Centrifugation-based isolation of myosin for measurement of its synthesis rate in small muscle samples.
Myosin is involved in muscle mobility which is particularly affected in many pathophysiological situations. It is composed of heavy (MHC) and light (MLC) chains and measurements of its specific fractional synthesis rate (FSR) are scarce, mostly because of difficulties in isolating this protein. Our aim was to isolate pure myosin from small rat gastrocnemius skeletal muscle samples by setting up a procedure compatible with determination of stable isotope incorporation into myosin using mass spectrometry detection, allowing calculation of its FSR. A centrifugation method was compared to a validated but time-consuming elution gel electrophoresis method. Statistical analysis by the Bland and Altman test revealed a tight relationship between both methods (r2 >0.97, p <0.0001). The purity of the myosin fractions using the two procedures was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition, the centrifugation procedure allowed simultaneous purification of MLC and MHC, whereas the elution gel electrophoresis technique resulted only in MHC isolation. Finally, the FSRs of myosin and MHC were found to be 0.114+/-0.026 and 0.140+/-0.029%/h, respectively (p not significant). In conclusion, the centrifugation method is a useful and reproducible procedure that results in sufficient amounts of pure myosin for reliable determinations of its own synthesis rate in vivo.
Zangarelli
A
A
Unité du métabolisme protéino-énergétique, UMR Université d'Auvergne/INRA, Centre de Recherche en Nutrition Humaine, CHU de Clermont-Ferrand, 63009 Clermont-Ferrand, France.
Walrand
S
S
Guillet
C
C
Gachon
P
P
Rousset
P
P
Giraudet
C
C
Picard
B
B
Boirie
Y
Y
D000818:Animals; D002498:Centrifugation / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D008297:Male; D018482:Muscle, Skeletal / Q000737:chemistry / Q000378:metabolism*; D009218:Myosins / Q000096:biosynthesis* / Q000302:isolation & purification*; D051381:Rats; D017208:Rats, Wistar
D009218:Myosins