Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.22.54 (calpain 3)
 430 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human muscle-specific calpain (CAPN3) was expressed in two heterologous systems: Sf9 insect cells and Escherichia coli cells. Polyclonal antibodies were prepared against peptides whose sequences were taken from the three unique regions of human CAPN3, namely NS, IS1, and IS2, which are not found in other members of the calpain family. Western blot analysis using these antibodies revealed that CAPN3 was well expressed in both systems. However, considerable rapid degradation of the expressed CAPN3 was observed in both Sf9 and E. coli cells. These antibodies were therefore also used to detect CAPN3 and its degradation products in human and rat muscles, as well as to detect the protein throughout the purification of the recombinant His-tagged human CAPN3 by Ni2+ affinity chromatography and by immunopurification over immobilized antibody. An alternative purification procedure was used for purification of all putative CAPN3 immunoreactive fragments by combining SDS-PAGE and hydroxyapatite chromatography. Two fragments of CAPN3 of approximately 55 kDa were purified, and their N-terminal amino acid sequencing demonstrated that cleavage of CANP3 occurred between residues 30-31 and 412-413, thus providing the first evidence for the localization of putative autolytic sites in this enzyme.
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PMID:Purification and identification of two putative autolytic sites in human calpain 3 (p94) expressed in heterologous systems. 1006 45

Tenderization of skeletal muscle in meat animals has been closely linked to the postmortem activity of the calpain proteolytic enzyme system, which includes the specific inhibitor calpastatin. Increased understanding of the skeletal muscle-specific calpain isoform p94 has prompted suggestions as to whether it too could have a role in the tenderization process. In this study, two groups of pigs were identified in which shear force measurements after 8 d of conditioning indicated a large variation in the tenderness of longissimus muscle. The quantity of p94 in the muscle was monitored by immunoblotting, using a porcine-specific polyclonal antibody raised against a recombinant peptide fragment generated as a fusion protein. The antiserum recognized a 94-kDa protein associated with myofibrils in skeletal but not cardiac muscle, as expected for this calpain isoform, although it could not be tested with the native protein because of the extreme instability of p94. In the first experiment, the mean shear force for the tough group was 6.71 +/- .28 kg (n = 12, SEM) and that of the tender group was 3.87 +/- .12 kg (n = 12), but there was no difference in the normalized absorbance of the immunopositive 94 kDa band on Western blots from samples collected at approximately 40 min postmortem. In the second experiment, the stability of p94 in chilled carcasses was investigated over 24 h, using a further two groups of 10 tough and 10 tender pigs of mean shear force values 5.36 +/- .14 kg and 2.81 +/- .15 kg, respectively. In tough and tender animals, there was a decline (P < .05) in the 94-kDa immunostaining material of mean half-lives of 13.8 and 12.9 h, respectively, although there was considerable variability. Despite this variability in half lives and shear force values, no correlation was seen between these factors. Thus, in porcine longissimus muscle, the variability in tenderness after 8 d of conditioning cannot be attributed to an underlying variability in p94.
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PMID:Relationship between skeletal muscle-specific calpain and tenderness of conditioned porcine longissimus muscle. 1022 62

Limb-girdle muscular dystrophy type 2A (LGMD2A) is an autosomal recessive disorder characterized mainly by symmetrical and selective atrophy of the proximal limb muscles. It derives from defects in the human CAPN3 gene, which encodes the skeletal muscle-specific member of the calpain family. This report represents a compilation of the mutations and variants identified so far in this gene. To date, 97 distinct pathogenic calpain 3 mutations have been identified (4 nonsense mutations, 32 deletions/insertions, 8 splice-site mutations, and 53 missense mutations), 56 of which have not been described previously, together with 12 polymorphisms and 5 nonclassified variants. The mutations are distributed along the entire length of the CAPN3 gene. Thus far, most mutations identified represent private variants, although particular mutations have been found more frequently. Knowledge of the mutation spectrum occurring in the CAPN3 gene may contribute significantly to structure/function and pathogenesis studies. It may also help in the design of efficient mutation-screening strategies for calpainopathies.
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PMID:Calpainopathy-a survey of mutations and polymorphisms. 1033 Mar 40

Sepsis is associated with a pronounced catabolic response in skeletal muscle, mainly reflecting degradation of the myofibrillar proteins actin and myosin. Recent studies suggest that sepsis-induced muscle proteolysis may reflect ubiquitin-proteasome-dependent protein breakdown. An apparently conflicting observation is that the ubiquitin-proteasome pathway does not degrade intact myofibrils. Thus, it is possible that actin and myosin need to be released from the myofibrils before they can be ubiquitinated and degraded by the proteasome. We tested the hypothesis that sepsis results in disruption of Z-bands, increased expression of calpains, and calcium-dependent release of myofilaments in skeletal muscle. Sepsis induced in rats by cecal ligation and puncture resulted in increased gene expression of micro-calpain, m-calpain, and p94 and in Z-band disintegration in the extensor digitorum longus muscle. The release of myofilaments from myofibrillar proteins was increased in septic muscle. This response to sepsis was blocked by treating the rats with dantrolene, a substance that inhibits the release of calcium from intracellular stores to the cytoplasm. The present results provide evidence that sepsis is associated with Z-band disintegration and a calcium-dependent release of myofilaments in skeletal muscle. Release of myofilaments may be an initial and perhaps rate-limiting component of sepsis-induced muscle breakdown.
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PMID:Sepsis stimulates release of myofilaments in skeletal muscle by a calcium-dependent mechanism. 1042 67

The muscle-specific calpain isoform p94 has high propensity to autocatalytic degradation, thus no significant amounts of the intact active protein have been available so far. As a result, aspects like its regulation (via Ca2+ and other factors) and its intracellular localization are unknown or obscure. In this work, large amounts of human p94 have been produced in insect cells using a recombinant baculovirus expression system. Although most of the protease was recovered in an insoluble and catalytically inactive form, the soluble fraction contained amounts of intact active p94 adequate for its characterization. His-tagged recombinant p94, obtained by the same expression system, was partially purified as an active product. Both the unmodified and the partially purified His-tagged p94 bound calcium with high affinity, and their autolytic activity required Ca2+. The sensitivity of the catalytic activity of the recombinant protease to Ca2+ was very high. In fact, p94 in soluble cell extracts autolysed to a significant extent even in the presence of submicromolar Ca2+ levels. Thus, in analogy to what demonstrated for the ubiquitous m- and micro-calpain isoforms, intracellular Ca2+ might be one of the factors controlling the activity of this muscle-specific calpain isoform.
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PMID:Expression, partial purification and functional properties of themuscle-specific calpain isoform p94. 1050 17

We have synthesized dextran derivatives called RGTAs (for regenerating agents) that were designed to mimic some of the properties of heparin or heparan sulfate to interact with and protect heparin binding growth factors. Some of these growth factors have been described to be involved in myogenesis control. In previous studies, we have shown that muscle regeneration in adults could be greatly enhanced in vivo by treatment with RGTA. Since muscle regeneration occurs through the activation of satellite cells, in the present study we have used primary cultures of rat satellite cells and treated them with the heparan sulfate analogue RGTA or heparin in order to stimulate their growth and differentiation. We also studied the effect of these substances on calpain (calcium-activated neutral proteases) expression in these cultures. Indeed, several reports, principally based on fetal myoblast cultures or myogenic cell lines, have suggested that calpains might be involved in myoblast fusion during myogenic differentiation. We therefore studied the expression of microcalpain (mu-calpain), millicalpain (m-calpain), and calpain 3 in the course of differentiation of these satellite cell cultures in the absence or in the presence of heparin or of a mimic compound (the RGTA RG1282). RGTA and heparin were shown to have a dual effect on satellite cell proliferation and differentiation: RGTA stimulated proliferation with a maximum dose effect at 1 microgam/ml. Heparin used at concentrations similar to those of RGTA was less efficient at stimulating proliferation. Both substances were shown, however, to induce precocious and enhanced differentiation of satellite cells. We showed by quantitative RT-PCR analysis that mu-calpain, m-calpain, and calpain 3 mRNAs were expressed in satellite cell cultures in proliferating myoblasts (day 3) and differentiating cultures (days 7 and 12). The level of mu-calpain mRNA was increased by a factor of 3 during differentiation of satellite cells, whereas the level of m-calpain mRNAs was slightly increased at day 12 only, and calpain 3 mRNA was slightly reduced in these differentiating cultures. Interestingly enough, RGTA and heparin, which both strongly increased differentiation, reduced the expression of the mu- and m-calpains and slightly increased that of calpain 3 in differentiating cultures. These results showed that there was no correlation between the extent of myoblast differentiation and the level of calpain expression in satellite cells grown in primary cultures and underscored the differences between these adult cells and fetal myoblasts.
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PMID:Studies on calpain expression during differentiation of rat satellite cells in primary cultures in the presence of heparin or a mimic compound. 1052 29

Members of the calpain proteinase family are present in all mammalian cells, although a novel calpain 94 kDa isoform is found almost exclusively in skeletal muscle. p94 is difficult to purify from muscle and recombinant p94 autolyses rapidly when expressed in COS cells. However, in vivo the enzyme may be stabilised by interaction with titin, which has two well-characterised binding sites for p94 at the N2- and M-lines. Both these titin subdomains are subject to muscle-specific alternative splicing, which could be related to p94 expression level or stability in muscles of different fibre type. In this study, porcine longissimus dorsi (LD), trapezius (TZ) and adductor longus (AL) were characterised as fast, intermediate and slow using commercially available specific anti-human fast- and slow-myosin heavy chain mAbs and also by conventional histochemistry. p94 was quantified both in whole muscle preparations and single fibres by western blotting using an anti-p94 antiserum generated by expressing a recombinant p94 sequence as a GST fusion protein antigen. SDS PAGE and immunoblotting revealed a single band of approximately 94 kDa with identical mobility in all muscle and fibre preparations. The intensity of the 94 kDa band was greater in LD (22 +/- 1.7 densitometric units mean +/- SEM, n = 3) than TZ and AL (10 +/- 2.3 and 6 +/- 0.9 units, respectively). Expressed as a ratio relative to actin immunoreactivity, p94 is present in all types of single fibres isolated from TZ, but at a significantly lower level (P < 0.01) in slow type I (0.08 +/- 0.01, n = 9), compared to fast IIA/IIB fibres (0.22 +/- 0.02, n = 26). No evidence was seen for rapid or variable rate of p94 degradation in either type of fibre. These data suggest a positive correlation between p94 expression level and fast glycolytic characteristics in porcine muscle.
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PMID:Fibre type-specific expression of p94, a skeletal muscle-specific calpain. 1053 22

p94, a muscle-specific member of the calpain family, also called calpain3 (CAPN3), has been identified as the gene product responsible for limb-girdle muscular dystrophy type 2A (LGMD2A). To elucidate the molecular mechanism of LGMD2A, the effects of missense point mutations found in LGMD2A on the unique properties of p94 were studied. All of the mutants examined to date lose their proteolytic activity against fodrin, a cytoskeletal protein, strongly suggesting that of the specific properties of p94, the loss of protease activity is the prime cause of LGMD2A. Studies of LGMD2A and p94 suggest a novel molecular mechanism for muscular dystrophy, showing that a combined pathologic and biochemical approach is effective.
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PMID:New aspect of the research on limb-girdle muscular dystrophy 2A: a molecular biologic and biochemical approach to pathology. 1063 25

Steers of known percentage Brahman (B) and Angus (A) breeding (100% A, n = 6; F1 B x A, n = 6; and 100% B, n = 6) were used to determine the effect of calcium chloride injection on the calpain proteinase system and meat tenderness. The steers were slaughtered in six replications (at either 9 or 14 mm of backfat, determined ultrasonically), with each breed type represented. Calpains and calpastatin activities were measured on fresh, prerigor longissimus muscle samples. Carcass data were collected after a 24-h chill, and the short loin (IMPS #180), top sirloin (IMPS #184), and top round (IMPS #168) were removed from both sides of each carcass. The cuts from the right side were then injected at 5% (wt/wt) with CaCl2 solution (2.2%). Longissimus muscle calpain and calpastatin activities were also measured at 48 h postmortem from the injected and control sides of each carcass. Warner-Bratzler shear force was measured on steaks from the three subprimals aged 1, 2, 5, 15, or 31 d. Marbling scores and USDA quality grades were higher (P<.05) in A than in F1 B x A and B carcasses. Calpastatin activity was higher (P<.05) in muscle from B than in muscle from A and F1 B x A steers, and postmortem storage (O vs 48 h) and CaCl2 injection reduced (P<.05) the activity of the calpains and calpastatin. Strip loin and top sirloin steaks from A and F1 B x A steers were more tender (P<.05) than steaks from B steers; however, top round steak tenderness did not differ (P>.05) across breed type. Calcium injection improved strip loin and top sirloin steak tenderness, but it did not affect top round steak tenderness. Collectively, these data show that CaC12 injection can be used to improve meat tenderness, with similar responses shown in cattle containing 0, 50, and 100% B inheritance. However, even with CaCl2 injection, B steaks are less tender than their A and F1 B x A counterparts.
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PMID:Calcium-activated tenderization of strip loin, top sirloin, and top round steaks in diverse genotypes of cattle. 1064 69

A defect of the gene for p94 (calpain 3), a skeletal muscle-specific calpain, is responsible for limb girdle muscular dystrophy type 2A (LGMD2A), or 'calpainopathy', which is an autosomal recessive and progressive neuromuscular disorder. To study the relationships between the physiological functions of p94 and the etiology of LGMD2A, we created transgenic mice that express an inactive mutant of p94, in which the active site Cys129 is replaced by Ser (p94:C129S). Three lines of transgenic mice expressing p94:C129S mRNA at various levels showed significantly decreased grip strength. Sections of soleus and extensor digitorum longus (EDL) muscles of the aged transgenic mice showed increased numbers of lobulated and split fibers, respectively, which are often observed in limb girdle muscular dystrophy muscles. Centrally placed nuclei were also frequently found in the EDL muscle of the transgenic mice, whereas wild-type mice of the same age had almost none. There was more p94 protein produced in aged transgenic mice muscles and it showed significantly less autolytic degradation activity than that of wild-type mice. Although no necrotic-regenerative fibers were observed, the age and p94:C129S expression dependence of the phenotypes strongly suggest that accumulation of p94:C129S protein causes these myopathy phenotypes. The p94:C129S transgenic mice could provide us with crucial information on the molecular mech-anism of LGMD2A.
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PMID:Myopathy phenotype of transgenic mice expressing active site-mutated inactive p94 skeletal muscle-specific calpain, the gene product responsible for limb girdle muscular dystrophy type 2A. 1081 21


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