Baltensperger K, Carafoli E, Chiesi M
Laboratory of Biochemistry, Swiss Federal Institute of Technology, Zürich.
Eur J Biochem. 1988 Feb 15;172(1):7-16. doi: 10.1111/j.1432-1033.1988.tb13848.x.
It has been proposed that the plasma membrane Ca2+ pump of smooth muscle tissues may be regulated by cGMP-dependent phosphorylation [Popescu, L. M., Panoiu, C., Hinescu, M. & Nutu, O. (1985) Eur. J. Pharmacol. 107, 393-394; Furukawa, K. & Nakamura, H. (1987) J. Biochem. (Tokyo) 101, 287-290]. This hypothesis has been tested on a smooth muscle sarcolemma preparation from pig thoracic aorta. The actomyosin-extracted membranes showed ATP-dependent Ca2+ uptake as well as cGMP-dependent protein kinase (G-kinase) activity. The molecular masses of the major protein substrates of the G-kinase (G1) and that of the Ca2+ pump were compared. Electrophoretic analysis of the phosphorylated intermediate of the sarcolemmal Ca2+-ATPase and the G1 phosphoprotein showed that these two proteins are not identical. The results were confirmed by using a 125I-calmodulin overlay technique and an antibody against human erythrocyte Ca2+-ATPase. Ca2+-uptake experiments with prephosphorylated membrane vesicles were carried out to elucidate possible effects of cGMP-dependent phosphorylation of membrane proteins on the activity of the Ca2+ pump. The cGMP-dependent phosphorylation was found to be extremely sensitive to temperature leading to very low steady-state phosphorylation levels at 37 degrees C. The difficulty was overcome by ATP[gamma S], which produced full and stable thiophosphorylation of G1 during the Ca2+-uptake experiments at 37 degrees C. However, the cGMP-dependent thiophosphorylation failed to influence the Ca2+-uptake properties of sarcolemmal vesicles. The results show that the Ca2+ pump of smooth muscle plasma membrane is not a direct target of the cGMP-dependent protein kinase and is not regulated by the cGMP-dependent phosphorylation of membrane proteins.
有人提出，平滑肌组织的质膜钙泵可能受环鸟苷酸依赖性磷酸化调节[波佩斯库，L.M.，帕诺尤，C.，海内斯库，M.和努图，O.（1985年）《欧洲药理学杂志》107卷，393 - 394页；古川，K.和中村，H.（1987年）《生物化学杂志》（东京）101卷，287 - 290页]。该假说已在猪胸主动脉平滑肌肌膜制备物上进行了验证。提取了肌动球蛋白的膜显示出ATP依赖性钙摄取以及环鸟苷酸依赖性蛋白激酶（G激酶）活性。比较了G激酶（G1）的主要蛋白质底物和钙泵的分子量。对肌膜钙 - ATP酶的磷酸化中间体和G1磷蛋白进行电泳分析表明，这两种蛋白质并不相同。使用125I - 钙调蛋白覆盖技术和抗人红细胞钙 - ATP酶抗体证实了结果。进行了预磷酸化膜囊泡的钙摄取实验，以阐明膜蛋白的环鸟苷酸依赖性磷酸化对钙泵活性可能产生的影响。发现环鸟苷酸依赖性磷酸化对温度极其敏感，导致在37℃时稳态磷酸化水平非常低。通过ATP[γS]克服了这一困难，在37℃的钙摄取实验中，ATP[γS]使G1产生了完全且稳定的硫代磷酸化。然而，环鸟苷酸依赖性硫代磷酸化未能影响肌膜囊泡的钙摄取特性。结果表明，平滑肌质膜的钙泵不是环鸟苷酸依赖性蛋白激酶的直接作用靶点，不受膜蛋白的环鸟苷酸依赖性磷酸化调节。
