De Lanerolle P, Strauss J D, Felsen R, Doerman G E, Paul R J
Department of Physiology and Biophysics, University of Illinois, Chicago 60680.
Circ Res. 1991 Feb;68(2):457-65. doi: 10.1161/01.res.68.2.457.
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)
我们采用免疫方法来研究肌球蛋白轻链磷酸化（MLC-Pi）在平滑肌收缩控制中的作用。我们的目的是在生理激活水平的Ca2+存在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。通过用木瓜蛋白酶消化从用火鸡砂囊MLCK免疫的山羊获得的免疫球蛋白G（IgG）分子来制备Fab片段。然后通过在MLCK-Sepharose 4B柱上进行色谱法纯化抗MLCK Fab。如蛋白质印迹分析所示，这些亲和纯化的Fab片段抑制从火鸡砂囊平滑肌纯化的MLCK的活性，并与各种哺乳动物平滑肌中的MLCK单特异性相互作用。在使用Triton X-100使其通透（去皮）的豚鼠结肠带中测试这些Fab片段对收缩特性的影响。将直径约100微米、长4毫米的去皮纤维安装用于等长测量，并浸入钙-乙二醇双乙酸盐缓冲液中。在松弛溶液（Ca2+小于1 nM）中用抗MLCK Fab预孵育75分钟的纤维，当转移到收缩溶液（Ca2+ = 0.5 microM）中时，产生的等长力约为平行对照收缩的25%。当在挛缩峰值时加入收缩溶液中时，抗MLCK Fab引起松弛，尽管存在Ca2+，但在约120分钟内松弛完成。当纤维与另一种针对人IgG Fc区域产生的亲和纯化小鼠Fab（对照Fab）孵育时，未观察到对等长力的显著影响。（摘要截短于250字）
