Identification of two calpastatin forms in rat skeletal muscle and their susceptibility to digestion by homologous calpains

Pontremoli, S.; Melloni, Edon; Viotti, Pier Luigi; Michetti, M.; Salamino, Franca; Horecker, B.L.

doi:10.1016/0003-9861(91)90247-g

Cited by 54 publications

(52 citation statements)

References 30 publications

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“…The above data at least indicate that in the gastrocnemius of tumour-bearing animals the setting of Ca 2+ -dependent proteolytic system was being shifted in such a way as to favour its activity. Moreover, since calpastatin itself is a specific calpain substrate in vivo (Pontremoli et al, 1991), its decrease also suggests that in this muscle there was an ongoing activation of the calpain system. To substantiate this inference, in the muscle of tumour-bearing animals we also measured how changed the level of the 130 kDa plasma-membrane Ca 2+ -ATPase, another yet unrelated calpain substrate, and compared it to the level of the sarcoplasmic 90 kDa Ca 2+ -ATPase, which has been reported to be resistant to calpain proteolysis (Yoshida et al, 1990).…”

Section: Resultsmentioning

confidence: 93%

“…To isolate calpastatin, the supernatant was dialysed against 50 mM Na acetate/acetic acid buffer, pH 6.7, containing 0.1 mM EDTA and 0.5 mM β-mercaptoethanol. The samples were then separately loaded on a DE32 column (1 × 10 cm) equilibrated in the dialysis buffer, and proteins eluted with 200 ml of a linear 0-0.35 M NaCl gradient (Pontremoli et al, 1991;Salamino et al, 1994a). To isolate calpain, the supernatant was dialysed against 50 mM Na borate/boric acid buffer, pH 7.5, containing 0.1 mM EDTA, and 0.5 mM β-mercaptoethanol.…”

Section: Isolation Of Calpastatin and Calpainmentioning

confidence: 99%

“…NaCl was then added to a 0.3 M final concentration and the samples separately loaded on a Phenyl Sepharose CL-4B column (1 × 4 cm) equilibrated in the dialysis buffer with 0.3 M NaCl added. Calpain activity was eluted with 20 ml of the same buffer without NaCl in 1 ml fractions (Pontremoli et al, 1991;Salamino et al 1994).…”

Section: Isolation Of Calpastatin and Calpainmentioning

confidence: 99%

“…Measuring the time-course of changes in substrate levels may thus provide an estimate of the calpain activity in vivo cumulated over time. In the present work we adopted this approach and selected two independent reporter substrates, namely, calpastatin, the endogenous calpain inhibitor that is cleaved by calpain itself (Pontremoli et al, 1991), and the 130 kDa Ca 2+ -ATPase associated with the plasma membrane (Salamino et al, , 1994b. As an internal control for the latter substrate, we also measured the levels of the sarcoplasmic 90 kDa ATPase, which is resistant to proteolysis by calpain (Yoshida et al, 1990).…”

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confidence: 99%

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Activation of Ca2+-dependent proteolysis in skeletal muscle and heart in cancer cachexia

et al. 2001

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Cachexia is a syndrome characterized by profound tissue wasting that frequently complicates malignancies. In a cancer cachexia model we have shown that protein depletion in the skeletal muscle, which is a prominent feature of the syndrome, is mostly due to enhanced proteolysis. There is consensus on the views that the ubiquitin/proteasome pathway plays an important role in such metabolic response and that cytotoxic cytokines such as TNFα are involved in its triggering (Costelli and Baccino, 2000), yet the mechanisms by which the relevant extracellular signals are transduced into protein hypercatabolism are largely unknown. Moreover, little information is presently available as to the possible involvement in muscle protein waste of the Ca 2+ -dependent proteolysis, which may provide a rapidly activated system in response to the extracellular signals. In the present work we have evaluated the status of the Ca 2+ -dependent proteolytic system in the gastrocnemius muscle of AH-130 tumour-bearing rats by assaying the activity of calpain as well as the levels of calpastatin, the natural calpain inhibitor, and of the 130 kDa Ca 2+ -ATPase, both of which are known calpain substrates. After tumour transplantation, total calpastatin activity progressively declined, while total calpain activity remained unchanged, resulting in a progressively increasing unbalance in the calpain/calpastatin ratio. A decrease was also observed for the 130 kDa plasma membrane form of Ca 2+ -ATPase, while there was no change in the level of the 90 kDa sarcoplasmic Ca 2+ -ATPase, which is resistant to the action of calpain. Decreased levels of both calpastatin and 130 kDa Ca 2+ -ATPase have been also detected in the heart of the tumour-bearers. These observations strongly suggest that Ca 2+ -dependent proteolysis was activated in the skeletal muscle and heart of tumour-bearing animals and raise the possibility that such activation may play a role in sparking off the muscle protein hypercatabolic response that characterizes cancer cachexia. © 2001 Cancer Research Campaign http://www.bjcancer.com

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Section: Resultsmentioning

confidence: 93%

Section: Isolation Of Calpastatin and Calpainmentioning

confidence: 99%

Section: Isolation Of Calpastatin and Calpainmentioning

confidence: 99%

mentioning

confidence: 99%

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Activation of Ca2+-dependent proteolysis in skeletal muscle and heart in cancer cachexia

et al. 2001

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“…Purification of -calpain and m-calpain from rat skeletal muscle was carried out as described (22). 1 unit of calpain activity is defined as the amount of enzyme producing 1 mol of free NH 2 groups in standard conditions (23).…”

Section: Methodsmentioning

confidence: 99%

Acyl-CoA-binding Protein Is a Potent m-Calpain Activator

Melloni

Averna

Salamino

et al. 2000

Journal of Biological Chemistry

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Acyl-CoA-binding protein, a 20-kDa homodimer that exerts many physiological functions, promotes activation of the classic calpain forms, most markedly that of the m-isozyme. This protein factor was purified from rat skeletal muscle and was also expressed in Escherichia coli. Both native and recombinant acyl-CoA-binding proteins show the same molecular properties and an identical capacity to decrease the [Ca 2؉ ] required for m-calpain activity. The binding of long-chain acyl-CoAs to acyl-CoA-binding protein does not modify the activating effect on calpains. Acyl-CoA-binding protein seems to be involved in the m-calpain regulation process, whereas the previously identified UK114 activator is a specific modulator of -calpain. Acyl-CoA-binding protein is proposed as a new component of the Ca 2؉ -dependent proteolytic system. A comparative analysis among levels of classic calpains and their activator proteins is also reported.How the calcium-dependent proteolytic system is activated is an intriguing question in terms of understanding its general properties, physiological functions, and involvement in the occurrence of tissue damage (1-4). Calpains, the enzymatic components of the system, are primarily regulated by calcium ions. The binding of Ca 2ϩ to calmodulin-like domains of the proteinases induces a conformational change that constitutes the initial limiting step of the overall sequential activation process (5). In this new conformation calpains undergo limited autoproteolysis that irreversibly removes the molecular constraints responsible for stabilizing the native inactive form (1-4). In their autoproteolyzed active forms, calpains acquire the highest affinity for calpastatin, which becomes the only negative modulator of calpain activity (6). In rat brain we have recently identified a protein factor, called UK114, which specifically interacts with -calpains and accelerates their activation steps, increasing the affinity for calcium ions (7). The reduced time of calpain activation is consistent with the proposed involvement of the Ca 2ϩ -dependent proteolytic system in specific signal transduction pathways, resulting, for instance, in granule secretion in various cell types (8) or in the conversion of protein kinase C into an autoproteolyzed form produced during the differentiation of murine erythroleukemia cells (9).The rat brain -calpain activator shows a strict specificity for all -calpain isoforms, whereas it has no effect on the classic m-calpains (10), the activation of which requires a calcium concentration at least two orders of magnitude higher than that required by -calpain. Despite reports indicating that free calcium ions can be accumulated in specific cell regions at high micromolar concentrations (11), the calcium requirement for m-calpain activation can apparently be satisfied only during tissue necrosis, a situation far removed from physiological conditions (12). We now report that a highly conserved protein, called acylCoA-binding protein (ACBP) 1 (13-15), shows a potent m-calpain-activating pr...

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Wetting properties of polystyrene ionomers treated with plasma source ion implantation

Kim

Hong

Nah³

et al. 2002

J of Applied Polymer Sci

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ABSTRACT:The wetting properties of polystyrene-based ionomers treated with plasma source ion implantation (PSII) were investigated by the measurement of water contact angles. When sulfonated ionomers were aged for a few days, the hydrophobic recovery for the ionomers became much slower than that for the nonionic polymers. However, when the samples were aged over 20 days, the water contact angle of the ionomers converged with that of the nonionic polymer. Thus, it was concluded that the ionic interaction between the ionic groups and the presence of ionic groups together resulted in the slow hydrophobic recovery and that the aging effect was significant for the ionomers. For the methacrylate ionomer of low ion content, on the other hand, it was found that the PSII treatment produced only a small change in hydrophobic recovery behavior. Thus, it was suggested that the low ionic content coupled with the small size of the ionic unit might cause changes only of a very insignificant degree in hydrophobic recovery behavior.

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Identification of two calpastatin forms in rat skeletal muscle and their susceptibility to digestion by homologous calpains

Cited by 54 publications

References 30 publications

Activation of Ca2+-dependent proteolysis in skeletal muscle and heart in cancer cachexia

Activation of Ca2+-dependent proteolysis in skeletal muscle and heart in cancer cachexia

Acyl-CoA-binding Protein Is a Potent m-Calpain Activator

Wetting properties of polystyrene ionomers treated with plasma source ion implantation

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