Incorporation of [ 14C]glycine into Micrococcus lysodeikticus membrane protein and effects of protein synthesis inhibitors

Abstract

14C]Glycine incorporation into Micrococcus lysodeikticus membranes and membrane protein showed a variable level of resistance to the inhibitory effects of chloramphenicol at 100 μg/ml, a concentration which inhibited the incorporation into the cytoplasmic protein fraction to the extent of 90–97%. Incorporation into the membrane protein in the presence of chloramphenicol ranged from 30 to 100% that observed for control (no chloramphenicol) membrane protein fractions. Only 1% of the total incorporation, both in the presence and absence of chloramphenicol, could be accounted for as N-terminal groups of the proteins. Over 80% of the incorporated [ 14C]glycine was recovered as glycine after acid hydrolysis and on high-voltage electrophoresis of pronase digests, the label migrated as free glycine and peptides. Fractionation of the membrane proteins after lipid extraction with n- butanol revealed that the “water-soluble protein” fraction was the least affected by chloramphenicol and that incorporation into the “water-insoluble protein” fraction was inhbited to the extent of 75%. No such differential inhibitory effect upon incorporation into cytoplasmic and membrane proteins was observed with puromycin (5 μg/ml) which inhibited the uptake into both cellular fractions to an equal extent (80% after 90 min preincubation with the inhibitor). Cycloheximide was ineffective on both growth and incorporation of [ 14C]glycine.