Yokoyama N, Wang J H
Department of Medical Biochemistry, University of Calgary, Alberta, Canada.
J Biol Chem. 1991 Aug 5;266(22):14822-9.
Calmodulin (CaM)-stimulated phosphatase in bovine brain or bovine lung CaM-binding protein fractions were fractionated on a heparin-Sepharose column into three activity peaks, designated in order of column three activity peaks, designated in order of column elution as the brain peak I (BPI), peak II (BPII), and peak III (BPIII) or the lung peak I (LPI), peak II (LPII), and peak III (LPIII) phosphatases, respectively. The pooled individual peak fractions were further purified on a fast protein liquid chromatography Superose 12 column. Analysis of the purified samples by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that they all contained major peptides corresponding to alpha and beta subunits of the brain CaM-stimulated phosphatase. The phosphatases had similar specific activities and were similarly stimulated by Ni2+, Mn2+, Mg2+ + Ca2+, and CaM. They showed differential reactivity on immunotransblots with an alpha subunit-specific monoclonal antibody VJ6, which reacted strongly toward BPI and weakly toward BPIII and LPI, but showed no reactivity toward BPII, LPII, and LPIII. Each of the alpha subunits of the purified phosphatases had a distinct V8 protease and chymotrypsin peptide map. The results suggest that both bovine brain and bovine lung contain multiple CaM-stimulated phosphatase isozymes. The suggestion of three mammalian brain CaM-stimulated phosphatase isozymes is in agreement with the results of recent molecular cloning studies (Kuno, T., Takeda, T., Hirai, M., Ito, A., Mukai, H., and Tanaka, C. (1989) Biochem. Biophys. Res. Commun. 165, 1352-1358; Guerini, D., and Klee, C.B. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9183-9187; da Cruz e Silva, E. F., and Cohen, P. T. W. (1989) Biochim. Biophys. Acta 1009, 293-296). The successful purification of the individual isozymes may facilitate the elucidation of molecular basis and physiological significance of the isozymes.
牛脑或牛肺钙调蛋白（CaM）结合蛋白组分中的CaM刺激磷酸酶在肝素 - 琼脂糖柱上分离为三个活性峰，按照柱洗脱顺序分别命名为脑峰I（BPI）、峰II（BPII）和峰III（BPIII），或肺峰I（LPI）、峰II（LPII）和峰III（LPIII）磷酸酶。将各个峰级分合并后，在快速蛋白质液相色谱Superose 12柱上进一步纯化。通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳对纯化样品进行分析，结果显示它们均含有与脑CaM刺激磷酸酶的α和β亚基相对应的主要肽段。这些磷酸酶具有相似的比活性，并且受到Ni2 +、Mn2 +、Mg2 + + Ca2 +和CaM的相似刺激。它们在免疫印迹上与α亚基特异性单克隆抗体VJ6表现出不同的反应性，VJ6对BPI反应强烈，对BPIII和LPI反应较弱，但对BPII、LPII和LPIII无反应。纯化磷酸酶的每个α亚基都有独特的V8蛋白酶和胰凝乳蛋白酶肽图谱。结果表明，牛脑和牛肺均含有多种CaM刺激的磷酸酶同工酶。关于三种哺乳动物脑CaM刺激磷酸酶同工酶的推测与最近的分子克隆研究结果一致（Kuno, T., Takeda, T., Hirai, M., Ito, A., Mukai, H., and Tanaka, C. (1989) Biochem. Biophys. Res. Commun. 165, 1352 - 1358; Guerini, D., and Klee, C.B. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9183 - 9187; da Cruz e Silva, E. F., and Cohen, P. T. W. (1989) Biochim. Biophys. Acta 1009, 293 - 296）。单个同工酶的成功纯化可能有助于阐明同工酶的分子基础和生理意义。
