Biosynthesis of peripheral nervous system myelin proteins in vitro

Myelin prepared from spinal roots and sciatic nerves of young rats (24 days of age) after incubation with [3H] amino acids in glucose-Krebs Ringer bicarbonate contained a considerable amount of radioactivity incorporated into the proteins. When these were separated on SDS-polyacrylamide gels, peaks of radioactivity were found to correspond to the P0, 23K, P1, and P2 proteins. Although very little Fast-Green-stained protein was visible in the high molecular weight region of the gels (> 27,000), much radioactivity was located in this area. Incorporation of [3H] amino acids into the myelin proteins continued up to 3 h and was dependent on glucose, but not on O2-CO2 replacement of air in the flask, whereas cycloheximide inhibited incorporation by 80%. Much less amino acid was incorporated into myelin protein of old rats. High molecular weight proteins appeared to be more metabolically active than other myelin proteins, as estimated by the percent of total counts incorporated/amount of Fast-Green staining, and their activity decreased less with age. The 23K and P1 proteins incorporated [3H] amino acid in approximate proportion to their content. P0 was less, and P2 somewhat more metabolically active than their contents would predict. [3H]fucose also was incorporated in vitro into P0 and P1 (probably 19K) proteins as well as into the high molecular weight proteins, while glucosamine served less well as a glycoprotein precursor. This preparation may be useful for examining further parameters of PNS myelin synthesis, assembly, and the effects of neurotoxic agents.