Effect of parathyroid hormone on rat renal cAMP-dependent protein kinase and protein kinase C activity measured using synthetic peptide substrates.
Effect of parathyroid hormone on rat renal cAMP-dependent protein kinase and protein kinase C activity measured using synthetic peptide substrates.
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使用合成肽底物测量甲状旁腺激素对大鼠肾 cAMP 依赖性蛋白激酶和蛋白激酶 C 活性的影响。
DOI：
10.1016/0003-9861(91)90343-h
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发表时间：
1991
影响因子：
3.9
通讯作者：
Armbrecht,HJ
中科院分区：
文献类型：
--
作者：
Nemani,R;Wongsurawat,N;Armbrecht,HJ
甲状旁腺激素(PTH)对肾皮质的作用主要由cAMP依赖的蛋白激酶(PKA)介导，蛋白激酶C(PKC)也有一定的作用。然而，目前检测亚细胞组分中PKA和PKC的方法不灵敏，需要大量的蛋白质。最近，有报道了一种灵敏的测定蛋白激酶活性的方法。该方法使用合成肽作为底物，并采用串联层析程序分离磷酸化肽。我们采用这种方法在大鼠肾皮质薄片上研究了PTH对PKA和PKC活性的影响。甲状旁腺素(2 5 0 Nm)在30 S内使胞质PKA活性增加4~5倍，并维持至少5min。PTH还能迅速刺激细胞膜部分的PKC活性，降低细胞质中的PKC活性。这些变化在30岁的S时最大，但与PKA的变化不同，它们在此后迅速下降。PTH只有在10 nM或更高浓度时才能显著激活PKC。这项研究表明，PTH确实激活了肾组织中的PKC，尽管激活的持续时间比PKA短得多。它还表明，合成肽和串联层析的组合可以作为生物样品中蛋白激酶活性的灵敏分析方法。
The actions of parathyroid hormone (PTH) on the renal cortex are thought to be mediated primarily by cAMP-dependent protein kinase (PKA) with some suggestion of a role for protein kinase C (PKC). However, present methods for assaying PKA and PKC in subcellular fractions are insensitive and require large amounts of protein. Recently, a sensitive method for measuring the activity of protein kinases has been reported. This method uses synthetic peptides as substrates and a tandem Chromatographic procedure for isolating the phosphorylated peptides. We have adapted this method to study the effect of PTH on PKA and PKC activity using thin slices of rat renal cortex. PTH (250 nm) stimulated cytosolic PKA activity four- to fivefold within 30 s, and PKA activity was sustained for at least 5 min. PTH also rapidly stimulated PKC activity in the membrane fraction and decreased PKC activity in the cytosol. These changes were maximal at 30 s, but unlike changes in PKA, they declined rapidly thereafter. PTH significantly activated PKC only at concentrations of 10 nmor greater. This study demonstrates that PTH does activate PKC in renal tissue, although the duration of activation is much less than for PKA. It also demonstrates that a combination of synthetic peptides with tandem chromatography can be used as a sensitive assay procedure for protein kinase activity in biological samples.