| It is generally accepted that the interaction between actin and phosphorylated myosin by myosin light chain kinase (MLCK) in a Ca2+-CaM dependent manner is the principal mechanism of regulation of smooth muscle contraction. However, this simple theory based on CDPM is not sufficient to explain all the aspects of smooth muscle contractile activity. It is known that the rise in [Ca2+]i and CDPM is in a very short time in physiological conditions, while the tension may keep much longer time without change in [Ca2+]i. The mechanism of this phenomenon is still unclear. It's supposed that other mechanisms different from CDPM by MLCK may be involved in the force remaining. Our study revealed that a constitutively active myosin light chain kinase fragment (MLCKF), trace amount of calponin (TAC) and protein kinase–cAMP dependent (PKA) can influence the function of smooth muscle myosin in a Ca2+-CaM independent way, which may be contribute to the sustained tension of smooth muscle. The results are valuable for the further investigation of mechanisms of Ca2+-independent regulation of smooth muscle contractile activity and for the further exploration of new medicines for diseases of smooth muscle.Part one The characterization of Ca2+-calmodulin independent and dependent phosphorylation of myosin light chains by a fragment from MLCK Myosin and MLCK were purified from fresh gizzard smooth muscle. The constitutively active MLCKF (61KD) was prepared by tryptic digestion of MLCK. Western blot was used to demonstrate the homogeneity of trypsin-digested MLCKF and intact MLCK. Recombinant-calmodulin (CaM) was purified from the E.coli expressed CaM. Phosphorylation of MLC20 was detected by Glycerol-PAGE and Scoin Image Software, and MgATPase activity of myosin was measured with spectrophotometry. The results indicated that 61KD MLCKF can phosphorylate MLC20 in Ca2+-CaM independent and dependent way. The Ca2+-CaM independent phosphorylation of myosin (CIPM) by MLCKF was more efficient than CIPM by MLCK and was less efficient than CDPM by MLCK in phosphorylating MLC20 and stimulating myosin MgATPase 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 than CDPM by MLCK; CDPM by MLCKF was more efficient than CDPM by MLCK in phosphorylating MLC20 and stimulating myosin MgATPase activity. These results suggested that MLCKF possibly plays a certain role in regulating smooth muscle contractile activity via Ca2+-CaM independent and dependent way; CIPM by MLCKF may be involved in keeping the tension of smooth muscle and CDPM by MLCKF may be contribute to the initial burst of smooth muscle contraction.Part two The Ca2+-calmodulin independently regulatory effects of trace amount of calponin on the smooth muscle myosins in different statesCalponin, a thin filament-associated protein, plays an important role in the regulation of smooth muscle contractility. Our recent study revealed a new phenomenon that trace amount of calponin (TAC) could influence the function of different states of myosin in a Ca2+-CaM independent manner, which attracted our attention and encouraged us to do the following study. The lowest calponin/myosin ratio we selected was 1/10000, and the myosins used in this assay were unphosphorylated myosin, CDPM and CIPM by MLCK, and CIPM by PKA. Binding assay, myosin phosphorylation determination, and MgATPase measurement were used in this study. Our data showed that in the absence of actin, TAC increased the precipitations of unphosphorylated myosin, CDPM and CIPM by MLCK, and stimulated the MgATPase activities of these myosins slightly but significantly even if calponin/myosin ratio is very low; 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, myosin and TAC, the increase of myosin precipitation was abolis...
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