Guanosine cyclic monophosphate-dependent protein kinase from foetal calf heart. Purification, general properties and catalytic subunit.
Guanosine cyclic monophosphate-dependent protein kinase from foetal calf heart. Purification, general properties and catalytic subunit.
复制标题
来自胎牛心脏的鸟苷环单磷酸依赖性蛋白激酶。
作者：
M. Shoji;J. Patrick;C. Davis;J. Kuo
从胎牛心脏中纯化了环GMP依赖性蛋白激酶，并对其一般性质和亚基结构进行了研究。经pH5.3-等电沉淀、DEAE-纤维素层析、Sephadex G-200过滤和羟基磷灰石处理，从心脏提取物中纯化了900倍以上的酶。纯化的心肌酶，从环AMP依赖性蛋白激酶的污染，表现出刺激性调节剂（或含有刺激性调节剂组分的粗调节剂）的绝对需求，其环GMP刺激活性。环腺苷酸依赖性蛋白激酶的抑制性调节剂（蛋白抑制剂）既不能刺激也不能抑制环鸟苷酸靶酶。该酶对8-溴环GMP、环GMP和环AMP的Ka值分别为0.013、0.033和3.0 μ M。环GMP依赖性酶的活性需要Mg 2+和Co 2+，最佳浓度分别为约30和0.5 mM。酶活性的最适pH范围为6至9。组蛋白通常是有效的底物蛋白。该酶表现出更大的亲和力组蛋白比环AMP依赖类蛋白激酶。全酶（表观摩尔重量）150 000）的心肌环GMP依赖性蛋白激酶解离成环GMP非依赖性催化亚基（表观mol. 60 000）通过环GMP和组蛋白。催化亚基需要刺激调节剂的活动，在环GMP存在下的全酶的情况下。
Cyclic GMP-dependent protein kinase was purified from foetal calf hearts, and its general properties and subunit structure were studied. The enzyme was purified over 900-fold from the heart extract by pH 5.3-isoelectric precipitation, DEAE-cellulose chromatography, Sephadex G-200 filtration and hydroxyapatite treatment. The purified myocardial enzyme, free from cyclic AMP-dependent protein kinase contamination, exhibited an absolute requirement of stimulatory modulator (or crude modulator containing the stimulatory modulator component) for its cyclic GMP-stimulated activity. Inhibitory modulator (protein inhibitor) of cyclic AMP-dependent protein kinase could not stimulate nor inhibit the cyclic GMP target enzyme. The enzyme had Ka values of 0.013, 0.033 and 3.0 micronM for 8-bromo cyclic GMP, cyclic GMP and cyclic AMP respectively. The cyclic GMP-dependent enzyme required Mg2+ and Co2+ for its activity, with optimal concentrations of about 30 and 0.5 mM respectively. The pH optimum for the enzyme activity ranged from 6 to 9. Histones were generally effective substrate proteins. The enzyme exhibited a greater affinity for histones than did the cyclic AMP-dependent class of protein kinase. The holoenzyme (apparent mol.wt. 150 000) of the myocardial cyclic GMP-dependent protein kinase was dissociated into a cyclic GMP-independent catalytic subunit (apparent mol.wt. 60 000) by cyclic GMP and histone. The catalytic subunit required the stimulatory modulator for its activity, as in the case of the holoenzyme in the presence of cyclic GMP.