Renata Dąbrowska

Functional domain of caldesmon

Limted proteolysis of caldesmon has been used in studying the structure‐function relationship of this protein. Digestion with α‐chymotrypsin yields three major fragments of 110, 80 and 40 kDa. Only the 40 kDa fragment preserves functional properties of the parent molecule: it binds to F‐actin, causes inhibition of actomyosin ATPase and binds to calmodulin in a Ca2+‐dependent manner. Its further degradation produces an 18 kDa polypeptide that also retains all these properties. Neither F‐actin nor calmodulin binding induces dramatic changes in susceptibility to chymotryptic cleavage and the sites of cleavage of caldesmon.

The origin of 30,000 dalton protein in troponin preparations

Received 17 November 197 2 1. Introduction It is already well established that the Ca2+ sensi- tivity of actomyosin is rendered by the regulatory protein system, composed of troponin and tropo- myosin [ 1,2] . Troponin appeared to consist of sever- al components which can be separated by DEAE- Sephadex chromatography [3,4] . We have recently shown [4,5] that out of three main constituents of troponin called: TN-C (mol. wt. 18,000), TN-I (mol. wt. 24,000) and TN-B (mol. wt. 39,000) (the average molecular weights and the nomenclature were taken from [5] ) two latter proteins are very susceptible to the proteolytic digestion. Thus, the other constituent of troponin corresponding to the protein of molecu- lar weight 13,000- 14,000 daltons, was found to be the product of catheptic degradation of TN-I, formed during preparation of troponin, namely during incuba- tion at pH 4.6 [4, S] . In addition, troponin prepara- tions contained usually variable amounts of another protein of molecular weight of about 30,000 daltons [4, 6-81 . We have recently found particularly large amounts of this protein in troponin preparations from slow and cardiac muscles [9] concominantly with considerable decrease of TN-B. The observation suggested that 30,000 component might derive from TN-B. This assumption obtained experimental evi- dence in the present work.

Caldesmon weakens the bonding between myosin heads and actin in ghost fibers

Earlier studies using polarized microphotometry have shown that caldesmon inhibits the alterations in structure and flexibility of actin in ghost fibers that take place upon the binding of myosin heads (Gałazkiewicz et al. (1987) Biochim. Biophys. Acta 916, 368-375). The present investigations, performed with an IAEDANS label attached to myosin subfragment 1 (S-1), revealed that this inhibition results from the weakening of the binding between myosin heads and actin as indicated by the caldesmon-induced increase in the random movement of S-1. Parallel experiments with actin labeled at Cys-374 demonstrated that this effect of caldesmon is transmitted to the C-terminus of the actin molecule resulting in a conformational adjustment in this region of the molecule.

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.

Actin and Thin-Filament-Associated Proteins in Smooth Muscle

Actin is a major component of both contractile and cytoskeletal structures in all eukaryotic cells [1–4]. It can exist either as a monomeric molecule (G-actin) or as a filamentous polymer (F-actin). Both forms can be reversibly transformed one into another depending on ionic conditions, temperature and the presence of other proteins. The only functional form of actin is F-actin.

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.

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.