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.     

The contractile and regulatory proteins of insect flight muscle

1. Myosin, actin and the regulatory proteins were prepared from insect flight muscle. 2. The light subunit composition of the myosin differed from that of vertebrate muscle myosin. The ionic strength and pH dependence of the myosin adenosine triphosphatase (ATPase) were measured. 3. Actin was associated with a protein of subunit molecular weight 55000 and was purified by gel filtration. Impure actin had protein bound at a periodicity of about 40nm. 4. Regulatory protein extracts had tropomyosin and troponin components of subunit molecular weight 18000, 27000 and 30000. Crude extracts of regulatory proteins inhibited the ATPase activity of desensitized or synthetic actomyosin; this inhibition was relatively insensitive to high Ca(2+) concentrations. Purified insect regulatory protein produced as much sensitivity to Ca(2+) as did the rabbit troponin-tropomyosin complex. 5. Synthetic actomyosins were made from rabbit and insect proteins. Actomyosins containing insect myosin had a low ATPase activity that was activated by tropomyosin. The Ca(2+) sensitivity of actomyosins containing insect myosin or actin, with added troponin-tropomyosin complex from rabbit, was comparable with that of rabbit actomyosin.     

Identification of high molecular weight proteins in squid muscle by Western blotting analysis and postmortem rheological changes

The high molecular weight protein connectin (also called titin) in Japanese common squid (Todarodes pacificus) mantle muscle was identified by western blotting analysis with 3B9, the mouse anti-chicken skeletal muscle connectin monoclonal antibody. Similarly to vertebrate samples, there exists connectin in invertebrate squid mantle muscle, and the amino acid sequences are assumed to resemble those present in the A band of vertebrate connectin, judging by the specificity of 3B9. Moreover, the connectin in squid muscle migrated in this study as a closely spaced doublet of alpha and beta (titins 1 and 2). Between 5 and 7 h post-mortem, the SDS PAGE patterns of the squid sample indicated a change of the doublet bands into a single beta-connectin band. Simultaneously, the rheological properties of the squid muscle changed substantially. This degradation of alpha-connectin into beta-connectin in the muscle can explain the critical change that occurs during the post-mortem tenderization of squid muscle.     

昆虫飞行肌细胞凋亡的分子生物学研究进展

细胞凋亡是多细胞生物体内由激素刺激,基因调控,蛋白调节的一个主动的程序化死亡过程,对生物体特定组织功能的发生、发展、衰老及退化等具有重要作用。而昆虫飞行肌细胞凋亡对昆虫迁飞行为尤为重要,它直接决定迁飞性昆虫能否选择更适宜的寄主植物和生活条件,进而影响到对农作物的危害地域和严重程度。本文在近年来昆虫细胞凋亡的研究基础上,从分子生物学的角度,综述了调控昆虫飞行肌细胞凋亡的相关基因和蛋白质的研究进展,探讨了昆虫飞行肌细胞凋亡发生与调控的分子生物学机制。     

Identification of a connecting filament protein in insect fibrillar flight muscle

A major component on sodium dodecyl sulfate-containing gels of solubilized isolated Z-discs, purified from honeybee flight muscle, migrates with an apparent molecular weight of 360,000. Antibodies to this high molecular weight polypeptide have been prepared by injecting rabbits with homogenized gel slices containing the protein band. With indirect immunofluorescence microscopy these antibodies are localized to a region extending from the edge of the Z-band to the A-band in shortened or stretched sarcomeres. Similarly, glycerinated flight muscle treated with antiserum and prepared for electron microscopy shows enhanced density from the ends of the thick filaments to the I-Z junction regardless of sarcomere length. Evidence indicates that antiserum is directed toward a structural protein of connecting filaments, which link thick filaments to the Z-band in insect fibrillar muscle, rather than to a thin filament component. In Ouchterlony double-diffusion experiments a single precipitin band is formed when antiserum is diffused against solubilized Z-discs; no reaction occurs between antiserum and proteins from native thin filaments prepared from honeybee flight muscle. Further, antibody stains the I-band in flight muscle fibrils from which thin filaments are removed. Finally, honeybee leg muscle myofibrils, in which connecting filaments have not been observed, are not labelled with antibody. Since antibody binds to the short projections which extend from the flat surfaces of isolated Z-discs, these projections are assumed to be remnants of connecting filaments and the source of the 360,000 M_r protein.The amino acid composition of this high molecular weight material, purified by Sepharose chromatography, is presented. The protein has been named “projectin”.     

A proline shuttle in insect flight muscle.

A proline shuttle for oxidation of extramitochondrial NADH was reconstituted from soluble and mitochondrial fractions of blowfly ($\text{Phormia}$$\text{regina}$) flight muscle. The soluble fraction catalyzed reduction of Δ′-pyrroline-5-carboxylate to proline via the action of Δ′-pyrroline-5-carboxylate reductase (EC 1.5.1.2). The reaction required NADH as hydrogen donor, NAD (P) H being ineffective in this regard. Mitochondria catalyzed regeneration of Δ′-pyrroline-5-carboxylate from proline via action of proline oxidase. The capacity of the shuttle to operate under conditions of possible competition for Δ′-pyrroline-5-carboxylate between Δ′-pyrroline-5-carboxylate reductase and Δ′-pyrroline-5-carboxylate dehydrogenase (EC 1.5.1.12) was incestigated. Results of these investigations indicate that dehydrogenase activity does not significantly interfere with shuttle activity.     

Ultrastructural localization of the high molecular weight proteins associated with in vitro-assembled brain microtubules

Microtubules isolated from brain extracts by in vitro assembly (1, 19, 23) are composed principally of two tubulins and two high molecular weight proteins (microtubule-associated proteins [MAPS] 1 and 2) (2,5,7,20). Recently, it was demonstrated that in vitro-assembled brain microtubules (neurotubules) are coated with filaments (5, 7) which are similar to the filaments attached to neurotubules in situ (4, 15, 21, 24, 25), and it was suggested that the filaments are composed of the higher molecular weight MAPs (5, 7, 12). In this study, microtubules were assembled in the presence and absence of the MAPs, and thin sections of the microtubules were examined by electron microscopy. The results show that the filaments only occur on microtubules assembled in the presence of the MAPs and it is therefore concluded that the filaments are composed of the high molecular weight MAP's.     

Ca-activation and stretch-activation in insect flight muscle

Asynchronous insect flight muscle is specialized for myogenic oscillatory work, but can also produce isometric tetanic contraction. In skinned insect flight muscle fibers from Lethocerus, with sarcomere length monitored by a striation follower, we determined the relation between isometric force (F(0)) at serial increments of [Ca(2+)] and the additional active force recruited at each [Ca(2+)] by a stretch of approximately 12 nm per half-sarcomere (F(SA)). The isometric force-pCa relation shows that 1.5-2 units of pCa are necessary to raise isometric force from its threshold (pCa approximately 6.5) to its maximum (F(0,max)). The amplitude of F(SA) depends only on the preceding baseline level of isometric force, which must reach at least 0.05 F(0,max) to enable stretch-activation. F(SA) rises very steeply to its maximum as F(0) reaches approximately 0.2 F(0,max), then decreases as F(0) increases so as to produce a constant sum (F(0) + F(SA)) = F(max). Thus Ca- and stretch-activation are complementary pathways that trigger a common process of cross-bridge attachment and force production. We suggest that stretch-induced distortion of attached cross-bridges relieves the steric blocking by tropomyosin of additional binding sites on actin, thereby enabling maximum force even at low [Ca(2+)].     

The paramyosin of insect flight muscle

Paramyosin has been extracted and purified from the flight muscle of the insects Lethocerus cordofanus, Lethocerus maximus (water bugs), Heliocopris japetus (dung beetle) and Pachnoda ephippiata (rosechafer beetle). The subunit molecular weight, estimated by sodium dodecyl sulphate electrophoresis, is 107,000 ± 6000. The intrinsic sedimentation constant is 3.17 S and circular dichroism measurements give about 87 % helix, showing that the molecule is likely to be a two-chain rod.The amino acid composition of insect paramyosins resembles that of molluscan and annelid paramyosins except that the Glu/Asp ratio is higher. The amino acid analysis of insect tropomyosin is also given. Electron microscopy of tactoids shows an axial periodicity of 732 ± 8 Å or 146 Å with fine structure differing from that of molluscan tactoids.The proportion of paramyosin in the myofibrils, estimated by densitometry of stained gels, is 6.3% in L. cordofanus and 9.5% in rosechafer, and the ratio of myosin to paramyosin in L. cordofanus is 8.2. The possibility that paramyosin is a core protein of the myosin filaments is discussed.     

The high molecular weight proteins released from cultured cells.

Studies have been performed with the serum-free culture medium taken from several fibroblast monolayer culture lines. A high molecular weight protein fraction was separated from the concentrated medium by sucrose density gradient centrifugation. Polyacrylamide gel electrophoresis was used to assess the degree of purification obtained. In the electron microscope the negatively stained high molecular weight proteins were found to closely resemble the alpha2-macroglobulins. The suggestion that these proteins from cultured cells resemble the cylindrical protein complex isolated from mammalian erythrocyte ghosts is not supported by this study. The results are discussed in the light of the extensive literature now available on the electron microscopy of high molecular weight proteins.     

Glycogen of high molecular weight from mammalian muscle

Glycogen of high molecular weight has been isolated from mammalian muscle, in contrast to the material of low molecular weight commonly described. The large polysaccharide is similar to liver glycogen in the structure of its individual beta-particles and also, partially, in the mode of assembly into the gross alpha-particles. The large particles may be disrupted by 2-mercaptoethanol, but not to the same extent as their liver counterparts.     

Immunolocalization of ubiquitin in degenerating insect flight muscle

Summary Ubiquitin was localized by immunofluorescence microscopy during post-mating histolysis of fibrillar flight muscle in female fire ants, Solenopsis spp. Normal muscles, as well as histolysing muscles from artificially inseminated and haemolymph-injected females contained ubiquitin in association with nuclei, Z-lines, myofilaments and mitochondria. However, the density of the ubiquitin immunoreaction was markedly increased in the nuclei, Z-lines and mitochondria of degenerating tissues 6, 12 and 24 h post treatment. At these times the heaviest immunoreactivity for ubiquitin was seen in association with the nuclei, Z-lines and mitochondria. Immuno-controls, incubated in the absence of the primary antibody, showed no similar immunostaining. When insemination was preceded by the injection of actinomycin D, muscle degradation was significantly depressed after a 24-h period. Also, ubiquitin immunofluorescence was markedly reduced in tissues pre-treated with actinomycin D. These observations suggest that insemination increases the ubiquitination of specific myofibrillar proteins destined for degradation.     

Detection of high molecular weight vinculin binding proteins in muscle and nonmuscle tissues with an electroblot-overlay technique

In this study, we examined binding of radiolabelled vinculin to proteins separated on sodium dodecyl sulfate-polyacrylamide gels, and then electrophoretically transferred onto nitrocellulose sheets. We detected saturable binding of vinculin to polypeptides with apparent Mr's of 215,000, 205,000 and 185,000 in a low ionic strength extract from chicken gizzard membranes. Binding of vinculin to proteins with apparent Mr's of 205,000, 185,000, and 165,000 in human platelets was also detected. In addition, we found that [125I]vinculin binds to unlabelled vinculin and to alpha-actinin, although these interactions appear to be of lower affinity than those with the higher molecular weight proteins.     

Identification of low molecular weight GTP-binding proteins and their sites of interaction in subcellular fractions from skeletal muscle

The presence of low molecular weight GTP-binding proteins was investigated in subcellular fractions from skeletal muscle. Skeletal muscle homogenate, transverse tubules, triads, sarcoplasmic reticulum membranes, and cytosol fractions were separated in sodium dodecyl sulfate-gel electrophoresis and blotted onto nitrocellulose. The presence of GTP-binding proteins was explored by incubation of these blots with [alpha-32P] GTP. GTP labeled two polypeptides of Mr = 23,000 and 29,000 in all the fractions examined. Binding of [alpha-32P]GTP was specific and dependent on Mg2+. The 23-kDa polypeptide was labeled to a higher extent with [alpha-32P]GTP than the 29-kDa polypeptide, although both were enriched in transverse tubule fractions. A GTP-binding polypeptide of 40 kDa was also enriched in transverse tubule preparations and identified as Gi alpha by immunostaining with anti-Gi alpha. Using a blot overlay approach and [alpha-32P]GTP-labeled cytosolic components, several polypeptides were identified that interact with the 23- and 29-kDa GTP-binding proteins. Among these components were polypeptides of Mr = 60,000, 47,000, 44,000, 42,000, and 38,000, which were mainly of cytosolic origin but also associated with triads and transverse tubule membranes. The 47-, 44-, 42-, and 38-kDa polypeptides were found to be structurally related to the glycolytic enzymes enolase, 3-phosphoglyceric phosphokinase, aldolase, and glycoeraldehyde-3-phosphate dehydrogenase, respectively. The purified glycolytic enzymes specifically bound the 23- and 29-kDa GTP-binding proteins under both denaturing and nondenaturing conditions. The association of the GTP-binding proteins with these polypeptides was resistant to detergents such as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), Triton X-100, and Tween. A 23-kDa GTP-binding protein purified from chromaffin cells bound to a 157-kDa polypeptide in triads and chromaffin cell membranes. The 157-kDa polypeptide was a minor component in these membranes and not related to the subunits of the dihydropyridine receptor. In view of the proposed function of low molecular weight GTP-binding proteins in processes such as membrane communication and secretion coupling, the association of these proteins with transverse tubules and triads in skeletal muscle is discussed in terms of a role in signal transmission.