INSERTION AND TURNOVER OF MACROPHAGE PLASMA-MEMBRANE PROTEINS

The composition, insertion, and turnover of externally disposed proteins on the macrophage plasma membrane were analyzed. Cells labeled with [35S]methionine were incubated with the nonpermeant reagent trinitrobenzene sulfonic acid to introduce the trinitrophenyl moiety on free amino groups of externally oriented membrane proteins. The cells were then incubated with rabbit anti-dinitrophenyl IgG and the immune complexes formed with the trinitrophenyl-proteins were isolated from detergent lysates of the cells by using fixed Staphylococcus aureus as the immunoadsorbent. Proteins isolated by this method were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The interval between the release of newly synthesized proteins from ribosomes and their appearance at the cell surface, where they became accessible to trinitrobenzene sulfonic acid, was studied in pulsechase experiments. The 'transit' time of four major membrane glycoproteins (48,000-310,000 M(r)) ranged from 36 to 55 min and their appearance on the cell surface occurred in a relatively synchronous fashion. The turnover of most proteins of molecular weight above 50,000 was very slow (t( 1/2 ) > 80 hr) and was rather synchronous. Two exceptions were the 310,000 M(r) protein, which was lost with a t ( 1/2 ) = 21 hr, and a major glycoprotein (M(r) 48,000), which exhibited more complex kinetics. Although the overall turnover of surface proteins was biphasic in nature, the rapid phase of protein loss was largely due to low molecular weight species.