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ISSN 1672-9145 Acta Biochim Biophys Sin 2004, 36(8): 529–536 CN 31-1940/Q

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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

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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]