Influence of osmotic stress on protein methylation in resealed erythrocytes.
Resealed erythrocytes are a useful means of targeting exogenous proteins or extending their activity. We tested human resealed erythrocytes as a model system for studying protein methyl esterification, a reaction involved in the processing of spontaneously deamidated/isomerized polypeptides. Our results show that resealed erythrocytes are still active in the metabolic processes that lead to the formation of methyl-esterified proteins. The methylation pattern of endogenous membrane proteins appeared to be similar to that of normal erythrocytes, with bands 2.1, 3, 4.1 and 4.2 as the major methyl acceptors. We detected methyl esterification of ovalbumin, as an exogenous substrate trapped within resealed erythrocytes. Methyl incorporation was almost completely inhibited by simultaneously loading red cells with adenosine and homocysteine thiolactone, in vivo precursors of the transmethylation inhibitor S-adenosylhomocysteine. We investigated the effects of repeated resealing procedures on methyl acceptability of endogenous membrane proteins. We found that methyl-incorporation levels increased, despite an apparent conserved protein composition of the membrane. This result suggests that osmotic stress to the membrane may be responsible for increased protein methylation due to the appearance of new sites or an increased accessibility of existing sites.
Ingrosso
D
D
Institute of Biochemistry of Macromolecules, School of Medicine, Second University of Naples, Italy.
Cotticelli
M G
MG
D'Angelo
S
S
Buro
M D
MD
Zappia
V
V
Galletti
P
P
D001665:Binding Sites; D001693:Biological Transport, Active; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000097:blood; D008715:Methionine / Q000378:metabolism; D008716:Methionine Adenosyltransferase / Q000097:blood; D008745:Methylation; D009997:Osmotic Pressure; D010047:Ovalbumin / Q000378:metabolism; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase; D011496:Protein Methyltransferases / Q000097:blood; D011506:Proteins / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000378:metabolism
D008565:Membrane Proteins; D011506:Proteins; D012436:S-Adenosylmethionine; D010047:Ovalbumin; D008715:Methionine; D011496:Protein Methyltransferases; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase; D008716:Methionine Adenosyltransferase