Cohen M E, Reinlib L, Watson A J, Gorelick F, Rys-Sikora K, Tse M, Rood R P, Czernik A J, Sharp G W, Donowitz M
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Proc Natl Acad Sci U S A. 1990 Nov;87(22):8990-4. doi: 10.1073/pnas.87.22.8990.
Ileal brush border membranes contain an endogenous Ca2+/calmodulin (CaM)-dependent protein kinase activity that modulates the activity of the apical membrane Na+/H+ exchanger. To further characterize this kinase, synapsin I, a substrate for Ca2+/CaM-dependent protein kinases, was added to preparations of ileal brush border membranes. In the presence of Ca2+/CaM, synapsin I was phosphorylated. Phosphopeptide mapping demonstrated that the addition of Ca2+/CaM to brush border membranes stimulated the phosphorylation of sites in synapsin I specific for Ca2+/CaM-dependent protein kinase II. Immunoblots containing brush border and microvillus membrane proteins were probed with an antibody that recognizes the 50-kDa subunit of rat brain Ca2+/CaM-dependent protein kinase II. This antibody labeled major and minor species of 50 and 53 kDa, respectively, with more labeling of the brush border than the microvillus membranes. Right-side-out ileal villus cell brush border vesicles were prepared containing CaM, ATP, and 350 nM free Ca2+. Na+/H+ exchange was inhibited by the presence of Ca2+/CaM/ATP within the vesicles. A 21-amino acid peptide inhibitor of CaM kinase II was enclosed within some vesicle preparations by freeze-thaw. The effect on Na+/H+ exchange of Ca2+/CaM/ATP was partially reversed by the inhibitor peptide. These studies demonstrate the presence of Ca2+/CaM-dependent protein kinase II in rabbit ileal villus cell brush border membranes. Based on the effect of a specific inhibitor peptide of Ca2+/CaM kinase II, it is concluded that this kinase inhibits brush border Na+/H+ exchange, which participates in the regulation of ileal Na+ absorption.
回肠刷状缘膜含有一种内源性钙/钙调蛋白（CaM）依赖性蛋白激酶活性，该活性可调节顶端膜钠/氢交换体的活性。为了进一步表征这种激酶，将突触素I（一种钙/钙调蛋白依赖性蛋白激酶的底物）添加到回肠刷状缘膜制剂中。在钙/钙调蛋白存在的情况下，突触素I发生了磷酸化。磷酸肽图谱分析表明，向刷状缘膜中添加钙/钙调蛋白可刺激突触素I中特定于钙/钙调蛋白依赖性蛋白激酶II的位点发生磷酸化。用识别大鼠脑钙/钙调蛋白依赖性蛋白激酶II的50 kDa亚基的抗体对含有刷状缘和微绒毛膜蛋白的免疫印迹进行检测。该抗体分别标记了50 kDa和53 kDa的主要和次要条带，刷状缘的标记比微绒毛膜更多。制备了含有钙调蛋白、ATP和350 nM游离钙的外翻回肠绒毛细胞刷状缘囊泡。囊泡中存在钙/钙调蛋白/ATP会抑制钠/氢交换。通过冻融将钙调蛋白激酶II的一种21个氨基酸的肽抑制剂包裹在一些囊泡制剂中。抑制剂肽部分逆转了钙/钙调蛋白/ATP对钠/氢交换的影响。这些研究证明了兔回肠绒毛细胞刷状缘膜中存在钙/钙调蛋白依赖性蛋白激酶II。基于钙/钙调蛋白激酶II特异性抑制剂肽的作用，得出结论：这种激酶抑制刷状缘钠/氢交换，而钠/氢交换参与回肠钠吸收的调节。
