Measurement of protein degradation by release of labelled 3-methylhistidine from skeletal muscle and non-muscle cells.
The rates of [3H]N(tau)-methylhistidine (3-MH) accumulation in the medium, following pulse labelling of cells for 48 h with [3H]methionine, were used to measure myofibrillar protein degradation. In fused C2C12 myotubes, incubation for 24 or 48 h after the labelling period gave rates of myofibrillar degradation of 38 and 42%/day. In a leucine free medium, these rates were similar; 40 and 47%/day, respectively. Using identical conditions +/- leucine, but in the absence of [3H]-methionine, rates of protein accretion and synthesis over 24-48 h were measured. From these data, rates of total protein degradation were calculated by difference and were similar to myofibrillar degradation rates. We have used the same pulse labelling protocol to assess whether the method is applicable to non-muscle cell lines based on the knowledge that 3T3 fibroblasts contain actin in the cytoskeleton. 3-MH was detected both in protein and upon its release into the medium. Actin degradation measured over a 48 h period gave a value half that obtained for total degradation, but the results suggest that the release of 3-MH by fibroblasts in vivo could be appreciable. The development of this methodology should provide a useful tool to investigate signalling mechanisms regulating actin degradation in a variety of cell types.
Thompson
M G
MG
Rowett Research Institute, Bucksburn, Aberdeen, United Kingdom.
Palmer
R M
RM
Thom
A
A
Mackie
S C
SC
Morrison
K S
KS
Harris
C I
CI
D016475:3T3 Cells; D000199:Actins / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D003470:Culture Media; D004151:Dipeptides / Q000032:analysis; D005347:Fibroblasts / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism; D007930:Leucine / Q000502:physiology; D008715:Methionine / Q000378:metabolism; D008762:Methylhistidines / Q000378:metabolism*; D051379:Mice; D009124:Muscle Proteins / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism*; D009210:Myofibrils / Q000378:metabolism; D014316:Tritium
D000199:Actins; D003470:Culture Media; D004151:Dipeptides; D008762:Methylhistidines; D009124:Muscle Proteins; D014316:Tritium; C028119:N(beta)-alanyl-1-methyl-histidine; D006639:Histidine; D008715:Methionine; D007930:Leucine; C028118:3-methylhistidine