Calmodulin-dependent multifunctional protein kinase. Evidence for isoenzyme forms in mammalian tissues.
Calmodulin-dependent multifunctional protein kinase. Evidence for isoenzyme forms in mammalian tissues.
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钙调蛋白依赖性多功能蛋白激酶。
DOI：
10.1111/j.1432-1033.1986.tb10502.x
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发表时间：
1986
期刊：
影响因子：
--
通讯作者：
Kelly,PT
中科院分区：
文献类型：
--
作者：
Shenolikar,S;Lickteig,R;Hardie,DG;Soderling,TR;Hanley,RM;Kelly,PT
从大鼠脑（表观分子质量640 kDa）、兔肝（300 kDa）和兔骨骼肌（700 kDa）中广泛纯化的钙/钙调素依赖性多功能蛋白激酶，对其结构、免疫学和酶性质进行了分析。与脑钙调蛋白依赖性蛋白激酶50 kDa催化亚基的亲和纯化多克隆抗体的免疫学交叉反应性证实了在蛋白激酶的所有亚基中存在共同的抗原决定因子。单维磷酸肽模式，由s消化自磷酸化蛋白激酶获得。用胰蛋白酶和凝乳胰蛋白酶联合消化的125i标记蛋白的二维指纹图谱显示，该肝酶的两个亚基（51 kDa和53 kDa）非常相似。骨骼肌钙调蛋白依赖性蛋白激酶的56 - kDa多肽和/或58 - kDa多肽之间存在类似的同一性。这些数据表明，肝脏和肌肉蛋白激酶的亚基可能是由部分蛋白水解或自磷酸化产生的。脑酶的50 - kDa和60 - kDa亚基的肽模式证实了这两个催化亚基代表不同的蛋白质产物。磷酸化肽图谱和二维肽指纹图谱的比较表明，脑钙调素依赖性蛋白激酶的50 - kDa和60 - kDa亚基与肝脏和肌肉多肽具有相当大的结构同源性。然而，在肝脏51‐kDa亚基、骨骼肌56‐kDa和大脑50‐kDa和60‐kDa多肽中观察到大量独特的肽，这表明同工酶形式的存在。所有钙调素依赖性蛋白激酶都能以3-5 mol磷酸/mol蛋白的化学计量量快速磷酸化突触蛋白I。通过胰蛋白酶/糜蛋白酶消化32p标记的突触素I获得的磷酸肽的二维分离表明，相同的肽被所有钙调素依赖性蛋白激酶磷酸化。这些数据代表了肝脏钙调蛋白依赖性蛋白激酶与从脑和骨骼肌分离的酶的首次结构和免疫学比较。研究结果表明，存在一个高度保守的钙调蛋白依赖多功能蛋白激酶家族，具有相似的结构、免疫和酶特性。单个催化亚基似乎代表了在哺乳动物组织中选择性表达的不同蛋白质产物或同工酶的表达。
Calcium/calmodulin‐dependent multifunctional protein kinases, extensively purified from rat brain (with apparent molecular mass 640 kDa), rabbit liver (300 kDa) and rabbit skeletal muscle (700 kDa), were analysed for their structural, immunological, and enzymatic properties. The immunological cross‐reactivity with affinity‐purified polyclonal antibodies to the 50‐kDa catalytic subunit of the brain calmodulin‐dependent protein kinase confirmed the presence of common antigenic determinants in all subunits of the protein kinases. One‐dimensional phosphopeptide patterns, obtained by digestion of the autophosphorylated protein kinases withS. aureusV8protease, and two‐dimensional fingerprints of the125I‐labelled proteins digested with a combination of trypsin and chymotrypsin, revealed a close similarity between the two subunits (51 kDa and 53 kDa) of the liver enzyme. Similar identity was observed between the 56‐kDa and/or 58‐kDa polypeptides of the skeletal muscle calmodulin‐dependent protein kinase. The data suggest that the subunits of the liver and muscle protein kinases may be derived by partial proteolysis or by autophosphorylation. The peptide patterns for the 50‐kDa and 60‐kDa subunits of the brain enzyme confirmed that the two catalytic subunits represented distinct protein products. The comparison of the phosphopeptide maps and the two‐dimensional peptide fingerprints, indicated considerable structural homology among the 50‐kDa and 60‐kDa subunits of the brain calmodulin‐dependent protein kinase and the liver and muscle polypeptides. However, a significant number of unique peptides in the liver 51‐kDa subunit, skeletal muscle 56‐kDa, and the brain 50‐kDa and 60‐kDa polypeptides were observed and suggest the existence of isoenzyme forms. All calmodulin‐dependent protein kinases rapidly phosphorylated synapsin I with a stoichiometry of 3–5 mol phosphate/mol protein. The two‐dimensional separation of phosphopeptides obtained by tryptic/chymotryptic digestion of32P‐labelled synapsin I indicated that the same peptides were phosphorylated by all the calmodulin‐dependent protein kinases. Such data represent the first structural and immunological comparison of the liver calmodulin‐dependent protein kinase with the enzymes isolated from brain and skeletal muscle. The findings indicate the presence of a family of highly conserved calmodulin‐dependent multifunctional protein kinases, with similar structural, immunological and enzymatic properties. The individual catalytic subunits appear to represent the expression of distinct protein products or isoenzymes which are selectively expressed in mammalian tissues.