Abstract
Methyl group turnover rates for specific methyl-accepting membrane proteins in intact irreversibly sickled cells (ISCs) have been determined. The turnover of methyl groups on all methyl acceptor membrane proteins carboxylmethylated in ISCs is not concerted but proceeds in an ordered sequence which is markedly different from that exhibited by unfractionated normal erythrocytes (AA). In ISCs methyl group turnover based on initial demethylation rate constants is most rapid for membrane polypeptides migrating in sodium dodecyl sulfate at 30,000-39,000 Da, 40,000-55,000 Da, polypeptides comigrating with cytoskeletal component band 4.1, and band 4.5. In contrast, initial methyl group turnover rates obtained after less than 20% of the total methyl groups are turned over in (AA) cells show most rapid demethylation rates for membrane polypeptides migrating at 40,000-55,000 Da, polypeptides comigrating with band 4.5, cytoskeletal components bands 2.1 and 4.1. Results also show significant differences between ISCs and most dense fractions from normal (AA) and nonsickle hemolytic anemias in the demethylation of cytoskeletal proteins, bands 2.1 and 4.1. These findings indicate qualitative differences in accessibility of methyl acceptor substrates to the methylating-demethylating enzyme activity in the cytosol of irreversibly sickled cells compared to discocytic control erythrocytes.
Highlights
More recent studiescarried out by Ro et al [13] and Green and Kalra [14] have investigated the possibility that postsynthetic methylesterification of erythrocyte membrane proteins in certain pathological states, sickle cell disease, maybe altered compared to that observed with clinically normal human erythrocytes
In thisstudy polypeptide substrates designated as peaks A, B, C, and D correspond to Coomassie blue staining bands 2.1, 3, 4.1 [11], and 4.5 [10, 13, 14], respectively; uncharacterized substrate proteins migrating in SDS-gels at 40,000-55,000 Da, 30,00039,000 Da, and 15,000-20,000 Da are designated as peaks E, F, and G, respectively
The toprofile (Fig. 1) fornormal (AA)erythrocytes showed major [3H] methyl incorporation into threeproteins, peaks D, E, F, and minor incorporation for higher molecular weight proteins comigrating with bands 2.1, 3, and 4.1, described previously as major substratesfor the protein methyl transferaseactivity in erythrocytes measured under different conditions, i.e. 3Hmethyl incorporation at 2.5 h [11].to profiles for irreversibly sickled cells (ISCs)-rich fractions showed major incorporation into peaks D, E, F, as seen in normal (AA) cells, and major [3H]methyl group incorporation into polypeptides comigrating with band 4.1 and peak B comigrating with band 3, and into low molecular weight polypeptides in contrast to normal (AA)red cells
Summary
Membrane Protein Methyl Group Turnover in Normal and Irreversibly Sickled Erythrocytes-The turnover of total membrane protein methyl groups in normal and 1%-rich erythrocyte preparations has been studied. Intact red cells were subjected to a short pulse (60 min) of ~-[rnethyl-~H]methionine (4 PM) and subsequent incubation with excess nonradiolabeledmethionine (4 mM) added at time (to)R. adiolabeled membranes prepared from red cell aliquots taken after incubation at 1, 2, and 4hafter to were analyzed by SDSelectrophoresis at pH 2.4. Membranes prepared from aliquots of 3H-methylated red cells taken after the 60-minute pulse (to) and at 1-, 2-, and 4-h intervals of the cold chase were analyzed bydodecyl sulfate-gel electrophoresis Letters A-F indicate the ranges over which radioactivity in gel slices was integrated to constitute single methylated species
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