Abstract
The level of carboxyl methylation of membrane proteins has been measured in intact human erythrocyte populations of different ages separated by density gradient centrifugation. Age separation was confirmed by measurement of cytosolic pyruvate kinase specific activity in each fraction. When cells of different ages were incubated with L-[methyl-3H]methionine, the steady state level of 3H radioactivity covalently bound to membrane proteins is observed to be at least 3-fold higher in older erythrocytes. Because the specific radioactivity of the methyl group donor S-adenosyl-L-[methyl-3H]methionine was identical in all age fractions, this represents an increase in the extent of modification of membrane proteins by carboxyl methylation. Of the three major methylated erythrocyte membrane proteins, this increase in carboxyl methylation with age is 4 to 7-fold for bands 2.1 and 3, while the increase in band 4.1 is 3 to 4-fold. This increase in the steady state level of methylation with age cannot be explained by changes in either the intrinsic rate of methyl transfer or by changes in the rate constant of methyl turnover. We, therefore, propose that the age-dependent change in carboxyl methylation is due to an increase in the number of available acceptor sites as the erythrocyte ages in vivo. Since methylation of acidic residues on erythrocyte membrane proteins has been detected exclusively on D-aspartic acid residues (McFadden, P. N., and Clarke, S. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2460-2464), these results are consistent with an accumulation of D-aspartic acid in membrane protein due to spontaneous racemization a the cell ages. The relationship of these observations to possible functions of erythrocyte membrane protein carboxyl methylation is discussed.
Highlights
The levelof carboxyl methylation of membrane pro- this enzymatic reactionhas been observed in many teins has been measured in intact human erythrocyte mammalian tissues
State level of 3Hradioactivity covalentlybound to mem- This reaction occurs in bacteria where methylation of spebrane proteins is observed to be at least %fold higher cific glutamyl residues of chemoreceptors has been shown to tsiobnhefrinaotohntlnsdeeiemnapreneeroitrwnhytecaythirslnergisaodrscobeeyunytiptenicscda.aotrBlhbneieoonecxrxaSyatue-llslnameatdtgeeohetfnehomfyssrploayaecdltciti-ioiffo~inicn-c.as[rOta,miodftehnittoihhosaeyfcrZmttei-hpve~rirmeHteye]--me-btcHeheioaniwinsnvesavor(el9evr,em1hd1yoi)dnr.erItonhcloyensfiasasicdsrtata,eptnetttash,&wteoifomitnmheptmhehteyehnltyhoelymsetleeasrnttieooorfnnsaoosffpaecatuhrkeatamsaptreoyatorathtxyiacilsbspse(ire8done)-. major methylated erythrocyte membrane proteins, this isolated from proteolytic digests of human erythrocyte memincrease in carboxyl methylation with age is 4 to 7-fold brane proteins [12]
Ithas been shown that the for bands 2.1 and 3,while the increase in band 4.1 is 3 aspartyl-P-methyl ester isolated in this way is entirely in the to 4-fold
Summary
From the Departmentof Chemistry a n d Biochemistry a n d The MolecularBiology Institute, University of California, Los Angeles, California 90024. Age separation was confirmed by it is freely reversible in the cell yielding methanol as the measurement of cytosolic pyruvate kinase specific ac- product [4,5,6] This reversibility suggests that this reaction tivity in each fraction. A. 79, 2460-2464), these resultsare consistent with an accumulation of Daspartic acid in membrane protein due to spontaneous racemization as thecell ages The relationship of these observations to possible functions of erythrocyte membrane protein carboxyl methylation is discussed. Since the specific radioactivity of the methyl group donor, AdoMet, was the same in all age fractions, this represents an increase in the steady state numboefrcarboxyl methyl esters We suggest that this increase is due to the exposure of new methylatable sites on membrane proteins. Approximateaverage cell age was calculatedfrom pyruvate kinase specific activity deviations from that of unfractionated cells using a first order half-timeof 29 days [26] and assuming an average age of 60 days for unfractionated cells
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
