Chicken Gizzard Muscle Research Articles

Possible involvement of neurite outgrowth factor (NOF) receptor in axonal outgrowth from developing chick retina

We report that an 82-kDa membrane protein from chicken gizzard muscles is a neurite outgrowth factor (NOF)-receptor interacting with a domain for neurite-promoting activity of NOF. We found that an 82-kDa membrane protein in the developing chicken retina exhibited the same properties as those of gizzard muscles. The neuritic response of the retinal neurons to NOF increased in 6- and 8-day embryos, reaching a maximum level on the 8th-day and then rapidly decreased with aging. In parallel with this, the amount of NOF receptor in the retinas was maximal in 8- and 11-day embryos and thereafter decreased with aging. The results suggest that the failure in the neuritic response of the retinal neurons to NOF with aging is due to the decrease in NOF receptor.

Calcium-Dependent Control of Caldesmon-Actin Interaction by S100 Protein1

Caldesmon from chicken gizzard muscle has been examined for ability to interact with S100 protein using sedimentation, low-shear viscosity, and affinity chromatography. Ca2+/S100 protein, like Ca2+/calmodulin, inhibited the binding of caldesmon to F-actin in a concentration-dependent manner and the inhibition was not observed in the absence of Ca2+. Caldesmon was bound to S100 protein-Sepharose in the presence of Ca2+ and released with EGTA, indicating that there is a direct interaction between caldesmon and S100 protein. The binding of S100 protein to caldesmon also relieved actomyosin Mg2(+)-ATPase inhibition by caldesmon. The molar ratio of S100 protein to caldesmon required for half-maximal restoration was about 0.3, a value less than that in the case of calmodulin. S100 protein, however, was less effective in terms of the maximal extent of the restoration. With respect to Ca2(+)-sensitivity, the restoration profiles were monophasic with a midpoint at 3 x 10(-5) M for S100 protein and 8 x 10(-6) M for calmodulin. The restoration by S100 protein was almost wholly inhibited by TFP, but not by W-7. Taken together, our results suggest that a Ca2(+)-binding protein other than calmodulin may regulate caldesmon-dependent cellular functions.

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.

Direct inhibition of contractile apparatus by analogues of amiloride in the smooth muscle of guinea-pig taenia caecum and chicken gizzard

The relaxant effects of amiloride and its analogues, benzamil, 5-( N', N'-diethyl)-amiloride (DEAM) and 5-( N-ethyl- N-isopropyl)-amiloride (EIAM), were investigated using smooth muscle of guinea-pig taenia caeci and chicken gizzard. High K +-induced contractions of intact taenia and gizzard were inhibited by these compounds (1–100 μM) with the order of potency; benzamil ≥ EIAM > DEAM > amiloride. Contractions of permealized taenia and gizzard were also inhibited by these compounds at concentrations 8–35 times higher than those needed to inhibit the contractions of intact tissues. These compounds inhibited 20 K myosin light chain (MLC) phosphorylation at the concentrations needed to inhibit the contraction in the permealized muscles. Calmodulin (CaM) activity, as monitored by erythrocyte membrane (Ca 2+ + Mg 2+)-ATPase and phosphodiesterase activities, was inhibited by DEAM and EIAM at similar concentrations as those to inhibit the MLC phosphorylation. Benzamil also inhibited CaM activity at concentrations 4–8 times higher than those required to inhibit MLC phosphorylation. However, amiloride failed to inhibit CaM activity. Among these compounds, amiloride and benzamil inhibited Ca 2+/CaM-independent MLC phosphorylation due to trypsin-treated MLC kinase. Taenia tissue gradually accumulated these compounds and the tissue/medium ratio exceeded 3.5–17 after a 3-hr incubation period. These results indicate that amiloride and its analogues inhibit smooth muscle contraction mainly by the direct inhibition of MLC phosphorylation. The inhibitory effect of amiloride may be attributable to the inhibition of MLC kinase, whereas the inhibitory effect of DEAM and EIAM may largely be attributable to the inhibition of CaM. Benzamil may inhibit contraction by the inhibition of both MLC kinase and CaM. Differences in the drug-sensitivity between intact and permealized tissues may be attributable to the difference in drug accumulation by the cell.

Complete amino-acid sequence of subfragment-2 in adult chicken skeletal muscle myosin.

Although the complete amino-acid sequence of the short subfragment-2 (short S-2) and the partial sequence of the hinge region derived from adult chicken skeletal muscle myosin have been reported previously, the sequence of the N-terminal portion of subfragment-2 (S-2) and the connective portion between the above two regions could not be determined. In this study, the amino-acid sequence of these undetermined portions were completely sequenced. Furthermore, overlaps of cyanogen bromide (CNBr) peptides in the hinge region were also isolated and sequenced. Peptides obtained by hydrolysis with dilute formic acid and by digestion with lysyl endopeptidase of S-2 were purified and sequenced. These results established the complete amino-acid sequence of S-2 composed of 429 amino-acid residues. This sequence of adult chicken skeletal muscle myosin was compared with that of chicken embryonic skeletal muscle, chicken gizzard muscle and rabbit cardiac muscle myosin (alpha-myosin heavy chain) and shows degrees of 96%, 38% and 84% sequence identities, respectively. The frequency with which hydrophobic residues are present at position "a" in seven-residues repeats of the hinge region was markedly reduced when compared to the short S-2 sequence of the chicken skeletal muscle myosin.

Antihypertensive compounds with potent protein kinases inhibitory activity

A new indorocarbazole antibiotic SF2370 has been found in the culture broth of Actinomadura sp. ; it has also been found to have the activities of antihypertension and diuresis. Derivatives of SF2370, NA0344, NA0345 and NA0346, when injected (0.1−1 mg/kg i.v.) significantly lowered the blood pressure of anesthetized normotensive rats and showed a long lasting antihypertensive action. In the case of p.o. administration to conscious restrained SHRs, measurement of blood pressure by the plethysmographic method indicated that 10 mg/kg of the compounds was enough to lower the blood pressure; the antihypertensive activities were found to remain more than 12 hrs after oral administration. By studying of the mode of antihypertensive action, these compounds dose dependently relaxed the isolated aortic preparation contracted by norepinephrine. In addition, it was found that protein kinase C activity of mice brain was inhibited by the compounds; the IC 50 values were in the range of 0.062 – 0.20 μM. Moreover, a superprecipitation of actomyosin from smooth muscle of chicken gizzard was inhibited by the compounds, having an IC 50 values of 0.31 – 0.72 μM.

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.

An 82-kilodalton membrane protein that inhibits the activity of neurite outgrowth factor.

Neurite outgrowth factor (NOF), an extracellular matrix glycoprotein of 700 kilodaltons (kDa), promoted neurite outgrowth from cultured ciliary ganglion (CG) neurons of chicken embryo. A fraction solubilized with Nonidet P-40 of chicken gizzard muscle membranes inhibited the neurite-promoting activity of NOF in a dose-dependent manner, but not that of laminin. Binding of CG neurons to the substratum and their survival were not affected by the extract. The inhibitory activity of the extract was abolished by treatment with trypsin or heat. The molecular size was determined to be about 82 kDa by ligand blotting. The active component was partially purified by column chromatography. It is suggested that this molecule interacts with the domain of NOF responsible for its neurite-promoting activity and may modulate NOF activity during development in vivo.

P-515 - Interaction of monoclonal antibodies with a neurite outgrowth factor from chicken gizzard extract

Abstract A neurite outgrowth factor (NOF), which promotes neurite outgrowth of cultured peripheral neurons, was purified to homogeneity from chicken gizzard extract and used as an immunogen for generating monoclonal antibodies. From 603 hybridoma clones, 55 positive hybridoma cell lines were detected. Four of them (IgG 1 ( k -chain) producers designated as 5-10A, M1-2G, 4-2C and 1-4D) were selected for further characterization. These antibodies immunoprecipitated 125 I-labelled gizzard NOF molecules, whereas they did not inhibit the biological activity of NOF (or NOF-like molecules) derived from the tissues tested. The subcellular distribution of NOF in chick embryonal tissues was investigated using monoclonal antibody 4-2C. Both ciliary ganglionic (CG) neurons and gizzard muscle tissues were stained. The NOF content of embryonic CG, measured by an immunoblot assay, increased about 20-fold during embryo-genesis from 6 to 15 days, whereas the sensitivity of neurons to NOF reciprocally decreased. At least two different sizes of NOFs (700 and 800 kDa) were found in CG of various ages, with the 800/700 kDa ratio gradually increasing with age. A mixture of these 4 monoclonal antibodies was used for immunoaffinity purification of NOF from chicken gizzard muscles.

Interaction of monoclonal antibodies with a neurite outgrowth factor from chicken gizzard extract.

A neurite outgrowth factor (NOF), which promotes neurite outgrowth of cultured peripheral neurons, was purified to homogeneity from chicken gizzard extract and used as an immunogen for generating monoclonal antibodies. From 603 hybridoma clones, 55 positive hybridoma cell lines were detected. Four of them (IgG1 (kappa-chain) producers designated as 5-10A, M1-2G, 4-2C and 1-4D) were selected for further characterization. These antibodies immunoprecipitated 125I-labelled gizzard NOF molecules, whereas they did not inhibit the biological activity of NOF (or NOF-like molecules) derived from the tissues tested. The subcellular distribution of NOF in chick embryonal tissues was investigated using monoclonal antibody 4-2C. Both ciliary ganglionic (CG) neurons and gizzard muscle tissues were stained. The NOF content of embryonic CG, measured by an immunoblot assay, increased about 20-fold during embryogenesis from 6 to 15 days, whereas the sensitivity of neurons to NOF reciprocally decreased. At least two different sizes of NOFs (700 and 800 kDa) were found in CG of various ages, with the 800/700 kDa ratio gradually increasing with age. A mixture of these 4 monoclonal antibodies was used for immunoaffinity purification of NOF from chicken gizzard muscles.

Troponin-T and glyceraldehyde-3-phosphate dehydrogenase share a common antigenic determinant

A 37 kDa protein in extracts of bovine aorta and equine platelets was observed on SDS-polyacrylamide gel electrophoretograms to react with polyclonal and monoclonal antibodies to rabbit skeletal troponin-T (TnT) by immunoblotting. Following purification by precipitation at pH 4.6 and several ion-exchange chromatographic steps, it has been identified as glyceraldehyde-3-phosphate dehydrogenase (G3PD) by amino acid analyses and NH2-terminal sequencing. By ELISA, the anti-troponin-T monoclonal antibody reacted with rabbit skeletal G3PD appreciably but 120-fold less specifically than with TnT. A cyanogen bromide fragment (CB2) of TnT (residues 71-151) reacted with the monoclonal antibody nearly as well as intact TnT. This cross-reactivity between G3PD and TnT can be ascribed to a weak homology in the amino acid sequences of the two proteins between residues 72-80 of TnT and residues 157-165 of G3PD. Other regions of limited sequence similarity in the two proteins are also present. We conclude that the identification of diffuse cytoplasmic indirect immunofluorescent staining observed with a monoclonal anti-TnT antibody in chicken gizzard muscle is probably attributable to cross-reactivity with G3PD.

Chicken ornithine transcarbamylase: its unexpected expression.

Ornithine transcarbamylase (OTC), one of the enzymes of the urea cycle, is detectable in some strains of chickens, although they have no functional urea cycle. The enzyme consists of three identical subunits of 36 kd and is present in mitochondria of the kidney. Using immunoabsorbent column chromatography, we found further evidence that the enzyme is detectable as a precursor form (40 kd) in chicken brain, heart, liver, pancreas, gizzard, small intestine, and breast muscle. When an extract of small intestine containing only precursor OTC was treated with a kidney extract, the precursor was converted into OTC. This suggests that there is a tissue-specific processing protease in the kidney which splits a peptide off the precursor, causing the expression of OTC activity in this organ. However, the reason why the enzyme or its precursor is expressed in these organs is not known. The results of this study suggest that, unlike mammals, chickens are more organ specific with regard to the ability to incorporate precursor OTC into mitochondria.

Stability properties of oxymyoglobin from chicken gizzard smooth muscle

1. 1. Oxymyoglobin (MbO 2) was isolated directly from the smooth muscle of chicken gizzard and was examined for its spectral and stability properties. 2. 2. When compared with sperm whale MbO 2 as a reference, chicken gizzard MbO 2 was found to be much more susceptible to autoxidation. Its pH-dependence was therefore analyzed in terms of an “acid-catalyzed three-state model”. 3. 3. The complete amino acid sequence of the myoglobin was also determined. Its hydropathy profile revealed that the region corresponding to the distal side of the heme iron appears to be less hydrophobic.

The effects of caldesmon on smooth muscle heavy actomeromyosin ATPase activity and binding of heavy meromyosin to actin.

Caldesmon was purified to homogeneity from both chicken gizzard and bovine aortic smooth muscles. Caldesmon purified from bovine aorta was slightly larger than caldesmon purified from chicken gizzards (Mr = 140,000) when the two were compared electrophoretically. Caldesmon bound tightly to actin saturating at a molar ratio of 1 caldesmon monomer per 6.6 actin monomers. Ca2+-calmodulin appeared to reduce the affinity of caldesmon for actin. Caldesmon was also a potent inhibitor of heavy actomeromyosin ATPase activity producing a maximal effect at a ratio of 1 caldesmon monomer per 7-10 actin monomers. This effect was also antagonized by Ca2+-calmodulin. While caldesmon inhibited heavy actomeromyosin ATPase activity, it greatly enhanced binding of both unphosphorylated and phosphorylated heavy meromyosin to actin in the presence of MgATP, reducing the Kd for binding by a factor of 40 for each form of heavy meromyosin. Although we did identify a Ca2+-calmodulin-stimulated "caldesmon kinase" activity in caldesmon preparations purified under nondenaturing conditions, we observed no effect of phosphorylation (2 mol of PO4/mol of caldesmon) on the capacity to inhibit heavy actomeromyosin ATPase activity. Our results suggest that caldesmon could serve some role in smooth muscle function by enhancing cross-bridge affinity while inhibiting actomyosin ATPase activity.

Ca 2+ 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°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 Ca 2+-dependent manner, indicating that the Ca 2+ sensitivity involves myosin or a protein associated with it. Partial loss of Ca 2+ 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 Ca 2+ dependence at higher temperatures or lower ionic strengths than does activation by skeletal muscle actin. The Ca 2+ dependence of the activity of phosphorylated heavy meromyosin is about half that of myosin and is affected differently by temperature, ionic strength and Mg 2+, being independent of temperature and optimal at lower concentrations of NaCl. Raising the concentration of Mg 2+ above 2–3 mM inhibits the activity of heavy meromyosin but stimulates that of myosin, indicating that Mg 2+ and Ca 2+ activate myosin at different binding sites.

The role of subunit autolysis in activation of smooth muscle Ca2+-dependent proteases.

Ca2+-dependent proteases isolated from chicken gizzard and bovine aortic smooth muscle were compared with respect to subunit autolysis and the role of autolysis in modulating enzyme activity. The protease isolated from chicken gizzard was a heterodimer consisting of 80,000- and 30,000-dalton subunits. The protease isolated under identical conditions from bovine aorta consisted of 75,000- and 30,000-dalton subunits. In the presence of Ca2+, both enzymes underwent autolysis of their 30,000-dalton subunits with conversion to an 18,000-dalton species. In addition, the 80,000-dalton subunit of the gizzard protease was degraded to a 76,000-dalton form. The Ca2+ concentrations required for autolysis of the 30,000-dalton subunits were different for the two enzymes (i.e. gizzard: K0.5 Ca2+ = 335 microM; aortic: K0.5 Ca2+ = 1,250 microM) although in both cases, stimulation of autolysis by Ca2+ exhibited positive cooperativity. When compared with respect to kinetics of substrate degradation, the native forms of the smooth muscle Ca2+-dependent proteases (gizzard, GIIa = 80,000/30,000-dalton heterodimer; bovine aortic, IIa = 75,000/30,000-dalton heterodimer) exhibited a lag phase in product appearance. On the other hand, the autolyzed forms (gizzard, GIIb = 76,000/18,000-dalton heterodimer; bovine aortic, IIb = 75,000/18,000-dalton heterodimer) exhibited linear rates of substrate degradation. These results were analyzed in terms of autolysis of the 30,000-dalton subunits as determined by the conversion of this subunit to its 18,000 dalton form. For both enzymes, the time course for the autolytic transition, 30,000----18,000 daltons, and Ca2+-dependence of the apparent rate constants for this transition were found to correlate well with the lag phase in enzymatic activity. No such correlation could be established for the 80,000----76,000 dalton autolytic transition of the high molecular mass subunit of the gizzard protease. Our results suggest that catalytic activity of the Ca2+-dependent proteases isolated from gizzard and bovine aortic smooth muscle requires autolysis of the 30,000-dalton subunit. The native or unautolyzed forms of these enzymes appear to be proenzymes that can be activated by autolysis.

Cross-reactivity of a monoclonal antibody to the catalytic subunit of chicken gizzard desmin-specific calpain

1. 1. The desmin-specific calpain I from chicken gizzard smooth muscle is a dimer of 83 and 35 kDalton subunits. 2. 2. A monoclonal antibody to the large subunit did not cross-react with chicken gizzard and hamster skeletal muscle calpain II, but it did recognize hamster skeletal muscle desmin-specific calpain I and the denatured calpain II from chicken gizzard smooth muscle. 3. 3. These results indicate that different desmin-specific calpains have similar large subunits which differ significantly from the large subunit of calpain II in the same tissue.

Phosphorylation of regulatory light chain a (RLC-a) in smooth muscle myosin of scallop, Patinopecten yessoensis.

One of the two regulatory light chains, RLC-a, of scallop smooth muscle myosin was fully phosphorylated by myosin light chain kinase of chicken gizzard muscle. The residue phosphorylated was Ser. It may be the Ser at number 11 from the N-terminal. The sequence of 9 residues around the Ser-11, QRATSNVFA, is identical with that around the phosphorylatable Ser of LC20 of chicken gizzard myosin. RLC-a was also phosphorylated slowly by cAMP-dependent protein kinase. The phosphorylation of RLC-a may be involved in the regulatory system for the catch contraction of scallop muscle.

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.

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.