Insulin-like growth factor I receptor analysis of satellite cell-derived myotube membranes established from two lines of Targhee rams selected for growth rate.
Ovine-derived fibroblasts were used to validate an insulin-like growth factor I (IGF-I) membrane-receptor binding assay system. Competitive binding using fibroblasts revealed that half-maximal inhibition of 125I-IGF-I binding by IGF-I was 2.3 nM. SDS-polyacrylamide gel electrophoresis analysis of specific protein-associated 125I-IGF-I was consistent with the migration of 125I-IGF-I-labeled Type I IGF receptor alpha-subunits at Mr 133,000 daltons. Further, the efficiency of two cell solubilization methods was examined and time-dependent binding equilibrium was determined for the membrane assay system. Satellite cell-derived myotubes were subsequently isolated from primary satellite cell cultures established from the semimembranosus muscles of high and low efficiency-of-gain (EOG) Targhee rams, and IGF-I receptor dynamics were measured. A membrane competitive binding study revealed that half-maximal inhibition of 125I-IGF-I binding was achieved by 1-ng IGF-I for low, and 10-ng IGF-I for high, EOG myotube membrane preparations. Kd values were similar between the high EOG (4.78 nM) and low EOG (2.95 nM) groups; however, receptor concentrations (Bmax) appeared to differ between groups. High EOG membrane receptor Bmax was 3.88 pmole/micrograms protein (19.87 pmole/micrograms DNA), whereas low EOG membrane receptor Bmax was 1.22 pmole/micrograms protein (9.28 pmole/micrograms DNA). These preliminary findings support the hypothesis that genetic selection for EOG results in altered satellite cell responsiveness to IGF-I.
Mathison
B D
BD
Department of Animal Sciences, Washington State University, Pullman 99164-6320.
Mathison
B A
BA
McNamara
J P
JP
Dodson
M V
MV
D000375:Aging; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D046508:Culture Techniques / Q000379:methods; D005347:Fibroblasts / Q000378:metabolism; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D024510:Muscle Development; D009132:Muscles / Q000166:cytology / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D017451:Receptors, Somatomedin; D012756:Sheep / Q000254:growth & development*; D013002:Somatomedins / Q000378:metabolism*; D013045:Species Specificity
D011956:Receptors, Cell Surface; D017451:Receptors, Somatomedin; D013002:Somatomedins; D007334:Insulin-Like Growth Factor I