Assembly of avian skeletal muscle myosins: evidence that homodimers of the heavy chain subunit are the thermodynamically stable form.
Using a double antibody sandwich ELISA we examined the heavy chain isoform composition of myosin molecules isolated from chicken pectoralis major muscle during different stages of development. At 2- and 40-d posthatch, when multiple myosin heavy chain isoforms are being synthesized, we detected no heterodimeric myosins, suggesting that myosins are homodimers of the heavy chain subunit. Chymotryptic rod fragments of embryonic, neonatal, and adult myosins were prepared and equimolar mixtures of embryonic and neonatal rods and neonatal and adult rods were denatured in 8 M guanidine. The guanidine denatured myosin heavy chain fragments were either dialyzed or diluted into renaturation buffer and reformed dimers which were electrophoretically indistinguishable from native rods. Analysis of these renatured rods using double antibody sandwich ELISA showed them to be predominantly homodimers of each of the isoforms. Although hybrids between the different heavy chain fragments were not detected, exchange was possible under these conditions since mixture of biotinylated neonatal rods and fluoresceinated neonatal rods formed a heterodimeric biotinylated-fluoresceinated species upon renaturation. Therefore, we propose that homodimers are the thermodynamically stable form of skeletal muscle myosin isoforms and that there is no need to invoke compartmentalization or other cellular regulatory processes to explain the lack of heavy chain heterodimers in vivo.
Kerwin
B
B
Department of Food Science and Technology, University of California, Davis 95616.
Bandman
E
E
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D002642:Chick Embryo; D002645:Chickens; D002918:Chymotrypsin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006146:Guanidines / Q000494:pharmacology; D024510:Muscle Development; D009132:Muscles / Q000378:metabolism* / Q000648:ultrastructure; D009218:Myosins / Q000276:immunology / Q000378:metabolism* / Q000648:ultrastructure; D013816:Thermodynamics
D000911:Antibodies, Monoclonal; D006146:Guanidines; D002918:Chymotrypsin; D009218:Myosins