pH 7.5。120，000分子量激酶的分离的催化亚基对pH显示出相同的反应。环AMP在10- 7 m范围内半最大地激活所有三种激酶，而环GMP在10- 6 m范围内半最大地激活激酶。8-甲硫基环腺苷酸（8-Methylthio-cyclicAMP，8-Methylthio-cyclicAMP）比环腺苷酸（cyclicAMP，8-Methylthio-cyclicAMP）对三种激酶的激活作用更强，提示三种激酶的催化亚基相似或相同。
Three protein kinases were separated from bovine epididymal spermatozoa by using DEAE-Sephadex ion exchange chromatography and Sephadex G-100 gel filtration. The molecular weights of these three kinases were estimated to be 120,000, 78,000 and 56,000 by the use of Sephadex G-100 gel filtration. The activity of all three kinases was greatly enhanced by cyclic adenosine 3′,5′-monophosphate (cyclic AMP). In the presence of 10-6mcyclic AMP, a catalytic subunit of molecular weight 30,000 to 35,000 was separated from each of the three protein kinases. Under these same conditions, cyclic AMP-binding proteins were located in fractions corresponding to molecular weights of 78,000, 35,000 to 40,000, and 17,000 to 18,000; the latter may result from proteolysis for it was not observed in fresh preparations. The cyclic AMP-binding protein of molecular weight 78,000 combined with the isolated catalytic subunits of all three protein kinases.The apparentKmfor casein (approximately 3 mg per ml) appeared to be neither different for the three kinases nor affected by the addition of cyclic AMP. The apparentKmfor ATP varied from 3.5 to 8.7 µm, but it appeared to be the same for all three protein kinases and also appeared to be unaffected by cyclic AMP. The isolated catalytic subunit of the kinase of molecular weight 120,000 exhibited aKmfor ATP of 5.5 µm, which agreed with theKmfor the intact kinase.Maximum kinase activity was at about pH 6.5 with casein as substrate and at about pH 7.5 with histone as substrate for all three kinases. The isolated catalytic subunit of the 120,000 molecular weight kinase showed identical responses to pH.Cyclic AMP activated all three kinases half-maximally in the 10-7mrange, whereas cyclic GMP activated the kinases half-maximally in the 10-6mrange. 8-Methylthio-cyclic AMP appeared to be more potent than cyclic AMP in activating the kinases.The data suggest that the catalytic subunits of the three kinases are either similar or identical.