Distribution of antibodies to the troponin complex,troponin-C and troponin-I in chicken skeletal muscle as determined by a simplified method for immuno-electron microscopy

Antisera against the troponin complex, troponin-C and troponin-I have been utilized to locate these proteins in normal, adult chicken skeletal muscle and in filaments prepared from chicken acetone dried powder. The antisera had been previously characterized by immunochemical methods and were employed to ascertain the distribution of the proteins by a simple method for immuno-electron microscopy. Glycerinated chicken breast muscle was treated with the antisera, unreacted antibody was washed from the muscle, and a goat anti-rabbit γ-globulin was added to enhance the electron density of the antigen-antibody complexes. A periodic distribution of anti-troponin-C at a mean interval of 389 Å was observed along the thin filaments in the sectioned tissue. Anti-troponin-I was deposited every 399 Å (P < 0.01). Thin filaments were prepared from acetone dried powder and reacted with the antisera. The anti-troponin-C was located every 386 Å; anti-troponin-I, every 399 Å (P < 0.01). Our technique for immuno-electron microscopy is compared with that used by others, and the significance of the findings is discussed.     

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”.     

Visualization of monoclonal antibody binding to tropomyosin on native smooth muscle thin filaments by electron microscopy

We have used three different monoclonal antibodies (LCK16, JLH2 and JLF15) to tropomyosin for the localization of tropomyosin molecules within smooth muscle thin filaments. Thin filaments were incubated with monoclonal antibodies and visualized by negative staining electron microscopy. All three monoclonal antibodies caused the aggregation of thin filaments into ordered bundles, which displayed cross-striations with a periodicity of 37 ± 1 nm. In contrast, conventional rabbit antiserum to tropomyosin distorted and aggregated the thin filaments without generating cross-striations. Therefore, monoclonal antibodies to tropomyosin allow us, for the first time, to observe directly the distribution of tropomyosin molecules along the thin filaments of smooth muscle cells. The binding sites of the antibodies to skeletal muscle tropomyosin were examined by decorating tropomyosin paracrystals with monoclonal antibodies. The LCK16 monoclonal antibody binds the narrow band of tropomyosin paracrystals, whereas the JLF15 antibody binds the wide band of tropomyosin paracrystals.     

The structure of A segments from glycerinated skeletal muscle.

The A band of skeletal muscle consists of an array of thick myosin-containing filaments along with non-myosin proteins such as C protein and M line protein. In order to study the arrangement of the myosin and non-myosin components, A segments which are aggregations of thick filaments held together at the M line were prepared from glycerinated chicken pectoral and rabbit psoas muscles and examined by electron microscopy. Details of the preparative technique and comparison of the morphologies of A segments and I segments are provided. The A segments from chicken pectoral muscle exhibited 11 to 12 stripes in each half lateral to the bare zone. Several less distinct bands as well as subdivisions of the individual stripes were also observed. The periodicity of the major stripes in the A segments was 424 +/- 10 A. The A segments prepared from rabbit psoas muscle had a periodicity of 432 +/- 13 A, but in contrast with chicken A segments, fewer rabbit A segments showed this periodicity. We conclude that A segments can be separated from glycerinated chicken and rabbit skeletal muscles and compare our results with those of others who prepared A segments from frog and rabbit skeletal muscles in the absence of glycerol.     

Detection and isolation of a 30 kDa abnormal protein in avian dystrophic muscle

We have studied the protein composition of the pectoralis superficialis muscle of genetically dystrophic (New Hampshire line 413) and normal control (line 412) chickens by one- and two-dimensional gel electrophoresis. A protein, referred to hereafter as the 30 kDa abnormal protein, was specifically detected in the affected muscle. It was purified to homogeneity, and its molecular properties were studied. It is a monomer with a molecular mass of approximately 30 kDa and an isoelectric point of about pI 8.4. We have screened by Western blotting a variety of muscles from line 412 and line 413 chickens for the presence of the 30 kDa protein. While the pattern of total protein is very similar in all cases, the 30 kDa protein was not detected in the pectoralis superficialis muscle of line 412 chickens. However, the immunoreactive bands were detected in the sartorius muscle and the tensor fasciae latae muscle from dystrophic and normal chickens. Interestingly, the immunoreactive bands of normal skeletal muscles are smaller in molecular weight than those of dystrophic skeletal muscles. To determine the early time sequence of the appearance of the abnormal protein, we studied muscles from embryos and post-hatched chickens at various ages. The abnormal protein was detected in dystrophic muscles as early as 15 days ex ovo and occurred throughout development up to six months ex ovo. Although the implication of the dystrophy-associated appearance of the 30 kDa protein in the affected muscle is not clear at present, it would be of particular interest to elucidate the biochemical functions of the 30 kDa protein in the affected muscle (pectoralis superficialis muscle) of genetically dystrophic chicken.     

The location and periodicity of a troponin-T-like protein in the myofibril of the horseshoe crab Limulus polyphemus

Localization studies show that a 59,000 M_r, protein from Limulus muscle, thought to be a troponin-T-like protein, is associated with thin filaments. Immunoelectron microscopy indicates that its periodicity on thin filaments is 380 to 390 Å, a distribution characteristic of proteins comprising the troponin complex.     

A Mechanistic Model of Ca Regulation of Thin Filaments in Cardiac Muscle

We present a model of Ca-regulated thin filaments in cardiac muscle where tropomyosin is treated as a continuous elastic chain confined in the closed position on the actin helix by electrostatic forces. The main distinction from previous works is that the intrinsic stress-free helical shape of the tropomyosin chain was taken into account explicitly. This results in the appearance of a new, to our knowledge, tension-like term in the energy functional and the equilibrium equation. The competitive binding of calcium and the mobile segment of troponin-I to troponin-C were described by a simple kinetic scheme. The values of dimensionless model parameters were estimated from published data. A stochastic Monte Carlo simulation of calcium curves has been performed and its results were compared to published data. The model explains the high cooperativity of calcium response of the regulated thin filaments even in the absence of myosin heads. The binding of myosin heads to actin increases the calcium sensitivity while not affecting its cooperativity significantly. When the presence of calcium-insensitive troponin-C was simulated in the model, both calcium sensitivity and cooperativity decreased. All these features were previously observed experimentally.     

A Mechanistic Model of Ca Regulation of Thin Filaments in Cardiac Muscle

We present a model of Ca-regulated thin filaments in cardiac muscle where tropomyosin is treated as a continuous elastic chain confined in the closed position on the actin helix by electrostatic forces. The main distinction from previous works is that the intrinsic stress-free helical shape of the tropomyosin chain was taken into account explicitly. This results in the appearance of a new, to our knowledge, tension-like term in the energy functional and the equilibrium equation. The competitive binding of calcium and the mobile segment of troponin-I to troponin-C were described by a simple kinetic scheme. The values of dimensionless model parameters were estimated from published data. A stochastic Monte Carlo simulation of calcium curves has been performed and its results were compared to published data. The model explains the high cooperativity of calcium response of the regulated thin filaments even in the absence of myosin heads. The binding of myosin heads to actin increases the calcium sensitivity while not affecting its cooperativity significantly. When the presence of calcium-insensitive troponin-C was simulated in the model, both calcium sensitivity and cooperativity decreased. All these features were previously observed experimentally.     

Ultrastructural features of cardiac muscle cells in a tarantula spider

The ultrastructural features of cardiac muscle cells and their innervation were examined in the tarantula spider Eurypelma marxi Simon. The cells are transversely striated and have an A band length of about three microns. H zones are indistinct and M lines are absent. Thick and thin myofilament diameters are approximately 200 and 70 Å respectively. Eight to 12 thin filaments usually surround each thick one. Accumulations of thick and thin myofilaments occur perpendicular to the bulk of the myofilaments in some cells. The Z line is discontinuous and thick filaments may pass through the spaces in the Z line. Extensive systems of sarcoplasmic reticulum and transverse tubules are present; these form numerous dyadic junctions in both A and I band regions. Sarcolemmal invaginations form Z line tubules; lateral extensions of the plasma membrane portion of these invaginations form dyads. Nerve branches of the cardiac ganglion make multiple neuromuscular synapses with at least some of the cardiac muscle cells. Both large granular and small agranular vesicles are present in the presynaptic terminals. Intercalated discs similar to those present in other arthropod hearts occur between the ends of adjacent cardiac muscle cells.     

Distribution of fast myosin heavy chain isoforms in thick filaments of developing chicken pectoral muscle

Colloidal gold-conjugated monoclonal antibodies were prepared to stage-specific fast myosin heavy chain (MHC) isoforms of developing chicken pectoralis major (PM). Native thick filaments from different stages of development were reacted with these antibodies and examined in the electron microscope to determine their myosin isoform composition. Filaments prepared from 12-d embryo, 10-d chick, and 1-yr chicken muscle specifically reacted with the embryonic (EB165), neonatal (2E9), and adult (AB8) antimyosin gold-conjugated monoclonal antibodies, respectively. The myosin isoform composition was more complex in thick filaments from stages of pectoral muscle where more than one isoform was simultaneously expressed. In 19-d embryo muscle where both embryonic and neonatal isoforms were present, three classes of filaments were found. One class of filaments reacted only with the embryonic antibody, a second class reacted only with the neonatal-specific antibody, and a third class of filaments were decorated by both antibodies. Similar results were obtained with filaments prepared from 44-d chicken PM where the neonatal and adult fast MHCs were expressed. These observations demonstrate that two myosin isoforms can exist in an individual thick filament in vivo. Immunoelectron microscopy was also used to determine the specific distribution of different fast MHC isoforms within individual filaments from different stages of development. The anti-embryonic and anti-adult antibodies uniformly decorated both homogeneous and heterogeneous thick filaments. The neonatal specific antibody uniformly decorated homogeneous filaments; however, it preferentially decorated the center of heterogeneous filaments. These observations suggest that neonatal MHC may play a specific role in fibrillogenesis.     

DIRECT ISOLATION OF NATIVE THIN FILAMENTS FROM EMBRYONIC MUSCLE CELLS*

A method is presented for the release of “native” thin filaments from 13-day old embryonic chick muscle without tryptic digestion or desoxycholate (DOC) solubilization of Z bands. The isolated filaments were 50–60 Å in diameter, of variable length, and formed “arrowhead-like” complexes with heavy meromyosin (HMM). In addition, the filaments interacted with purified myosin to form actomyosin as effectively as action extracted from an acetone powder of muscle. The Mg⁺⁺-dependent ATPase activity and extent of superprecipitation of the synthetic actomyosin required a low concentration of Ca⁺⁺, strongly suggesting the presence of troponin and tropomyosin on the thin filaments isolated from muscle at this stage of embryogenesis. The native thin filaments were more sensitive to trypsin than synthetic F-actin prepared from an acetone powder based on measurements of flow birefrengence, viscosity and the ability to activate myosin ATPase.     

Specificity of desmin to avian and mammalian muscle cells.

The extent of invariance and heterogeneity in desmin, the major component of the muscle form of 100 Å filaments, has been investigated in avian and mammalian muscle and nonmuscle cells with two-dimensional gel electrophoresis and indirect immunofluorescence. Desmin from chick, duck and quail, smooth, skeletal and cardiac muscle cells is resolved into two isoelectric variants, α and β, with each possessing the same charge and electrophoretic mobility in all three avian species irrespective of muscle type. Guinea pig and rat muscle desmin resolves into only one variant; it also possesses the same charge and electrophoretic mobility in the two mammalian species, but it is more acidic and slower in electrophoretic mobility than the two avian variants.In immunofluorescence, desmin is localized together with α-actinin along myofibril Z lines. Antibodies to chick smooth muscle desmin, prepared against the protein purified by preparative SDS gel electrophoresis prior to immunization, cross-react with myofibril Z lines in all three avian species. These antibodies do not cross-react with either rat or guinea pig myofibril Z lines. Similarly, they do not cross-react with avian or mammalian nonmuscle cells grown in tissue culture and known to contain cytoplasmic 100 Å filaments.These results demonstrate that desmin is highly conserved within avian muscle cells and within mammalian muscle cells. It is, however, both biochemically and immunologically distinguishable between avian and mammalian muscle cells, and between muscle and nonmuscle cells. We conclude that there are biochemically and immunologically specific forms of desmin for avian and mammalian muscle cells. Furthermore, within a particular vertebrate species, there are at least two separate classes of 100 Å filaments: the muscle class whose major component is desmin, and the nonmuscle class whose major component is distinct from desmin. Taking into consideration the immunological specificity reported by other laboratories for the 100 Å filaments in glial cells, for neurofilaments and for the epidermal 80 Å keratin filaments, we propose that a given vertebrate species contains at least four major distinguishable classes of 100 Å filaments: muscle 100 Å filaments (desmin filaments), glial filaments, neurofilaments and epidermal keratin filaments.     

The organization and fine structure of the muscles of the scolex of the cysticercoid of Hymenolepis microstoma

The organization and fine structure of the muscles of the scolex of the cysticercoid of Hymenolepis microstoma are described. The contractile apparatus consists of thick (175–325 Å diameter × 1.4 μm) and thin (60–80 Å diameter × 1 μm) filaments. The thick filaments are occasionally attached to the thin filaments by cross bridges. The thin filaments are attached to the dense bodies or to a dense zone at the sarcolemma at muscle insertions. In contracted muscle the thick filaments appear as quasi-hexagonal arrays or in lines. Each thick filament is surrounded by an orbit of up to 12 thin filaments, which in turn may be shared by adjacent thick filaments. Thin filaments may be present in quasi-rectangular or hexagonal groupings indicating some low order degree of actin lattice. The fusiform dense bodies (1,500 Å × 900 Å), consisting of up to 25 discrete substructures, are distributed uniformly throughout the myofiber and/or attached to the sarcolemma at attachment plaques. The sarcoplasmic reticulum, consisting of a presumed anastomosing network of tubules is structurally connected to the sarcolemma by periodic deposits of electron opaque material. Sarcoplasmic extensions of the myofiber(s) contain the nucleus, Golgi complexes, rough endoplasmic reticulum, ribosomes, β-glycogen, mitochondria and membrane bound electron dense structures. Upon activation of the metacestode, groups of α-glycogen and enlargement of the rough endoplasmic reticulum were observed. Microtubules which were conspicuously absent from the sarcoplasm of the unactivated worms appeared adjacent to the myofibers in activated worms.     

The stoichiometry and location of troponin I- and troponin C-like proteins in the myofibril of the bay scallop, Aequipecten irradians.

Localization and quantification studies were carried out on bay-scallop (Aequipecten irradians) striated-muscle troponin C- and troponin I-like proteins. Indirect immunofluorescence microscopy of scallop myofibrils stained with either rabbit anti-(scallop troponin I) or anti-(scallop troponin C) antibodies shows staining of all I-bands observed. The results of quantification studies using sodium dodecyl sulfate poly-acrylamide-gel electrophoresis of untreated scallop myofibrils, washed scallop myofibrils, and isolated scallop thin filaments indicate an actin/tropomyosin/troponin-C molar rationn of 7:1:1. The molar ratio for troponin I could not be determined in untreated myofibrils because of interfering bands; in washed myofibrils a value of 0.6 mol of troponin I/mol of tropomyosin was found. Purified scallop troponin C binds Ca2+ and interacts with scallop troponin I to relieve troponin I-induced inhibition of actomyosin ATPase. Although scallop troponin C is an acidic protein, it appears to be less acidic than troponin C from higher organisms. A calmodulin-like protein has been isolated from scallop striated muscle that activates bovine brain phosphodiesterase to the same extent as does brain calmodulin. Its amino acid composition and its electrophoretic mobility on alkaline 6 M-urea/polyacrylamide gels differs from that of scallop troponin C, and it appears not to be associated with thin filaments.     

Myofibrillar protein degradation in the chicken. 3-Methylhistidine release in vivo and in vitro in normal and genetically muscular-dystrophic chickens

Myofibrillar protein degradation was measured in 4-week-old normal (line 412) and genetically muscular-dystrophic (line 413) New Hampshire chickens by monitoring the rates of 3-methylhistidine excretion in vivo and in vitro. A method of perfusing breast and wing muscles was developed and the rate of 3-methylhistidine release in vitro was measured between 30 and 90min of perfusion. During this perfusion period, 3-methylhistidine release from the muscle preparation was linear, indicating that changes in 3-methylhistidine concentration of the perfusate were the result of myofibrillar protein degradation. Furthermore, the viability of the perfused muscle was maintained during this interval. After 60min of perfusion, ATP, ADP and creatine phosphate concentrations in pectoral muscle were similar to muscle freeze-clamped in vivo. Rates of glucose uptake and lactate production were constant during the perfusion. In dystrophic-muscle preparations, the rate of 3-methylhistidine release in vitro (nmol/h per g of dried muscle) was elevated 2-fold when compared with that in normal muscle. From these data the fractional degradation rates of myofibrillar protein in normal and dystrophic pectoral muscle were calculated to be 12 and 24% respectively. Daily 3-methylhistidine excretion (nmol/day per g body wt.) in vivo was elevated 1.35-fold in dystrophic chickens. Additional studies revealed that the anti-dystrophic drugs diphenylhydantoin and methylsergide, which improve righting ability of dystrophic chickens, did not alter 3-methylhistidine release in vitro. This result implies that changes in myofibrillar protein turnover are not the primary lesion in avian muscular dystrophy. From tissue amino acid analysis, the myofibrillar 3-methylhistidine content per g dry weight of muscle was similar in normal and dystrophic pectoral muscle. More than 96% of the 3-methylhistidine present in pectoral muscle was associated with the myofibrillar fraction. Dystrophic myofibrillar protein contained significantly less 3-methylhistidine (nmol/g of myofibrillar protein) than protein from normal muscle. This observation supports the hypothesis that there may be a block in the biochemical maturation and development of dystrophic muscle after hatching. Free 3-methylhistidine (nmol/g wet wt.) was elevated in dystrophic muscle, whereas blood 3-methylhistidine concentrations were similar in both lines. In summary, the increased myofibrillar protein catabolism demonstrated in dystrophic pectoral muscle correlates with the increased lysosomal cathepsin activity in this tissue as reported by others.     

The levels of creatine kinase and adenylate kinase in the plasma of dystrophic chickens reflect the rates of loss of these enzymes from the circulation

The rates of loss of adenylate kinase and creatine kinase from the circulation after intravenous injection of homogenous chicken skeletal muscle enzymes were examined to determine the role of plasma clearance rates in determining the plasma levels of these enzymes in normal and dystrophic chickens. The rapid clearance of adenylate kinase activity (average half-life of 5 min) and the slower biphasic clearance of creatine kinase activity (average half-lives of 0.95 and 11 hr) are consistent with the elevation of creatine kinase but not adenylate kinase in the blood plasma of dystrophic chickens compared to normal chickens. The rates of clearance of these enzymes were similar in normal chickens compared to dystrophic chickens. Radioiodinated enzymes were cleared at similar, but slightly more rapid rates than the loss of enzyme activity. The loss of adenylate kinase activity from the circulation may be due in part to inactivation since adenylate kinase activity is rapidly inactivated in serum in vitro, and because no increase in adenylate kinase activity is observed in the most specific sites of clearance of the radioiodinated enzyme, the liver and spleen. The comparison of enzyme activities in press juices to the activities in high-ionic-strength homogenates of muscle tissue from normal and dystrophic muscle, indicates that adenylate kinase activity is not associated with intracellular structures to the extent that would prohibit release from dystrophic muscle tissue. These results, and those presented previously with regard to plasma levels and clearance rates of AMP aminohydrolase and pyruvate kinase in normal and dystrophic chickens (11) support our hypothesis that the rates of loss of muscle enzyme activities from the circulation are important in determining the circulating levels of muscle enzymes in dystrophic chickens. Furthermore, from the measurement of plasma levels and clearance rates of creatine kinase, it was estimated that the efflux rate of creatine kinase from dystrophic muscle tissue is 2.0% of the total breast muscle creatine kinase per day.     

Elevation of calmodulin in avian muscular dystrophy

In an attempt to understand the mechanism of calcium accumulation in myopathies, changes in the major calcium-binding protein, calmodulin, was studied in genetically dystrophic chickens. Measurements by radioimmunoassay revealed an increase in the calmodulin concentration of dystrophic chicken muscles. Poly A-containing RNA(s) of fast and slow muscles from the normal and dystrophic chicks were hybridized with [32P]-labeled calmodulin cDNA probe by the dot-hybridization technique. Densitometric scan of the autoradiogram showed that the calmodulin mRNA levels of dystrophic fast muscles (pectoralis and posterior latissimus dorsi) were approximately two-fold higher than those of the corresponding normal muscles. No significant change in calmodulin and calmodulin messenger RNA of slow muscle (ALD) was found in dystrophic chickens. Our results suggest that increased calcium flux within the dystrophic muscle may be modulated by calmodulin.     

Connectin filaments in stretched skinned fibers of frog skeletal muscle

Indirect immunofluorescence microscopy of highly stretched skinned frog semi-tendinous muscle fibers revealed that connectin, an elastic protein of muscle, is located in the gap between actin and myosin filaments and also in the region of myosin filaments except in their centers. Electron microscopic observations showed that there were easily recognizable filaments extending from the myosin filaments to the I band region and to Z lines in the myofibrils treated with antiserum against connectin. In thin sections prepared with tannic acid, very thin filaments connected myosin filaments to actin filaments. These filaments were also observed in myofibrils extracted with a modified Hasselbach-Schneider solution (0.6 M KCl, 0.1 M phosphate buffer, pH 6.5, 2 mM ATP, 2 mM MgCl2, and 1 mM EGTA) and with 0.6 M Kl. SDS PAGE revealed that connectin (also called titin) remained in extracted myofibrils. We suggest that connectin filaments play an important role in the generation of tension upon passive stretch. A scheme of the cytoskeletal structure of myofibrils of vertebrate skeletal muscle is presented on the basis of our present information of connectin and intermediate filaments.     

Changes in the Levels and Forms of Cholinesterases in the Blood Plasma of Normal and Dystrophic Chickens

Abstract: Acetylcholinesterase (AChE) and pseudocholinesterase (°ChE) were analysed in the blood plasma of developing chickens, both normal and those with inherited muscular dystrophy. The amounts and the molecular forms of each were examined. °ChE concentration rises in the plasma of normal and dystrophic chicks at the end of embryonic development and is maintained after hatching at a constant, relatively high level, accounting for 90-95% of total cholinesterase activity in normal plasma. This level is maintained in normal and dystrophic chickens. In embryonic plasma of both normal and dystrophic chicks, on the other hand, the levels of AChE are higher than those of °ChE. Immediately after hatching the AChE level decreases rapidly in normal plasma, reaching a very low level by 2-3 weeks ex ovo. The AChE level in plasma from dystrophic birds, although less than normal from day 19 in ovo to 2 weeks ex ovo, subsequently increases to peak around 4 months at levels 15-20-fold of those in normal birds. There is virtually no enzyme of either type in the erythrocytes of normal or dystrophic chickens. The changes of AChE in plasma were correlated with the alterations of AChE in dystrophic fast-twitch muscles, suggesting that the latter pool is a precursor of the plasma AChE. Both the AChE and the °ChE in plasma exist in multiple molecular forms, which are similar to certain of those found previously in the muscles of these birds. The major form (60-80%) of both enzymes in the plasma is the M form (sedimentation coefficient ≥11 S) in all cases, but it is accompanied by certain other forms. In no case is there any of the heaviest form (H₂, 19-20 S) of AChE or of °ChE found in normal and dystrophic muscle, which is attached at the synapses in normal muscle. The pattern of forms of plasma °ChE is constant at all ages, and in normal and dystrophic chickens. The pattern of forms of AChE in the plasma, in contrast, varies with age and with dystrophy in a characteristic manner. The sedimentation coefficients and the amounts of the enzymes in fast-twitch muscle of dystrophic animals are compared with those of the plasma forms, and an interpretation is given of the characteristic patterns of AChE and of χE in their blood.     

The involvement of sarcotubular membranes in genetic muscular dystrophy

Microsomal preparations from breast muscle of normal and dystrophic chickens are characterized with regard to ultrastructural features, protein composition, Ca²⁺ transport and ATPase activity.Dystrophic muscle yields a greater microsomal dry weight, with a reduced protein to lipid ratio. This is related to the presence of a considerable number of low density microsomes, in addition to seemingly normal microsomes. The low density microsomes display a reduced number of protein particles on freeze fracture faces.Electrophoretic analysis reveals nearly identical patterns in normal and dystrophic microsomes. Furthermore, normal and dystrophic microsomes sustain equal rates of Ca²⁺ transport and ATPase, demonstrating an identical protein specific activity. However, the dystrophic microsomes have a lower capacity to retain transported Ca²⁺.The high yield of low density microsomes with reduced capacity for Ca²⁺ uptake is attributed to the presence of membranes proliferated in the junctional and tubular sarcomere regions of the dystrophic muscle. It is suggested that proliferation of such membranes accounts for the altered excitation-contraction coupling and cable properties of genetically dystrophic muscle.