Antibodies against a nonapeptide of polyomavirus middle T antigen: cross-reaction with a cellular protein(s).

Antibodies were raised against the sequence Glu-Glu-Glu-Glu-Tyr-Met-Pro-Met -Glu, which represents a part of the middle T antigen of polyomavirus that is considered to be important in inducing the phenotype of transformed cells. The antibodies reacted with native as well as denatured middle T antigens. In addition, the antibodies immunoprecipitated a cellular protein with an apparent molecular weight of 130,000 (130K) from mouse and rat cells. In some cases, a 33K protein was also immunoprecipitated. Immunoprecipitation of middle T antigen as well as 130K and 33K proteins was blocked by the peptide. The antibodies labeled microfilaments of untransformed mouse, rat, human, and chicken cells by immunofluorescence. This labeling was also blocked by the peptide. The labeling pattern and distribution under a variety of conditions were indistinguishable from those of anti-actin antibodies, although no evidence has been obtained to indicate that the anti-peptide antibodies react with actin. The 130K protein migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis slightly slower than chicken gizzard vinculin (130K) and slightly faster than myosin light-chain kinase of chicken smooth muscle (130K). Neither of these proteins absorbed the anti-peptide antibodies. The 33K protein does not seem to be tropomyosin (32K to 40K).     

α-Actinin: Immunofluorescent localization of a muscle structural protein in nonmuscle cells

Antibodies specific for the skeletal muscle structural protein α-actinin are used to localize this protein by indirect immunofluorescence in nonmuscle cells. In cultured nonmuscle cells, α-actinin is localized along or between actin filament bundles producing an almost regular periodicity. The protein is also detected in the form of fluorescent plaques at some ends of actin filament bundles, as well as in a filamentous form in some overlap areas of cells. In spreading rat embryo cells, α-actinin assumes a focal distribution which corresponds to the vertices of a highly regular actin filament network. The results suggest that α-actinin may be involved in the organization of actin filament bundles, in the attachment of actin filaments to the plasma membrane, and in the assembly of actin filaments in areas of cell to cell contact.     

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.     

A new simple method of preparing actin from chicken gizzard

A simple method for preparing actin from chicken gizzard was described. This method takes advantage of a property of gizzard tropomyosin, that is, that it does not form Mg paracrystals readily.     

Use of actin isoform-specific antibodies to probe the domain structure in three smooth muscles

We investigated the location of actin isoforms in relation to each other and to filament attachment sites by studying the edge-to-edge distribution of both immunofluorescence and immunogold probes in smooth muscle cells from three sources. Antibodies to alpha- or alpha,gamma-actin labeled uniformly across smooth muscle cells from each source. Antibodies to beta-cytoplasmic actin were concentrated on and near dense bodies, especially in gizzard smooth muscle, but were also located throughout the filament compartment. Double immunofluorescent labeling with antibodies to alpha- or alpha/gamma- and to beta-actin shows overlap of label at dense bodies and attachment plaques. Double immunofluorescent labeling with antibodies to alpha-actinin and to beta-actin identified dense bodies and attachment plaques as sites of colocalization. Immunogold labeling with anti-desmin was most prominent near dense bodies in the gizzard and was widely dispersed in vas deferens and arterial smooth muscle cells. Our results indicate that there is extensive overlap between the locations of contractile and cytoskeletal elements and, thus, do not support the two-domain model of smooth muscle structure. Tissue-specific organizational motif differences were seen when gizzard, vas deferens, and artery were compared and suggest that one model may not apply to these three smooth muscles.     

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.     

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.     

The removal of extracellular fibronectin from areas of cell-substrate contact

Immunofluorescent labeling for fibronectin was largely excluded from sites of closest contact between spreading chicken gizzard fibroblasts and the substratum. This was observed by double immunofluorescent labeling of fixed cells for fibronectin and vinculin, a smooth muscle intracellular protein that is specifically associated with focal adhesion plaques, in conjunction with interference-reflection microscopy. When the cells were plated on a fibronectin-coated substratum they adhered to its surface and rapidly spread on it. The immunofluorescent labeling for fibronectin in those cultures (after fixation and triton permeabilization) was usually absent from the newly formed, vinculin-containing focal adhesion plaques. We have found, however, that the accessibility to the cell-substrate gap at the focal adhesion plaques is limited and therefore a more direct approach was adopted. We have found that cells spreading on a substrate coated with rhodamine-labeled fibronectin progressively removed the underlying protein from the substrate. The removal of fibronectin involved at least two distinct mechanisms. Part of the substrate-associated fibronectin was removed from small areas and displaced toward the cell center. The arrowhead-shaped areas from which fibronectin was removed often coincided with vinculin-rich focal contacts. We observed, however, many areas where focal contacts were found over unperturbed fibronectin carpet, as well as fibronectin-free areas with no overlapping focal contacts. The possibilities that fibronectin is actively displaced from areas of cell-substrate contact, that the focal adhesion plaques are transiently associated with these areas and their implications on the dynamics of cell spreading and locomotion are discussed. The second route of fibronectin removal from the substrate was endocytosis. The rhodamine-labeled fibronectin was found in the cells in a partial or transient association with clathrin-containing structures.     

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).     

The functional properties of full length and mutant chicken gizzard smooth muscle caldesmon expressed in Escherichia coli

Wild type chicken gizzard caldesmon (756 amino acids) was expressed in a T7 RNA polymerase-based bacterial expression system at a yield of 1 mg pure caldesmon per litre bacterial culture. A mutant composed of amino acids 1-578 was also constructed and expressed. The wild type and mutant caldesmon were purified and compared with native chicken gizzard caldesmon. Native and wild type expressed caldesmon were indistinguishable in assays for inhibition of actin-tropomyosin activation of myosin ATPase, reversal of inhibition by Ca²⁺-calmodulin and binding to actin, actin-tropomyosin, Ca²⁺-calmodulin, tropomyosin and myosin. The mutant missing the C-terminal 178 amino acids had no inhibitory effect and did not bind to actin or Ca²⁺-calmodulin. It bound to tropomyosin with a 5-fold reduced affinity and to myosin with a greater than 10-fold reduced affinity.     

Distribution of actin and tropomyosin in Cryptosporidium muris

Actin and tropomyosin of Cryptosporidium muris were localized by immunogold labeling. Two kinds of antibodies for actin labeling were used. The polyclonal antibody to skeletal muscle (chicken back muscle) actin was labeled on the pellicle and cytoplasmic vacuoles of parasites. The feeder organelle has showed a small amount of polyclonal actin antibody labeling as well. Whereas the monoclonal antibody to smooth muscle (chicken gizzard muscle) actin was chiefly labeled on the filamentous cytoplasm of parasites. The apical portion of host gastric epithelial cell cytoplasm was also labeled by smooth muscle actin together. The polyclonal antibody to tropomyosin was much more labeled at C. muris than host cells, so it could be easily identified even with low magnification (×2,000). The tropomyosin was observed along the pellicle, cytoplasmic vacuoles, and around the nucleus also. The skeletal muscle type actin seems to play a role in various cellular functions with tropomyosin in C. muris; on the other hand, the smooth muscle type actin was located mainly on the filamentous cytoplasm and supported the parasites'' firm attachment to host cells. Tropomyosin on the pellicle was thought to be able to stimulate the host as a major antigen through continuous shedding out by the escape of sporozoites or merozoites from their mother cells.     

人骨骼肌α辅肌动蛋白（α－actinin）的提取及其抗血清的制备和鉴定

本文提取人骨骼肌α辅肌动蛋白（α－ａｃｔｉｎｉｎ）是综合了文献报导有关提取兔肌α－ａｃｔｉｎｉｎ的和提取鸡胗α－ａｃｔｉｎｉｎ的方法,稍加修改而确定的。用Ｈａｓｓｅｌｂａｃｈ－Ｓｃｈｎｅｉｄｅｒ缓冲液提取骨骼肌中的肌球蛋白后,将残余物经硼酸－缓冲液提取、匀浆及高速离心去掉肌动蛋白和肌原纤维的其它成份,上清加硫酸铵至３０％,３５％饱合度所得的沉淀用２２０ｍｍｏｌ／ＬＴｒｉｓ－乙酸溶解、透析、离心后经ＤＥ－５２柱层析可得电泳纯。α－ａｃｔｉｎｉｎ。将人骨骼肌α－ａｃｔｉｎｉｎ纯化制品免疫了三只大耳白纯种家兔,两个多月后,三只兔子都产生免抗人骨骼肌α－ａｃｔｉｎｉｎ的特异抗血清,用双向免疫扩散法和酶联免疫吸附试验（ＥＬＩＳＡ）测定,产生的抗体效价较高,用双扩散法测定效价为１：３２,用ＥＬＩＳＡ测定,用比率法判断结果,效价最高者为１：１００,０００左右,经免疫电镜观察结果证实,上述抗血清可以满足进一步实验要求。     

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.     

Development of myofibrils in the gizzard of chicken embryos

Summary The intracellular distributions of major muscle proteins, myosin, actin, tropomyosin, -actinin, and desmin, in smooth muscle cells of chicken gizzard at various stages of embryogenesis were investigated by immunofluorescence-labeling of enzyme-dispersed cells cultured up to three hours. These muscle proteins, except some part of myosin, were organized into fibrous structures as soon as synthesis and accumulation of proteins started. As for myosin, a considerable amount of it was dispersed in soluble cytoplasm as well. On the other hand, Ca⁺⁺-dependent contractility was detected with detergent-extracted myoblasts and glycerinated tissue from embryos older than 7 days. Although the nascent myofibrils bear a resemblance to stress fibers, the former could be distinguished from the latter by their high stability in dispersed, spherical cells. The above findings, therefore, show that the synthesis of contractile proteins is followed by immediate assembly of them into functional myofibrils without undergoing any intermediate structure. Based on these findings, the mechanism of myofibril formation in developing smooth muscle cells is discussed.     

Smooth muscle cells of the chicken aortic arch differ from those in the gizzard and the femoral artery in the distribution of F-actin, α-actinin and filamin

Summary The distribution of F-actin, -actinin and filamin in smooth muscle cells of the chicken was examined by immunofluorescent and immunoelectron microscopy. Those from the gizzard, the femoral artery and the aortic arch were compared. F-Actin labeled by NBD-phallacidin was seen diffusely distributed in the sarcoplasm in the gizzard and the femoral artery, but in the aorta it was observed as streaks and spots, with unstained areas in between. Epon sections of the aortic arch showed that bundles of thin myofilaments run in various directions interspersed with areas mostly occupied by intermediate filaments. -Actinin labelling occurred in dense plaques along the sarcolemma in all the muscles examined. While dense bodies in the sarcoplasm were common and labelled for -actinin in the gizzard and the femoral artery, hardly any were seen in the aortic arch and little labelling for -actinin was observed in the sarcoplasm. Filamin was concentrated along the periphery of dense bodies and plaques in the gizzard and the femoral artery, but it was seen diffusely in the sarcoplasm of the aortic muscle. After chemical skinning of the latter, filamin labelling persisted only in the F-actin bundles, and other areas became negative. The present results show that smooth muscle cells of the aortic arch contrast with those of the gizzard and even with those of the femoral artery in the distribution of F-actin, -actinin and filamin. The mechanisms of contraction and/or stress maintenance in the aortic smooth muscle may be different from those in other smooth muscles.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Calcium sensitivity of contractile proteins from chicken gizzard muscle.

Actin, myosin, and "native" tropomyosin (NTM) were separately isolated from chicken gizzard muscle and rabbit skeletal muscle. With various combinations of the isolated contractile proteins, Mg-ATPase activity and superprecipitation activity were measured. It was thus found that gizzard myosin and gizzard NTM behaved differently from skeletal myosin and skeletal NTM, whereas gizzard actin functioned in the same wasy as skeletal actin. It was also found that gizzard myosin preparations were often Ca-sensitive, that is, that the two activities of gizzard myosin plus actin without NTM were activated by low concentrations of Ca2+. The Mg-ATPase activity of a Ca-insensitive preparation of gizzard myosin was not activated by actin even in the presence of Ca2+. When Ca-sensitive gizzard myosin was incubated with ATP (and Mg2+) in the presence of Ca2+, a light-chain component of gizzard myosin was phosphorylated. The light-chain phosphorylation also occurred when Ca-insensitive myosin was incubated with gizzard NTM and ATP (plus Mg2+) in the presence of Ca2+. In either case, the light-chain phosphorylation required Ca2+. Phosphorylated gizzard myosin in combination with actin was able to exhibit superprecipitation, and Mg-ATPase of the phosphorylated gizzard myosin was activated by actin; the actin activation and superprecipitation were found to occur even in the absence of Ca2+ and NTM or tropomyosin. The phosphorylated light-chain component was found to be dephosphorylated by a partially purified preparation of gizzard myosin light-chain phosphatase. Gizzard myosin thus dephosphorylated behaved exactly like untreated Ca-insensitive gizzard myosin; in combination with actin, it did not superprecipitate either in the presence of Ca2+ or in its absence, but did superprecipitated in the presence of NTM and Ca2+. Ca-activated hydrolysis of ATP catalyzed by gizzard myosin B proceeded at a reduced rate after removal of Ca2+ (by adding EGTA), whereas that catalyzed by a combination of actin, gizzard myosin, and gizzard NTM proceeded at the same rate even after removal of Ca2+. However, addition of a partially purified preparation of gizzard myosin light-chain phosphatase was found to make the recombined system behave like myosin B. Based on these findings, it appears that myosin light-chain kinase and myosin light-chain phosphatase can function as regulatory proteins for contraction and relaxation, respectively, of gizzard muscle.     

Immunofluorescence microscopy of a myopathy : α-Actinin is a major constituent of nemaline rods

A biopsy of skeletal muscle taken from a child with the clinical symptoms of congenital nemaline myopathy was studied. Light and electron microscopy revealed rod-like structures within the muscle fibres, and thus confirmed the clinical diagnosis. Indirect immunofluorescence, using specific antibodies against actin and desmin (both derived from chicken gizzard) as well as against α-actinin and tropomyosin (both from porcine skeletal muscle) revealed that the rods consist of massive accumulations of α-actinin. Desmin seems to be peripherally associated with the rods. Anti-actin and anti-tropomyosin did not stain the rods; however, a masking effect could not be ruled out. These findings support previous hypotheses that nemaline rods can be taken to be lateral-polymers of normal Z-disks.     

In vitro labeling of proteins by reductive methylation: Application to proteins involved in supramolecular structures

Actin and tropomyosin, purified from both muscle and brain, and α-actinin, purified from muscle, have been labeled in vitro by reductive methylation to specific activities of greater than 10⁵ dpm/μg protein. Actin so modified bound DNase I and polymerized identically to unmodified actin. Furthermore, the spectral properties of actin did not change after labeling. The interactions of labeled tropomyosin and α-actinin with F-actin were nearly identical to those of the unmodified proteins. These modified proteins comigrated with their unmodified counterparts in both SDS-containing polyacrylamide gels and isoelectric focusing gels. The labeled actin was quantitatively extracted from SDS-containing polyacrylamide gels (yield > 98% of radioactivity applied demonstrating that all of the radioactivity was protein bound. The reductive methylation procedure worked well at pH 8.0–8.5 in either pyrophosphate buffer or Bicine buffer using formaldehyde with [³H]-sodium borohydride as the reducing agent. The procedure could also be performed at pH 7.0 in phosphate buffer using [¹⁴C]-formaldehyde with sodium cyanoborohydride as the reducing agent. Proteins so labeled are ideal for use in quantitative experiments involving protein-protein interactions.     

Mode of degradation of myofibrillar proteins by rabbit muscle cathepsin D

The mode of degradation of myofibrillar proteins by the action of highly purified rabbit muscle cathepsin D (EC 3.4.23.5) was studied using SDS-polyacrylamide gel electrophoresis. Cathepsin D optimally degraded myosin heavy chain, α-actinin, tropomyosin, troponin T and troponin I at around pH 3. It did not degrade actin or troponin C. Degradation of myosin heavy chain produced four major fragments of 155 000, 130 000, 110 000 and 90 000 daltons. Troponin T was hydrolyzed to 33 000-, and 20 000- and 11 000-dalton fragments. Troponin I was degraded into fragments of 13 000 and 11 000 daltons. Degradation of α-actinin and tropomyosin was not as rapid as that of mysoin and troponins T and I. Tropomyosin gave a fragment of 30 000 daltons, but α-actinin showed no distinct band of this fragment on gels.     

Vascular smooth muscle caldesmon

Caldesmon, a major actin- and calmodulin-binding protein, has been identified in diverse bovine tissues, including smooth and striated muscles and various nonmuscle tissues, by denaturing polyacrylamide gel electrophoresis of tissue homogenates and immunoblotting using rabbit anti-chicken gizzard caldesmon. Caldesmon was purified from vascular smooth muscle (bovine aorta) by heat treatment of a tissue homogenate, ion-exchange chromatography, and affinity chromatography on a column of immobilized calmodulin. The isolated protein shared many properties in common with chicken gizzard caldesmon: immunological cross-reactivity, Ca2+-dependent interaction with calmodulin, Ca2+-independent interaction with F-actin, competition between actin and calmodulin for caldesmon binding only in the presence of Ca2+, and inhibition of the actin-activated Mg2+-ATPase activity of smooth muscle myosin without affecting the phosphorylation state of myosin. Maximal binding of aorta caldesmon to actin occurred at 1 mol of caldesmon: 9-10 mol of actin, and binding was unaffected by tropomyosin. Half-maximal inhibition of the actin-activated myosin Mg2+-ATPase occurred at approximately 1 mol of caldesmon: 12 mol of actin. This inhibition was also unaffected by tropomyosin. Caldesmon had no effect on the Mg2+-ATPase activity of smooth muscle myosin in the absence of actin. Bovine aorta and chicken gizzard caldesmons differed in several respects: Mr (149,000 for bovine aorta caldesmon and 141,000 for chicken gizzard caldesmon), extinction coefficient (E1%280nm = 19.5 and 5.0 for bovine aorta and chicken gizzard caldesmon, respectively), amino acid composition, and one-dimensional peptide maps obtained by limited chymotryptic and Staphylococcus aureus V8 protease digestion. In a competitive enzyme-linked immunosorbent assay, using anti-chicken gizzard caldesmon, a 174-fold molar excess of bovine aorta caldesmon relative to chicken gizzard caldesmon was required for half-maximal inhibition. These studies establish the widespread tissue and species distribution of caldesmon and indicate that vascular smooth muscle caldesmon exhibits physicochemical differences yet structural and functional similarities to caldesmon isolated from chicken gizzard.