Purification and characterization of pregnant sheep myometrium myosin light chain kinase.
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.
Pato
M D
MD
Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada.
Lye
S J
SJ
Kerc
E
E
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
D000906:Antibodies; D009219:Myosin-Light-Chain Kinase; D014357:Trypsin