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

Abstract

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, alpha-bungarotoxin. About half the receptors which are unavailable for interaction with extracellular alpha-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.