Interaction between chicken gizzard caldesmon and tropomyosin

Chicken gizzard muscle caldesmon has been examined for ability to interact with tropomyosin from chicken gizzard muscle by using fluorescence enhancement of tropomyosin labeled with dansyl chloride (DNS) and affinity chromatography. The binding of caldesmon to tropomyosin was regulated by Ca2+ and calmodulin, i.e., at low ionic strength most of the caldesmon bound to tropomyosin-Sepharose 4B was co-eluted by adding calmodulin only in the presence of Ca2+, but not in its absence. This regulation by Ca2+ and calmodulin was also suggested by fluorescence measurements. Actin- and calmodulin-binding sites on the caldesmon molecule were located in the 38K fragment (Fujii, T., Imai, M., Rosenfeld, G.C., & Bryan, J. (1987) J. Biol. Chem. 262, 2757-2763). When 38K-enriched fraction was applied to the tropomyosin-Sepharose, the 38K fragment was retained by the column and could be eluted by adding Ca2+ and calmodulin.     

Properties of carboxypeptidase A-treated chicken gizzard tropomyosin

Chicken gizzard tropomyosin was digested with carboxypeptidase A at the weight ratios of enzyme to substrate 1:200 and 1:50. Removal of about 16 C-terminal amino acid residues per tropomyosin molecule, at lower enzyme concentration, caused reversion of the effect on skeletal actomyosin ATPase activity from activating to inhibiting without an influence on polymerizability and actin-binding ability. Removal of about 26 C-terminal amino acid residues per molecule, at higher enzyme concentration, resulted in loss of polymerizability and actin binding ability. Digestion of gizzard tropomyosin with carboxypeptidase A has no dramatic effect on its binding to troponin T. The results show that not only the existence of head-to-tail overlapping regions but also their length is important for the functional properties of chicken gizzard tropomyosin.     

Accumulation of tropomyosin in developing chicken gizzard smooth muscle

Smooth muscle of chicken embryonic gizzards has been shown to contain 9 tropomyosin isoforms (E1, E2, E3, E4, E5, E6, E7, E8, and E9) in addition to alpha and beta isoforms (Hosoya et al. (1989) J. Biochem. 105, 712-717). At the early stages of development, the amount of these isoforms was larger than those of alpha and beta isoforms. However, they gradually decreased at later stages and finally disappeared completely after hatching. By using two-dimensional gel electrophoresis and an image analyzing system, we examined the process of tropomyosin accumulation in gizzard smooth muscle development. The accumulation patterns of tropomyosin isoforms and their relative molar ratios to actin in embryonic development were different from those in the stages after hatching. The relative molar ratio of tropomyosin to actin in the thin filament preparation of embryonic gizzards was lower than that of adult, and it gradually increased in the course of embryonic development.     

Thermal unfolding equilibria in homodimeric chicken gizzard tropomyosin coiled coils

CD studies are presented on thermal unfolding of coiled-coil homodimers of two genetic variant chains of chicken gizzard tropomyosin (CG-Tm). The experiments include the effects of cross-linking both isoforms and the dependence on protein concentration of unfolding in both reduced isoforms, variables not examined in extant work. The general shapes of the unfolding curves for singly cross-linked species depend on whether the crosslink is at C190 (its site on one isoform) or at C36 (its site on the other). These curves are compared with extant ones for various cross-linked species of rabbit tropomyosin. The comparison supports the view that the unfolding behavior of cross-linked species results from a complex interaction of strain at the cross-link, local variations in structural stability, and loop entropy. The observed concentration dependence of the transition temperature for the uncross-linked (reduced) species of CG-Tm is very small (2.9°C) for one variant homodimer and unobservably small (< 2°C) for the other in the 100-fold concentration range (～ 0.01–1.0 mg/mL) accessible here. These experimental values of ΔT_m are much smaller than are predicted from extant values of the van't Hoff transition enthalpies, calling the latter into question. © 1994 John Wiley & Sons, Inc.     

Cooperativity of actin-activated ATPase of gizzard heavy meromyosin in the presence of gizzard tropomyosin

The mechanism for the potentiation of the actin-activated ATPase of smooth muscle myosin by tropomyosin is investigated using smooth muscle actin, tropomyosin, and heavy meromyosin. In the presence of tropomyosin, an increase in Vmax occurs with no effect on KATPase and Kbinding at 20 mM ionic strength. Utilizing N-ethylmaleimide-treated subfragment-1, which forms rigor complexes with actin in the presence of ATP but does not have ATPase activity, experiments were carried out to determine if the tropomyosin-actin complex exists in both the turned-off and turned-on forms as in the skeletal muscle system. At both 60 and 100 mM ionic strengths, the presence of rigor complexes on the smooth muscle actin filament containing bound tropomyosin causes a 2-3-fold increase in Vmax and about a 3-fold increase in KATPase, resulting in about a 4-fold increase in ATPase activity at moderate actin concentration. The increase in KATPase is correlated with an increase in Kbinding. The finding that rigor complexes increase Vmax and the binding constant for heavy meromyosin to tropomyosin-actin at an ionic strength close to physiological conditions indicates that the tropomyosin-actin complex can be turned on by rigor complexes in a cooperative manner. However, in contrast to the situation in the skeletal muscle system, the increase in KATPase is associated with a corresponding increase in Kbinding. Furthermore, there is only a 3-fold increase in KATPase in the smooth muscle system rather than a 10-fold increase as in the skeletal muscle system.     

Domain mapping of chicken gizzard caldesmon

Limited proteolysis, affinity chromatography, and immunoblotting have been used to define the domains of chicken gizzard caldesmon, caldesmon120, that interact with calmodulin, F-actin, and a monoclonal antibody prepared using human platelet caldesmon. Treatment of caldesmon120 with chymotrypsin produces groups of fragments near 100, 80, 60, 38, and 20 kDa. Further digestion produces peptides between 40 and 50 kDa. The 100- and 80-kDa peptides cross-react with the monoclonal antibody; the smaller polypeptides do not. The kinetics of cleavage and the antibody studies indicate that the 38- and 80-kDa fragments are the two major pieces of the 120-kDa protein. The 38-kDa fragment, purified by high performance liquid chromatography, and several of its subfragments at 21 and 25 kDa sediment with F-actin, bind to calmodulin-Sepharose in the presence of Ca2+, and are displaced from F-actin by Ca2+-calmodulin. The 80-kDa fragments did not interact with F-actin or calmodulin. We have tentatively placed the 38-kDa fragment at the C-terminal using polyclonal antibodies selected against a beta-galactosidase-caldesmon120 fusion protein produced by a lambda gt11 lysogen. The 38-, 25-, and 21-kDa fragments cross-react with these antibodies; the 80- and 60-kDa fragments do not. Caldesmon77 from human platelets also cross-reacts with these selected antibodies. The results suggest that interacting calmodulin and F-actin binding sites are localized on a 38-kDa C-terminal fragment of caldesmon. The smallest subfragment of this peptide that binds to both F-actin and calmodulin-Sepharose is about 21 kDa. The monoclonal antibody epitope is tentatively localized near the N-terminal of caldesmon77 and must be within 50 kDa of the N-terminal on caldesmon120.     

Rapid purification of chicken gizzard alpha-actinin

A method of purification has been developed which yields highly purified α-actinin and requires less than one day to complete. The α-actinin is extracted from washed chicken gizzard muscle with water at 37°. Actin and a 55,000 dalton protein are quantitatively precipitated from the extract with 20 mM MgCl₂. The α-actinin is subsequently precipitated from the extract by 30% (NH₄)₂SO₄ and fractionated on DEAE cellulose. Spontaneous protein aggregation is prevented by adding 10% glycerol.     

5'-nucleotidase from bull seminal plasma, chicken gizzard and snake venom is a zinc metalloprotein

Using flame atomic absorption spectrometry the tight association of zinc to three different purified 5'-nucleotidases at a molar ratio of 2 could be proven. These 5'-nucleotidases purified from bull seminal plasma (BSP), chicken gizzard (CG) and snake venom (SV) are thus zinc metalloproteins. Removal of zinc results in the loss of their AMPase activity, which could be fully restored after readdition of zinc at a molar ratio of 2, for BSP and CG, and 1.5, for SV 5'-nucleotidase. Reactivation of their AMPase activity after the removal of zinc could also be obtained by addition of cobalt and copper ions, which were found to also bind with a molar ratio of 2 to the three 5'-nucleotidases tested.     

Glycosaminoglycans from chicken muscular stomach or gizzard

Glycosaminoglycans (GAGs) were prepared from the muscular stomach or gizzard of the chicken. The content of GAGs on a dry weight basis contains 0.4 wt.% a typical value observed for a muscle tissue. The major GAG components were chondroitin-6-sulfate and chondroitin-4-sulfate (~64 %) of molecular weight 21–22 kDa. Hyaluronan (~24 %) had a molecular weight 120 kDa. Smaller amounts (12 %) of heparan sulfate was also present which was made of more highly sulfated chains of molecular weight of 21-22 kDa and a less sulfated low molecular weight (< 10 kDa) heterogeneous partially degraded heparan sulfate. Chicken gizzard represents an inexpensive and readily available source of muscle tissue-derived GAGs.     

Amino acid sequence of chicken gizzard gamma-tropomyosin

Chicken gizzard muscle tropomyosin has been fractionated into its two major components, beta and gamma and the amino acid sequence of the gamma component established by the isolation and sequence analysis of fragments derived from cyanogen bromide cleavage and tryptic digestions. Despite its much slower mobility on sodium dodecyl sulfate-polyacrylamide electrophoretic gels, it has the same polypeptide chain length (284 residues) as the alpha and beta components of rabbit skeletal muscle. Evidence for microheterogeneity of the chicken gizzard component was detected both on electrophoretic gels and in the sequence analysis. The gamma component is more closely related to rabbit skeletal alpha-tropomyosin than to the beta component. While the protein is highly homologous to the rabbit skeletal tropomyosins, significant sequence differences are observed in two regions; between residues 42-83 and 258-284. In the latter region (COOH-terminal) the alterations in sequence are very similar to those seen in platelet tropomyosin when compared with the skeletal proteins.     

Reaction of phenylglyoxal with chicken gizzard myosin

Modification of chicken gizzard myosin with phenyl[2-14C]-glyoxal inhibited the K+-ATPase (ATP phosphohydrolase, EC 3.6.1.32) activity as a function of time. During the 2.5 and 15 min interval 3.2 mol of the reagent were incorporated per 4.7 X 10(5) g protein and the K+-ATPase activity was 50% inhibited. Phenylglyoxal reacted with arginine residues of gizzard myosin in a mol ratio of two to one, phenylglyoxal to arginine as determined spectrophotometrically. The modification was limited to the subfragment 1 heavy chain and rod-like regions and none of the light chains were lost. The inhibition of the ATPase activity occurred when the subfragment 1 region was modified predominantly. The same results were obtained when the myosin was phosphorylated and then incubated with phenylglyoxal. Substrate MgATP2- or MgADP enhanced the inactivation of gizzard myosin; there was an increase in the incorporation of the reagent and a change in the distribution into the heavy chains. Approx. 0.5 mol of the nucleotide was bound to 4.7 X 10(5) g of phenylglyoxal myosin. Conformational changes, induced by these modifications, were responsible for the inhibition of enzymic activity. Arginine residues of gizzard myosin are necessary for the maintenance of the ATPase activity of this contractile protein.     

Properties of caldesmon isolated from chicken gizzard.

Chicken gizzard smooth muscle contains two major calmodulin-binding proteins: caldesmon (11.1 microM; Mr 141 000) and myosin light-chain kinase (4.6 microM; Mr 136 000), both of which are associated with the contractile apparatus. The amino acid composition of caldesmon is distinct from that of myosin light-chain kinase and is characterized by a very high glutamic acid content (25.5%), high contents of lysine (13.6%) and arginine (10.3%), and a low aromatic amino acid content (2.4%). Caldesmon lacked myosin light-chain kinase and phosphatase activities and did not compete with either myosin light-chain kinase or cyclic nucleotide phosphodiesterase (both calmodulin-dependent enzymes) for available calmodulin, suggesting that calmodulin may have distinct binding sites for caldesmon on the one hand and myosin light-chain kinase and cyclic nucleotide phosphodiesterase on the other. Consistent with the lack of effect of caldesmon on myosin phosphorylation, caldesmon did not affect the assembly or disassembly of myosin filaments in vitro. As previously shown [Ngai & Walsh (1984) J. Biol. Chem. 259, 13656-13659], caldesmon can be reversibly phosphorylated. The phosphorylation and dephosphorylation of caldesmon were further characterized and the Ca2+/calmodulin-dependent caldesmon kinase was purified; kinase activity correlated with a protein of subunit Mr 93 000. Caldesmon was not a substrate of myosin light-chain kinase or phosphorylase kinase, both calmodulin-activated protein kinases.     

Differentiation of smooth muscle cells in chicken gizzard

During development of the chicken gizzard, a thick layer of undifferentiated cells (mesenchymal cells) is constructed, and the cells differentiate into smooth muscle cells or connective tissues. We found that the differentiation of smooth muscle cells occurred first near the outer surface of the gizzard and the differentiated area spread to the inside of the gizzard. Therefore, we assumed that the differentiation of most of the smooth muscle cells in the gizzard is induced by differentiated smooth muscle itself. When undifferentiated cells from gizzard of 7-day-old embryo (Hamburger and Hamilton's stages 26-27) were cultured on a coverglass coated with extract of gizzard that contained differentiated smooth muscle cells, the cells attached to the coverglass and differentiated into smooth muscle cells. On the other hand, extract of gizzard from 7-day-old embryo did not induce the differentiation of smooth muscle cells, though it induced the attachment of cells. We found that activity for the differentiation of smooth muscle cells appeared when differentiated smooth muscle cells appeared in developing gizzard. Gizzard contained higher activity for the differentiation of smooth muscle cells than the other tissues. Transforming growth factor-beta (TGF-beta), which induces the differentiation of vascular smooth muscle cells, did not induce the differentiation of smooth muscle cells in gizzard, though extract of aorta induced the differentiation of smooth muscle cells in gizzard. The results obtained here support evidence that the differentiation of most of the smooth muscle cells in gizzard is induced by a self-catalytic mechanism in which differentiated smooth muscle itself induces the differentiation of smooth muscle cells.     

Properties of talin from chicken gizzard smooth muscle

This paper describes the structural and biochemical characterization of talin, a protein localized to various cellular sites where bundles of actin filaments attach to the plasma membrane. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein has a molecular mass of 225,000 +/- 5,000 daltons. Hydrodynamic measurements at protein concentrations less than 0.72 mg/ml indicate a monomeric protein with a native molecular mass of 213,000 +/- 15,000 daltons. Sedimentation equilibrium experiments indicate self-association at protein concentrations of 0.72 mg/ml and higher. The data suggest that this self-association is a simple monomer:dimer equilibrium over the range of concentrations observed. At low protein concentrations where talin is a monomer, the Stokes radius and sedimentation coefficient vary with ionic strength. Under low ionic strength conditions (5-20 mM NaCl), talin has a Stokes radius of 6.5 nm and a sedimentation value of 9.4, suggesting an asymmetric globular molecule; whereas under high ionic strength conditions (200 mM NaCl), the Stokes radius increases to 7.7 nm and the sedimentation coefficient decreases to 8.8, suggesting a more elongated protein. This conformation change is confirmed by electron microscopy which reveals a more globular protein at low ionic strength which unfolds to become an elongated flexible molecule as the ionic strength is increased to physiological and higher levels. The amino acid composition of talin indicates a low level of aromatic residues, consistent with its relatively low extinction coefficient, talin has an isoelectric point between pH 6.7 and 6.8 based on isoelectric focusing. The detailed purification of talin is described.     

Light chains of chicken embryonic gizzard myosin.

1. Myosin from gizzards of 15-day-old chicken embryos was highly purified by ammonium sulfate fractionation in the presence of ATP and MgCl2, ultra-centrifugation and Sepharose 4B chromatography. 2. The myosin composed of heavy and three light chains as determined by sodium dodecyl sulfate (SDS) gel electrophoresis. The molecular weights of the light chains were 23,000 (L23), 20,000 (L20), and 17,000 (L17), respectively. The amount of L23 light chain decreased and disappeared, and the L17 light chain increased steadily in the course of development. The amount of L20 light chain did not change. 3. ATPase activity of the embryonic myosin was essentially the same as that of adult myosin. The change in the light chain pattern in the course of development did not correlate to the ATPase activity. 4. Antigenicity of the heavy chains in the embryonic myosin was the same as that of the adult heavy chains. However, antibodies to light chains were not detected in the antibodies to either the embryonic or adult myosins.     

Tropomyosin isoforms in developing chicken gizzard smooth muscle

In the embryonic smooth muscle of chicken gizzards we found 4 high-Mr-type and 5 low-Mr-type tropomyosin isoforms in addition to alpha- and beta-isoforms reported already. The criteria by which they were identified as tropomyosin isoforms were as follows: 1) anomalous reduction of electrophoretic mobility in the presence of urea, 2) cross reactivity with antisera against tropomyosins, 3) inclusion in a tropomyosin preparation obtained by the usual method for tropomyosin purification, and 4) binding ability to skeletal muscle actin. At the early stages of development, the amounts of these isoforms were larger than those of alpha- and beta-isoforms, but they gradually decreased at later stages and finally disappeared completely after hatching. Our previous study of gizzard smooth muscle showed that the amount ratio of accumulated tropomyosin to gamma-actin was reasonably constant in the development after hatching, while, at the earlier embryonic stages (7-14 d of incubation), it was lower than expected. The isoforms found in this study were present in amounts large enough to bring the ratio at the earlier stages up to the constant amount ratio observed after hatching. Therefore, the coordinate accumulation of actin and tropomyosin was suggested to occur even at the embryonic stages.     

Limited proteolysis of chicken gizzard 5'-nucleotidase.

Chicken gizzard 5'-nucleotidase represents an ectoenzyme which is linked to the plasma membrane via a phosphatidylinositol glycan. We have characterized the possible domain-like organization of 5'-nucleotidase by limited proteolysis. A hydrophobic proteolytic fragment carrying the intact C-terminus, as well as two major hydrophilic products, were identified. We developed procedures for specific radiolabelling of the active center of 5'-nucleotidase. This allowed us to locate the catalytic site within hydrophilic fragments obtained after limited proteolysis. We demonstrate that removal of N-linked carbohydrate chains increases the sensitivity of 5'-nucleotidase to proteolytic attack, indicating that sugar moieties protect against proteolysis. 5'-Nucleotidase represents a binding protein for components of the extracellular matrix. The interaction between 5'-nucleotidase and the laminin/nidogen complex unmasked proteolytic cleavage sites in the C-terminal portion of the enzyme. This resulted in the specific production of a hydrophilic form of 5'-nucleotidase. In summary, we have further characterized chicken gizzard 5'-nucleotidase: (1) the protein is organized into two domain-like structures, (2) the N-terminal domain harbors the active center; (3) N-linked carbohydrates protect the protein against proteolytic degradation; (4) interaction with components of the extracellular matrix alters the conformation of 5'-nucleotidase.     

Troponin C-like protein in chicken gizzard muscle.

1. A troponin C-like protein was prepared from frozen chicken gizzard by preparative polyacrylamide gel electrophoresis and its apparent molecular weight was estimated to be about 15,500 daltons. 2. In urea gel electrophoresis, the mobility of the troponin C-like protein increased slightly in the presence of Ca2+, like that of skeletal muscle troponin C. On the other hand, the mobility of the the troponin C-like protein in glycerol gel electrophoresis, unlike that of skeletal muscle troponin C, was significantly decreased by Ca2+. 3. In alkaline gel electrophoresis, the troponin C-like protein formed a Ca2+-dependent complex with troponin I or troponin T from skeletal muscle. 4. The troponin C-like protein could neutralize the inhibitory effect of skeletal muscle troponin I on the Mg2+-activated ATPase of actomyosin from rabbit skeletal muscle, but could not confer Ca2+-sensitivity on the actomyosin in the presence of troponin I and troponin T from skeletal muscle.     

Structure and function of chicken gizzard myosin.

In our previous study (Onishi, H., Susuki, H., Nakamura, k., and Watanabe, S. J. Biochem. 83, 835-847, 1978), we found it to be characteristic of chicken gizzard myosin that thick filaments of gizzard myosin are readily disassembled by a stoichiometric amount of ATP (3 mol of ATP per mol of myosin), and that the ATPase activity of gizzard myosin in the ATP-disassembled state is much lower than that of gizzard myosin disassembled by a high concentration of KCl. We now report the following findings: (1) Thick filaments of (unphosphorylated) gizzard myosin can be in a bipolar structure or in a non-polar structure, depending on the method of preparing the thick filaments. (2) Thick filaments of (unphosphorylated) gizzard myosin in either the bioplar or the non-polar structure are readily disassembled by ATP. (3) Addition of rabbit skeletal C-protein does not confer ATP resistance on thick filaments of (unphosphorylated) gizzard myosin. (4) Unphosphorylated) gizzard myosin in the ATP-disassembled state is in a dimeric form as determined by ultracentrifugation. Moreover, 0.2 M KCl-dissociated gizzard myosin in monomeric form is converted to a dimeric form by ATP. (5) The Mg-ATPase activity of (unphosphorylated) gizzard myosin is much lower in its dimeric form (less than one-tenth) than in its monomeric form. The activity depression observed around 0.15 M KCl is therefore due to the formation of myosin dimers. (6) Skeletal L-meromyosin can increase the very low activity of (unphosphorylated) gizzard myosin ATPase at low ionic strength (0.13 M KCl) by forming ATP-resistant hybrid filaments with (unphosphorylated) gizzard myosin, preventing the formation of myosin dimers. (7) Gizzard myosin in which one of the light-chain components is phosphorylated by myosin light-chain kinase can form thick filaments which are resistant to the disassembling action of ATP. (8) Even in the presence of ATP, thick filaments of phosphorylated gizzard myosin do not disassembled into myosin dimers. Accordingly, the ATPase activity of phosphorylated gizzard myosin does not show activity depression at low ionic strength.     

Amino acid sequence of chicken gizzard beta-tropomyosin. Comparison of the chicken gizzard, rabbit skeletal, and equine platelet tropomyosins

Chicken gizzard beta-tropomyosin has the same chain length (284 residues) as other muscle tropomyosins, and is most closely related to the beta component of rabbit skeletal muscle. The majority of the amino acid substitutions are restricted to two regions of the structure, residues 185-216 and 258-284. The altered sequences at the COOH-terminal ends (residue 258-284) of the two gizzard components are very similar to each other and to those in platelet tropomyosin and can be correlated with the reduced affinity of interaction of all three tropomyosins with skeletal troponin T and its T1 fragment. The virtually identical NH2-terminal sequences of all four muscle tropomyosin chains indicates that the gizzard proteins' greater ability to polymerize head-to-tail is due to the sequence changes at its COOH terminus. On the other hand, the weaker head-to-tail aggregation of the platelet protein must be due to its NH2-terminal sequence alterations. Examination of the distribution of amino acids and the frequency of their substitution in the a to g positions of the repeating pseudoheptapeptide for all five tropomyosin sequences (four muscle and one platelet) emphasizes the importance of Glu residues at position e. Examination of those features of the muscle sequences implicated in the stabilization of their coiled-coil structures and in their interactions with F-actin suggest only marginal differences among them, with the possible exception of the chicken gizzard gamma component.