title: Effects of antibodies to myosin light chain kinase on contractility and myosin phosphorylation in chemically permeabilized smooth muscle.
abstract: 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)
authors.affiliation: Department of Physiology and Biophysics, University of Illinois, Chicago 60680.
authors.forename: P
authors.initials: P
authors.lastname: De Lanerolle
authors.forename: J D
authors.initials: JD
authors.lastname: Strauss
authors.forename: R
authors.initials: R
authors.lastname: Felsen
authors.forename: G E
authors.initials: GE
authors.lastname: Doerman
authors.forename: R J
authors.initials: RJ
authors.lastname: Paul
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000494:pharmacology*; D002118:Calcium / Q000494:pharmacology; D006168:Guinea Pigs; D007140:Immunoglobulin Fab Fragments / Q000276:immunology / Q000502:physiology; D009130:Muscle, Smooth / Q000378:metabolism / Q000502:physiology*; D009219:Myosin-Light-Chain Kinase / Q000276:immunology*; D009218:Myosins / Q000378:metabolism*; D010539:Permeability; D010766:Phosphorylation; D014661:Vasoconstriction
chemical_list: D000906:Antibodies; D007140:Immunoglobulin Fab Fragments; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D009219:Myosin-Light-Chain Kinase; D009218:Myosins; D002118:Calcium