Labeling of pigeon erythrocyte membrane proteins by low-level reductive methylation

Abstract

The reductive methylation procedure of G.E. Means and R. E. Feeney (1968) Biochemistry 7, 2192–2201) was adapted for 3H-labeling of membrane proteins using pigeon erythrocyte membrane. Usably high 3H incorporation into protein was obtained, e.g., 28 μCi/mg protein with 83 nmol (input) H 2CO/mg protein, B 3H 4 − at 10 Ci/mmol, and a B 3H 4 −/H 2CO ratio of 0.34. With this low H 2CO/protein ratio, methylation did not perturb ATP-dependent 45Ca 2+ uptake, Na +-dependent [ 14C]glycine uptake, membrane vesicle sealing, or isoelectric focusing patterns of methylated membrane proteins. The labeled membrane proteins were shown to be good tracers for the unlabeled proteins by using two-dimensional isoelectric focusing x sodium dodecyl sulfate gel electrophoresis.