Peripheral nervous system myelin assembly in vitro: perturbation by the ionophore monensin.

Abstract

Abstract: Sciatic nerves from 13‐day‐old rats were incubated in vitro with [35S]methionine in the presence or absence of 0.22 μM monensin and total paniculate and myelin fractions prepared. The total particulate was further subfractionated by continuous density gradient centrifugation, after which the maximal specific activities of three marker enzymes, 2′,3′‐cyclic nucleotide phospho‐diesterase (myelin), 5′‐nucleotidase (plasma membrane), and cerebroside sulphotransferase were recovered at 0.72, 0.82, and 0.92 M sucrose, respectively. The radiolabelled proteins present in the gradient subtractions were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) and fluorography, and bands corresponding to the P0 and myelin basic proteins were identified by co‐migration with unlabelled myelin marker proteins on both one‐dimensional SDS‐PAGE and two‐dimensional nonequilibrium isoelectric focussing/SDS‐PAGE systems. Following a 90‐min incubation with [35S]methionine, newly synthesized myelin basic proteins were recovered in fractions between 0.5 and 0.7 M sucrose; this distribution was unaltered by monensin. In contrast, the distribution of newly synthesized P0 protein across the gradients was influenced by monensin: a bimodal distribution across the control gradients with peaks of recovery of 0.60 and 0.82 M sucrose was altered to give a single peak at an intermediate density of 0.72 M sucrose. The total proportions of newly synthesized P0 and myelin basic proteins (MBP) present across the entire gradients were calculated from the fluorograms, and the ratio was found to be 2.8 P0: (LBP + SBP), in both the presence and absence of the ionophore. However, only 70% and 50% of the control levels of MBP and P0 were recovered with a purified myelin fraction after incubation with monensin. The results are discussed with reference to different intracellular transport processes for the P0 glycoprotein and the MBP within the Schwann cell, and also to the differential compartmentation of the sites of synthesis and membrane export within the Golgi body.