Document Detail


Reverse reaction of smooth muscle myosin light chain kinase. Formation of ATP from phosphorylated light chain plus ADP.
MedLine Citation:
PMID:  3522566     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Incubation of smooth muscle phosphorylated heavy meromyosin in the presence of myosin light chain kinase, calmodulin, ADP, and Ca2+ results in a decrease of the protein-bound phosphate. The dephosphorylation is not due to phosphatase activity and is dependent on the presence of ADP and the active ternary myosin light chain kinase complex. Using 32P-labeled phosphorylated 20,000-dalton light chains as the phosphate donor, the formation of ATP from ADP can be demonstrated. This reaction requires the presence of Ca2+, calmodulin, and myosin light chain kinase. These results indicate that myosin light chain kinase can catalyze a reverse reaction and form ATP from ADP and phosphorylated substrate. The rate of the reverse reaction, kcat/KLC approximately 0.21 min-1 microM-1, is considerably slower than the forward reaction under similar conditions and is therefore detectable only at relatively high concentrations of myosin light chain kinase. For the reverse reaction, KmADP is approximately 30 microM and ATP is a competitive inhibitor, KIATP approximately 88 microM. For the forward reaction, measured with both isolated light chains and intact myosin, KmATP is approximately 100 microM and ADP is a competitive inhibitor, KiADP approximately 140 microM (myosin) and 120 microM (light chains). Thus, the affinity of ATP for the forward and reverse reactions is similar, but the affinity of ADP is higher for the reverse reaction. From the light chain dependence of the two reactions, the following was calculated: forward, Km = 5 microM, kcat = 1720 min-1, and reverse, Km = 130 microM, kcat = 27 min-1. In contrast to the data obtained with isolated light chains, it is suggested that, with intact myosin as substrate, the Km term is primarily responsible for determining the rate of the reverse reaction. With light chains phosphorylated at serine 19 and threonine 18, it was shown that both sites act as a phosphate donor, although the reverse reaction for threonine 18 is slower than that for serine 19.
Authors:
M Ikebe; D J Hartshorne
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  261     ISSN:  0021-9258     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  1986 Jun 
Date Detail:
Created Date:  1986-07-28     Completed Date:  1986-07-28     Revised Date:  2009-11-19    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  8249-53     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Adenosine Diphosphate / metabolism*
Adenosine Triphosphate / biosynthesis*
Animals
Calcium / metabolism
Calmodulin / metabolism
Cattle
Kinetics
Molecular Weight
Muscle, Smooth / enzymology*
Myosin Subfragments / metabolism
Myosin-Light-Chain Kinase
Protein Kinases / metabolism*
Turkeys
Grant Support
ID/Acronym/Agency:
HL 20984/HL/NHLBI NIH HHS; HL 23615/HL/NHLBI NIH HHS
Chemical
Reg. No./Substance:
0/Calmodulin; 0/Myosin Subfragments; 56-65-5/Adenosine Triphosphate; 58-64-0/Adenosine Diphosphate; 7440-70-2/Calcium; EC 2.7.-/Protein Kinases; EC 2.7.11.18/Myosin-Light-Chain Kinase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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