Characterization and purification of a Ca2+ ion-activated neutral proteinase inhibitor in rabbit skeletal muscle

This paper describes the isolation, purification and properties of a specific inhibitor of calcium-activated neutral proteinase (CaANP) in rabbit skeletal muscle. The inhibitor was a thermo-acid-stable protein degraded by trypsin and chymotrypsin and seemed to contain two polypeptide chains with molecular weights of 70 000 and 13 000 daltons. Maximal inhibitory activity was obtained at neutral pH. High salt concentrations were needed to suppressinhibition. Inhibitor concentration had no effect on the optimal Ca++ ion levels for CaANP. These experiments also show that enzyme inhibitor association was instantaneous and did not need any incubation.     

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.     

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.     

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     

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.     

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.     

Fish muscle cytoskeletal network: its spatial organization and its degradation by an endogenous serine proteinase

The extraction of white croaker skeletal myofibrils with KI rendered a residue in which a network of longitudinal and transverse filaments could be observed by scanning electron microscopy. A trypsin-like serine proteinase isolated from the same muscle was able to produce a complete and rapid disruption of the network, while major myofibrillar proteins were only slightly modified. This fact suggests that the disassembly of the cytoskeletal network may be an early event in the proteolysis of myofibrils. Desmin was not attacked by the proteinase under the assayed conditions, which indicates that some other unidentified component of the network would be the primary target of the action of the enzyme on myofibrils.     

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.     

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.     

Partial purification and characterization of cysteine proteinase from metamorphosing tadpole tails of Rana catesbeiana

• 1.1. A leupeptin-sensitive proteinase was partially purified from regressing tadpole tails by acetone factionation and column chromatography on S-Sepharose.
• 2.2. The enzyme degraded hemoglobin and myoglobin at pH 3.0. The enzyme also hydrolyzed Z-Phe-Arg-MCA and Boc-Val-Leu-Lys-MCA at pH 4.0.
• 3.3. The enzyme activity was inhibited by leupeptin, egg cystatin, E-64 and monoiodoacetic acid and was activated by l-cysteine.
• 4.4. The enzyme degraded myosin and actin in myofibrils of tadpole tails.
• 5.5. The enzyme belongs to the cysteine proteinase and is possibly involved in tail degradation during the metamorphosis of tadpoles.

     

Identification of a myofibril-bound serine protease and its endogenous inhibitor in mouse skeletal muscle

Myofibrillar proteins, like all other intracellular proteins, are in a dynamic state of continual degradation and resynthesis. The proteolytic system responsible for degrading myofibrillar proteins in skeletal muscle is not well defined. A proteolytic activity associated to myofibrils was found in mouse skeletal muscle, as show electrophoretic patterns, and denominated by us, as protease M. During incubation of whole myofibrils at 37 degrees C, myosin heavy chain, alpha actinin, actin and troponin T suffered degradation. These effects were inhibited selectively by serine protease inhibitors (soybean trypsin inhibitor, di-isopropyl phosphofluoridate, phenylmethanesulfonyl fluoride). Using myofibrils as protease M source, azocaseinolytic activity was also detected. Endogenous inhibitor and various compounds effects on protease M activity were also quantified by trichloroacetic acid soluble products formation, using radiolabeled myofibrils. An endogenous trypsin inhibitor isolated from the muscle cytoplasmic fraction could inhibit protease M activity on myofibrillar proteins and on azocasein. While K(+) increased protease M activity, the presence of Ca(2+) did not show any effect. Data presented in this study suggest that reported protease M may be implicated in myofibrillar degradation in vivo and isolated endogenous inhibitor may provide a mechanism to control its action in mouse skeletal muscle.     

The effect of recombinant caspase 3 on myofibrillar proteins in porcine skeletal muscle

The objective of this study was to investigate the potential role of the caspase protease family in meat tenderisation by examining if caspase 3 was capable of causing myofibril protein degradation. Full-length human recombinant caspase 3 (rC3) was expressed in Escherichia coli and purified. The rC3 was active in the presence of myofibrils isolated from porcine longissimus dorsi muscle (LD) and retained activity in a buffer system closely mimicking post mortem conditions. The effect of increasing concentrations of rC3, incubation temperature, as well as incubation time on the degradation of isolated myofibril proteins were all investigated in this study. Myofibril protein degradation was determined by SDS-PAGE and Western blotting. There was a visible increase in myofibril degradation with a decrease in proteins identified as desmin and troponin I and the detection of protein degradation products at approximately 32, 28 and 18 kDa with increasing concentrations of rC3. These degradation products were analysed using MALDI-TOF mass spectrometry and identified to occur from the proteolysis of actin, troponin T and myosin light chain, respectively. The production of these degradation products was not inhibited by 5 mM EDTA or semi-purified calpastatin but was inhibited by the caspase-specific inhibitor Ac-DEVD-CHO. The temperature at which isolated myofibrils were incubated with rC3 was also found to affect degradation, with increasing incubation temperatures causing increased desmin degradation and cleavage of pro-caspase 3 into its active isoform. Incubation of isolated myofibrils at 4°C for 5 days with rC3 resulted in the visible degradation of a number of myofibril proteins including desmin and troponin I. This study has shown that rC3 is capable of causing myofibril degradation, hydrolysing myofibril proteins under conditions that are similar to those found in muscle in the post mortem conditioning period.     

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.     

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.     

Quantitative Assay for CASF (Ca2+-activated sarcoplasmic factor) Activity,and Effect of CASF Treatment on ATPase Activities of Rabbit Myofibrils

The ability of CASF (Ca²⁺-activated sarcoplasmic factor), a proteolytic enzyme that has recently been isolated from muscle and that removes Z-disks from myofibrils, to remove soluble material from myofibrils and to alter the Mg²⁺-modified ATPase activity of myofibrils was studied. A new assay involving determination of soluble material released from myofibrils was developed to measure CASF activity quantitatively. Optimum pH and optimum Ca²⁺ concentration for CASF activity as determined by this new assay were 7.0 and 1 mm, respectively. Proteolytic activity of CASF on myofibrils was prevented completely by excess EDTA. CASF treatment of myofibrils at CASF to myofibril ratios of 1: 20 by weight for 30 min caused a 20~25% increase in Mg²⁺-modified ATPase activity. CASF treatment for 360 min under these same conditions caused a decrease in Mg²⁺-modified ATPase activity at the highest ionic strengths used in this study (46.7 and 66.7 mm KCI). The increase in Mg²⁺-modified ATPase activity may originate from CASF degradation of troponin, whereas the decrease in Mg²⁺- modified ATPase activity may be due to CASF destruction or release of α-actinin from myofibrils. Digestion of myofibrils by CASF causes in the myofibrils (degradation of Z-lines, increase of ATPase activity) that are very similar to the changes caused by postmortem storage.     

Degradation of rat cardiac myofibrils and myofibrillar proteins by a myosin-cleaving protease.

The degradation of rat cardiac myofibrils and their constituent proteins with a myosin-cleaving protease was studied. Electrophoretograms of the digestion products of myofibrils showed that myosin,M-protein, C-protein, and troponin were degraded, but actin and tropomyosin were not. Degradation of these constituents resulted in losses of the Mg2+-ATPase activity and its Ca2+-sensitivity of myofibrils. Incubation of myofibrils with the protease induced the release of alpha-actinin without degradation. Susceptibilities of myosin, actin, troponin, and alpha-actinin purified from rat and pig hearts to the protease were essentially identical to those of the assembled forms in myofibrils. Although the purified tropomyosin was readily degraded into five fragments with the protease, the tropomyosin assembled in myofibrils and actin-tropomyosin complex were insusceptible to the protease. Digestion of myosin in the filamentous state with the protease resulted in the disappearance of myosin heavy chain and light chain 2, producing two fragments having molecular weights of 130,000 and 94,000 which originated from the degradation of heavy chain. The Ca2+- and EDTA-ATPase activities of the degradation products remained unchanged during incubation for 22 h. The actin-activated ATPase activity of myosin was reduced by 30% during incubation for 6 h, and recovered to the original level on adding actin to give a ratio of actin to myosin of 2:1. The pH optima for degradation of myosin in the soluble and filamentous states were 8.5 and 7.0, respectively. The results indicate that cardiac myosin in the filamentous state was more readily degraded with the protease than the myosin in the soluble state.     

Lysosomal (vacuolar) proteinases of yeast are essential catalysts for protein degradation, differentiation, and cell survival

Mutants deficient in the vacuolar (lysosomal) endopeptidases proteinase yscA and proteinase yscB of the yeast Saccharomyces cerevisiae exhibit a drastically reduced protein degradation rate under nutritional stress conditions. The differentiation process of sporulation is considerably disturbed by the absence of the two endopeptidases. Also under vegetative growth conditions and under conditions of false protein synthesis, the two vacuolar endopeptidases exhibit some effect on protein degradation, which is, however, much less pronounced as found under starvation conditions. Proteinase yscA deficiency leads to rapid cell death when glucose-grown cells starve for nitrogen or other nutrients. Whereas overall protein degradation is affected in the endopeptidase mutants, degradation of two distinct false proteins analyzed is not altered in the absence of proteinase yscA and proteinase yscB. Also catabolite inactivation and degradation of fructose-1,6-bisphosphatase is not affected to a greater extent in the endopeptidase-deficient strains.     

Proteasomes are tightly associated to myofibrils in mature skeletal muscle

Proteasomes are the major actors of nonlysosomal cytoplasmic protein degradation. In particular, these large protein complexes (about 2500 kDa) are considered to be responsible for muscular degradation during skeletal muscle atrophy. Despite their unusual and important size, they are widely described as soluble and mobile in the cytoplasm. In mature skeletal muscle, we have previously observed a sarcomeric distribution of proteasomes, as revealed by the distribution of α1/p27K, a subunit of the 20S core-particle (prosome) of proteasome. Here, we extend these observations at the electron microscopic level in vivo. We also show that this sarcomeric pattern is dependent of the extension of the sarcomere. Using isolated myofibrils, we demonstrate that proteasomes are still attached to the myofibrils after the isolation procedure, and reproduce the observations made in vivo. In addition, the extraction of actin by gelsolin largely removes proteasomes from isolated myofibrils, but some of them are held in place after this extraction, showing a sarcomeric disposition in the absence of any detectable actin, and suggesting the existence of another molecular partner for these interactions. From these results, we conclude that most of detectable 20S proteasomes in skeletal muscle cells is tightly attached to the myofibrils.     

Probing of the structural stability of vimentin and desmin-type intermediate filaments with Ca2+-activated proteinase, thrombin and lysine-specific endoproteinase Lys-C

Intermediate filaments (IFs) reconstituted from purified, delipidated vimentin and desmin as well as respective protofilaments were subjected to degradation by Ca2+-activated neutral thiol proteinase, thrombin and lysine-specific endoproteinase Lys-C, respectively. The breakdown products were analyzed by SDS-polyacrylamide gel electrophoresis and negative stain electron microscopy. While Ca2+-activated proteinase and thrombin caused rapid and complete degradation of IFs with kinetics not significantly different from those of the degradation of protofilaments, lysine-specific endoproteinase did not exert any electron microscopically detectable effect on filament structure. Although both types of subunit proteins were truncated at their non-alpha-helical, C-terminal polypeptides by this proteinase, they were still able to assemble into 10 nm filaments. Closer electron microscopic inspection of IFs treated with Ca2+-activated proteinase revealed numerous ruptures along the filaments already at very early stages of digestion. SDS-polyacrylamide gel electrophoresis of the processed filaments in conjunction with previous biochemical characterizations of the breakdown of protofilaments by Ca2+-activated proteinase showed that these inhomogeneities primarily arose from degradation of the arginine-rich, non-alpha-helical N-termini of the filament proteins. These findings demonstrate that, although the N-terminus of vimentin and desmin is essential for filament stability, it is still highly susceptible to proteolytic attack in particular and very likely to posttranslational modification in general. Such structural modifications of the N-termini of IF proteins might exert great influences on the intracellular distribution and molecular organization of IFs in various physiological and pathological conditions.