Localization of the charge differences in the actins of rabbit skeletal muscle and chicken gizzard by two-dimensional gel electrophoretic analysis of tryptic fragments.

Partial tryptic cleavage products of pure actin from rabbit skeletal muscle and chicken gizzard are compared by two-dimensional electrophoresis in polyacrylamide gels with respect to isoelectric point and molecular weight. While the intact polypeptides (Mr 42,000) have different isoelectric points, two large cleavage products (Mr 35,000) generated from both both actin species have identical isoelectric points and identical molecular weights. These relatively trypsin-resistant cleavage products are presumably identical to the known "core actin" fragments which lack the aminoterminal region of the polypeptide chain. Therefore the differences that are responsible for the different isoelectric points of rabbit skeletal muscle actin and chicken gizzard actin seem to be restricted to the aminoterminal part of the actin polypeptide chains as was proposed on the basis of partial amino acid sequence data.     

Characterization of chicken skeletal muscle myosin light chain kinase. Evidence for muscle-specific isozymes

Myosin light chain kinase purified from chicken white skeletal muscle (Mr = 150,000) was significantly larger than both rabbit skeletal (Mr = 87,000) and chicken gizzard smooth (Mr = 130,000) muscle myosin light chain kinases, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Km and Vmax values with rabbit or chicken skeletal, bovine cardiac, and chicken gizzard smooth muscle myosin P-light chains were very similar for the chicken and rabbit skeletal muscle myosin light chain kinases. In contrast, comparable Km and Vmax data for the chicken gizzard smooth muscle myosin light chain kinase showed that this enzyme was catalytically very different from the two skeletal muscle kinases. Affinity-purified antibodies to rabbit skeletal muscle myosin light chain kinase cross-reacted with chicken skeletal muscle myosin light chain kinase, but the titer of cross-reacting antibodies was approximately 20-fold less than the anti-rabbit skeletal muscle myosin light chain kinase titer. There was no detectable antibody cross-reactivity against chicken gizzard myosin light chain kinase. Proteolytic digestion followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or high performance liquid chromatography showed that these enzymes are structurally very different with few, if any, overlapping peptides. These data suggest that, although chicken skeletal muscle myosin light chain kinase is catalytically very similar to rabbit skeletal muscle myosin light chain kinase, the two enzymes have different primary sequences. The two skeletal muscle myosin light chain kinases appear to be more similar to each other than either is to chicken gizzard smooth muscle myosin light chain kinase.     

The Complete Amino Acid Sequence of Actins from Bovine Aorta, Bovine Heart, Bovine Fast Skeletal Muscle, and Rabbit, Slow Skeletal Muscle

Complete amino acid sequences for four mammalian muscle actins are reported: bovine skeletal muscle actin, bovine cardiac actin, the major component of bovine aorta actin, and rabbit slow skeletal muscle actin. The number of different actins in a higher mammal for which full amino acid sequences are now available is therefore increased from two to five. Screening of different smooth muscle tissues revealed in addition to the aorta type actin a second smooth muscle actin, which appears very similar if not identical to chicken gizzard actin. Since the sequence of chicken gizzard actin is known, six different actins are presently characterized in a higher mammal.
The two smooth muscle actins—bovine aorta actin and chicken gizzard actin—differ by only three amino acid substitutions, all located in the amino-terminal end. In the rest of their sequences both smooth muscle actins share the same four amino acid substitutions, which distinguish them from skeletal muscle actin. Cardiac muscle actin differs from skeletal muscle actin by only four amino acid exchanges. No amino acid substitutions were found when actins from rabbit fast and slow skeletal muscle were compared.
In addition we summarize the amino acid substitution patterns of the six different mammalian actins and discuss their tissue specificity. The results show a very close relationship between the four muscle actins in comparison to the nonmuscle actins. The amino substitution patterns indicate that skeletal muscle actin is the highest differentiated actin form, whereas smooth muscle actins show a noticeably closer relation to nonmuscle actins. By these criteria cardiac muscle actin lies between skeletal muscle actin and smooth muscle actins.     

The complete amino acid sequence of actins from bovine aorta, bovine heart, bovine fast skeletal muscle, and rabbit slow skeletal muscle. A protein-chemical analysis of muscle actin differentiation.

Complete amino acid sequences for four mammalian muscle actins are reported: bovine skeletal muscle actin, bovine cardiac actin, the major component of bovine aorta actin, and rabbit slow skeletal muscle actin. The number of different actins in a higher mammal for which full amino acid sequences are now available is therefore increased from two to five. Screening of different smooth muscle tissues revealed in addition to the aorta type actin a second smooth muscle actin, which appears very similar if not identical to chicken gizzard actin. Since the sequence of chicken gizzard actin is known, six different actins are presently characterized in a higher mammal. The two smooth muscle actins--bovine aorta actin and chicken gizzard actin--differ by only three amino acid substitutions, all located in the amino-terminal end. In the rest of their sequences both smooth muscle actins share the same four amino acid substitutions, which distinguish them from skeletal muscle actin. Cardiac muscle actin differs from skeletal muscle actin by only four amino acid exchanges. No amino acid substitutions were found when actins from rabbit fast and slow skeletal muscle were compared. In addition we summarize the amino acid substitution patterns of the six different mammalian actins and discuss their tissue specificity. The results show a very close relationship between the four muscle actins in comparison to the nonmuscle actins. The amino substitution patterns indicate that skeletal muscle actin is the highest differentiated actin form, whereas smooth muscle actins show a noticeably cloer relation to nonmuscle actins. By these criteria cardiac muscle actin lies between skeletal muscle actin and smooth muscle actins.     

玉米花粉肌动蛋白与兔骨骼肌肌动蛋白的比较研究

本文对玉米花粉肌动蛋白和兔骨骼肌肌动蛋白进行了比较研究。玉米花粉肌动蛋白与兔骨骼肌肌动蛋白具有相同的分子量(42KD)。玉米花粉肌动蛋白可与兔抗鸡胃肌动蛋白抗血清产生免疫沉淀反应。玉米花粉肌动蛋白与兔骨骼肌肌动蛋白的氨基酸组成以及胰蛋白酶水解所得到的肽谱都相似。它们的羧基未端氨基酸顺序完全一致,其顺序都是Lys.Cys.Phe(COOH)。它们的圆二色谱基本相同,由圆二色谱计算得到的二级结构数据也相近。以上的结果表明了玉米花粉肌动蛋白与兔骨骼肌肌动蛋白的相似性。     

Identification of a high molecular weight actin-binding protein in skeletal muscle.

A large polypeptide having a molecular weight of 240,000 as determined by electrophoresis in the presence of sodium dodecyl sulfate has been identified in whole cell homogenates from chick skeletal muscle myoblasts and the rat myoblast L6 cell line. A similar polypeptide was identified in both thigh and breast chicken skeletal muscle, but the latter contained less of this protein per g of tissue. Antibodies made to gizzard filamin (an actin-binding protein having a molecular weight of 240,000) cross-reacted with the partially purified Mr = 240,000 protein from chicken skeletal muscle. With use of the indirect immunofluorescence technique, the filamin antibody localized in the Z-line region of chicken skeletal muscle myofibrils. These results indicate that skeletal muscle contains a filamin-like protein that may form an integral part of the myofibril structure.     

An antiactin antibody that distinguishes between cytoplasmic and skeletal muscle actins

We elicited antibodies in rabbits to actin purified from body wall muscle of the marine mollusc, Aplysia californica. We found that this antiactin has an unusual specificity: in addition to reacting with the immunogen, it recognizes cytoplasmic vertebrate actins but not myofibrillar actin. Radioimmunoassay showed little or no cross-reaction with actin purified from either chicken gizzard or rabbit skeletal muscle. Immunocytochemical studies with human fibroblasts and L6 myoblasts revealed intense staining of typical cytoplasmic cables. Myofibrils were not stained after treatment of human and frog skeletal muscle with the antibody, although the distribution of immunofluorescence suggested that cytoplasmic actin is associated with membrane systems in the muscle fiber. The antibody may therefore be especially suited for studying the localization of cytoplasmic actin in skeletal muscle cells even in the presence of a great excess of the myofibrillar form.     

Comparison of the effects of smooth and skeletal muscle actins on smooth muscle actomyosin Mg2+-ATPase

Actin has been purified from smooth muscle (chicken gizzard) by two different procedures and its activation of smooth muscle myosin Mg2+-ATPase activity compared with that achieved with rabbit skeletal muscle actin. The procedure of Pardee and Spudich (Methods Enzymol. (1982) 85, 164-181) for the purification of rabbit skeletal muscle actin is readily applicable to the isolation of chicken gizzard actin, enabling large quantities to be purified in two days. Smooth muscle actin could be successfully stored as F-actin at -80 degrees C and survived freezing and thawing at least twice. Smooth muscle actin activated myosin Mg2+-ATPase to a higher level than its skeletal muscle counterpart (77.9 nmol Pi/min/mg myosin vs 48.1 nmol Pi/min/mg myosin).     

Identification of amino acid substitutions differentiating actin isoforms in their interaction with myosin

Various aspects of actin--myosin interaction were studied with actin preparations from two types of smooth muscle: bovine aorta and chicken gizzard, and from two types of sarcomeric muscle: bovine cardiac and rabbit skeletal. All four preparations activated the Mg2+-ATPase activity of skeletal muscle myosin to the same Vmax, but the Kapp for the smooth muscle preparations was higher. At low KCl, pH 8.0 and millimolar substrate concentrations the Kapp values differed by a factor of 2.5. This differential behaviour of the four actin preparations correlates with amino acid substitutions at positions 17 and 89 of actin polypeptide chain, differentiating the smooth-muscle-specific gamma and alpha isomers from cardiac and skeletal-muscle-specific alpha isomers. This correlation provides evidence for involvement of the NH2-terminal portion of the actin polypeptide chain in the interaction with myosin. The differences in the activation of myosin ATPase by various actins were sensitive to changes in the substrate and KCl concentration and pH of the assay medium. Addition of myosin subfragment-1 or heavy meromyosin in the absence of nucleotide produced similar changes in the fluorescence of a fluorescent reagent N-(1-pyrenyl)-iodoacetamide, attached at Cys-374, or 1,N6-ethenoadenosine 5'-diphosphate substituted for the bound ADP in actin protomers in gizzard and skeletal muscle F-actin. The results are consistent with an influence of the amino acid substitutions on ionic interactions leading to complex formation between actin and myosin intermediates in the ATPase cycle but not on the associated states.     

Ca2+ dependence of the ATPase activity of phosphorylated smooth muscle myosin: effects of tropomyosin and actin

Calcium ions produce a 3-4-fold stimulation of the actin-activated ATPase activities of phosphorylated myosin from bovine pulmonary artery or chicken gizzard at 37 degrees C and at physiological ionic strengths, 0.12-0.16 M. Actins from either chicken gizzard or rabbit skeletal muscle stimulate the activity of phosphorylated myosin in a Ca2+-dependent manner, indicating that the Ca2+ sensitivity involves myosin or a protein associated with it. Partial loss of Ca2+ sensitivity upon treatment of phosphorylated gizzard myosin with low concentrations of chymotrypsin and the lack of any change on similar treatment of actin supports the above conclusion. Although both actins enhance ATPase activity, activation by gizzard actin exhibits Ca2+ dependence at higher temperatures or lower ionic strengths than does activation by skeletal muscle actin. The Ca2+ dependence of the activity of phosphorylated heavy meromyosin is about half that of myosin and is affected differently by temperature, ionic strength and Mg2+, being independent of temperature and optimal at lower concentrations of NaCl. Raising the concentration of Mg2+ above 2-3 mM inhibits the activity of heavy meromyosin but stimulates that of myosin, indicating that Mg2+ and Ca2+ activate myosin at different binding sites.     

Chicken-gizzard actin. Interaction with skeletal-muscle myosin

Interaction of actin from chicken gizzard and from rabbit skeletal muscle with rabbit skeletal muscle myosin was compared by measuring the rate of superprecipitation, the activation of the Mg-ATPase and inhibition of K-ATPase activity of myosin and heavy meromyosin, and determination of binding of heavy meromyosin in the absence of ATP. Both the rate of superprecipitation of the hybrid actomyosin and the activation of myosin ATPase by gizzard actin are lower than those obtained with skeletal muscle actin. The activation of myosin Mg-ATPase by the two actin species also shows different dependence on substrate concentration: with gizzard actin the substrate inhibition starts at lower ATP concentration. The double-reciprocal plots of the Mg-ATPase activity of heavy meromyosin versus actin concentration yield the same value of the extrapolated ATPase activity at infinite actin concentration (V) for the two actins and nearly double the actin concentration needed to produce half-maximal activation (Kapp) in the case of gizzard actin. A corresponding difference in the abilities of the two actin species to inhibit the K-ATPase activity of heavy meromyosin in the absence of divalent cations was also observed. The results are discussed in terms of the effect of substitutions in the amino acid sequence of gizzard and skeletal muscle actins on their interaction with myosin.     

Potentiation of actomyosin ATPase activity by filamin

It was found that thin filaments from chicken gizzard muscle activate skeletal muscle myosin Mg2+-ATPase to a greater extent than does the complex of chicken gizzard actin and tropomyosin. The protein factor responsible for this additional activation has been now identified as the high Mr actin binding protein, filamin.     

Effects of various amino acid replacements on the conformational stability of G-actin

Circular dichroic spectra of native, EDTA-treated and heat-denatured G-actin from chicken gizzard smooth muscle are virtually the same as those of rabbit skeletal muscle actin. The rates of changes produced by EDTA or heat in the secondary structure are, however, higher in the case of gizzard actin. Similar differences were found in the rates of inactivation as measured by loss of polymerizability during incubation with EDTA or Dowex 50. The results are explicable in terms of local differences in the conformation at specific site(s) important for maintaining the native state of actin monomer. Involvement of the ATP binding site was shown by measuring the equilibrium constant for the binding of ATP to the two actins. Difference in the conformation of some additional site(s) is indicated by a higher rate constant of inactivation of nucleotide-free actin observed for gizzard actin. No significant difference was found in the equilibrium constant for the binding of Ca2+ at the single high-affinity site in gizzard and skeletal muscle actin. Comparison of inactivation kinetics of actin from chicken gizzard, rabbit skeletal, bovine aorta, and bovine cardiac muscle suggests that the amino acid replacements Val-17----Cys-17 and/or Thr-89----Ser-89 have a destabilizing effect on the native conformation of G-actin. The results indicate that deletion of the acidic residue at position 1 of the amino acid sequence has no effect on the conformation of the ATP binding site and the high-affinity site for divalent cation as well.     

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.     

Molecular properties and functions in vitro of chicken smooth-muscle alpha-actinin in comparison with those of striated-muscle alpha-actinins

alpha-Actinin purified from chicken gizzard smooth muscle was characterized in comparison with alpha-actinins from chicken striated muscles, or fast-skeletal muscle, slow-skeletal muscle, and cardiac muscle. The gizzard alpha-actinin molecule consisted of two apparently identical subunits with a molecular weight of 100,000 on SDS-polyacrylamide gel electrophoresis, as do striated-muscle alpha-actinins. Its isoelectric points in the presence of urea were similar to the striated-muscle counterparts. Despite these similarities, distinctive amino acid sequences between smooth-muscle alpha-actinin and striated-muscle alpha-actinins were revealed by peptide mapping using limited proteolysis in SDS. Gizzard alpha-actinin was immunologically distinguished from striated-muscle alpha-actinins. Gizzard alpha-actinin formed bundles of gizzard F-actin as well as of skeletal-muscle F-actin, but could not form any cross-bridges between adjacent actin filaments under conditions where skeletal-muscle alpha-actinin could. Temperature-dependent competition between gizzard alpha-actinin and tropomyosin on binding to gizzard thin filaments was demonstrated by electron microscopic observations. Gizzard alpha-actinin promoted Mg2+-ATPase activity of reconstituted skeletal actomyosin, gizzard acto-skeletal myosin, and gizzard actomyosin. This promoting effect was depressed by the addition of gizzard tropomyosin. These findings imply that, despite structural differences between gizzard and striated-muscle alpha-actinin molecules, they function similarly in vitro, and that gizzard alpha-actinin can interact not only with smooth-muscle actin (gamma- and beta-actin) but also with skeletal-muscle actin (alpha-actin).     

Phosphorylation of filamin and other proteins in cultured fibroblasts.

Incubation of subcellular fractions of fibroblasts with [³²P]ATP demonstrated 10 phosphoproteins whose phosphorylation can be increased by cyclic AMP or cyclic AMP-dependent protein kinase. One of these phosphoproteins, MW 240,000, resembles the actin binding protein, filamin, and can be selectively precipitated by antibodies to chicken gizzard filamin. Furthermore chicken gizzard filamin can be phosphorylated by skeletal muscle protein kinase and cyclic AMP stimulates this reaction.     

Isolation and localization from chicken gizzard of an inhibitory protein for Mg2+-activated skeletal muscle actomyosin ATPase.

An inhibitory protein for Mg2+-activated actomyosin ATPase from rabbit skeletal muscle was prepared from frozen chicken gizzard and purified by DEAE-Sephadex chromatography and gel filtration. 2. The inhibition by this protein was released by the addition of skeletal muscle troponin C and was independent of gizzard tropomyosin. 3. Localization of the inhibitory protein in gizzard muscle tissue and gizzard thin filaments was demonstrated by immunohistological techniques and immunodiffusion tests.     

A binding activity of actin with human C1q

A direct interaction of actin with C1q was demonstrated by two in vitro assays using purified human C1q and actins from rabbit skeletal muscle, chicken gizzard muscle and ascaris body wall. Every actin gave rise to a precipitation line with human C1q in agarose gel diffusion. A direct binding of actin with human C1q was ascertained by a binding assay system using radio-labelled rabbit actin and paper discs coated with human C1q. This binding of rabbit actin to C1q was inhibited by addition of unlabelled homologous and heterologous actins in the assay system. Results indicated that such interactions of actins with the human C1q were beyond species specificity.     

Isolation, characterization and immunocytochemical localization of caldesmon-like protein from molluscan striated muscle

A 140-kDa polypeptide present in the striated muscle of Pecten maximus and Sepia officinalis was purified to homogeneity and its main properties were investigated using biochemical and cytochemical approaches. The protein was found to be similar to chicken gizzard caldesmon. It is a heat-stable protein. It cross-reacts immunologically with anti-(gizzard caldesmon) antibody, binds to calmodulin-Sepharose in a Ca2+-dependent manner, cosediments with F-actin filaments and acts in the absence and presence of tropomyosin as a potent inhibitor of rabbit skeletal actomyosin Mg2+-ATPase. The immunocytochemistry of ultrathin sections revealed, at the light microscopy resolution level, that caldesmon-like protein is present in all types of muscles hitherto examined from invertebrates and vertebrates. However, according to the distribution and the intensity of the fluorescent reaction, we concluded that, under our experimental conditions, caldesmon is not homogeneously distributed and not located in the myofibrillar bands of striated muscles but rather in the sarcoplasmic elements, at the periphery of the fibres.     

Co-localization of immunoreactive forms of calponin with actin cytoskeleton in platelets, fibroblasts, and vascular smooth muscle

Calponin is an actin binding protein found in the smooth muscle cells of chicken gizzard. The localization of the protein was examined in bovine platelets, mouse fibroblasts, and the smooth muscle cells of the bovine aorta. Immunoblotting of whole platelet lysates revealed that the antibody to chicken gizzard calponin recognized two proteins with apparent molecular masses of 37 and 23 kDa in the resting state and an additional high-molecular-weight component (approximately 40 kDa) in the activated state. The localizations of calponin and caldesmon, and the correlation of their localizations with that of the actin cytoskeleton were analyzed by immunofluorescence microscopy using appropriate antibodies and rhodamine-phalloidin. In resting bovine platelets, calponin exhibited the same distribution as actin filaments, which are organized in a characteristic wheel-like structure. A similar distribution was observed with the anti-caldesmon antibody. Colocalization of calponin and actin were shown in activated platelets and along stress fibers of both fibroblasts and smooth muscle cells. These results suggest not only a cytoskeletal role associated with microfilaments but also a regulatory role of these proteins for actin-myosin interaction.