Immune response of rabbits to native G-actins.

Actins are highly conserved proteins and are therefore claimed to be not very immunogenic without prior denaturation or chemical modification. We have obtained in rabbits high-titered antibodies to "native" G-actins from chicken and man, and assayed their cross-reaction using an enzyme immunoassay, Western blotting and immunohistochemistry. The antigens differ in their ability to induce antibody formation (chicken gizzard actin [(beta), gamma] greater than chicken skeletal actin [alpha] = human platelet actin [beta, (gamma)]). Antibodies to skeletal actin [alpha] are muscle-specific and mainly directed against the homologous region comprising the N-terminus (residues 1-226). Antibodies to gizzard actin [(beta), gamma] cross-react, to a lesser extent, with the alpha and beta, (gamma) isoforms. They show no regional specificity within the homologous antigen. Antibodies to the tryptic core fragment (residues 69-374) of skeletal actin react with fragments comprising the C-terminal part of muscular actins. Antibodies to platelet actin [beta, (gamma)] cross-react with muscular actins, recognizing not the native, but slightly degraded molecules. Platelet actin induces the formation of high-titered albumin antibodies for hitherto unknown reasons.

Purification, biological properties and partial sequence analysis of 67-kDa calcimedin and its 34-kDa fragment from chicken gizzard.

Calcimedin is a group of proteins, originally isolated from chicken gizzard, which are able to bind to several hydrophobic matrices in the presence of Ca2+. Although the molecular properties have been partially discovered, the physiological functions of calcimedins have not yet been clearly defined. In this study, we describe the isolation and characterization of 67-kDa calcimedin and its 34-kDa fragment from chicken gizzard. Both structural and functional studies establish that 67-kDa calcimedin is a member of the calpactin/lipocortin family: it displays phospholipase A2 inhibitory activity, Ca2(+)-dependent F-actin binding and phospholipid binding activity similar to those of calpactins (lipocortins). By comparing the sequence of 67-kDa calcimedin with the predicted sequence of 67-kDa calelectrin, we concluded that the primary structure of these 67-kDa proteins is highly conserved. In particular, the sequences GLGTDEGAIIXVLTQR and EGAGTDESTLIEIMATR conform with the annexin consensus sequence which is characteristic of the calpactin/lipocortin family. A 34-kDa fragment of 67-kDa calcimedin was also purified and their relatedness has been confirmed by antibody cross-reactivity. The sequence data further support that the 34-kDa fragment is derived from the C-terminal portion of 67-kDa calcimedin by limited proteolysis. The 34-kDa fragment, which contains the annexin consensus sequence, preserves the phospholipase A2 inhibitory activity, and binds F-actin and phospholipids.

KN-62, 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazi ne, a specific inhibitor of Ca2+/calmodulin-dependent protein kinase II.

1-[N,O-Bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpipera zine (KN-62), a selective inhibitor of rat brain Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM kinase II) was synthesized and its inhibitory properties in vitro and in vivo were investigated. KN-62 inhibited phosphorylation of exogenous substrate (chicken gizzard myosin 20-kDa light chain) by Ca2+/CaM kinase II with Ki value of 0.9 microM, but no significant effect up to 100 microM on activities of chicken gizzard myosin light chain kinase, rabbit brain protein kinase C, and bovine heart cAMP-dependent protein kinase type II. KN-62 also inhibited the Ca2+/calmodulin-dependent autophosphorylation of both alpha (50 kDa) and beta (60 kDa) subunits of Ca2+/CaM kinase II dose dependently in the presence or absence of exogenous substrate. Kinetic analysis indicated that this inhibitory effect of KN-62 was competitive with respect to calmodulin. However, KN-62 did not inhibit the activity of autophosphorylated Ca2+/CaM kinase II. Moreover, Ca2+/CaM kinase II bound to a KN-62-coupled Sepharose 4B column, but calmodulin did not. These results suggest that KN-62 affects the interaction between calmodulin and Ca2+/CaM kinase II following inhibition of this kinase activity by directly binding to the calmodulin binding site of the enzyme but does not affect the calmodulin-independent activity of already autophosphorylated (activated) enzyme. We examined the effect of KN-62 on cultured PC12 D pheochromocytoma cells. KN-62 suppressed the A23187 (0.5 microM)-induced autophosphorylation of the 53-kDa subunit of Ca2+/CaM kinase in PC12 D cells, which was immunoprecipitated with anti-rat forebrain Ca2+/CaM kinase II polypeptides antibodies coupled to Sepharose 4B, thereby suggesting that KN-62 could inhibit the Ca2+/CaM kinase II activity in vivo.

Structural and functional features of the alpha 3 chain indicate a bridging role for chicken collagen VI in connective tissues.

Type VI collagen is a component of 100 nm long periodic filaments with a widespread distribution around collagen fibers and on the surface of cells. It is an unusual collagen constituted by three distinct chains, one of which (alpha 3) is much larger than the others and is encoded by a 9-kb mRNA. The amino acid sequence of the alpha 3(VI) deduced from the present cDNA clones specifies for a multidomain protein of at least 2648 residues made of a short collagenous sequence (336 residues), flanked at the N-terminus by nine 200 residue long repeating motifs and at the C-terminus by two similar motifs that share extensive identities with the collagen-binding type A repeats of von Willebrand factor. Type VI collagen and alpha 3(VI) fusion proteins bound to insolubilized type I collagen in a specific, time-dependent, and saturable manner. The alpha 3(VI) chain has three Arg-Gly-Asp sequences in the collagenous domain, and cell attachment was stimulated by the triple helix of type VI collagen and by alpha 3(VI) fusion proteins containing Arg-Gly-Asp sequences. This function was specifically inhibited by the Arg-Gly-Asp-Ser synthetic peptide. The type I collagen-binding and the cell-attachment properties of the alpha 3(VI) chain provide direct information for the role of type VI collagen in connective tissues.

Assembly properties of two CNBr fragments of avian desmin that correspond to the headpiece domain and helix 1B.

To study how different domains of the muscle-specific intermediate filament protein, desmin, contribute to its polymerization, two of its CNBr fragments were examined as to their oligomeric structure under assembly conditions. One of these, D88, covers residues 1-88 and represents almost the entire headpiece; the other, D109, covers residues 145-254, and includes the entire Helix 1B and part of linker L12 of the intact molecule. Chemical cross-linking followed by SDS-PAGE, and analytical gel filtration, revealed that in 10 mM Tris-HCl, pH 8.5, conditions that favor tetramerization of intact desmin D88 formed only dimers. D109, on the other hand, formed primarily a dimeric species but low levels of trimeric and tetrameric species were also detectable. These data are consistent with the proposal that, during assembly of intact protein molecules into IF, the headpiece and Helix 1 contribute to dimerization of two polypeptides into a parallel, in-register coiled-coil. However, additional interactions, including headpiece-to-rod binding and hydrophobic interaction along the entire rod domain, are required to stabilize the tetramers and full-size IF.

Kinetic evidence for insertion of actin monomers between the barbed ends of actin filaments and barbed end-bound insertin, a protein purified from smooth muscle.

An actin polymerization-retarding protein was isolated from chicken gizzard smooth muscle. This protein copurified with vinculin on DEAE-cellulose and gel filtration columns. The polymerization-retarding protein could be separated from vinculin by hydroxylapatite chromatography. The isolated polymerization-retarding protein lost its activity within a few days, but was stable for weeks when it was not separated from vinculin. We termed the polymerization-retarding protein "insertin". Because of the instability of the isolated insertin, we investigated the effect of insertin-vinculin on actin polymerization. Insertin-vinculin retarded nucleated actin polymerization maximally fivefold. Polymerization at the pointed ends of gelsolin-capped actin filaments was not affected by insertin-vinculin, suggesting that insertin-vinculin binds to the barbed ends, but not to the pointed ends, of actin filaments. Retarded polymerization was observed even if the actin monomer concentration was between the critical concentrations of the ends of treadmilling actin filaments. As at this low monomer concentration the pointed ends depolymerize, monomers appeared to be inserted at the barbed ends between the terminal subunit and barbed end-bound insertin molecules. Insertin-vinculin was found not to increase the actin monomer concentration to the value of the pointed ends. These observations support the conclusion that insertin is not a barbed end-capping protein but an actin monomer-inserting protein. According to a quantitative analysis of the kinetic data, all observations could be explained by a model in which two insertin molecules were assumed to bind co-operatively to the barbed ends of actin filaments. Actin monomers were found to be inserted between the barbed ends and barbed end-bound insertin molecules at a rate of about 1 x 10(6) M-1 s-1. Insertin may be an essential part of the machinery of molecules that permit treadmilling of actin filaments in living cells by insertion of actin molecules between membranes and actin filaments.

Two isoforms of smooth muscle myosin regulatory light chain in chicken gizzard.

We isolated a cDNA clone for a new isoform of chicken smooth muscle myosin regulatory light chain (MRLC) from a cDNA library of embryonic chicken gizzard. The deduced amino acid sequence was different in 10 amino acid residues from the previously reported polypeptide sequences of chicken gizzard MRLC. The in vitro transcription/translation product from the cDNA comigrated with a minor isoform of chicken gizzard MRLC (L20-B) in a two-dimensional gel electrophoresis. This isoform was detected only in the embryonic gizzard and was slightly more acidic than the predominant isoform (L20-A). The partial polypeptide sequence of L20-A was confirmed to be identical to the previously reported MRLC sequence. Nevertheless, Northern blot analysis showed that L20-B-related mRNAs were present in both the embryonic and adult gizzard. Non-denaturing pyrophosphate polyacrylamide gel electrophoresis showed that the in vitro transcription/translation product could be associated with native myosin when mixed and coprecipitated in a low-ionic-strength buffer with adult chicken gizzard myosin. Moreover, the coprecipitated translation product was phosphorylated in vitro by chicken gizzard myosin light chain kinase apparently more rapidly than L20-A on the native myosin heavy chain. From these findings, we concluded that at least two isoforms of smooth muscle MRLC exist in chicken gizzard and that their expression may be regulated translationally depending on the developmental stage.

Caldesmon has two calmodulin-binding domains.

Chicken gizzard caldesmon was cleaved with chymotrypsin or CNBr, and the calmodulin-binding fragments were isolated using an affinity column. Limited chymotryptic digestion gives rise to a 38 kDa calmodulin-binding fragment (CT40) as described previously (Szpacenko, A. & Dabrowska, R., FEBS Lett. 202, 182-186, 1986; Fujii, T., Imai, M., Rosenfeld, G. C. & Bryan, J., J. Biol. Chem. 261, 16155-16160, 1987; Yazawa, M., Yagi, K. & Sobue, K., J. Biochem. 102, 1065-1073, 1987). In the case of CNBr cleavage a 37 kDa calmodulin-binding fragment (CB40) was obtained. Both CT40 and CB40 contain a reactive thiol group, but these thiols are apparently in different environments as judged by the responses of attached fluorescent labels to calmodulin-binding. A comparison of the N-terminal sequences of CB40 and CT40 with the complete sequence of caldesmon shows that the two calmodulin-binding fragments in fact originate from different parts of the parent molecule. Thus there exist two calmodulin-binding sites in caldesmon, one in the N-terminal half and the other in the C-terminal half of the molecule. This is consistent with the recent finding that up to two calmodulin molecules can be crosslinked to each caldesmon molecule (Wang, C.-L.A., Biochem. Biophys. Res. Commun., 156, 1033-1038, 1988).

Type VI collagen: high yields of a molecule with multiple forms of alpha 3 chain from avian and human tissues.

A differential extraction procedure followed by molecular sieve column chromatography for the isolation of large quantities of the tissue form of type VI collagen is described. Recovery of the protein was more than 60% from both chick gizzard and human placenta. On reduced NaDodSO4-gels chick type VI collagen migrated as two major bands at Mr = 140,000 and 150,000 that were present in a 1:1 ratio and five less intense bands between Mr = 230,000 and 180,000. By immunoblotting with a polyclonal antibody against the pepsinized form of chick type VI collagen, all these bands were stained. Furthermore, the amino acid composition of the five higher Mr polypeptides indicated that they all contained hydroxyproline and hydroxylysine. In the chick type VI collagen molecule the five bands of higher Mr belong to the alpha 3 chain since they were recognized by monoclonal antibodies specific for the chick Mr = 260,000 alpha 3 chain. On examination of antigenic activity by solid-phase radioimmunobinding, densitometry of stained NaDodSO4 polyacrylamide gels, and protein content type VI was found to be an abundant collagen since it accounted for up to 0.1% of the tissue wet weight. The yields per tissue wet weight and the migration pattern of human type VI collagen polypeptides were similar to those of the chick. Agarose/polyacrylamide composite gels indicated that the molecular size of the tissue form of type VI collagen molecules under non-reduced conditions corresponded to a basic type of tetrameric molecule.

Biochemical and theoretical approach to localization of metal-ion-binding sites in the actin primary structure.

The number of Ca2+ ions bound at sites other than the single high-affinity site in CaCl2-induced polymers of rabbit skeletal muscle, chicken gizzard, and bovine aorta actin was determined. The polymer of skeletal muscle and aorta actin contained 4 mol Ca2+/mol, whereas gizzard actin only 3 mol weakly bound Ca2+/mol monomer. This difference correlates with the deletion in smooth muscle gamma-actin of one out of four NH2-terminal acidic residues typical of skeletal and smooth muscle alpha-actin isoforms, suggesting that this additional acidic residue in alpha-actins is involved in the weak binding of cations which is essential for polymerization. This experimental result, as well as a theoretical analysis of the actin primary structure, argue against the implication of the NH2-terminal acidic residues in the high-affinity site for divalent cation. The analysis of the actin primary structure aimed at identification of sequences resembling the known Ca2+-binding patterns has revealed the absence of an EF-hand Ca2+-binding site. The best match was obtained between the sequence of the 292-301 segment and that of Ca2+ site in lectins. However, in the light of experimental data discussed, it is more plausible that the actual high-affinity Ca2+ site in actin involves sequentially distant residues from the NH2- and COOH-terminal portions of the polypeptide chain.

Characteristics of the myosin and tropomyosin binding regions of the smooth muscle caldesmon.

Limited digestion of caldesmon by alpha-chymotrypsin generates mainly 110, 80, 60, 38, and 28 kDa fragments. Affinity chromatography of these fragments on columns immobilized with myosin, HMM, or tropomyosin showed that the bound fraction from these columns was similar and it contained 110, 80, 60 and 28 kDa fragments. These fragments did not bind to myosin filaments, acto-HMM, actin or tropomyosin-actin in the solution, and they had no effect on the actin-activated ATPase of HMM. In contrast, the flow-through fraction from these affinity columns inhibited the actin-activated ATPase. Binding studies revealed that the 38 kDa fragment and its break down products bound to actin and tropomyosin-actin, and they were released partially from actin by calmodulin with a concomitant increase in the ATPase activity. These results indicate that, unlike the actin binding domain, the myosin and tropomyosin binding domains require the caldesmon molecule to be intact in order to exert their effects on the protein-protein interaction.

A Ca2(+)-sensitive glycoprotein, GP90, associated with the cytoskeleton from brain and gizzard.

We describe here the expression during development, tissue distribution and molecular properties of GP90: a major concanavalin A (ConA)-binding glycoprotein present in the neuronal membrane skeleton from chicken brain. GP90 is co-isolated with, and has a similar developmental profile to contactin (previously called GP130). In whole brain, GP90 undergoes rapid synthesis between embryonic days 10 and 12. Unlike contactin, it is not restricted to nervous tissue and is quite abundant in gizzard, where there are two antigenically related proteins of 100K and 90K (K = 10(3) Mr). In both brain and gizzard GP90 and (GP100) are enriched in the membrane skeleton fraction. Trypsinization of live cells suggest that GP90 from gizzard is related to GP100 by the removal of a polypeptide chain. GP90 from both neurones and gizzard cells is protected from proteolysis by the presence of extracellular Ca2+. In the absence of Ca2+ a soluble fragment of approximately 70K can be released from the surface of cells indicating that a large fraction of GP90 is extracellular. Deglycosylation of GP90 from brain using endoglycosidase F demonstrates the presence of at least five carbohydrate chains and a polypeptide chain of approximately 80K. Immunofluorescence studies show that GP90 is exposed on the surface of cultured neurones, gizzard cells and most glial cells with the exception of Schwann cells. It is observed in clusters or patches even when cells are prefixed, suggesting this may be the normal distribution of GP90.

Caldesmon structure and function: sequence analysis of a 35 kilodalton actin- and calmodulin-binding fragment from the C-terminus of the turkey gizzard protein.

We have determined the amino acid sequence of a 35 kDa proteolytic fragment ("CaD35") derived from the C-terminus of turkey gizzard caldesmon. This 239-residue peptide contains binding sites for actin and calmodulin. Residues 1-96 of CaD35 comprise "CaD15", an actin-binding subfragment which we previously showed to resemble the tropomyosin-binding segment of troponin T. The remainder of the CaD35 sequence shows no significant similarity to other proteins. Residues 111-128 may form a basic, amphipathic helix which interacts with calmodulin.

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.

A comparison of two methods to establish the prevalence of lead shot ingestion in mallards (Anas platyrhynchos) from The Netherlands.

Two collection methods for screening the mallard (Anas platyrhynchos) population in the Netherlands for the ingestion of spent lead shot were compared. One method consisted of examination of gizzards from mallards shot by hunters (n = 2,859) and the other method consisted of examination of gizzards from mallards caught in duck traps (n = 865). The 95% confidence interval of lead shot ingestion in the mallard population estimated by the first method was 1.7 to 2.9% and by the second method 1.1 to 3.1%. These values were not significantly different. From the numbers of lead pellets embedded in the gizzard wall in hunter-killed and trapped mallards it was estimated that at least 22 to 68% of the trapped ducks had been hit by lead shot previously, but survived. Furthermore, this study shows that it is reasonable to assume that a substantial part of the pellets which are identified (in this study and other studies) as ingested, may well have been shot into the gizzard lumen at some time before the birds were actually killed. To avoid lead poisoning in mallards and in raptors depredating waterfowl hit by lead shot, a change to steel shot is advocated.

Caldesmon: anomalous electrophoretic behaviour in polyacrylamide gel.

In SDS gels caldesmon (Mr = 140 kDa) and myosin light chain kinase (Mr = 130 kDa) migrate as a closely separated doublet. When glycerol is added to the gel caldesmon is characterized by an anomalous migration. In fact under this latter condition, the distance between caldesmon and myosin light chain kinase is enhanced by two-three times. The nature of putative caldesmon and myosin light chain kinase was confirmed by physicochemical, enzymatic and immunological methods.

Analytical determination of methylated histidine in proteins: actin methylation.

The methylation of histidine in actin from various muscle and nonmuscle sources has been studied by formation of phenylthiocarbamyl derivatives and subsequent reverse-phase high-pressure liquid chromatographic separation and analysis of actin hydrolyzates. All the actin species examined were found to contain 3-methylhistidine. This method has also been used in assays for the enzyme(s) responsible for methylation of rabbit skeletal muscle actin and to investigate the formation of other methylated residues in vitro. 3-Methyl-histidine is the major methylation product in this in vitro reaction.

Amino acid sequence of a 15 kilodalton actin-binding fragment of turkey gizzard caldesmon: similarity with dystrophin, tropomyosin and the tropomyosin-binding region of troponin T.

We have determined the amino acid sequence of a 15 kDa actin-binding fragment of turkey gizzard caldesmon. The 96-residue fragment contains 29 acidic and 29 basic residues, and is predicted to have an extended helical conformation stabilized by numerous internal salt bridges. CaD15 bears some resemblance to dystrophin, tropomyosin and several other proteins, but is most strikingly similar to the tropomyosin-binding segment of troponin T.

A hydrophobic region on myosin light chains modulated by divalent cations.

A hydrophobic region was detected on several types of myosin light chain by enhancement of the quantum yield of 1-anilino-8-naphthalenesulfonate (ANS) fluorescence. The character of this non-polar region was altered by the binding of Ca2+ or Mg2+ to the light chain, the quantum yield of the ANS being increased, and its emission maximum undergoing a blue-shift. These changes enabled the binding of divalent cations to the myosin light chains to be monitored. When Ca2+ was bound to gizzard regulatory light chain, a biphasic enhancement of light-chain-bound ANS fluorescence occurred, the first phase taking place in the micromolar range and the second in the millimolar range of free Ca2+ concentration. Enhancement of protein-bound ANS fluorescence as divalent cations were bound was also observed with other types of myosin light chain.

Fluorescence from pyrene-labeled native and reconstituted chicken gizzard tropomyosins.

Sulfhydryl groups at Cys-36 on the beta chain and at Cys-190 on the gamma chain of chicken gizzard tropomyosin were reacted with the pyrene-containing sulfhydryl-specific reagents N-(1-pyrenyl)iodoacetamide and N-(1-pyrenyl)maleimide. Tropomyosin prepared and labeled under nondenaturing conditions displayed significant pyrene monomer emission but low levels of pyrene excimer fluorescence. In contrast, tropomyosin subjected to denaturation and renaturation prior to labeling, or labeled in the denatured state prior to renaturation, displayed considerable excimer emission. Furthermore, labeling of isolated beta or gamma chains in denaturant, followed by reconstitution, gave separate samples of beta beta- and gamma gamma-tropomyosin that exhibited even greater pyrene excimer to monomer emission ratios. As pyrene excimers can form only when an excited pyrene is immediately adjacent to a ground state pyrene, i.e., when the labeled Cys residues on the two chains in a tropomyosin coiled coil share the same cross section, these results support conclusions based upon chemical crosslinking studies [C. Sanders, L. D. Burtnick, and L. B. Smillie (1986) J. Biol. Chem. 261, 12774-12778] that native gizzard tropomyosin exists predominantly as a beta gamma-heterodimer. In addition, the low degree of labeling of native gizzard tropomyosin and the differences in degrees of labeling of beta beta- and gamma gamma-tropomyosins in the absence of denaturants reflect on the accessibilities of the sulfhydryl groups in these tropomyosin isoforms. Circular dichroism measurements indicate that the labeled proteins form stable coiled coil structures that have thermal stabilities comparable to that of the native protein.