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Blebbistatin is a myosin II-specific inhibitor. However, the mechanism and tissue specificity of the drug are not well understood. Blebbistatin blocked the chemotaxis of vascular smooth muscle cells (VSMCs) toward sphingosylphosphorylcholine (I[C.sub.50] = 26.1 [+ or -] 0.2 and 27.5 [+ or -] 0.5 [micro]M for GbaSM-4 and A7r5 cells, respectively) and platelet-derived growth factor BB (I[C.sub.50] = 32.3 [+ or -] 0.9 and 31.6 [+ or -] 1.3 [micro]M for GbaSM-4 and A7r5 cells, respectively) at similar concentrations. Immunofluorescence and fluorescent resonance energy transfer analysis indicated a blebbistatin-induced disruption of the actin-myosin interaction in VSMCs. Subsequent experiments indicated that blebbistatin inhibited the [Mg.sup.2+]-ATPase activity of the unphosphorylated (I[C.sub.50] = 12.6 [+ or -] 1.6 and 4.3 [+ or -] 0.5 [micro]M for gizzard and bovine stomach, respectively) and phosphorylated (I[C.sub.50] = 15.0 [+ or -] 0.6 [micro]M for gizzard) forms of purified smooth muscle myosin II, suggesting a direct effect on myosin II motor activity. It was further observed that the [Mg.sup.2+]-ATPase activities of gizzard myosin II fragments, heavy meromyosin (I[C.sub.50] = 14.4 [+ or -] 1.6 [micro]M) and subfragment 1 (I[C.sub.50] = 5.5 [+ or -] 0.4 [micro]M), were also inhibited by blebbistatin. Assay by in vitro motility indicated that the inhibitory effect of blebbistatin was reversible. Electron-microscopic evaluation showed that blebbistatin induced a distinct conformational change (i.e., swelling) of the myosin II head. The results suggest that the site of blebbistatin action is within the S1 portion of smooth muscle myosin II. Boyden chamber; fluorescence resonance energy transfer; ATPase; in vitro motility assay; electron microscopy

Research has been conducted on the calcium binding protein 40 (CBP40). The expression of the CBP40 recombinant proteins in Escherichia coli in investigating Ca (super)2+ -binding properties is described.

Blebbistatin is a myosin II-specific inhibitor. However, the mechanism and tissue specificity of the drug are not well understood. Blebbistatin blocked the chemotaxis of vascular smooth muscle cells (VSMCs) toward sphingosylphosphorylcholine (IC50 = 26.1 ± 0.2 and 27.5 ± 0.5 µM for GbaSM-4 and A7r5 cells, respectively) and platelet-derived growth factor BB (IC50 = 32.3 ± 0.9 and 31.6 ± 1.3 µM for GbaSM-4 and A7r5 cells, respectively) at similar concentrations. Immunofluorescence and fluorescent resonance energy transfer analysis indicated a blebbistatin-induced disruption of the actin-myosin interaction in VSMCs. Subsequent experiments indicated that blebbistatin inhibited the Mg2+-ATPase activity of the unphosphorylated (IC50 = 12.6 ± 1.6 and 4.3 ± 0.5 µM for gizzard and bovine stomach, respectively) and phosphorylated (IC50 = 15.0 ± 0.6 µM for gizzard) forms of purified smooth muscle myosin II, suggesting a direct effect on myosin II motor activity. It was further observed that the Mg2+-ATPase activities of gizzard myosin II fragments, heavy meromyosin (IC50 = 14.4 ± 1.6 µM) and subfragment 1 (IC50 5.5 ± 0.4 µM), were also inhibited by blebbistatin. Assay by in vitro motility indicated that the inhibitory effect of blebbistatin was reversible. Electron-microscopic evaluation showed that blebbistatin induced a distinct conformational change (i.e., swelling) of the myosin II head. The results suggest that the site of blebbistatin action is within the Sl portion of smooth muscle myosin II. [ABSTRACT FROM AUTHOR]