Robert Makuch

The effects of caldesmon extraction on mechanical properties of skinned smooth muscle fibre preparations

The role of caldesmon in the regulation of smooth muscle contraction was investigated in chemically skinned smooth muscle fibres from the guineapig taenia coli. A 19-kDa C-terminal fragment of caldesmon gave a minor (<5%) reduction of force in fully thiophosphorylated fibres, but reduced force by about 50% at intermediate activation levels without affecting the level of light chain phosphorylation. An extraction procedure was developed using incubation in solutions containing high Mg2+ concentrations. Protein analysis revealed a selective decrease in the amount of caldesmon in the fibres. Maximal active force per cross-sectional area was unaffected. The Ca2+ dependence of active force was shifted towards lower Ca2+ concentrations and became less steep. The effects of extraction of caldesmon could in part be reversed by incubation in a solution containing purified caldesmon. The results are consistent with the hypothesis that caldesmon in smooth muscle thin filaments inhibits force generation and plays a role in regulating cooperative attachment of cross-bridges at sub-maximal levels of activation in smooth muscle.

The importance of C‐terminal amino acid residues of actin to the inhibition of actomyosin ATPase activity by caldesmon and troponin I

Proteolytic elimination of three C‐terminal amino acid residues from actin weakens its interaction with caldesmon and troponin I and, in consequence, lowers the inhibitory effects of both proteins on actomyosin ATPase activity. These results prove the importance of C‐terminal extremity of actin to the overall interaction of this protein with caldesmon and troponin I.

Characterization of chicken gizzard calcyclin and examination of its interaction with caldesmon

Using a procedure developed to purify calcyclin from mouse Ehrlich ascites tumor cells calcyclin was purified from smooth muscle of chicken gizzard. Chicken gizzard calcyclin bound to phenyl-Sepharose in a calcium dependent manner as did mouse EAT cells and rabbit lung calcyclin but appeared to be more acidic than its mammalian counterparts as revealed by ion exchange chromatography on Mono Q. Chicken gizzard calcyclin bound 45Ca2+ on nitrocellulose filters and exhibited a shift in electrophoretic mobility on urea-PAGE depending on Ca2+ concentration. Crosslinking experiments with BS3 showed that chicken gizzard calcyclin was able to form noncovalent dimers. As indicated by a decrease in maximum tryptophan fluorescence emission of caldesmon (about 14% at 1:1 molar ratio) and displacement of calmodulin from its complex with caldesmon, chicken gizzard calcyclin binds caldesmon. This binding was, however, much weaker than that of calmodulin and could not influence the interaction of caldesmon with actin. In consequence, calcyclin was unable to reverse the inhibitory effect of caldesmon on actin-activated Mg(2+)-ATPase activity of myosin in the presence of Ca2+.

Lys-373 of actin is involved in binding to caldesmon.

Limited proteolysis of actin with trypsin removes its two or three C‐terminal amino acid residues [Proc. Natl. Acad. Sci. USA 81 (1984) 3680–3684]. Carboxypeptidase B‐treatment of G‐ and F‐actin previously digested with trypsin revealed that in the first case preferential release of three and in the second two C‐terminal amino acid residues takes place. Tryptic removal of three but not two C‐terminal amino acid residues of actin causes weakening of its interaction with caldesmon and lowering of the caldesmon‐induced inhibitory effect on actomyosin ATPase activity. Therefore, it is concluded that the third amino acid residue from the C terminus of actin, Lys‐373, is important for the interaction with caldesmon.

Location of the calmodulin- and actin-binding domains at the C-terminus of caldesmon.

Digestion of caldesmon with carboxypeptidase Y is accompanied by loss of its ability to inhibit actomyosin ATPase activity and to bind actin and calmodulin. Similarly, carboxypeptidase Y digestion of a terminal 40 kDa chymotryptic fragment of caldesmon abolishes its inhibition of the actomyosin ATPase and binding to actin and calmodulin. This represents the first direct demonstration that these functional domains of caldesmon are located close to the carboxy‐terminus of the molecule.

Phosphatidylserine liposomes can be tethered by caldesmon to actin filaments

Rotary shadowing electron microscopy revealed that attachment of caldesmon to phosphatidylserine (PS) liposomes was mainly through its C-terminal end. To determine the PS-binding sites of caldesmon, we have made use of synthetic peptides covering the two C-terminal calmodulin binding sites and a recombinant fragment corresponding to the N-terminal end of the C-terminal domain that contains an amphipathic helix. Interactions of these peptides with the PS liposomes were studied by nondenaturing gel electrophoresis and fluorescence spectroscopy. The results showed that both calmodulin-binding sites of caldesmon were able to interact with PS. The affinity (Kd) of PS for these sites was in the range of 1.8-14.3 x 10(-5) M, compared to 0.69 x 10(-5) M for the whole caldesmon molecule. Fragments located outside of calmodulin-binding sites bound PS weakly (3.85 x 10(-4) M) and thus may contain a second class of lipid-binding sites. Binding of PS induced conformational changes in regions other than the C-terminal PS-binding sites, as evidenced by the changes in the susceptibility to proteolytic cleavages. Most significantly, the presence of caldesmon greatly increased binding of PS to F-actin, suggesting that caldesmon may tether PS liposomes to actin filaments. These results raise the possibility that caldesmon-lipid interactions could play a functionally important role in the assembly of contractile filaments near the membranes.

Visualization of caldesmon binding to synthetic filaments of smooth muscle myosin.

We have used synthetic filaments of unphosphorylated chicken gizzard myosin with a compact, highly ordered structure under relaxing conditions (in the absence of Ca2+ and in the presence of ATP) to visualize the mode of caldesmon binding to myosin filaments by negative staining and immunogold electron microscopy. We demonstrate that the addition of caldesmon to preformed myosin filaments leads to the appearance of numerous smooth projections curving out from the filament surface. The addition of caldesmon or its N-terminal fragment resulted in the partial masking of myosin filament periodicity. However, it did not change the inner structure of the filaments. It is demonstrated that most caldesmon molecules bind to myosin filaments through the N-terminal part, while the C-terminal parts protrude from the filament surface, as confirmed by immunoelectron microscopy visualization. Together with the available biochemical data on caldesmon binding to both actin and myosin and electron microscopic observations on the mode of caldesmon attachment to actin filaments with the C-termini of the molecules curving out from the filaments, the visualization of caldesmon attachment to myosin filaments completes the scenario of actin to myosin tethering by caldesmon.

Polymerization of actin induced by actin-binding fragments of caldesmon

Our earlier studies revealed that caldesmon causes assembly of G-actin into polymers morphologically indistinguishable from those formed in the presence of salt (Gałazkiewicz, B., Belagyi, J. and Dabrowska, R. (1989) Eur. J. Biochem. 181, 607-614). In this work we have investigated the effect of actin-binding fragments of caldesmon on actin polymerization process followed by measurements of the changes in fluorescence of pyrenyl conjugated with G-actin and ATP hydrolysis. The results indicate that C-terminal 34 kDa fragment of caldesmon containing two actin-binding sites and 19 kDa containing high-affinity binding site have similar capability to polymerize actin to that of intact molecule. Binding of each of these fragments to G-actin causes bypassing of nucleation phase. The 11.5 kDa fragment comprising low affinity actin-binding site has much lower potency to polymerize actin. Conformation of actin monomers in filaments formed upon 19 kDa fragment and that formed upon 11.5 kDa fragment differs. The former fragment seems to resemble more conformation of monomers in filaments formed upon intact caldesmon than the latter one.

Caldesmon inhibits both force development and transition of actin monomers from "OFF" to "ON" conformational state by changing its position in thin filaments.

We have investigated the effect of caldesmon on the actin conformational state and its position at force generation in glycerinated fibers upon transformation from relaxation to rigor. F‐actin and caldesmon were labeled with TRITC‐phalloidin or acrylodan, respectively, and the orientation and mobility of the probes were calculated. Transition from relaxation to rigor was accompanied by force development and by the changes in orientation and mobility of TRITC‐phalloidin that were typical for actin monomer transformation from the “OFF” to the “ON” conformational state. In the presence of caldesmon, both the force developed by the fibers and the changes in the orientation and mobility of TRITC‐phalloidin were markedly decreased. In contrast, the orientation and mobility of acrylodan change essentially showed the displacement of the caldesmon molecules and the changes in its mobility. The results are evidence that structure and/or mode of the attachment of caldesmon to actin modulates both the force production and transition of actin monomers from “OFF” to “ON” conformations in the ATPase cycle.