THE ACTIVATION OF EXPRESSED CGMP-DEPENDENT PROTEIN-KINASE ISOZYME-I-ALPHA AND ISOZYME-I-BETA IS DETERMINED BY THE DIFFERENT AMINO-TERMINI
THE ACTIVATION OF EXPRESSED CGMP-DEPENDENT PROTEIN-KINASE ISOZYME-I-ALPHA AND ISOZYME-I-BETA IS DETERMINED BY THE DIFFERENT AMINO-TERMINI
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DOI：
10.1111/j.1432-1033.1991.tb16509.x
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发表时间：
1991-12-18
期刊：
影响因子：
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通讯作者：
HOFMANN, F
中科院分区：
文献类型：
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作者：
RUTH, P;LANDGRAF, W;HOFMANN, F
牛cGMP依赖蛋白激酶I-α和I-β同工酶的基因只在其氨基末端区域(氨基酸分别为1-89和1-104)上有所不同。每种重组同工酶(ri-α和ri-β)都在COS-7细胞中瞬时表达，并与从牛气管中纯化的cGMP激酶同工酶P-I和P-II进行了比较。P-I、P-II、Ri-α和Ri-β亚基的分子质量约为75 kDa。Ri-α和Ri-β的S20和w值分别为7.6和7.2，表明它们是以二聚体全酶形式存在的。特异性抗体免疫染色显示P-I和Ri-α以及P-II和Ri-β在免疫学上是不可区分的。P-I、P-II、Ri-α和Ri-β具有相同的催化活性。而ri-α和ri-β分别在0.1mU-M和1.3mU-McGMP和0.3mU-M和12-mU-M8-溴鸟苷3‘，5’-(环)磷酸(Br8-cGMP)时半最大激活。P-I和P-II的表观K(A)值有相似的变化。P-i和ri-α结合2molcGMP/摩尔亚单位到高亲和力(位点1)和低亲和力(位点2)的cGMP结合部位。P-Ⅱ和ri-β仅在两个低亲和力结合位点(位点2)结合2molcGMP/m ol酶亚基，k-1分别为0.92min-1和4.8min-1。这些结果表明，从I-α氨基末端结构域到I-β结构域的改变通过改变结合位点1的结合性质使cGMP的表观K(A)值增加10倍。cGMP同工酶的差异表达可能是体内抑制cGMP水平长期升高的一个重要机制。
cDNA of bovine cGMP-dependent protein kinase (cGMP kinase) isozymes I-alpha and I-beta differ only in their amino-terminal domains (amino acids 1-89 and 1-104, respectively). Each recombinant isozyme (rI-alpha and rI-beta) was transiently expressed in COS-7 cells and its properties were compared with the cGMP kinase isozymes P-I and P-II purified from bovine trachea. The subunit of P-I, P-II, rI-alpha and rI-beta had a molecular mass of about 75 kDa. rI-alpha and rI-beta had S20,w values of 7.6 and 7.2, respectively, indicating that they were present as dimeric holoenzymes. Immunostaining with specific antibodies showed that P-I and rI-alpha, and P-II and rI-beta, were immunologically indistinguishable. P-I, P-II, rI-alpha and rI-beta had the same catalytic activity. However, rI-alpha and rI-beta were half-maximally activated at 0.1-mu-M and 1.3-mu-M cGMP, and 0.3-mu-M and 12-mu-M 8-bromoguanosine 3',5'-(cyclic)phosphate (Br8-cGMP), respectively. P-I and P-II had a similar shift in their apparent K(A) values. P-I and rI-alpha bound 2 mol cGMP/mol subunit to high-affinity (site 1) and low-affinity (site 2) cGMP-binding sites. The exchange rates were 0.005-0.009 min-1 for site 1 and 3.7 min-1 for site 2. In contrast, P-II and rI-beta bound 2 mol cGMP/mol enzyme subunit at only two low-affinity binding sites (site 2) with k-1 values of 0.92 min-1 and 4.8 min-1. These results suggest that a change from the I-alpha amino-terminal domain to that of I-beta increases the apparent K(A) value for cGMP 10-fold by altering the binding properties of binding site 1. The differential expression of the cGMP kinase isozymes could be an important mechanism in vivo to dampen the effect of long-term elevation of cGMP level.