EFFECTS OF ANTIBODIES TO MYOSIN LIGHT CHAIN KINASE ON CONTRACTILITY AND MYOSIN PHOSPHORYLATION IN CHEMICALLY PERMEABILIZED SMOOTH-MUSCLE

We have used an immunological approach to investigate the role of myosin light chain phosphorylation (MLC-P(i)) 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-mu-m 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+ < 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-mu-M). 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). MLC-P(i), quantitated by an immunoblotting assay, was increased from basal values of 0.13 +/- 0.03 mol P(i)/mol 20-kDa myosin light chain (n = 4) to 0.62 +/- 0.01 (n = 9) in contracting solution. MLC-P(i), however, was lowered to only 0.36 +/- 0.01 (n = 7) after 120 minutes in the presence of anti-MLCK Fab in contrast to the complete inhibition of force. Moreover, anti-MLCK Fab also relaxed a contraction induced by ATP-gamma-S without any apparent decrease in MLC-P(i). These data suggest that antibodies inhibiting specific contractile protein function can be used as functional probes in the skinned fiber model system. In addition to inhibiting MLCK catalytic activity, anti-MLCK Fab may relax smooth muscle by a mechanism involving a structural role for MLCK.