Casein Zymography for Analysis of Calpain-1 and Calpain-2 Activity

Biswas, Ashim Kumar; Tandon, S.

doi:10.1007/978-1-4939-8988-1_3

Cited by 6 publications

(4 citation statements)

References 8 publications

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“…Results of calpain‐2 indicated that there were considerable variations in its concentration in thigh and breast and muscles, irrespective of sex of different chicken species. In comparison to calpain‐1, calpain‐2 is much more stable at broader range of pH and its concentration might go up to 10 times compared to the calpain‐1 (Biswas & Tandon, 2019a). For this, concentration of calpain‐2 was observed considerably ( p < 0.01) elevated than calpain‐1 in various muscle samples.…”

Section: Resultsmentioning

confidence: 99%

Casein zymography‐based detection and one‐step purification for simultaneous quantification of calcium‐induced endogenous proteases in breast and thigh muscles from different chicken breeds

Khoja

Arsalan

Biswas

et al. 2022

Food Processing Preservation

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Identification and separation of endogenous proteases are important to find out their role in changes of biochemical process in post‐mortem muscle. Diverse studies were conducted to find out the activity of these enzymes but only a very few studies were carried out on avian species. In this study, a simple casein zymography method was explained for detection of calpain‐1 and calpain‐2 with the standardization of extraction buffer and that were quantified biochemically after one‐step separation and purification on anion exchange chromatography. DEAE Sephacel matrix was used to purify calpain‐1, calpain‐2, and calpastatin. SDS‐PAGE analysis showed native and autolyzed forms of calpains. Biochemical test indicated concentrations of calpain‐1, calpain‐2, and calpastatin being higher in breast than thigh muscles across all four breeds of chicken. A simple, effective, and low‐cost extraction, purification, and separation technique was developed for accurate identification of calpain‐1and calpain‐2 on casein zymography method and quantification by biochemical test. Novelty Impact Statement Identification and isolation of endogenous proteases are necessary to determine their significance in the PM aging of chicken meat from various breeds. In this study, a straightforward casein zymography approach for in‐vitro detection of calpain‐1 and calpain‐2 activity is devised. This will aid in the optimization of PM aging duration for meat tenderization.

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Section: Resultsmentioning

confidence: 99%

Casein zymography‐based detection and one‐step purification for simultaneous quantification of calcium‐induced endogenous proteases in breast and thigh muscles from different chicken breeds

Khoja

Arsalan

Biswas

et al. 2022

Food Processing Preservation

Self Cite

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“…Calpains are the main enzymes responsible for proteolysis and highly depends on Ca 2+ accumulation in the sarcoplasm, as it is under this condition which calpains are activated and autolyzed, and the brighter band on the casein gels indicates the extractable calpain-1 is not autolyzed in native postmortem muscles ( Biswas and Tandon, 2019 ). The extractable calpain-1 activity of lamb chops declined during postmortem storage time illustrating that the autolysis proceeded in all treatments during postmortem storage ( Du et al, 2017 ).…”

Section: Discussionmentioning

confidence: 99%

The Influence of Vacuum Packaging of Hot-Boned Lamb at Early Postmortem Time on Meat Quality during Postmortem Chilled Storage

Zhao¹,

Chen²,

Bruce³

et al. 2022

Food Sci Anim Resour

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To evaluate the effects of early postmortem vacuum packaging (VP) on meat quality during postmortem chilled storage, hot-boned lamb was vacuum-packaged at 1, 6, 12, 24, and 48 h postmortem and stored around 2°C until 168 h postmortem, with lamb packaged in plastic wrap as the control (aerobic packaging). Intramuscular pH decline was delayed when lamb was vacuum packaged at 1, 6, and 12 h postmortem (p<0.05). The lamb vacuum-packaged at 1 h postmortem (VP-1h group) had significantly lower shear force values and purge losses accompanied by lower free thiol group values than other treatments during postmortem storage and was also higher in extractable calpain-1 activity by 6 h postmortem (p<0.05). Free thiol group concentrations were significantly higher after VP at 6 and 12 h postmortem (p<0.05). Packaging lamb under vacuum very early postmortem produced the lowest shear force and purge loss, likely by slowing heat loss and muscle temperature decline, implying that lamb quality is improved by VP when applied very early postmortem. This was at the expense of protein oxidation, which was unrelated to other meat quality measurements, most likely because potential contracture during hot boning confounded its impact. Further research is required to understand the implications of the interaction between protein oxidation, VP, and hot boning on the acceptability of lamb.

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“…The main principle is that the enzymes in the lysates are separated by the polyacrylamide gel containing casein. Casein molecules in zymography gels act as substrates for calpain in solutions containing calcium ions, and their degradation reflects enzymatic activity [ 35 , 36 ]. In resolving gel, 0.21 mg casein was copolymerized with 4.9 ml ddH 2 O, 2.5 ml 1.5 mol/L Tris–HCl (pH 8.8), 2.5 ml polyacrylamide solution (4:0.16), 40 μl ammonium persulfate solution (APS), and 28 μl N,N,N’,N’-Tetramethylethylenediamine (TEMED).…”

Section: Methodsmentioning

confidence: 99%

Fam20c regulates the calpain proteolysis system through phosphorylating Calpasatatin to maintain cell homeostasis

Liu

Zhang

et al. 2023

J Transl Med

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Background The family with sequence similarity 20-member C (FAM20C) kinase, a Golgi casein kinase, which is responsible for phosphorylating the majority of the extracellular phosphoproteins within S-x-E/pS motifs, and is fundamentally associated with multiple biological processes to maintain cell proliferation, biomineralization, migration, adhesion, and phosphate homeostasis. In dissecting how FAM20C regulates downstream molecules and potential mechanisms, however, there are multiple target molecules of FAM20C, particularly many phenomena remain elusive, such as changes in cell-autonomous behaviors, incompatibility in genotypes and phenotypes, and others. Methods Here, assay for transposase-accessible chromatin using sequencing (ATAC-seq), RNA sequencing (RNA-seq), proteomics, and phosphoproteomics were performed in Fam20c-dificient osteoblasts and to facilitate an integrated analysis and determine the impact of chromatin accessibility, genomic expression, protein alterations, signaling pathway, and post translational modifcations. Results By combining ATAC-seq and RNA-seq, we identified TCF4 and Wnt signaling pathway as the key regulators in Fam20c-dificient cells. Further, we showed Calpastatin/Calpain proteolysis system as a novel target axis for FAM20C to regulate cell migration and F-actin cytoskeleton by integrated analysis of proteomics and phosphoproteomics. Furthermore, Calpastatin/Calpain proteolysis system could negatively regulate the Wnt signaling pathway. Conclusion These observations implied that Fam20c knockout osteoblasts would cause cell homeostatic imbalance, involving changes in multiple signaling pathways in the conduction system.

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Casein Zymography for Analysis of Calpain-1 and Calpain-2 Activity

Cited by 6 publications

References 8 publications

Casein zymography‐based detection and one‐step purification for simultaneous quantification of calcium‐induced endogenous proteases in breast and thigh muscles from different chicken breeds

Casein zymography‐based detection and one‐step purification for simultaneous quantification of calcium‐induced endogenous proteases in breast and thigh muscles from different chicken breeds

The Influence of Vacuum Packaging of Hot-Boned Lamb at Early Postmortem Time on Meat Quality during Postmortem Chilled Storage

Fam20c regulates the calpain proteolysis system through phosphorylating Calpasatatin to maintain cell homeostasis

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