Kinetics of biosynthesis of acetylcholine receptor and subsequent incorporation into plasma membrane of cultured chick skeletal muscle

20% of the acetylcholine receptors in cultured chick skeletal muscle remain unbound following long-term growth of muscle in medium containing a potent, essentially irreversible receptor-blocking agent, α-bungarotoxin. About half the receptors which are unavailable for interaction with extracellular α-bungarotoxin are newly synthesized molecules which presumably are being processed and transported to the plasma membrane. When the muscle cultures are switched to a medium containing ²H, ¹³C, ¹⁵N-amino acids, these receptors are rapidly labeled, the fraction of labeled molecules beginning to plateau at 3 hr. Few labeled receptors appear in the plasma membrane during the first 3 hr of labeling with ²H, ¹³C, ¹⁵N-amino acids. After 3.5 hr of labeling, virtually all the receptors being incorporated into the plasma membrane are labeled receptors. The kinetics of labeling of the "pool" and "surface" receptors with ²H, ¹³C, ¹⁵N-amino acids confirm the "precursor-product" type relationship of pool and surface acetylcholine receptors. In this study, receptors synthesized in medium containing ²H, ¹³C, ¹⁵N-amino acids were resolved from ¹H, ¹²C, ¹⁴N-receptors by velocity sedimentation in sucrose-deuterium oxide and sucrose-H₂O gradients, and their densities were estimated from sedimentation rates in shallow gradients of various average density. Estimated densities were 1.32 g/cm³ for ¹H, ¹²C, ¹⁴N-receptors and 1.41 g/cm³ for ²H, ¹³C, ¹⁵N-receptors. This density difference corresponds to 80% substitution of normal aminoacyl residues by ²H, ¹³C, ¹⁵N-residues in the denser receptor.