Effects of antibodies to myosin light chain kinase on contractility and myosin phosphorylation in chemically permeabilized smooth muscle.
Effects of antibodies to myosin light chain kinase on contractility and myosin phosphorylation in chemically permeabilized smooth muscle.
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肌球蛋白轻链激酶抗体对化学透化平滑肌收缩性和肌球蛋白磷酸化的影响。
DOI：
10.1161/01.res.68.2.457
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发表时间：
1991
影响因子：
20.1
通讯作者：
Paul,RJ
中科院分区：
文献类型：
--
作者：
DeLanerolle,P;Strauss,JD;Felsen,R;Doerman,GE;Paul,RJ
我们利用免疫学方法研究了肌球蛋白轻链磷酸化（MLC-Pi）在平滑肌收缩性控制中的作用。我们的目的是在Ca2+生理激活水平存在的情况下特异性抑制肌球蛋白轻链激酶（MLCK），以便揭示其他假定的Ca2（+）依赖性调节系统。用火鸡胗MLCK免疫山羊，用木瓜蛋白酶酶切获得免疫球蛋白G （IgG）分子，制备Fab片段。Anti-MLCK Fab在MLCK-Sepharose 4B色谱柱上纯化。这些亲和纯化的Fab片段抑制从火鸡胗平滑肌中纯化的MLCK的活性，并通过Western blot分析证明与多种哺乳动物平滑肌中的MLCK单特异性相互作用。我们用Triton X-100测试了这些Fab片段对可渗透（剥皮）的豚鼠大肠带绦虫的收缩性能的影响。剥皮后的纤维直径约为100微米，长约4毫米，安装在等距测量中，浸入钙- egta缓冲液中。与抗mlck Fab在松弛溶液（Ca2+小于1 nM）中预孵育75分钟的纤维，在转移到收缩溶液（Ca2+ = 0.5微米）时，产生了约25%的平行控制收缩的等长力。当将抗mlck Fab添加到收缩溶液中时，尽管存在Ca2+，但仍可在约120分钟内完成松弛。当纤维与另一种亲和纯化的小鼠Fab（对照Fab）孵育时，未观察到对等长力的显著影响。（摘要删节250字）
We have used an immunological approach to investigate the role of myosin light chain phosphorylation (MLC-Pi) in the control of contractility in smooth muscle. Our aim was to specifically inhibit myosin light chain kinase (MLCK) in the presence of physiologically activating levels of Ca2+ so that other putative Ca2(+)-dependent regulatory systems could be unmasked. Fab fragments were prepared by papain digestion of immunoglobulin G (IgG) molecules obtained from goats immunized with turkey gizzard MLCK. Anti-MLCK Fab was then purified by chromatography on an MLCK-Sepharose 4B column. These affinity-purified Fab fragments inhibit the activity of MLCK purified from turkey gizzard smooth muscle and interact monospecifically with MLCK in various mammalian smooth muscles as demonstrated by a Western blot analysis. The effect of these Fab fragments on the contractile properties was tested in guinea pig taenia coli made permeable (skinned) using Triton X-100. Skinned fibers, approximately 100 microns in diameter and 4 mm long, were mounted for isometric measurements and immersed in calcium-EGTA buffers. Fibers preincubated with anti-MLCK Fab in relaxing solution (Ca2+ less than 1 nM) for 75 minutes developed about 25% of the isometric force of a parallel control contraction when transferred to contracting solution (Ca2+ = 0.5 microM). When added to contracting solution at the peak of a contracture, anti-MLCK Fab elicited a relaxation that was complete in about 120 minutes despite the presence of Ca2+. No significant effect on isometric force was observed when fibers were incubated with another affinity-purified mouse Fab raised against the Fc region of human IgG (control Fab).(ABSTRACT TRUNCATED AT 250 WORDS)
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影响因子：
20.1
作者：
R. Paul;G. Doerman;C. Zeugner;J. C. Riiegg
通讯作者：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
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影响因子：
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作者：
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DOI：
--
发表时间：
1980
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
deLanerolle,P;Stull,JT
通讯作者：
Stull,JT