Use of casein zymography to measure the activities of … of casein zymography to measure the ......
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Arch. Tierz., Dummerstorf 49 (2006) Special Issue, 90 Danish Institute of Agricultural Sciences, P.O Box 50, 8830-DK Tjele, Denmark M. THERKILDSEN, I. L. SØRENSEN, and N. OKSBJERG Use of casein zymography to measure the activities of μ- and mM calcium-dependent calpains during myogenesis in primary porcine satellite cell cultures (preliminary data) (Anwendung von Casein-Zymographie zur Messung von den Aktivitäten der μ- und mM Kalzium-abhängigen Kalpaine während der Myogenese in primären Satellitenzellkulturen von Schweinen (vorläufige Daten)) Muscle cells in cultures cease to proliferate and start to differentiate morphologically (fusion of cells) and biochemically (expression of muscle specific proteins) following a reduction of growth factors in the medium. Several pieces of evidence suggest that the calpain system (μ- and mM calpains, their inbibitor calpastatin, and muscle specific calpain p94), especially the mM calpain, is involved in the fusion processes. Measuring the activity of μ- and mM calpains by purification requires large amounts of cells. Measurement of calpain activities by use of casein zymography, which is more sensitive, may be an alternative. Consequently, we have studied μ- and mM calpain activities by use of casein zymography during myogenesis of porcine satellite cells. Porcine satellite cells were isolated from 12-week old pigs and seeded in 24-well plates. Cells were grown in growth medium until 80% confluence after which the medium was replaced with a differentiation medium. Cells were harvested at 50% and 80% confluence and the days after changing the medium were designated d1, and d2, d3, and d 4. Following homogenisation zymography was carried out on none- denaturated 12.5% separating gels with 0.5% casein incorporated. The activities were quantified as density of bands. Results: Developmental stages 50% 80% d 1 d 2 d 3 d 4 mM-Calpain 345 369 835 827 868 659 μM Calpain nt nt 326 329 524 574 CK, mU/well 1.71 3.04 3.90 9,79 15,9 14,4 The data show that mM calpain activity was low in proliferating cells but increased to high levels preceding the fusion of cells (creatine kinase activity) and stayed high in differentiated cultures. The activity of μM calpain was not detectable in proliferating cells but increased steadily during differentiation cultures. In conclusion, the increased activity preceding fusion indicates that at least mM calpain is required in the fusion of muscle cells during myogenesis. Corresponding Author M. THERKILDSEN Abteilung für Lebensmittelwissenschaft Postfach 50 DK-8830 Tjele Dänemark E-Mail: [email protected]
Transcript of Use of casein zymography to measure the activities of … of casein zymography to measure the ......
Arch. Tierz., Dummerstorf 49 (2006) Special Issue, 90
Danish Institute of Agricultural Sciences, P.O Box 50, 8830-DK Tjele, Denmark
M. THERKILDSEN, I. L. SØRENSEN, and N. OKSBJERG
Use of casein zymography to measure the activities of µ- and mM calcium-dependent calpains during myogenesis in primary porcine satellite cell cultures (preliminary data) (Anwendung von Casein-Zymographie zur Messung von den Aktivitäten der µ- und mM Kalzium-abhängigen Kalpaine während der Myogenese in primären Satellitenzellkulturen von Schweinen (vorläufige Daten))
Muscle cells in cultures cease to proliferate and start to differentiate morphologically (fusion of cells) and biochemically (expression of muscle specific proteins) following a reduction of growth factors in the medium. Several pieces of evidence suggest that the calpain system (µ- and mM calpains, their inbibitor calpastatin, and muscle specific calpain p94), especially the mM calpain, is involved in the fusion processes. Measuring the activity of µ- and mM calpains by purification requires large amounts of cells. Measurement of calpain activities by use of casein zymography, which is more sensitive, may be an alternative. Consequently, we have studied µ- and mM calpain activities by use of casein zymography during myogenesis of porcine satellite cells. Porcine satellite cells were isolated from 12-week old pigs and seeded in 24-well plates. Cells were grown in growth medium until 80% confluence after which the medium was replaced with a differentiation medium. Cells were harvested at 50% and 80% confluence and the days after changing the medium were designated d1, and d2, d3, and d 4. Following homogenisation zymography was carried out on none-denaturated 12.5% separating gels with 0.5% casein incorporated. The activities were quantified as density of bands. Results: Developmental stages
50% 80% d 1 d 2 d 3 d 4
mM-Calpain 345 369 835 827 868 659 µM Calpain nt nt 326 329 524 574 CK, mU/well 1.71 3.04 3.90 9,79 15,9 14,4
The data show that mM calpain activity was low in proliferating cells but increased to high levels preceding the fusion of cells (creatine kinase activity) and stayed high in differentiated cultures. The activity of µM calpain was not detectable in proliferating cells but increased steadily during differentiation cultures. In conclusion, the increased activity preceding fusion indicates that at least mM calpain is required in the fusion of muscle cells during myogenesis.
Corresponding Author M. THERKILDSEN Abteilung für Lebensmittelwissenschaft Postfach 50 DK-8830 Tjele Dänemark
E-Mail: [email protected]
