Abstract
We have measured the rate of accumulation of amino acid residues in human erythrocyte membrane and cytosolic proteins which give D-aspartic acid upon acid hydrolysis. These residues would include D-aspartic acid, D-asparagine, as well as the beta-transpeptidation product, D-isoaspartic acid. Measurements made using age (density) fractionated cells indicate that racemization at these residues occurs on membrane proteins with a t1% (the time required to convert 1% to the D configuration) of about 38.6 days. Fractionation of membrane components revealed a faster rate of racemization for intrinsic proteins than for extrinsic proteins. On the other hand, significant age-dependent racemization was not detected for cytosolic proteins, and the calculated t1% value for these proteins is at least 4 times larger. These results suggest that in the 120-day life span of an erythrocyte, significant racemization of membrane (but not cytosolic) proteins can occur. We have also determined that the rates of accumulation of these residues for erythrocyte membrane and cytosolic proteins incubated in vitro are similar to those observed in vivo. These observations are discussed in terms of the possible cellular metabolism of racemized proteins.
Highlights
We have measured threate of accumulation of aminostudieshad indicated that enzymes present in these cells acidresidues in human erythrocytemembraneand could catalyze the formation of protein D-aspartic acid pcytosolicproteins whichgive D-aspartic acidupon acid methyl esters and L-isoaspartic acid a-methyl esters (5-10)
These results suggested that these methyltransferases recogacid, D-asparagine, as well as the &transpeptidation nize chemically altered aspartyl residues and that themethzwtmcmpaeaogriieitnotnezimhdfoasi(.ntubgdaicrouOeatatnrnn,tlnas% ftDetitohtihoy-cre(ieno)tssiohnemof)troeathraprfsteaeicopinsrmbtnaiihsodoereiaunuctnnirtteacpsedstrrq,eaooeud3cvtcii8ecredcsi.ueaen.6idrgllsleMsnsdtdtoioaheifanniayaccdssonfamu.aiFnncrsfevratetomaeetmraercrebgttextrirehno1taa-ratn% tdnsietaneerttopmsioapfoieccanrntredhopodameetfrceeoeinDiu--n-tssinpslTypdgiprlreshaooomgutttnriese.oat,iiadnnninatsrehtieisoayoapundcnosdtriosooftslfohnyirbpasmclartoeotaeutttthhselidoieawnnbtsnoeuoecttflhhotdaneectchttefchaeliuliersnsimnestinuarrgsebleatflsetlesiiepduotcuocnthiemtbnoshorfeeeattDshnariebd-dtpaouhastleehpiizsreareoira(rrt5rtiae,csmcse1ealeo0mcetfi-cad1itbtzi2hoiev)ned-e
Racemization was not detected for cytosolic proteins, Mechanistic considerations further suggest that racemizaand the calculated t l % value for these proteins is at tion andisomerization-prone intermediates may beimportant least 4 times larger. These results suggest that in the in the formation of D-aspartyl and L-isoaspartyl residues in
Summary
To examine whether significant protein racemization reactions can occur more generally in mammalian tissues and whether acell can limitthe accumulation of racemized aspartate and its derivatives, we chose to studythis process in human erythrocytes. The membrane and cytosolicproteins of these cells are synthesized in erythrocyte precursors; mature cells exist in the bloodstream for 120 days with essentially no protein synthesis or degradation. We were interestedin the erythrocyte system because recent
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