title: Purification and characterization of pregnant sheep myometrium myosin light chain kinase.
abstract: Myosin light chain kinase (MLCK) has been purified from the myometrium of pregnant sheep. The Mr of the enzyme was determined from SDS-polyacrylamide gels to be 160,000. It requires Ca2+ and calmodulin for activation, and phosphorylates the 20,000-Da light chains of myosin at a rapid rate. The specific activity for the myosin light chains from turkey gizzards and rabbit uterine muscle are 7.7 and 5.4 mumol/min/mg, respectively. The Km for the former substrate is 40 microM and the Vmax of the reaction is 19 mumol/min/mg. Polyclonal antibodies raised against the enzyme cross-reacted with pregnant sheep myometrium (psm), turkey gizzard (tg), and chicken gizzard MLCK. Affinity purification of the antibodies on tg-MLCK Sepharose resulted in the preparation of two fractions of antibodies with different reactivity toward these proteins. Fraction A antibodies which did not bind to the affinity column cross-reacted only with psm-MLCK while Fraction B antibodies which bound to the column cross-reacted with all three proteins. Western blots of extracts of turkey gizzards, human myometrium, and various tissues from sheep showed cross-reactivity of both fractions of antibodies with a 160,000-Da protein in the extracts of sheep smooth muscles. Only Fraction B antibodies cross-reacted with a protein (130,000 Da) in turkey gizzards and human myometrium extracts. Prolonged tryptic digestion of psm-MLCK produced large fragments Mr approximately 60,000 which appears to be similar to that formed from tg-MLCK, and some smaller peptides. Fraction A antibodies cross-reacted with the small peptides while Fraction B antibodies cross-reacted with the large fragments but not vice versa. Further analysis of the tryptic peptides suggests that the epitopes of Fraction A antibodies are localized in a peptide which appears to be in the NH2-terminal region of the molecule.
authors.affiliation: Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada.
authors.forename: M D
authors.initials: MD
authors.lastname: Pato
authors.forename: S J
authors.initials: SJ
authors.lastname: Lye
authors.forename: E
authors.initials: E
authors.lastname: Kerc
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology; D015153:Blotting, Western; D002852:Chromatography, Ion Exchange; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007700:Kinetics; D009215:Myometrium / Q000201:enzymology*; D009219:Myosin-Light-Chain Kinase / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D009928:Organ Specificity; D011247:Pregnancy; D012756:Sheep; D014357:Trypsin; D014590:Uterine Contraction
chemical_list: D000906:Antibodies; D009219:Myosin-Light-Chain Kinase; D014357:Trypsin