Protein kinase activity associated with pancreatic zymogen granules.
Protein kinase activity associated with pancreatic zymogen granules.
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与胰腺酶原颗粒相关的蛋白激酶活性。
DOI：
10.1042/bj2270743
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发表时间：
1985
期刊：
影响因子：
--
通讯作者：
Williams,JA
中科院分区：
文献类型：
--
作者：
Burnham,DB;Munowitz,P;Thorn,N;Williams,JA
采用等渗透Percoll梯度法从大鼠胰腺中制备纯化的酶原颗粒。在[γ - 32p]ATP存在的情况下，Ca2+诱导了几种颗粒蛋白的磷酸化，最明显的是mr - 13000蛋白，而添加环状AMP则没有影响。当添加磷脂酰丝氨酸时，Ca2+增加了其他蛋白质的磷酸化，对Mr为62 000的蛋白质的影响最大。纯化颗粒也能磷酸化外源底物。Ca2+诱导的富含赖氨酸的组蛋白磷酸化在磷脂酰丝氨酸的存在下增强了3倍以上，并且以混合组蛋白为底物显示了环amp激活的蛋白激酶活性。内源和外源蛋白半最大磷酸化所需的游离Ca2+和环AMP浓度分别为1-3微米和57纳米。用0.25 M-KCl处理颗粒导致磷脂酰丝氨酸依赖激酶活性释放到高速颗粒上清中。相反，Ca2+活化激酶活性的颗粒蛋白底物对KCl提取具有抗性，并且实际上存在于纯化的颗粒膜中。经KCl处理后，环AMP活化的激酶活性未被提取。综上所述，酶原颗粒中整体膜蛋白的磷酸化可由一种或多种Ca2+活化蛋白激酶诱导。这种反应是一种潜在的机制，通过Ca2+介导的分泌物调节外分泌胰腺的胞吐。
Purified zymogen granules were prepared from rat pancreas by using an iso-osmotic Percoll gradient. In the presence of [gamma-32P]ATP, phosphorylation of several granule proteins was induced by Ca2+, most notably a Mr-13 000 protein, whereas addition of cyclic AMP was without effect. When phosphatidylserine was also added, Ca2+ increased the phosphorylation of additional proteins, with the largest effect on a protein of Mr 62 000. Purified granules were also able to phosphorylate exogenous substrates. Ca2+-induced phosphorylation of lysine-rich histone was enhanced over 3-fold in the presence of phosphatidylserine, and cyclic AMP-activated protein kinase activity was revealed with mixed histone as substrate. The concentrations of free Ca2+ and cyclic AMP required for half-maximal phosphorylation of both endogenous and exogenous proteins were 1-3 microM and 57 nM respectively. Treatment of granules with 0.25 M-KCl resulted in the release of phosphatidylserine-dependent kinase activity into a high-speed granule supernatant. In contrast, granule-protein substrates of Ca2+-activated kinase activity were resistant to KCl extraction, and in fact were present in purified granule membranes. Kinase activity activated by cyclic AMP was not extracted by KCl treatment. It is concluded that phosphorylation of integral membrane proteins in the zymogen granule can be induced by one or more Ca2+-activated protein kinases. Such a reaction is a potential mechanism by which exocytosis may be regulated in the exocrine pancreas by Ca2+-mediated secretagogues.