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PubMed1992

Non-enzymatic weakening of myofibrillar structures during conditioning of meat: calcium ions at 0.1 mM and their effect on meat tenderization.

The tenderness of meat is set by the properties of connective tissue and myofibrils. Skeletal muscle connective tissues become firm with chronological aging concomitantly with the increase in intermolecular non-reducing cross-links of collagen, and this process toughens meat, however, connective tissues hardly change during conditioning of meat. Therefore, the tenderization of meat during post mortem aging, or to put it more precisely, during post rigor aging, stems for the most part from changes in myofibril structures. My research derives its origin on findings of two kinds of post mortem changes in myofibril structures; i) fragmentation of myofibrils; and ii) restoration of rigor-shortened sarcomeres. These results were published in 1967 [1], and were, thereafter proved by many workers to be closely related to meat tenderization. I report in this paper the essential molecular mechanisms of these phenomena, and of structural changes in connectin or titin filaments. All of them are non-enzymatically induced by 0.1 mM calcium ion, which is the ultimate concentration of sarcoplasmic calcium ion in post mortem muscles.

Calcium↗
PubMed2005

The impact of gastrocnemius muscle cell changes in chronic venous insufficiency.

OBJECTIVE: To investigate the pathological and metabolic changes in the gastrocnemius muscle in patients with chronic vein insufficiency (CVI). METHOD: Thirty-six patients with varicose veins were investigated by ambulatory venous pressure (AVP) and duplex ultrasonography. Twelve age and height-matched controls were used for comparison. Patients and controls consented to participate in this study. Twenty-one patients with primary vein varicose (group AI) and 15 patients (group AII) with primary deep venous valve incompetence (DVI) underwent biopsies of the gastrocnemius muscle during operation. Adductor biopsies obtained from the same limbs served as a control group (group B) and specimens from controls subjects without venous disease served as the second control group (group C). All the specimens were investigated by superoxide dismutase (SOD), nitric oxide (NO), Na+-K+-ATPase, Ca2+-ATPase and lactic acid (LD) determinations. Samples were subjected to light and electron microscopy following H & E staining, special ATPase, cytochrome oxidase/succinate dehydrogenase (COX/SDH) stains. RESULTS: Normal muscle architecture was seen following H & E, ATPase and COX/SDH staining and normal cell metabolism was observed in specimens of groups B and C. In group A, pathological changes were encountered in the gastrocnemius muscle including disseminated myofibril atrophy, cell denaturation and necrosis, inflammatory cell infiltration, proliferation and dilation of interfascicular veins. ATPase staining (pH 9.4) demonstrated grouping of atrophic fibres, especially type I myofibril grouping, accompanied by moderate to severe atrophy of type II muscle fibres. However, no patient had selective type I fibre atrophy. Enhanced enzymatic activity in single or multiple myofibrils was demonstrated by COX/SDH staining in approximately half of the specimens in group AII. In group AII, electron microscopy showed swelling, myelin figure denaturation of mitochondria, disruption of the myofibrils and increased lipid droplets in the gastrocnemius muscle. Increased concentration of LD was found in most specimens from group A patients. There were also reductions of SOD, NO, biochemical activity of Na+-K+-ATPase, Ca2+-ATPase with increasing concentration of LD in these patients, most prominently in group AII. We found correlation between AVP assessments and the biochemical measurements as well as morphological appearances of the gastrocnemius muscle. CONCLUSION: Venous hypertension results in pathophysiological changes in the gastrocnemius muscles of patients with DVI, associated with decreased calf pump function.

Adult↗
PubMed2005

Establishment of cardiac cytoarchitecture in the developing mouse heart.

Cardiomyocytes are characterized by an extremely well-organized cytoarchitecture. We investigated its establishment in the developing mouse heart with particular reference to the myofibrils and the specialized types of cell-cell contacts, the intercalated discs (ICD). Early embryonic cardiomyocytes have a polygonal shape with cell-cell contacts distributed circumferentially at the peripheral membrane and myofibrils running in a random orientation in the sparse cytoplasm between the nucleus and the plasma membrane. During fetal development, the cardiomyocytes elongate, and the myofibrils become aligned. The restriction of the ICD components to the bipolar ends of the cells is a much slower process and is achieved for adherens junctions and desmosomes only after birth, for gap junctions even later. By quantifying the specific growth parameters of prenatal cardiomyocytes, we were able to identify a previously unknown fetal phase of physiological hypertrophy. Our results suggest (1) that myofibril alignment, bipolarization and ICD restriction happen sequentially in cardiomyocytes, and (2) that increase of heart mass in the embryo is not only achieved by hyperplasia alone but also by volume increase of the individual cardiomyocytes (hypertrophy). These observations help to understand the mechanisms that lead to the formation of a functional heart during development at a cellular level.

Adherens Junctions↗
PubMed2006

Focal adhesion kinase is essential for costamerogenesis in cultured skeletal muscle cells.

A central question in muscle biology is how costameres are formed and become aligned with underlying myofibrils in mature tissues. Costameres are composed of focal adhesion proteins, including vinculin and paxillin, and anchor myofibril Z-bands to the sarcolemma. In the present study, we investigated the process of costamere formation ("costamerogenesis") in differentiating primary mouse myoblasts. Using vinculin and paxillin as costameric markers, we found that two additional focal adhesion components, alpha5beta1 integrin and focal adhesion kinase (FAK), are associated with costameres. We have characterized costamerogenesis as occurring in three distinct stages based on the organizational pattern of these costameric proteins. We show that both costamerogenesis and myofibrillogenesis are initiated at sites of membrane contacts with the extracellular matrix and that their maturation is tightly coupled. To test the importance of FAK signaling in these processes, we analyzed cells expressing a dominant negative form of FAK (dnFAK). When cells expressing dnFAK were induced to differentiate, both costamerogenesis and myofibrillogenesis were disrupted although the expression of constituent proteins was not inhibited. Likewise, inhibiting FAK activity by reducing FAK levels using an siRNA approach also resulted in an inhibition of costamerogenesis and myofibrillogenesis. The relationship between costamere and myofibril formation was tested further by treating myotube cultures with potassium or tetrodotoxin to block contraction and disrupt myofibril organization. This also resulted in inhibition of costamere maturation. We present a model of costamerogenesis whereby signaling through FAK is essential for both normal costamerogenesis and normal myofibrillogenesis which are tightly coupled during skeletal myogenesis.

Animals↗
PubMed1984

Changes in cardiac ultrastructure and myofibrillar proteins during ischemia in dogs, with special reference to changes in Z lines.

This study was designed to examine whether or not coronary ligation produces changes in myofibrillar proteins, especially Z line proteins including alpha-actinin and the 55 kDa protein, in the dog. Changes in ultrastructure of the myocardium after coronary ligation were also studied. A branch of the left anterior descending coronary artery was ligated for a period of 3, 6, 24 or 72 h. Coronary ligation produced ultrastructural changes in Z lines (characterized by broader and wavy Z lines) as well as changes in glycogen granules, mitochondria, and nuclei. Myofibrils were isolated from the myocardium that had been made ischemic by coronary ligation. The yield of myofibrils was reduced as the ischemic period was increased. Phase microscopic examination of the isolated myofibrils from the ischemic myocardium revealed that there were spread Z lines with wider I bands, these changes being essentially the same as those observed by the electron microscope in the ischemic myocardium. The effect of coronary ligation on the myofibrillar proteins was studied by means of polyacrylamide gel electrophoresis. In the isolated myofibrils from the myocardium 3 h after coronary ligation, there were decreases in the percentage of actin, alpha-actinin and the 55 kDa protein, and these changes progressively increased with lengthening of the ischemic period. These results suggest that coronary ligation decreases actin, alpha-actinin and the 55 kDa protein, in association with ultrastructural changes of the Z lines.

Actinin↗
PubMed1983

Quantitative determination of myosin and actin in rabbit skeletal muscle.

The myosin and actin content of muscle tissue and purified myofibrils from rabbit psoas muscle has been determined. Myofibrils were purified using Percoll gradients, which allowed rapid separation from nuclei and connective tissue proteins. Myosin and actin were quantitated by amino acid analysis of the appropriate bands from sodium dodecyl sulfate/polyacrylamide gels. Muscle tissue contained 94 and 619 nmol/g wet weight of myosin and actin, respectively, while myofibrils had 0.82 and 5.37 mumol/g protein. Thus myosin contributed 43% and actin 22% of the myofibril protein mass. The value of 2.5 myosins per 14.3 nm repeat as calculated from these results suggests that thick filament models with mixtures of two and three crossbridges per repeat should be considered.

Actins↗
PubMed2000

Kinetics of the initial steps of rabbit psoas myofibrillar ATPases studied by tryptophan and pyrene fluorescence stopped-flow and rapid flow-quench. Evidence that cross-bridge detachment is slower than ATP binding.

The kinetics of the tryptophan fluorescence enhancement that occurs when myofibrils (rabbit psoas) are mixed with Mg-ATP were studied by stopped-flow in different solvents (water, 40% ethylene glycol, 20% methanol) at 4 degrees C. Under relaxing conditions (low Ca(2+)) in water (mu = 0.16 M, pH 7.4) and at high ATP concentrations, the transient was biphasic, giving a k(fast)(max) of 230 s(-)(1) and a k(slow)(max) of 15 s(-)(1). The kinetics of the two phases were compared with those obtained by chemical sampling using [gamma-(32)P]ATP and quenching in acid (P(i) burst experiments: these give unambiguously the ATP cleavage kinetics), or cold Mg-ATP (cold ATP chase: ATP binding kinetics). k(slow) is due to ATP cleavage, as with S1. Interestingly, k(fast) is slower than the ATP binding kinetics. Instead, this constant appears to report ATP-induced cross-bridge detachment from actin because (1) it was identical to the fluorescence transient obtained on addition of ATP to pyrene-labeled myofibrils; (2) when the initial filament overlap in the myofibrils was decreased, the amplitude of the fast phase decreased; (3) there was no fluorescent enhancement upon the addition of ADP to myofibrils. This is different from the situation with S1 or actoS1 where there was also a fast fluorescent ATP-induced transient but whose kinetics were identical to those of the tight ATP binding. To increase the time resolution and to confirm our results, we also carried out transient kinetics in ethylene glycol and methanol. We interpret our results by a scheme in which a rapid equilibrium between attached (AM.ATP) and detached (M.ATP) states is modulated by the fraction of myosin heads in rigor (AM) during the time of experiment.

Adenosine Triphosphatases↗
PubMed1992

Ca(2+)-activated myofibrillar ATPase: transient kinetics and the titration of its active sites.

The transient kinetics of rabbit psoas Ca(2+)-activated myofibrillar Mg(2+)-ATPase were studied in a buffer of near physiological ionic strength at 4 degrees C by the rapid flow quench technique. The initial ATP binding steps were studied by the ATP chase and the cleavage and release of products steps were studied by the Pi burst method. The data obtained were interpreted by the simple scheme [formula; see text] represents the myosin heads with or without actin interaction. The constants obtained with myofibrils (where the molecules are highly organized) were compared with those with myosin subfragment 1 (S1) and cross-linked acto-S1 (where the molecules are dispersed in solution). Myofibrils appear to bind ATP as tightly as do S1 and cross-linked acto-S1. This suggests that with them k-2 less than kcat much less than k2, and it is proposed that the ATP chase method can be used to titrate the ATPase sites in myofibrils. The results of titration and single-turnover experiments revealed that myofibrils may contain partially active myosin heads. It is proposed that these heads bind ATP loosely without hydrolysis, as found with S1 [Tesi, C., N. Bachouchi, N., Barman, T., & Travers, F. (1989) Biochimie 71, 363-372]. There were large Pi bursts with the three preparations, showing that with all of them the release of products step (k4) is rate limiting.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1976

A Ca2+-activated protease possibly involved in myofibrillar protein turnover. Partial characterization of the purified enzyme.

The purified Ca2+-activated protease (CAF) isolated from porcine skeletal muscle and capable of removing Z-disks from intact myofibrils is optimally active on either myofibril or casein substrates at pH 7.5 and in the presence of 1 mM Ca2+ and at least 2 mM 2-mercaptoethanol. No CAF activity is detected when 1 mM Mg2+, Mn2+, Ba2+, Co2+, Ni2+, and Fe2+ are added singly. When added with 1 mM Ca2+, Co2+, Cu2+, Ni2+, and Fe2+ inhibit, whereas Mg2+, Mn2+, and Ba2+ have no effect on CAF activity. CAF is irreversibly inhibited by iodoacetate but is unaffected by soybean trypsin inhibitor. S0/20,W=5.90 S, and sedimentation equilibrium molecular weight - 112 000 for purified CAF. Because purified CAF migrates as two polypeptide chains with molecular weights of 80 000 and 30 000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the CAF molecule must consist of one each of these two polypeptide chains. Approximate molecular dimensions of 38 X 220 A can be calculated for CAF from calibrated gel permeation column data or from S0/20,W and the molecular weight. Amino acid composition and physical properties of purified CAF distinguish it from the known catheptic enzymes and from other proteases found in blood or in granulocytes. Purified CAF removes Z-disks the 400-A periodicity associated with troponin in the I band and partly degrades M lines but causes no other ultrastructurally detectable effects when incubated with myofibrils. These results agree with the earlier finding that purified CAF degrades troponin, tropomyosin, and C-protein but has no effect on myosin, actin, or alpha-actinin, and suggest that CAF may have a physiological role in disassembly of intact myofibrils during metabolic turnover of myofibrillar proteins.

Amino Acids↗
PubMed2002

Cardiac titin: molecular basis of elasticity and cellular contribution to elastic and viscous stiffness components in myocardium.

Myocardium resists the inflow of blood during diastole through stretch-dependent generation of passive tension. Earlier we proposed that this tension is mainly due to collagen stiffness at degrees of stretch corresponding to sarcomere lengths (SLS) > or = 2.2 microns, but at shorter lengths, is principally determined by the giant sarcomere protein titin. Myocardial passive force consists of stretch-velocity-sensitive (viscous/viscoelastic) and velocity-insensitive (elastic) components; these force components are seen also in isolated cardiac myofibrils or skinned cells devoid of collagen. Here we examine the cellular/myofibrillar origins of passive force and describe the contribution of titin, or interactions involving titin, to individual passive-force components. We construct force-extension relationships for the four distinct elastic regions of cardiac titin, using results of in situ titin segment-extension studies and force measurements on isolated cardiac myofibrils. Then, we compare these relationships with those calculated for each region with the wormlike-chain (WLC) model of entropic polymer elasticity. Parameters used in the WLC calculations were determined experimentally by single-molecule atomic force-microscopy measurements on engineered titin domains. The WLC modelling faithfully predicts the steady-state-force vs. extension behavior of all cardiac-titin segments over much of the physiological SL range. Thus, the elastic-force component of cardiac myofibrils can be described in terms of the entropic-spring properties of titin segments. In contrast, entropic elasticity cannot account for the passive-force decay of cardiac myofibrils following quick stretch (stress relaxation). Instead, slower (viscoelastic) components of stress relaxation could be simulated by using a Monte-Carlo approach, in which unfolding of a few immunoglobulin domains per titin molecule explains the force decay. Fast components of stress relaxation (viscous drag) result mainly from interaction between actin and titin filaments; actin extraction of cardiac sarcomeres by gelsolin immediately suppressed the quickly decaying force transients. The combined results reveal the sources of velocity sensitive and insensitive force components of cardiomyofibrils stretched in diastole.

Actin Cytoskeleton↗
PubMed2004

Effect of sarcomere length on step size in relaxed rabbit psoas muscle.

Recent experiments have shown that shortening and stretching of sarcomeres in single activated and unactivated myofibrils occur in stepwise fashion (Yang et al. (1998) Biophys J 74: 1473-1483; Blyakhman et al. (2001) Biophys J 81: 1093-1100; Yakovenko et al. (2002) Am J Physiol Cell Physiol 283: 735-742). Here, we carried out measurements on single myofibrils from rabbit psoas muscle to investigate steps in unactivated specimens in more detail. Activated and unactivated myofibrils were released and stretched in ramp-like fashion. The time course of length change in the single sarcomere was consistently stepwise. We found that in the unactivated myofibrils, step size depended on initial sarcomere length, diminishing progressively with increase of initial sarcomere length, whereas in the case of activated sarcomeres, step size was consistently 2.7 nm.

Animals↗
PubMed1978

Kinetics and regulation of the myofibrillar adenosine triphosphatase.

1. The steady-state kinetic behaviour of the ATPase (adenosine triphosphatase) of intact myofibrils was studied in the presence of both high and low concentrations of Ca2+ (0.25 mM and less than 10 nM respectively). 2. Kinetic data were collected over the initial linear phase of the assay, which lasts for 20--60s. To obtain consistent data we found it necessary to use either fresh myofibril preparations or preparations that had been stored in the presence of thiol compounds. 3. When assayed in the presence of 0.25 mM-Ca2+, the myofibrillar ATPase obeyed Michaelis-Menten kinetics over the range 0.03--5.0 mM-MgATP (Km 16 +/- 6 micrometer, V 0.4 +/- 0.1 mumol/min per mg). 4. At low Ca2+ concentrations (less than 10 nM) the myofibrillar ATPase displayed pronounced substrate inhibition, which was not observed at high Ca2+ concentrations. Thus increasing the MgATP concentration had the net effect of decreasing the ATPase activity at low Ca2+ relative to that at high Ca2+. This preferential effect of MgATP on the low-Ca2+ ATPase may be important in Ca2+ control. 5. The substrate inhibition that was observed at low Ca2+ was lost on storage or thiol modification of the myofibrils. 6. Under physiological conditions (2 mM-MgATP, I 0.15, pH 7.0), the ATPase of fresh and thiol-protected myofibrils displayed approx. 100-fold activation by Ca2+.

Adenosine Triphosphatases↗
PubMed1982

Effects of taxol and Colcemid on myofibrillogenesis.

To determine the relationship between thin filaments, Z-bands, microtubules, intermediate filaments (IFs), T-tubules, and sarcoplasmic reticulum (SR) during myofibrillogenesis, myotubes were selectively depleted of their myofibrils with 12-tetradecanoylphorbol 13-acetate (TPA) and then were allowed to regenerate in (i) normal medium, (ii) taxol, and (iii) Colcemid. Myofibrils assembled in normal medium formed typical A-, I-, Z-, M-, and H-bands and associated IFs, T-tubules, and SR. Myofibrils assembled in taxol formed "A-bands" of aligned thick filaments interdigitating with long microtubules and "I-bands" consisting only of microtubules. These unprecedented sarcomeres lacked thin filaments, Z-bands, and associated IFs and SR. "Solitary A-bands," consisting exclusively of laterally aligned bipolar thick filaments 1.6 microM in length without either thin filaments or microtubules, were observed. Myofibrils assembled in Colcemid formed all myofibrillar components in the absence of microtubules but these did not achieve rigorous lateral alignment. Colcemid and taxol induced the formation of patchy Z-bands that invariably served as insertion sites for thin filaments, irrespective of the presence or absence of adjacent thick filaments. Z-bands may function as actin-organizing centers for each sarcomere.

Actins↗
PubMed1997

Acceleration of post-mortem changes in Tsaiya duck (Anas platyrhynchos) breast muscle by lactic acid marination.

1. The effect of lactic acid marination at 5 degrees C on post mortem changes in breast muscle pectoralis major of spent layer Tsaiya duck was studied. 2. Myofibrils were prepared from 0.1 M and 0.2 M lactic acid marinated muscle and control (non-marinated samples) sampled at 0, 1, 3, 7 and 14 d post mortem. 3. Changes in myofibril fragmentation index (MFI), myofibrillar proteins and Z-line structure were examined. 4. Marination of duck breast muscle in lactic acid at 5 degrees C enhanced fragmentation of myofibrils and degradation of myofibrillar proteins and Z-line structure as compared to control samples. 5. In summary, lactic acid marination at 5 degrees C can accelerate the post mortem degradation of myofibrils in Tsaiya duck breast muscle.

Actinin↗
PubMed1985

Does actin bind to the ends of thin filaments in skeletal muscle?

We examined whether or not purified actin binds to the ends of thin filaments in rabbit skeletal myofibrils. Phase-contrast, fluorescence, and electron microscopic observations revealed that actin does not bind to the ends of thin filaments of intact myofibrils. However, in I-Z-I brushes prepared by dissolving thick filaments at high ionic strength, marked binding of actin to the free ends, i.e., the pointed ends, of thin filaments was observed when actin was added at an early phase of polymerization. As the polymerization of actin proceeded, the binding efficiency decreased. The critical actin concentration for this binding was higher than that for polymerization in solution. The binding of G-actin was not observed at low ionic strength. On the basis of these results, we suggest that a particular structure suppressing the binding of actin is present at the free ends of thin filaments in intact myofibrils and that a part of the end structure population is eliminated or modified at high ionic strength so that further binding of actin becomes possible. The myofibril and I-Z-I brush appear to be useful systems for studies aimed at elucidating the organizational mechanisms of actin filaments in vivo.

Actins↗
PubMed1985

Distributions of vimentin and desmin in developing chick myotubes in vivo. II. Immunoelectron microscopic study.

The distribution of the intermediate filament proteins vimentin and desmin in developing and mature myotubes in vivo was studied by single and double immunoelectron microscopic labeling of ultrathin frozen sections of iliotibialis muscle in 7-21-d-old chick embryos, and neonatal and 1-d-old postnatal chicks. This work is an extension of our previous immunofluorescence studies of the same system (Tokuyasu, K. T., P. A. Maher and S. J. Singer, 1984, J. Cell Biol., 98:1961-1972). In immature myotubes of 7-11-d embryos, significant labeling for desmin and vimentin was found only in intermediate filaments, and these proteins coexisted in the same individual filaments. Each of the two proteins was present in irregular clusters along the entire length of a filament. No exclusively vimentin- or desmin-containing filaments were observed at this stage. In the early myotubes, the intermediate filaments were essentially all longitudinally oriented, even when they contained three times as much desmin as vimentin. No special relationship was recognized between the dispositions of the filaments and the organization of the myofibrils. Occasionally, several myofibrils were already aligned in lateral registry at this early stage, but labeling for desmin and vimentin was largely absent at the level of the Z bands. Instead, the Z bands appeared to be covered by elements of the sarcoplasmic reticulum. The confinement of intermediate filaments to the level of the Z bands occurred in the myotubes of later embryos after the extensive lateral registry of the Z bands. Thus, intermediate filaments are unlikely to play a primary role in producing the lateral registration of myofibrils during myogenesis, but may be important in determining the polarization of the early myotube and the alignment of its organelles. Throughout the development of myotubes, desmin and vimentin remained in the form of intermediate filaments, although the number of filaments per unit volume of myotube appeared to be reduced as myofibrils increased in number in maturing myotubes. This observation indicated that the transverse orientation of intermediate filaments in mature myotubes does not result from the de novo polymerization of subunits from Z band to Z band, but a continuous shifting of the positions and directions of intact filaments.

Animals↗
PubMed1988

Intracellular targeting of isoproteins in muscle cytoarchitecture.

Part of the muscle creatine kinase (MM-CK) in skeletal muscle of chicken is localized in the M-band of myofibrils, while chicken heart cells containing myofibrils and BB-CK, but not expressing MM-CK, do not show this association. The specificity of the MM-CK interaction was tested using cultured chicken heart cells as "living test tubes" by microinjection of in vitro generated MM-CK and hybrid M-CK/B-CK mRNA with SP6 RNA polymerase. The resulting translation products were detected in injected cells with isoprotein-specific antibodies. M-CK molecules and translation products of chimeric cDNA molecules containing the head half of the B-CK and the tail half of the M-CK coding regions were localized in the M-band of the myofibrils. The tail, but not the head portion of M-CK is essential for the association of M-CK with the M-band of myofibrils. We conclude that gross biochemical properties do not always coincide with a molecule's specific functions like the participation in cell cytoarchitecture which may depend on molecular targeting even within the same cellular compartment.

Amino Acid Sequence↗
PubMed1989

Visualization of the polarity of isolated titin molecules: a single globular head on a long thin rod as the M band anchoring domain?

TII, the extractable form of titin, was purified from myofibrils and separated by high resolution gel permeation chromatography into two fractions (TIIA and TIIB). Novel specimen orientation methods used before metal shadowing and EM result in striking pictures of the two forms. Molecules layered on mica become uniformly oriented when subjected to centrifugation. TIIB comprises a very homogeneous fraction. All molecules reveal a single globular head at one end on a long and very thin rod of uniform diameter. The lengths of the rods have a very narrow distribution (900 +/- 50 nm). TIIA molecules seem lateral oligomers of TIIB, attached to each other via the head regions. While dimers are the predominant species, trimers and some higher oligomers can also be discerned. Mild proteolysis destroys the heads and converts TIIA and TIIB into TIIB-like rods. Similar molecules also result from titin purified from myofibrils by certain established purification schemes. Headless titin molecules show in gel electrophoresis only the TII band, while head bearing molecules give rise to two additional polypeptides at 165 and 190 kD. Immunoelectron microscopy of myofibrils identifies both titin-associated proteins as M band constituents. We speculate that in the polar images of TII the globular head region corresponds to the M band end of the titin molecules. This hypothesis is supported by immunoelectron micrographs of TIIB molecules using titin antibodies of known epitope location in the half sarcomere. This proposal complements our previous immunoelectron microscopic data on myofibrils. They showed that epitopes present only on the nonextractable TI species locate to the Z line and its immediately adjacent region (Fürst, D. O., M. Osborn, R. Nave, and K. Weber. 1988. J. Cell Biol. 106:1563-1572). Thus, the two distinct ends of the titin molecule attach to Z and M band material respectively.

Animals↗