Characterization of the phosphatase activity of a baculovirus-expressed calcineurin A isoform.
Characterization of the phosphatase activity of a baculovirus-expressed calcineurin A isoform.
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DOI：
10.1016/s0021-9258(19)49628-2
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发表时间：
1992-08
期刊：
影响因子：
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通讯作者：
Brian A. Perrino;Y. Fong;D. Brickey;Y. Saitoh;Y. Ushio;K. Fukunaga;E. Miyamoto;T. Soderling
中科院分区：
文献类型：
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作者：
Brian A. Perrino;Y. Fong;D. Brickey;Y. Saitoh;Y. Ushio;K. Fukunaga;E. Miyamoto;T. Soderling
用含有大鼠钙调磷酸酶A亚型cDNA的重组杆状病毒感染Sf 9细胞，通过钙调蛋白-琼脂糖亲和层析纯化钙调磷酸酶A。Sf 9表达的钙调磷酸酶A在EDTA（0.9 nmol/min/mg）存在下具有低的基础磷酸酶活性，其被Mn 2+刺激3-5倍。钙调素使Mn ~（2+）刺激活性增加3-5倍。牛脑钙调磷酸酶B使A亚基活性增加10-15倍，钙调蛋白进一步刺激重建的A和B亚基活性10-15倍（644 nmol/min/mg）。钙调磷酸酶A对32 P-RII pep（CaN的肽底物（DLDVPIPGRFDRRVSVAAE））的Km为111 μ M，有或没有钙调蛋白，钙调蛋白使Vmax增加约4倍。重构钙调磷酸酶A加B对32 P-RII pep的Km为20 μ M，钙调蛋白使Vmax增加18倍而不影响Km。CaN A467-492是一种来自钙调磷酸酶的合成自抑制肽（ITSFEEAKGLDRINERMPPRRDAMP），可抑制重组酶和A亚基的Mn 2 +/钙调素刺激活性，IC 50分别为25 μ M和90 μ M。用纯化的B亚基和钙调素重建钙调磷酸酶A的磷酸酶活性，可能有助于比较研究B亚基和钙调素对钙调磷酸酶A活性的调节。
Calcineurin A was purified by calmodulin-Sepharose affinity chromatography from Sf9 cells infected with recombinant baculovirus containing the cDNA of a rat calcineurin A isoform. The Sf9-expressed calcineurin A has a low basal phosphatase activity in the presence of EDTA (0.9 nmol/min/mg) which is stimulated 3-5-fold by Mn2+. Calmodulin increased the Mn2+ stimulated activity 3-5-fold. Bovine brain calcineurin B increased the A subunit activity 10-15-fold, and calmodulin further stimulated the activity of reconstituted A and B subunits 10-15-fold (644 nmol/min/mg). The Km of calcineurin A for 32P-RII pep (a peptide substrate (DLDVPIPGRFDRRVSVAAE) for CaN), was 111 microM with or without calmodulin, and calmodulin increased the Vmax about 4-fold. The Km of reconstituted calcineurin A plus B for 32P-RII pep was 20 microM, and calmodulin increased the Vmax 18-fold without affecting the Km. CaN A467-492, a synthetic autoinhibitory peptide (ITSFEEAKGLDRINERMPPRRDAMP) from calcineurin, inhibited the Mn2+/calmodulin-stimulated activities of the reconstituted enzyme and the A subunit with IC50's of 25 microM and 90 microM, respectively. The reconstitution of the phosphatase activity of an expressed isoform of calcineurin A by purified B subunit and calmodulin may facilitate comparative studies of the regulation of calcineurin A activity by the B subunit and calmodulin.