Epitope mapping of monoclonal antibodies against caldesmon and their effects on the binding of caldesmon to Ca++/calmodulin and to actin or actin-tropomyosin filaments.

The effects of monoclonal anti-caldesmon antibodies, C2, C9, C18, C21, and C23, on the binding of caldesmon to F-actin/F-actin-tropomyosin filaments and to Ca++/calmodulin were examined in an in vitro reconstitution system. In addition, the antibody epitopes were mapped by Western blot analysis of NTCB (2-nitro-5-thiocyanobenzoic acid) and CNBr (cyanogen bromide) fragments of caldesmon. Both C9 and C18 recognize an amino terminal fragment composed of amino acid residues 19 to 153. The C23 epitope lies within a fragment ranging from residues 230 to 386. Included in this region is a 13-residue repeat sequence. Interestingly this repetitive sequence shares sequence similarity with a sequence found in nuclear lamin A, a protein which is also recognized by C23 antibody. Therefore, it is likely that the C23 epitope corresponds to this 13-residue repeat sequence. A carboxyl-terminal 10K fragment contains the epitopes for antibodies C2 and C21. Among these antibodies, only C21 drastically inhibits the binding of caldesmon to F-actin/F-actin-tropomyosin filaments and to Ca++/calmodulin. When the molar ratio of monoclonal antibody C21 to caldesmon reached 1.0, a maximal inhibition (90%) on the binding of caldesmon to F-actin filaments was observed. However, it required double amounts of C21 antibody to exhibit a maximal inhibition of 70% on the binding of caldesmon to F-actin-tropomyosin filaments. These results suggest that the presence of tropomyosin in F-actin enhances caldesmon's binding. Furthermore, C21 antibody also effectively inhibits the caldesmon binding to Ca++/calmodulin. The kinetics of C21 inhibition on caldesmon's binding to Ca++/calmodulin is very similar to the inhibition obtained by preincubation of caldesmon with free Ca++/calmodulin. This result suggests that there is only one Ca++/calmodulin binding domain on caldesmon and this domain appears to be very close to the C21 epitope. Apparently, the Ca++/calmodulin-binding domain and the actin-binding domain are very close to each other and may interfere with each other. In an accompanying paper, we have further demonstrated that microinjection of C21 antibody into living chicken embryo fibroblasts inhibit intracellular granule movement, suggesting an in vivo interference with the functional domains [Hegmann et al., 1991: Cell Motil. Cytoskeleton 20:109-120].     

Identification by monoclonal antibodies and characterization of human platelet caldesmon

Actin-based gels were prepared from clarified high-salt extracts of human platelets by dialysis against physiological salt buffers. The gel was partially solubilized with 0.3 M KCl. Mice were immunized with the 0.3 M KCl extract of the actin gel, and hybridomas were produced by fusion of spleen cells with myeloma cells. Three hybridomas were generated that secrete antibodies against an 80-kD protein. These monoclonal antibodies stained stress fibers in cultured cells and cross-reacted with proteins in several tissue types, including smooth muscle. The cross-reacting protein in chicken gizzard smooth muscle had an apparent molecular weight of 140,000 and was demonstrated to be caldesmon, a calmodulin and actin-binding protein (Sobue, K., Y. Muramoto, M. Fujita, and S. Kakiuchi, Proc. Natl. Acad. Sci. USA, 78:5652-5655). No proteins of molecular weight greater than 80 kD were detectable in platelets by immunoblotting using the monoclonal antibodies. The 80-kD protein is heat stable and was purified using modifications of the procedure reported by Bretscher for the rapid purification of smooth muscle caldesmon (Bretscher, A., 1985, J. Biol. Chem., 259:12873-12880). The 80-kD protein bound to calmodulin-Sepharose in a Ca++-dependent manner and sedimented with actin filaments, but did not greatly increase the viscosity of F-actin solutions. The actin-binding activity was inhibited by calmodulin in the presence of calcium. Except for the molecular weight difference, the 80-kD platelet protein appears functionally similar to 140-kD smooth muscle caldesmon. We propose that the 80-kD protein is platelet caldesmon.     

Amino acid sequence studies on cyanogen bromide peptides of chicken caldesmon which bind to calmodulin

Three major calmodulin-binding cyanogen bromide peptides (fragments A, B, and D) were isolated from chicken gizzard muscle caldesmon and their amino acid sequences were determined. The molecular masses of fragments A, B, and D were estimated to 16, 12, and 9 kDa, respectively, by SDS-urea polyacrylamide gel electrophoresis. Fragment A was composed of 102 amino acid residues and contained homoserine at the C terminus. The amino acid sequence from the 37th residue of fragment A corresponds to the N-terminal sequence of the 15 kDa peptide which was obtained by thrombin digestion [Mornet, D., Audemard, E., & Derancourt, J. (1988) Biochem. Biophys. Res. Commun. 154, 564-571]. Thrombin 15 kDa peptide binds to F-actin but does not bind to calmodulin. Thus the N-terminal 36 residues and the C-terminal part from the 37th residue of fragment A are supposed to bind to calmodulin and F-actin, respectively. The sequences of fragments B and D were identical, but fragment D was composed of 64 amino acid residues and ended with tryptophan, whereas fragment B was of 98 or 99 amino acid residues and ended with proline. Both fragments B and D are supposed to be the C-terminal peptides of chicken caldesmon. Fragment B had heterogeneous sequences at the C-terminal region. These results can explain the reported heterogeneity of chicken caldesmon in charge and molecular mass.     

Phosphorylation of high-Mr caldesmon by protein kinase C modulates the regulatory function of this protein on the interaction between actin and myosin

High-Mr caldesmon, which is involved in smooth muscle contraction, was phosphorylated by protein kinase C. By chymotryptic digestion, actin- and calmodulin-binding assays and immunoprecipitation with the antibody to the C-terminal 35-kDa fragment, we have identified that all phosphate groups are incorporated exclusively into this fragment, which is the functional domain for binding actin and calmodulin. Phosphorylation of high-Mr caldesmon and its C-terminal 35-kDa fragment reduced their binding abilities to both F-actin and calmodulin. Further, their inhibitory effects on the actin-activated ATPase activity of gizzard myosin were also reversed in proportion to the degree of phosphorylation. These results suggest that phosphorylation of high-Mr caldesmon by protein kinase C, which is restricted within the C-terminal 35-kDa domain, results in the modulation of its activity in the smooth muscle actin--myosin interaction.     

Alternative model for the internal structure of laminin

A monoclonal antibody to laminin, LMN-1, was generated by immunizing rats with laminin from the EHS tumor and fusing the rat spleen cells with mouse NS-1 myeloma cells. Laminin fragments were generated by proteolytic digestion with thrombin, thermolysin, and chymotrypsin. Monoclonal antibody binding fragments were identified by immunoblotting. Fragments which bound monoclonal antibody LMN-1 included a 440-kilodalton (kDa) chymotrypsin fragment and thermolysin fragments of 440 and 110 kDa. These fragments could also be generated from within a 600-kDa thrombin fragment. Digestion of the 440-kDa chymotrypsin fragment with thermolysin generated the 110-kDa antibody binding fragment and a 330-kDa nonbinding fragment. Immunoblotting was performed on extracts of PYS-2 cells and EHS cells using polyclonal and monoclonal antibodies to laminin. Polyclonal antibodies stained the intact 850-kDa complex and the 200- and 400-kDa subunits, while monoclonal LMN-1 stained only the 400-kDa subunit and the complete molecule. Rotary shadowing of monoclonal LMN-1 bound to laminin molecules indicated that the binding site was within the long arm of laminin. Changes in the model of the internal organization of the laminin molecule are proposed, based on the binding of LMN-1 to the 400-kDa subunit and specific proteolytic fragments. The locations of the major thrombin and chymotrypsin fragments in the model are rotated 180 degrees relative to the previously described model [Ott, U., Odermatt, E., Engel, J., Furthmayr, H., & Timpl, R. (1982) Eur. J. Biochem. 123, 63-72] to include part of the 400-kDa subunit of laminin.     

Mapping of caldesmon: relationship between the high and low molecular weight forms

Caldesmon is a widely distributed contractile protein that occurs in both a high molecular weight [120-150-kilodalton (kDa)] and a low molecular weight (71-80-kDa) form, depending on the tissue. The structural relationship between these two forms was examined by mapping techniques. Partial cyanogen bromide cleavage in conjunction with sodium dodecyl sulfate gel electrophoresis was used to construct a map of the cleavage points and determine the relative position of the fragments in a high molecular weight caldesmon from chicken gizzard (caldesmon125). By use of this map, markers for different regions of the protein were obtained: Antibodies directed toward certain areas were prepared by affinity purification, and specific 125I-labeled tryptic peptides were found to originate from terminal cyanogen bromide fragments. Mapping of a lower molecular weight form of caldesmon (caldesmon72 from chicken liver) revealed the presence of sequences located in both ends of caldesmon125. A terminal 38-kDa fragment of both proteins was apparently identical on the basis of arrangement of cleavage sites, antibody reactivity, and iodopeptide mapping. Fragments from the other end of both proteins exhibited an identical pattern of peptides. These results show that it is sequences located in the central area of caldesmon125 which are missing in caldesmon72, indicating that the smaller molecule is not simply a proteolytic product of the larger. The two forms of caldesmon may be derived from separate genes or by alternative splicing from a single gene.     

Localization of the calmodulin- and the actin-binding sites of caldesmon

Expression of the C-terminal third of chicken gizzard caldesmon in Escherichia coli, using the Nagai vector (Nagai, K., and Th?gersen, H.V. (1987) Methods Enzmol. 153, 461-481), produces a cII-caldesmon fusion protein (27 kDa) with caldesmon sequence beginning at Lys579. Degradation during purification yields five peptides with molecular masses of 24, 22, 19 (two peptides), and 15 kDa. The 24-kDa peptide begins at Phe581; the 22-kDa peptide begins at Leu597, the two 19-kDa peptides begin at Phe581 and Val629, respectively; the 15-kDa peptide also begins at Val629. We estimate that the 15-kDa and one of the 19-kDa peptides end near Leu710. Site-directed mutagenesis was used to produce truncated peptides with known C termini; one peptide (17 kDa) terminates at Asn675. Digestion of the fragments with chymotrypsin generates a second 15-kDa fragment that begins at Ser666 (15K'). All of the peptides, with the exception of 15K', bind Ca(2+)-calmodulin-Sepharose and share a common 37-amino acid peptide between Val629 and Ser666, suggesting this contains the calmodulin binding site. Comparison with published sequences (Takagi, T., Yazawa, M., Ueno, T., Suzuki, S., and Yagi, K. (1989) J. Biochem. (Tokyo) 106, 778-783 and Bartegi, A., Fattoum, A., Derancourt, J., and Kassab, R. (1990) J. Biol. Chem. 265, 15231-15238) for other calmodulin-binding fragments further restricts the binding site to 7 residues, Trp-Glu-Lys-Gly-Asn-Val-Phe, between Trp659 and Ser666. All of the fragments, except the two 15-kDa peptides, co-sediment with F-actin, indicating that there are two segments in the C-terminal third of caldesmon that can interact with F-actin: one between Leu597 and Val629, the other between Arg711 and Pro756. Although separated in the primary sequence, these domains may interact with the calmodulin-binding region in the folded structure.     

Mechanism for the inhibition of acto-heavy meromyosin ATPase by the actin/calmodulin binding domain of caldesmon.

Caldesmon, an actin/calmodulin binding protein, inhibits acto-heavy meromyosin (HMM) ATPase, while it increases the binding of HMM to actin, presumably mediated through an interaction between the myosin subfragment 2 region of HMM and caldesmon, which is bound to actin. In order to study the mechanism for the inhibition of acto-HM ATPase, we utilized the chymotryptic fragment of caldesmon (38-kDa fragment), which possesses the actin/calmodulin binding region but lacks the myosin binding portion. The 38-kDa fragment inhibits the actin-activated HMM ATPase to the same extent as does the intact caldesmon molecule. In the absence of tropomyosin, the 38-kDa fragment decreased the KATPase and Kbinding without any effect on the Vmax. However, when the actin filament contained bound tropomyosin, the caldesmon fragment caused a 2-3-fold decrease in the Vmax, in addition to lowering the KATPase and the Kbinding. The 38-kDa fragment-induced inhibition is partially reversed by calmodulin at a 10:1 molar ratio to caldesmon fragment; the reversal was more remarkable in 100 mM ionic strength at 37 degrees C than in 20 or 50 mM at 25 degrees C. Results from these experiments demonstrate that the 38-kDa domain of caldesmon fragment of myosin head to actin; however, when the actin filament contains bound tropomyosin, caldesmon fragment affects not only the binding of HMM to/actin but also the catalytic step in the ATPase cycle. The interaction between the 38-kDa domain of caldesmon and tropomyosin-actin is likely to play a role in the regulation of actomyosin ATPase and contraction in smooth muscle.     

Localization and characterization of a 7.3-kDa region of caldesmon which reversibly inhibits actomyosin ATPase activity.

Cleavage of caldesmon with chymotrypsin yields a series of fragments which bind both calmodulin and actin and inhibit the binding of myosin subfragments to actin and the subsequent stimulation of ATPase activity. Several of these fragments have been purified by cation exchange chromatography and their amino-terminal sequences determined. The smallest fragment has a molecular mass of about 7.3 kDa and extends from Leu597 to Phe665. This polypeptide inhibits the actin-activated ATPase of myosin S-1; this inhibition is augmented by smooth muscle tropomyosin and relieved by Ca(2+)-calmodulin. The binding of the 7.3-kDa fragment to actin is competitive with the binding of S-1 to actin. Thus, this polypeptide has several of the important features characteristic of intact caldesmon. However, although an intact caldesmon molecule covers between six and nine actin monomers, the 7.3-kDa fragment binds to actin in a 1:1 complex. Comparison of this fragment with others suggests that a small region of caldesmon is responsible for at least part of the interaction with both calmodulin and actin.     

Further characterization of calpain-mediated proteolysis of the human erythrocyte plasma membrane Ca2+-ATPase

The membrane-bound form and a solubilized and purified form of the Ca2+-ATPase from human erythrocyte have been proteolyzed under controlled conditions by highly purified Ca2+-dependent neutral cysteine-protease, calpain I, in the absence and in the presence of the calmodulin-calcium complex. In the absence of calmodulin the 136-kDa enzyme was transformed into a group of fragments of 125-124 kDa, followed by the slower formation of a second group of fragments of 82-80 kDa. These heterogeneous fragments were capable of forming an acylphosphate intermediate. The 125- and 82-kDa minor components of each heterogeneous group of fragments (125-124 and 82-80 kDa) were capable of binding calmodulin, whereas the 124- and the 80-kDa major components did not. In the presence of calmodulin, however, the native enzyme was transformed into a 127-kDa fragment followed by the slower formation of an 85-kDa fragment. Both fragments (127 and 85 kDa) formed an acylphosphate intermediate and were capable of binding calmodulin. The presence of calmodulin during calpain action effectively protected the Ca2+-ATPase from proteolytic activation (K.K.W. Wang, A. Villalobo, and B.D. Roufogalis (1988) Arch. Biochem. Biophys. 260, 696-704) and prevented the formation of the calmodulin-insensitive 124- and 80-kDa fragments. Smaller fragments not capable of forming the acylphosphate intermediate were also produced, in particular a 39-37 kDa doublet band retaining the capacity to bind calmodulin. In contrast to the membrane-bound form, the purified form of the Ca2+-ATPase was proteolyzed by calpain at a slower rate.     

Phosphorylation of smooth muscle caldesmon by mitogen-activated protein (MAP) kinase and expression of MAP kinase in differentiated smooth muscle cells.

Smooth muscle caldesmon was phosphorylated in vitro by sea star p44mpk up to 2.0 mol of phosphate/mol of protein at both Ser and Thr residues. The phosphorylation sites were contained mainly in the COOH-terminal 10-kDa cyanogen bromide fragment which houses the binding sites for calmodulin, tropomyosin, and F-actin. Tryptic peptide maps of 32P-labeled caldesmon by p44mpk and p34cdc2 showed that while both enzymes recognized similar sites of phosphorylation, they have different preferred sites. Phosphorylation of caldesmon attenuated slightly its interaction with actin and had no effect on its binding to calmodulin and tropomyosin. Smooth muscle cell extracts from chicken gizzard and rat aorta contained 42- and 44-kDa proteins, respectively, which were cross-reactive with an antibody to sea star p44mpk. Immunoprecipitates from gizzard and aorta cell extracts, generated with the p44mpk antibody, possessed kinase activities toward myelin basic protein as well as caldesmon. These results suggest that MAP kinase may have functions in the differentiated smooth muscle cells distinct from those involved in the cell cycle.     

Structural analysis of rod GTP-binding protein, Gt. Limited proteolytic digestion pattern of Gt with four proteases defines monoclonal antibody epitope.

The epitope of monoclonal antibody (mAb 4A), which recognizes the alpha subunit of the rod G protein, Gt, has been suggested to be both at the carboxyl terminus (Deretic, D., and Hamm, H.E. (1987) J. Biol. Chem. 262, 10839-10847) and the amino terminus (Navon, S.E., and Fung, B.K.-K. (1988) J. Biol. Chem. 263, 489-496) of the molecule. To characterize further the mAb 4A binding site on alpha t and to resolve the discrepancy between these results limited proteolytic digestion of Gt or alpha t using four proteases with different substrate specificities has been performed. Endoproteinase Arg-C, which cleaves the peptide bond at the carboxylic side of arginine residues, cleaved the majority of alpha t into two fragments of 34 and 5 kDa. The alpha t 34-kDa fragment in the holoprotein, but not alpha t-guanosine 5'-O-(3-thiotriphosphate), was converted further to a 23-kDa fragment. A small fraction of alpha t-GDP was cleaved into 23- and 15-kDa fragments. Endoproteinase Lys-C, which selectively cleaves at lysine residues, progressively removed 17 and then 8 residues from the amino terminus, forming 38- and 36-kDa fragments. Staphylococcus aureus V8 protease is known to remove 21 amino acid residues from the amino-terminal region of alpha t, with the formation of a 38-kDa fragment. L-1-Tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin cleaved alpha t progressively into fragments of known amino acid sequences (38, then 32 and 5, then 21 and 12 kDa) and a transient 34 kDa fragment. The binding of mAb 4A to proteolytic fragments was analyzed by Western blot and immunoprecipitation. The major fragments recognized by mAb 4A on Western blots were the 34- and 23-kDa fragments obtained by endoproteinase Arg-C and tryptic digestion. Under conditions that allowed sequencing of the 15- and 5-kDa fragments neither the 34- nor the 23-kDa fragments could be sequenced by Edman degradation, indicating that they contained a blocked amino terminus. The smallest fragment that retained mAb 4A binding was the 23-kDa fragment containing Met1 to Arg204. Thus the main portion of the mAb 4A antigenic site was located within this fragment, indicating that the carboxyl-terminal residues from Lys205 to Phe350 were not required for recognition by the antibody. Additionally, the antibody did not bind the 38- and 36-kDa or other fragments containing the carboxyl terminus, showing that the amino-terminal residues from Met1 to Lys17 were essential for antibody binding to alpha t.     

Characterization of the carboxyl-terminal 10-kDa cyanogen bromide fragment of caldesmon as an actin-calmodulin-binding region

A pair of 10-kDa peptides, designated CB-a and CB-b, was isolated by calmodulin-Sepharose chromatography from a total CNBr digest of turkey gizzard caldesmon. CB-a encompasses the COOH-terminal segment of residues 659-756, according to the sequence of adult chicken gizzard caldesmon (Bryan, J., Imai, M., Lee, R., Moore, P., Cook, R.G., and Lin, W.G. (1989) J. Biol. Chem. 264, 13873-13879), whereas CB-b comprises the same structure but was a few amino acids shorter at its COOH terminus. Both peptides cosedimented with F-actin, and their binding was increased by smooth muscle tropomyosin. The Kd values were 1.3 and 0.5 microM, in the absence and presence of tropomyosin, respectively, with a maximum binding capacity of 6.9 actins/mol of peptides. The CB-a/CB-b fragments inhibited, in a tropomyosin-sensitive and Ca2(+)-calmodulin-dependent manner, the skeletal actomyosin subfragment 1 ATPase activity to a level close but not identical to that observed for the parent caldesmon. Ca2(+)-calmodulin was selectively cross-linked to either caldesmon or the CNBr peptides with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide producing 1:1 covalent complexes that were retained neither by phenyl-Sepharose nor by immobilized calmodulin. Moreover, the cross-linked caldesmon bound weakly to F-actin and did not inhibit the actomyosin subfragment 1 ATPase in the absence of Ca2+. The results suggest that the CB-a/CB-b peptide region contains major regulatory determinants of caldesmon.     

Epitopes on proliferating cell nuclear antigen recognized by human lupus autoantibody and murine monoclonal antibody

The immune epitopes of proliferating cell nuclear antigen (PCNA), also called cyclin, were analyzed by determining the reactivity between PCNA peptide fragments and anti-PCNA antibodies from lupus patients, murine monoclonal antibody (19A2), and rabbit anti-NH2-terminal peptide antibody. Limited digestion of PCNA/cyclin with Staphylococcus aureus V8 protease resulted in several peptide fragments. Five fragments of 30, 20, 15, 14, and 13 kDa were reactive with rabbit anti-NH2-terminal peptide antibody denoting that they contained the NH2-terminal peptide. The 30- and 20-kDa fragments reacted with 19A2 but the others did not. Lupus sera reacted with 17- and 15-kDa peptide fragments allowing their classification into three groups. Two of eight sera (type A) reacted only with the 17-kDa fragment. Two others (type B) reacted with both the 17- and 15-kDa fragments and the remaining four sera (type C) reacted only with the 15-kDa fragment. The sera reacting with the 15-kDa fragment also reacted with the 20-kDa fragment, but the sera reactive only with the 17-kDa fragment did not, indicating that the 17-kDa fragment was not a degradation product of 20-kDa fragments. The 19A2 epitope resided in the region between 15 and 20 kDa from the NH2 terminus, whereas there was at least one distinct epitope on each 15- and 17-kDa peptide, which were recognized by lupus autoantibodies.     

Caldesmon inhibits the cooperative turning-on of the smooth muscle heavy meromyosin by tropomyosin-actin

The 38-kDa chymotryptic fragment of caldesmon, which possesses the actin/calmodulin binding domain, was purified and utilized to study the mechanism for the inhibition of acto-myosin ATPase by caldesmon. The intact caldesmon inhibited the acto-HMM ATPase although it caused an increase in the binding of HMM to actin, presumably due to the interaction between the S-2 region of HMM and the caldesmon located on the actin filament. The 38-kDa fragment, which lacks the S-2 binding domain, inhibited both the acto-HMM ATPase and the HMM binding to actin. The ATPase and the HMM binding to actin decreased in parallel on increasing the 38-kDa fragment bound to actin. In the presence of tropomyosin, the ATPase activity fell more rapidly than did the HMM binding to actin. Binding of intact caldesmon or 38-kDa fragment to actin inhibited the cooperative turning-on of tropomyosin-actin by NEM.S-1, which forms rigor complexes in the presence of ATP. The absence of cooperative turning-on of the acto-HMM ATPase by rigor complexes in the presence of 38-kDa fragment was associated with an inhibition of the binding of HMM to tropomyosin-actin. Addition of NEM.S-1 to tropomyosin-actin-caldesmon caused a gradual decrease in the caldesmon-induced binding of HMM to actin. The calmodulin restored the caldesmon-induced binding of HMM to tropomyosin-actin, but it had only a slight effect on the acto-HMM ATPase. These data suggest that the cooperative turning-on of the smooth muscle tropomyosin-actin by rigor bonds is modulated by the interaction of caldesmon, tropomyosin, and calmodulin on the thin filament.     

Conformational change of turkey-gizzard caldesmon induced by specific chemical modification with carbodiimide

Water soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide was used to internally cross-link carboxyl and lysyl groups of caldesmon. The modification did not involve the two cysteines of the molecule which were previously labelled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine. The modified caldesmon exhibited a smaller Stokes radius (4.0 nm instead of 6.3 nm) and its electrophoretic mobility corresponded to an apparent molecular mass of approximately 82 kDa, appreciably lower than that of the native molecule (120 kDa), but more similar to the reported true molecular mass of 86,974 Da of chicken-gizzard caldesmon (Bryan, J., Imai, M., Lee, R., Moore, P., Cook. R. G. & Lin, W. (1989) J. Biol. Chem. 264, 13,873-13,879). Comparative circular dichroism analysis indicated a decrease of the alpha-helix content from 43% to 36% resulting from the chemical modification. The 1H-NMR spectra of the native and modified caldesmon showed that the covalent cross-linking affected mainly the central and N-terminal parts of the molecule. The C-terminal part, rich in aromatic amino acids, was unmodified by the carbodiimide treatment. This was also corroborated by the continued ability of the modified caldesmon to bind to actin and calmodulin, and by the property of the 90-kDa proteolytic N-terminal fragment to give an internally cross-linked species of 60 kDa. Using electron microscopy, the modified protein was shown to have a more compact shape and a reduced capacity to induce tight and long F-actin bundles. These conformational changes were obtained when the carbodiimide reaction was conducted at pH 6.0 and were not observed at pH 8.0. This suggests that local variation of the pH might affect the conformation of caldesmon which changes from an elongated to more compact shape, stabilized by electrostatic interactions. It is proposed that the flexibility of caldesmon might be involved in the regulatory function of this protein in the smooth muscle and might favour tightly packed F-actin bundles or weaker interactions between actin filaments.     

Mapping of distinct structural domains on microtubule-associated protein 2 by monoclonal antibodies

Monoclonal antibodies against microtubule-associated protein 2 (MAP2) were prepared and their specificity was verified by visualization of the antigens using the antibody overlay technique and by radioimmunoassay. MAP2 was cleaved by alpha-chymotrypsin to generate a series of high-molecular-mass fragments ranging between 270 and 140 kDa. The precursor-product relationship of these fragments was suggested from the rate of their appearance and from the analysis of the tryptic peptide map of each fragment. A group of monoclonal antibodies was found to react predominantly with the intact 270-kDa MAP2 molecule and a fragment having a mass of 240 kDa and to some extent with a 215-kDa fragment. Another group of monoclonal antibodies reacted with an antigenic determinant which was located on the 270-kDa molecule as well as on fragments as small as 140 kDa. None of the two groups of monoclonal antibodies reacted with the microtubule-binding domain of MAP2. These results suggest that one group of antibodies reacts with sites located at or dependent upon a terminal 60-kDa domain(s) distal to the microtubule-binding site of MAP2. The second group of antibodies, which can still bind to smaller proteolytic products, appear to be associated with the central region of the MAP2 molecule. Indirect immunofluorescence experiments with the antibody preparations indicated that at least some of the antigenic determinants are exposed when MAP2 is associated with microtubules in the cell body and neurite outgrowths of differentiated rat brain neuroblastoma B104 cells.     

Actin-caldesmon-myosin-subfragment-1 ternary complex viewed by electron microscopy. Competitive actin binding region for caldesmon and myosin subfragment-1

An earlier electron microscopic study using different caldesmon forms complexed with actin revealed that the aggregates produced display regular periodic striation after antibody labeling of the 35-kDa caldesmon fragment. This approach provides further evidence that a caldesmon fragment, even as small as 15 kDa, can induce actin filaments to assemble into bundles. The observed difference in the compactness of these structures, depending on the use of the 15-kDa fragment instead of the 35-kDa fragment, suggests the existence of more than one actin-binding site in the caldesmon molecule. In this study, the caldesmon-induced process of F-actin association was investigated in the presence of skeletal myosin subfragment-1, using light-scattering methods, cosedimentation experiment and electron microscopic techniques. We show that the actin-caldesmon association is partially destabilized in the presence of subfragment-1 and this leads to a ternary complex formation. Immunogold labelling of the actin filaments still reveals the presence of caldesmon within this structure. This latter result strengthens the hypothesis that actin has a site(s) able to bind both caldesmon and myosin subfragment-1, as detected by recent NMR observations. This evidence is discussed with respect to the regulatory function of caldesmon during smooth muscle contraction.     

Mapping of the microvillar 110K-calmodulin complex (brush border myosin I). Identification of fragments containing the catalytic and F-actin-binding sites and demonstration of a calcium ion dependent conformational change

In intestinal microvilli, the 110K-calmodulin complex is the major component of the cross-bridges which connect the core bundle of actin filaments to the membrane. Our previous work showed that the 110-kDa polypeptide can be divided into three functional domains: a 78-kDa fragment that contains the ATPase activity and the ATP-reversible F-actin-binding site, a 12-kDa fragment required for binding calmodulin molecules, and a terminal 20-kDa domain of unknown function [Coluccio, L. M., & Bretscher, A. (1988) J. Cell Biol. 106, 367-374]. By analysis of limited alpha-chymotryptic cleavage products, we now show that the molecular organization is very similar to that described for the S1 fragment of myosin. The catalytic site was identified by photoaffinity labeling with [5,6-3H]UTP, and fragments binding F-actin were identified by cosedimentation assays. Cleavage of the 78-kDa fragment yielded major fragments of 32 and 45 kDa, followed by cleavage of the 45-kDa fragment to a 40-kDa fragment. Of these, only the 32-kDa fragment was labeled by [5,6-3H]UTP. Physical characterization revealed that the 45- and 32-kDa fragments exist as a complex that can bind F-actin, whereas the 40-kDa/32-kDa complex cannot bind actin. We conclude that the catalytic site is located in the 32-kDa fragment and the F-actin-binding site is present in the 45-kDa fragment; the ability to bind actin is lost upon further cleavage of the 45-kDa fragment to 40 kDa. Peptide sequence analysis revealed that the 45-kDa fragment lies within the molecule and suggests that the 32-kDa fragment is the amino terminus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Two different adenylyl cyclases in brain distinguished by monoclonal antibodies

Four monoclonal antibodies, raised against the 115-kDa adenylyl cyclase from bovine brain [Pfeuffer, E. et al. (1985) EMBO J. 4, 3675-3679] have been selected and designated BBC-1 to BBC-4. BBC-1 and BBC-3 are highly specific for the 115-kDa enzyme from bovine brain. The two other antibodies, BBC-2 and BBC-4, recognize an additional 150-kDa adenylyl cyclase in bovine brain, but also in brain tissue from other species. In membranes from lung and myocardium (bovine and rabbit) only the 150-kDa species is detected by the crossreacting antibodies BBC-2 and BBC-4. The two adenylyl cyclases from brain can be separated by calmodulin-Sepharose: only the enzyme of 115 kDa but not that of 150 kDa was retained by the affinity resin and could be stimulated by Ca2+/calmodulin. The data obtained with these antibodies of defined specificity provide for the first time direct evidence for the presence of two distinct adenylyl cyclase species in brain tissue.