The characterization of Ca(2+)-calmodulin independent phosphorylation of myosin light chains by a fragment from myosin light chain kinase.
A constitutively active myosin light chain kinase (MLCK) fragment (MLCKF) was found to phosphorylate myosin light chains (MLC(20)) in a Ca(2+)-CaM independent way more effectively than the intact MLCK. The MLCKF was prepared by tryptic digestion of MLCK. Western blot was used to demonstrate the homogeneity of trypsin-digested MLCKF and intact MLCK. Phosphorylation of MLC(20) was detected by Gly-PAGE and Scoin Image Software, and Mg(2+)-ATPase activity of myosin was measured with spectrophotometry. Our results indicated that Ca(2+)-CaM independent phosphorylation of myosin (CIPM) by MLCKF was more efficient than CIPM by MLCK and less efficient than Ca(2+)-CaM dependent phosphorylation of myosin (CDPM) by MLCK in phosphorylating MLC(20) and stimulating myosin Mg(2+)-ATPase activity; both CIPM by MLCKF and CIPM by MLCK were less influenced by the rise of incubation-temperature, the prolonging of incubation-time, the increase of ionic strength of KCl and less sensitive to MLCK inhibitor ML-9   1-(5-chloronaphthalene-1-sulfonyl) -1H-hexahydro-1,4-diazepine   than CDPM by MLCK. The differences were statistically significant ((P)<0.01, or (P)<0.05). The results may be valuable to further investigating the mechanisms of sustained tension characterized by less energy consumption.
Yang
Jing-Xian
JX
Department of Pharmacology, Dalian Medical University, Dalian 116027, China.
Wang
Xiao-Ming
XM
Tang
Ze-Yao
ZY
Chen
Hua
H
Xu
Hong
H
Lin
Yuan
Y
D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006868:Hydrolysis; D018994:Myosin Light Chains / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011189:Potassium Chloride / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors
D002147:Calmodulin; D018994:Myosin Light Chains; D010446:Peptide Fragments; D011189:Potassium Chloride; D009219:Myosin-Light-Chain Kinase; D002118:Calcium