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ISSN
1672-9145
Acta Biochim Biophys Sin
2004, 36(8): 529–536
CN 31-1940/Q
Influence of Trace Amount of Calponin on Smooth Muscle Myosin in
Different States
Jing-Xian Yang,
Xiao-Hua Feng, Ying Zhang, and Yuan
Lin*
Department of Pharmacology, Dalian Medical University, Dalian 116027,
China
Abstract Calponin
(CaP), a thin filament-associated protein, is thought to be involved in
modulating smooth muscle contractile activity, but the role and mechanism keep unknown.
In this study, trace amount of calponin (TAC) was found to obviously influence
myosin in different states in Ca2+-independent manner, suggesting
a high efficient interaction between TAC and myosin. In this assay, the lowest ratio of CaP vs. myosin was
1:10,000, with the concentration of CaP 10,000-fold lower than that used
previously. Myosin phosphorylation, myosin Mg2+-ATPase
activity and protein binding activity were detected to determine the effects of
TAC on the myosin in different states. The amount of precipitated myosin that
bound to TAC was used as the index to determine the interaction between myosin
and TAC in binding assay. Our data showed that in the absence of actin, TAC
significantly increased the precipitation of unphosphorylated myosin, Ca2+-dependently or independently phosphorylated myosin by MLCK, and
stimulated the Mg2+-ATPase activities of these myosins slightly
but significantly. However, no obvious change of precipitation of myosin
phosphorylated by PKA was observed, indicating the relatively selective effect
of TAC. In the presence of actin, the increase of myosin precipitations was
abolished, and no obvious change of actin precipitations and actin-activated
myosin Mg2+-ATPase activities were observed implicating the high efficiency of
TAC on myosin being present in the absence of actin. Although we can not give
conclusive comments to our results, we propose that the high efficiency of
TAC-myosin interaction is present when actin is dissociated from myosin, even
if CaP/myosin ratio is very low; this high efficient interaction can be
abolished by actin. However, why and how TAC can possess such a high efficiency
to influence myosin and how the physiological significance of the high
efficiency of TAC is in regulating the interaction between myosin and actin
remain to be investigated.
Key words trace amount of calponin;
protein binding; phosphorylation; myosin; Mg2+-ATPase
—————–
Received: April 1, 2004 Accepted: June 14,
2004
This work was supported by a grant from the National Natural Science
Foundation of China (No. 30070203)
Abbreviations: CaM, calmodulin; MLCK, myosin light chain kinase;
CaP, calponin; TAC, trace amount of calponin; MLC20-P,
myosin light chain phosphorylation; PKA, protein kinase 3‘:5‘-cAMP-dependent;
cAMP, adenosine 3‘:5‘-cyclic monophosphate; DTT, dithiothreitol; EGTA, ethylene glycol
bis (2-aminoethyl ether)-N,N,N‘,N‘-tetraacetic acid; PMSF,
phenylmethyl sulfonyl fluoride; CDPM, Ca2+-CaM
dependently phosphorylated myosin; CIPM, Ca2+-CaM
independently phosphorylated myosin
*Corresponding author: Tel/Fax, 86-411-84720652; E-mail, [email protected] & [email protected]