Cross-linking of smooth muscle caldesmon to the NH2-terminal region of skeletal F-actin.

A Bartegi,A Fattoum,R Kassab

doi:10.1016/s0021-9258(19)39966-1

A Bartegi, A Fattoum + Show 1 more

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https://doi.org/10.1016/s0021-9258(19)39966-1

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Abstract

The cross-linking of the F-actin-caldesmon complex with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide in the presence of N-hydroxysuccinimide generated four major adducts which were identified on polyacrylamide gels. By cross-linking 3H-actin to 14C-caldesmon, these were found to represent 1:1 cross-linked complexes of actin and caldesmon displaying different electrophoretic mobilities. Tropomyosin did not noticeably affect the cross-linking process. The same four fluorescent species resulting from the cross-linking of caldesmon to F-actin labeled with N-[7-(dimethylamino)-4-methyl-3-coumarinyl]maleimide were subjected separately to partial cleavages with hydroxylamine or cyanogen bromide. These treatments yielded fluorescent 41- and 37-kDa fragments, respectively, from each cross-linked entity indicating unambiguously that caldesmon was cross-linked only to the NH2-terminal actin stretch of residues 1-12. This region is also known to serve for the carbodiimide-mediated cross-linking of the myosin subfragment-1 heavy chain (Sutoh, K. (1982) Biochemistry 21, 3654-3661). A covalent caldesmon-F-actin conjugate containing a protein molar ratio close to 1:19 was isolated following dissociation of uncross-linked caldesmon. It showed a low level of activation of the ATPase activity of skeletal myosin subfragment-1, and the binding of Ca2(+)-calmodulin to the derivative did not cause the reversal of the ATPase inhibition. In contrast, the reversible binding of caldesmon to F-actin cross-linked to myosin subfragment-1 did not inhibit the accelerated ATPase of the complex. The overall data point to the dual involvement of the actin's NH2 terminus in the inhibitory binding of caldesmon and in actomyosin interactions in the presence of ATP.

Highlights

The cross-linking of the F-actin-caldesmon complex with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide in the presence of N-hydroxysuccinimide generated four major adducts which were identified on polyacrylamide gels
The quantitative determinations of the relative amounts of actin and caldesmon in each of the four major products of the reaction indicated that crosslinking takes place between one actin and one caldesmon
The covalent union of actin to caldesmon in each cross-linked complex identified involves the acidic NH*-terminal actin segment of residues 1-12. Electrostatic contacts between this region and the actin-binding domain of caldesmon could play a significant role in the recognition between the two proteins as suggested by the reported decrease by moderate salt concentrations of the binding affinity of caldesmon to F-actin, Caldesmon is to be added to the long list of actin-binding proteins whose interaction at or near the NHP-terminal actin peptide has been revealed by specific EDC-catalyzed cross-linking

Summary

Introduction

The cross-linking of the F-actin-caldesmon complex with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide in the presence of N-hydroxysuccinimide generated four major adducts which were identified on polyacrylamide gels. Did not give any cross-linking product (Fig. IA, lane t2) and the addition of F-actin to EDC-NHS-activated caldesmon did not generate any new protein band (data not shown).

Results

Conclusion