Minocherhomjee A V, Roufogalis B D
Biochem J. 1982 Sep 15;206(3):517-25. doi: 10.1042/bj2060517.
Purified protein kinase (cyclic AMP-dependent) inhibitor (PKI) from bovine heart stimulated Ca(2+)+Mg(2+)-stimulated ATPase activity in human erythrocytes, the stimulation being maximal at 2mug/0.6ml. By contrast, PKI from rabbit skeletal muscle had no effect. Bovine heart PKI stimulated Ca(2+)+Mg(2+)-stimulated ATPase by increasing the Ca(2+)-sensitivity of the enzyme. This contrasted with the stimulation by calmodulin, which increased the maximum velocity of the Ca(2+)+Mg(2+)-dependent ATPase in addition to its effect on the Ca(2+)-sensitivity. Both membrane-bound and Triton X-100-solubilized Ca(2+)+Mg(2+)-stimulated ATPase activities were stimulated by PKI, indicating that the stimulation did not require an intact membrane structure. At low Ca(2+) concentration the stimulation by PKI and saturating concentrations of calmodulin were additive, suggesting that the two effectors acted by distinct mechanisms. Although 5mum-cyclic AMP inhibited Ca(2+)+Mg(2+)-stimulated ATPase activity by about 20% when measured at low ATP concentrations, probably by stimulation of phosphorylation by an endogenous protein kinase, the stimulation by PKI (about 100%) was not solely due to its antagonism of the protein kinase. This interpretation was supported by a number of observations. First, modification of arginine residues of bovine heart PKI abolished its inhibition of cyclic AMP-dependent protein kinase, but had no effect on the stimulation of Ca(2+)+Mg(2+)-stimulated ATPase. Secondly, trifluoperazine (20mum) antagonized the stimulation of Ca(2+)+Mg(2+)-dependent ATPase by PKI, similarly to its antagonism of calmodulin stimulation, but it did not affect the inhibition of protein kinase by PKI. We conclude that different mechanisms are involved in the inhibition of protein kinase and the stimulation of Ca(2+)+Mg(2+)-stimulated ATPase by PKI.
从牛心脏中纯化得到的蛋白激酶（环磷酸腺苷依赖性）抑制剂（PKI）可刺激人红细胞中Ca(2+)+Mg(2+)-刺激的ATP酶活性，在2μg/0.6ml时刺激作用最大。相比之下，来自兔骨骼肌的PKI则没有作用。牛心脏PKI通过增加该酶对Ca(2+)的敏感性来刺激Ca(2+)+Mg(2+)-刺激的ATP酶。这与钙调蛋白的刺激作用形成对比，钙调蛋白除了影响Ca(2+)敏感性外，还增加了Ca(2+)+Mg(2+)-依赖性ATP酶的最大反应速度。膜结合的和经Triton X-100增溶的Ca(2+)+Mg(2+)-刺激的ATP酶活性均受到PKI的刺激，这表明该刺激作用并不需要完整的膜结构。在低Ca(2+)浓度下，PKI的刺激作用与饱和浓度的钙调蛋白的刺激作用具有加和性，这表明这两种效应物的作用机制不同。尽管在低ATP浓度下测定时，5μM-环磷酸腺苷可使Ca(2+)+Mg(2+)-刺激的ATP酶活性抑制约20%，这可能是通过刺激内源性蛋白激酶的磷酸化作用实现的，但PKI的刺激作用（约为100%）并非仅仅是由于其对蛋白激酶的拮抗作用。这一解释得到了多项观察结果的支持。首先，对牛心脏PKI的精氨酸残基进行修饰消除了其对环磷酸腺苷依赖性蛋白激酶的抑制作用，但对Ca(2+)+Mg(2+)-刺激的ATP酶的刺激作用没有影响。其次，三氟拉嗪（20μM）可拮抗PKI对Ca(2+)+Mg(2+)-依赖性ATP酶的刺激作用，类似于其对钙调蛋白刺激作用的拮抗，但它并不影响PKI对蛋白激酶的抑制作用。我们得出结论，PKI对蛋白激酶的抑制作用和对Ca(2+)+Mg(2+)-刺激的ATP酶的刺激作用涉及不同的机制。
