Hormone-responsive alkaline proteinase in rat skeletal muscle is not a mast cell-derived enzyme

Proteinase activity was determined in myofibrils from intact rat skeletal muscle and from skeletal muscle myocytes grown in culture. In vivo administration of the mast cell degranulator compound 48/80 abolished the alkaline proteinase activity in myofibrils obtained from normal or streptozotocin-diabetic rats. Exposure of myocytes to compound 48/80 in cell cultures had no effect on their myofibrillar proteinase activity, nor did it affect the rate of overall protein degradation in these cells. Co-incubation of cultured mast cells (line P815Y) with myocytes followed by sonication of the cell mixture resulted in a marked reduction of the proteinase activity in the pellet fraction, suggesting that the mast cells contain inhibitor(s) of myofibrillar proteinase activity. It is suggested that the myofibril-bound alkaline proteinase activity is not a mast cell-derived enzyme but a genuine component of muscle cells. The in vivo 48/80-induced reduction of muscle myofibrillar proteinase activity appears to be due to release of a soluble inhibitory activity rather than removal of mast cell proteinase from the tissue by degranulation.     

Action of a serine proteinase from fish skeletal muscle on myofibrils

The action of a serine proteinase from fish skeletal muscle on myofibrils was studied. The enzyme was able to destroy the structural integrity of myofibrils, and to degrade both their major contractile and cytoskeletal constituent proteins. Proteolysis could be completely prevented by the addition of a trypsin inhibitor isolated from the same muscle.     

Characterization of an alkaline proteinase of fish muscle

1. The alkaline proteinase showing pH optimum 8.0 from white croaker (Sciaena schlegeli) skeletal muscle was purified electrophoretically homogeneously (2000-fold) using a combination of DEAE-cellulose chromatography, hydroxylapatite chromatography and Ultrogel AcA 34 gel filtration. 2. It was stable for 1 hr at 50 degrees C. The molecular weight of the enzyme was estimated to be 430,000 by gel filtration, with the enzyme composed of four kinds of subunits, the chain molecular weights of which were 45,000, 48,000, 51,000 and 57,000. 3. From the effects of inhibitors, the enzyme was identified as cysteine proteinase. ATP and Cu2+ inhibited the activity 50% at 10 mM and 70% at 0.1 mM, respectively. 4. Thus the enzyme was characterized as a high molecular weight, heat-stable, alkaline cysteine proteinase (HAP). 5. The enzyme showed hardly any activity below 50 degrees C but considerable activity at around 60 degrees C against myofibrils, digesting myosin heavy chain, actin and tropomyosin. With the addition of 5 M urea the enzyme hydrolyzed myofibrils well at around 30 degrees C.     

Neutral calcium dependent proteinase from rabbit skeletal muscle: activity on myofibrillar proteins

Experiments have been carried out to explore the proteolytic cleavage of rabbit skeletal myofibrils by a calcium dependent neutral proteinase (CaANP). Polyacrylamide gel elctrophoresis on great slabs showed the ability of CaANP to degrade myofibrils more readily than supposed. Besides the hydrolysis of troponin T and the apparition of degradation product of 30,000 molecular weight, the activity of this enzyme is obvious too on some components of the M-line and on heavy subunits of tropomyosin as well as on three unidentified proteic fractions. The variety of the degradation products which appear suggest that the specificity of CaANP is not as selective as presumed. The participation of this proteinase in the postmorten evolution of muscle and its intervention in the turnover of myofibrillar proteins is discussed.     

Purification and some physico-chemical and enzymic properties of a calcium ion-activated neutral proteinase from rabbit skeletal muscle

Ca²⁺-activated neutral proteinase was purified from rabbit skeletal muscle by a method involving DEAE-Sephacel chromatography, affinity chromatography on organomercurial–Sepharose and gel filtration on Sephacryl S-200 and Sephadex G-150. The SDS (sodium dodecyl sulphate)/polyacrylamide-gel-electrophoresis data show that the purified enzyme contains only one polypeptide chain of mol.wt. 73000. The purification procedure used allowed us to eliminate a contaminant containing two components of mol.wt. about 30000 each. Whole casein or α₁-casein were hydrolysed with a maximum rate at 30°C, pH7.5, and with 5mm-CaCl₂, but myofibrils were found to be a very susceptible substrate for this proteinase. This activity is associated with the destruction of the Z-discs, which is caused by the solubilization of the Z-line proteins. The activity of the proteinase in vitro is not limited to the removal of Z-line. SDS/polyacrylamide-gel electrophoresis on larger plates showed the ability of the proteinase to degrade myofibrils more extensively than previously supposed. This proteolysis resulted in the production of a 30000-dalton component as well as in various other higher- and lower-molecular-weight peptide fragments. Troponin T, troponin I, α-tropomyosin, some high-molecular-weight proteins (M protein, heavy chain of myosin) and three unidentified proteins are degraded. Thus the number of proteinase-sensitive regions in the myofibrils is greater than as previously reported by Dayton, Goll, Zeece, Robson & Reville [(1976) Biochemistry 15, 2150–2158]. The Ca²⁺-activated neutral proteinase is not a chymotrypsin- or trypsin-like enzyme, but it reacted with all the classic thiol-proteinase inhibitors for cathepsin B, papain, bromelain and ficin. Thus the proteinase was proved to have an essential thiol group. Antipain and leupeptin are also inhibitors of the Ca²⁺-activated neutral proteinase.     

Proteolytic and physicochemical mechanisms involved in meat texture development.

Development in meat texture is a complex process originating very likely from a softening of the structural elements, especially myofibrils. This process probably involves two sets of mechanisms: 1) an enzymatic mechanism involving at least two of the three proteolytic systems so far identified and present in this tissue, namely lysosomal (cathepsins) and calcium dependent (calpains) proteinases; 2) a physicochemical mechanism based on the important post mortem rise in muscle osmotic pressure which could be twice as high as in live animals. Despite the large progress in muscle enzymology, the nature of the proteinases responsible for the post mortem proteolysis associated with the development in meat texture is still not clearly established. In the present review, data obtained from two different approaches attempting to answer this question were analysed. The first one was based on the identification of a set of structural and biochemical changes associated with meat texture development and to examine which proteolytic system or proteinase would be able to reproduce them when incubated with either myofibrils or muscle fibres as substrate. The second tentatively relates the rate and the extent of the changes in meat texture to the proteolytic equipment of the tissue. The first approach led to the conclusion that changes in muscle proteins and structure can be only explained by considering a synergistic action of both lysosomal and calcium-dependent proteinases. From the second, it was concluded that the process of meat texture development did not depend on the proteinase levels but was related to their initial potential efficiency assessed by measurement of the enzyme/inhibitor ratio. With respect to the physicochemical mechanisms, the post mortem rise in muscle osmotic pressure was shown to be responsible for some biochemical changes occurring in myofibrils. This was further substantiated by the fact that the greatest osmotic pressure values were observed in muscles exhibiting highest tenderising rate. On the other hand we provide evidence suggesting that the substrate, namely myofibrils, might constitute an important limiting step of the efficiency of both types of mechanism. Taken together, the findings presented emphasize that improvement of our knowledge in this field will greatly depend on the development of basic research on these different topics notably: 1) the mechanisms by which proteinases activities are regulated in living and post mortem muscles; and 2) the myofibrillar structure, especially in slow-twitch or type I muscles.     

Glucocorticoids in intracellular catabolism of myosin and actin

The catabolic effect of glucocorticoids on the structural proteins of contractile apparatus seems to be realized through the increased alkaline proteinase activity and accelerated synthesis of light enzyme subunits. The administration of large amounts of glucocorticoids increased the excretion of 3-methylhistidine in rats by 60%. Digestion of isolated myofibrils with alkaline proteinase resulted in the degradation of myosin heavy chain and actin. The turnover rate of actin and myosin heavy chain was decreased.     

Fish skeletal muscle contains a novel serine proteinase with an unusual subunit composition.

Proteinase I, an enzyme previously shown to be able to degrade contractile and cytoskeletal elements of white-croaker (Micropogon opercularis) myofibrils, was purified to apparent homogeneity by chromatography on DEAE-Sephacel, octyl-Sepharose CL 4B and arginine-Sepharose 4B. Its Mr was determined to be 269,000 by Sephacryl S-300 gel filtration. Under denaturing conditions, the enzyme dissociated into two subunits with Mr 20,000 and 15,500, in a molar ratio of 1.8:1. Proteinase I showed a pH optimum of 8.5. The enzyme was strongly inhibited by several serine proteinase inhibitors, whereas inhibitors of the other types of proteinases did not affect, or only scarcely affected, its activity. Several N-terminal-blocked 4-methyl-7-coumarylamide substrates having either arginine or lysine residues adjacent to the fluorogenic group were efficiently hydrolysed by the enzyme. These results indicate that proteinase I is a trypsin-like serine proteinase. The enzyme appears to be distinct from other proteinases previously described in skeletal muscle, and might be involved in the catabolism of myofibrillar proteins.     

Cytoskeletal filaments in embryonic chick myocardial cells as revealed by the quick-freeze deep-etch method combined with immunocytochemistry

Summary The three-dimensional organization of cytoskeletal filaments associated with the myofibrils and sarcolemma of the myocardial cells of early chick embryos was studied by the rapid-freeze deep-etch method combined with immunocytochemistry. In the endoplasmic region of saponin-treated myocardial cells, 12–14 nm filaments formed a loose network surrounding nascent myofibrils. These 12–14 nm filaments attached to the myofibrils and some of them converged into Z disc regions. In the non-junctional cytocortical region thinner 8–11 nm filaments composed a dense network just beneath the sarcolemma. In myofibril terminating regions at the sarcolemma, i.e., the fascia adherens, 3–5 nm cross-bridges were observed among the thin filaments. In Triton-permeabilized and myosin subfragment 1 (S1)-treated samples, subsarcolemmal 8–11 nm filaments proved to be S1-decorated actin filaments under which there was a loose network of S1-undecorated filaments. Subsarcolemmal S1-decorated actin filaments had mixed polarity and attached to the sarcolemma at one end. A loose network of S1-undecorated filaments among myofibrils in the endoplasmic region was revealed to consist of desmin-containing intermediate filaments after immuno-gold staining for desmin. These networks connecting myofibrils with sarcolemma were assumed to play an important role in integrating and transmitting the contractile force of individual myofibrils within early embryonic myocardial cells.     

Cytoskeletal ultrastructure and lipid composition of I-Z-I fraction in muscle from pre- and post-spawned female hake (Merluccius hubbsi)

Myofibrils from pre- and post-spawned female hake were depleted of thin and thick filaments by KI-treatment and the cytoskeletal ultrastructure of the preparations was investigated by electron microscopy. Irrespective of the gonadal stage of the fish, transmission (TEM) and scanning (SEM) electron micrographs showed an extensive network of filaments connecting Z-structures, which appeared as two circular rings held together. Z-structures of KI-treated myofibrils from post-spawned hake were more compact than those from pre-spawned fish. This ultrastructural difference was absent when KI-treated myofibrils from hake in both gonadal conditions were prepared in the presence of a proteinase inhibitor cocktail (1 mM EDTA+1 mM PMSF+1 mM iodoacetic acid). The total lipid (TL) content of the I-Z-I fraction from fish in pre-spawned condition was about 3.7 mg/g I-Z-I and non-polar lipids (NPL) represent 86.5% of TL. TL and NPL contents of the I-Z-I fraction from post-spawned hake were about 50% and 78.2% lower than those obtained from this fraction in pre-spawning fish. No significant changes (p>0.05) were observed in phospholipids (PL) and acidic phospholipids (AP). No significant differences (p<0.01) were observed among the corresponding lipid fractions of I-Z-I purified from pre- and post-spawning fish in the presence of a protease inhibitor cocktail.     

Calcium-dependent proteolysis of calcium-binding proteins

In several myopathic disorders, the internal muscle cell calcium concentration increases significantly as compared to normal muscle cells. We report that in the presence of elevated calcium levels, the calcium-binding proteins troponin C and calmodulin are protected from digestion by the chymotrypsin-like serine proteinase that co-purifies with isolated myofibrils. Degradation of the 67k calcimedin in the presence of calcium shows altered major cleavage fragments while degradation of myosin is unaffected by the presence of calcium. A role for this serine proteinase in muscle-wasting diseases is suggested.     

The pattern of organization of intermediate filaments and their asymmetrical association with dense bodies in smooth muscle of an ascidian Halocynthia roretzi

Summary An extensive network of intermediate filaments that interconnected cytoplasmic dense bodies and connected the dense bodies to the cell surface was revealed in double-fixed, tannic acid-stained preparations of ascidian smooth muscle. The filament network ran through spaces in the continuous network of myofibrils, connecting them longitudinally, obliquely and transversely to form an intimately associated, dual network. In their transverse passage, the intermediate filaments ran across myofibrils along I-zones exclusively, interconnecting successive dense bodies.The pattern of attachment of intermediate filaments to dense bodies was predominantly one-sided. The filaments, which themselves were not incorporated into the contractile apparatus, remained folded or unfolded between myofibrils and between sarcomere-like structures in synchrony with the contraction-relaxation cycles.These results suggest that the intermediate filaments mechanically maintain the organization and arrangement of myofibrils via an intimate association with the myofibrils in the regions of the dense bodies, in such a way that the filaments do not impede muscle function.Based on these observations, a new model for the network of intermediate filaments in smooth muscle cells is proposed.     

Myonase is localized in skeletal muscle myofibrils

A novel chymotrypsin-like proteinase termed myonase was previously purified from MDX-mouse skeletal muscle [Hori et al. (1998) J. Biochem. 123, 650-658]. Western blots and immunohistochemical analyses showed that myonase was present within myocytes of both MDX-mouse and control mouse, and subcellular fractionation showed that it was associated with myofibrils. No significant difference was observed on Western blots between the amounts of myonase in myofibrils of MDX-mouse and control mouse, but the amount of myonase recoverable as a pure protein was 5-10-fold more when MDX-mouse was the source of the skeletal muscle. Myofibrils also possessed an endogenous inhibitor of myonase, whose inhibitory activity at physiological pH (pH 7.4) depended on salt concentration, stronger inhibition being observed at a low salt concentration. Inhibition at alkaline pH (pH 9) was weak and independent of salt concentration. Myonase in myofibrils was partially released at neutral pH by a high salt concentration (>0.6 M NaCl). However, even at 4 M NaCl, more than 80% of myonase remained within the myofibrils. Under alkaline conditions, release of myonase from myofibril was more extensive. At pH 12, myonase was almost completely present in the soluble fraction. Release of myonase under these conditions coincided with the solubilization of other myofibrillar proteins.     

Myofibrillar creatine kinase: reversible binding to contractile proteins, stoichiometric ratio to myosin and its functional role

Rat heart myofibrils were isolated and purified in three different media: sucrose medium; EGTA medium; EGTA+ATP medium. All preparations were characterized by similar Ca2+-sensitive ATPase activities and were practically free of mitochondrial and sarcolemmal contaminations. However, they contained different amounts of creatine kinase. In preparations which showed the most intact ultrastructure, the activity of creatine kinase was 0.99 +/- 0.12 IU/mg. It was found that creatine kinase can be bound to myofibrils in a reversible manner with Kd = 0.16 mg/ml = 1.8 X 10(-6) M; the creatine kinase/myosin ratio was estimated to be approximately 1:10. The localization of creatine kinase was found to be a basis for the high turnover rate of ATP in the coupled creatine kinase and ATPase reactions occurring in cardiac myofibrils.     

Purification and characterization of a proteinase inhibitor from white croaker skeletal muscle (Micropogon opercularis)

A trypsin proteinase inhibitor has been purified to homogeneity from the skeletal muscle of white croaker (Micropogon opercularis). Previously, we had described the occurrence in fish muscle of a serine protease (proteinase I) which showed a great capacity to degrade whole myofibrils in vitro and an endogenous inhibitor that prevented the action of the protease, both on natural and artificial substrates. In this paper, we report the purification and further biochemical characterization of the endogenous trypsin inhibitor. The purification was carried out by DEAE-Sephacel, Con A-Sepharose, Sephacryl S-300 and Mono Q. Throughout the purification procedure, trypsin inhibitory activity was assayed using azocasein as substrate. The molecular mass of the inhibitor was 65 kDa, as estimated by SDS-PAGE and gel filtration. The trypsin inhibitor is a glycoprotein, as deduced by the fact that it binds to Con A-Sepharose and stains with PAS and showed a wide range of pH stability (from 5 to 11). The thermal stability of the inhibitor considerably decreased at temperatures >60 degrees C. Assays of the inhibitor against various proteases indicated that it is highly specific for serine proteases, since it did not inhibit proteases belonging to any other groups. The inhibitor was able to inhibit the endogenous target enzyme (proteinase I) in a dose-dependent manner, with a 50% inhibition at a molar ratio close to 1. The present work contributes to improving our understanding of the physiological role of the proteinase I-inhibitor system in muscle protein breakdown, as well as its influence on post mortem proteolysis.     

Stringent requirement for Ca2+ in the removal of Z-lines and alpha-actinin from isolated myofibrils by Ca2+-activated neutral proteinase

Treatment of isolated myofibrils with Ca2+-activated neutral proteinase (CANP) results in specific removal of Z-line and of alpha-actinin. To investigate the ionic requirement for these processes, we measured Z-line removal by phase-contrast and interference microscopy and alpha-actinin removal by sodium dodecyl sulphate/polyacrylamide-gel electrophoretic analysis of myofibrillar proteins. The proteolytic digestion of native purified proteins was measured directly on polyacrylamide gels and by the fluorescamine technique. We found that the removal of Z-line and alpha-actinin as well as the release of proteolytic degradation products from isolated myofibrils by CANP occur only in the presence of Ca2+; Sr2+, Ba2+, Mn2+, Mg2+, Co2+ and Zn2+ are all ineffective. In contrast with this stringent requirement for Ca2+, the proteolytic activity of CANP measured with denatured casein, native and denatured haemoglobin, native actin and tropomyosin also occurs in the presence of other bivalent cations, in the following order: Ca2+ greater than Sr2+ greater than Ba2+. These data suggest that only Ca2+ can produce the conformational change in myofibrils that renders them susceptible to the action of CANP, whereas its proteolytic activity is stimulated by several bivalent ions.     

Degradation of myofibrillar proteins by a calpain-like proteinase in the arm muscle of Octopus vulgaris

The effects of a calpain-like proteinase (CaDP) isolated from the arm muscle of Octopus vulgaris on the myofibrils and myofibrillar proteins isolated from the same tissue were examined. Our studies clearly showed that treatment of intact myofibrils with CaDP in the presence of 5 mM Ca²⁺ results in the degradation of the major myofibrillar proteins myosin, paramyosin, and actin. From the isolated α- and β-paramyosins only β-paramyosin is degraded by CaDP in the presence of 5 mM Ca²⁺ producing three groups of polypeptides of 80, 75, and 60 kDa, respectively. The degradation rate depends on the proteinase to substrate ratio, temperature, and time of proteolysis and is inhibited by the endogenous CaDP inhibitory factor (CIF), as well as by various known cysteine proteinase inhibitors (E-64, leupeptin, and antipain). From the other myofibrillar proteins examined myosin, but not actin, is degraded by CaDP; myosin heavy chain (MHC, 200 kDa) is degraded by CaDP producing four groups of polypeptides of lower molecular masses (155, 125, 115, and 102 kDa, respectively); the degradation rate depends on the incubation time and the proteinase to substrate ratio. Furthermore, CaDP undergoes limited autolysis in the presence of both the exogenous casein and the endogenous β-paramyosin producing two large active fragments of 52 and 50.6 kDa, respectively; CIF reversibly inhibits this CaDP autolysis. Accepted: 26 May 2000     

Localization of paratropomyosin in skeletal muscle myofibrils and its translocation during postmortem storage of muscles

Using polyclonal antibodies against paratropomyosin, which is believed to modify the actin-myosin interaction in postrigor skeletal muscles, we studied the localization of paratropomyosin in chicken breast muscle myofibrils. Intact myofibrils stained with fluorescent antibodies showed that paratropomyosin was exclusively located at the A-I junction region of sarcomeres. In stretched myofibrils (3.7 micron in sarcomere length), the approximate width of the fluorescent stripes and their relation to the A band remained constant. Removal of the A band from myofibrils led to loss of stainability. During postmortem storage of muscles, on the other hand, paratropomyosin was translocated from its original position at the A-I junction region onto thin filaments. The translocation of paratropomyosin was successfully induced with a calcium ion concentration of 10(-4) M in the presence of protease inhibitors. We therefore conclude that in postrigor muscles, paratropomyosin is released from the A-I junction region following the increase in the sarcoplasmic calcium ion concentration to 10(-4) M, and then binds to thin filaments, which results in weakening of rigor linkages formed between actin and myosin.     

Unusual organization of desmin intermediate filaments in muscular dysgenesis and TTX-treated myotubes

Cytoskeletal intermediate filaments were studied in muscular dysgenesis (mdg) and tetrodotoxin-treated inactive mouse embryo muscle cultures during myofibrillogenesis. Both muscular dysgenesis and tetrodotoxin-treated muscles are characterized in vitro by a total lack of contractile activity and an abnormal development of myofibrils. We studied the organization of the microtubule and intermediate filament networks with immunofluorescence, using anti-tubulin, anti-vimentin, and anti-desmin antibodies during normal and mdg/mdg myogenesis in vitro. Mdg/mdg myotubes present a heterogeneous microtubule network with scattered areas of decreased microtubule density. At the myoblast stage, cells expressed both vimentin and desmin. After fusion only desmin expression is revealed. In mutant myotubes the desmin network remains in a diffuse position and does not reorganize itself transversely, as it does during normal myogenesis. The absence of a mature organization of the desmin network in mdg/mdg myotubes is accompanied by a lack of organization of myofibrils. The role of muscle activity in the organization of myofibrils and desmin filaments was tested in two ways: (i) mdg/mdg myotubes were rendered active by coculturing with normal spinal cord cells, and (ii) normal myotubes were treated with tetrodotoxin (TTX) to suppress contractions. Mdg/mdg innervated myotubes showed cross-striated myofibrils, whereas desmin filaments remained diffuse. TTX-treated myotubes possessed disorganized myofibrils and a very unusual pattern of distribution of desmin: intensively stained desmin aggregates were superimposed upon the diffuse network. We conclude, on the basis of these results, that myofibrillar organization does not directly involve intermediate filaments but does need contractile activity.     

Rapid purification of secretory acid proteinase from Candida albicans and its characterization.

Secretory acid proteinase from C. albicans was purified from culture supernatant to apparent homogeneity by ion-exchange chromatography. Two isozymes of the proteinase were resolved using a novel chromatofocusing method. The enzyme, which appears to be a glycoprotein, consists of a single polypeptide chain with glutamine at the N-terminus. Its molecular weight is about 45,000 and isoelectric point is pH 4.6. At pH 5, the proteinase is stable at 45 degrees C for at least 15 min. It has a broad substrate specificity. With BSA as a substrate, Km was determined to be 1.6 x 10(-4) M. The enzyme is inhibited by pepstatin and thus is a carboxyl proteinase. It undergoes autocatalytic digestion at or below pH 5.0. The kinetics of induction of proteinase by various proteins are also reported.