Protein kinase C as a measure of transbilayer phosphatidylserine asymmetry.
The endogenous phosphatidylserine of normal erythrocytes is confined to the cytoplasmic leaflet of the membrane. However, under pathologic conditions transmembrane asymmetry can be altered and cytofacial phosphatidylserine may appear on the cell surface. A sensitive alternative method for the measurement of the exposed phosphatidylserine content of erythrocyte membrane was developed using the activation of exogenous protein kinase C. Erythrocytes containing exogenous phosphatidylcholine incorporated into the outer membrane monolayer do not stimulate protein kinase C activity more than untreated cells. In contrast, red cells that have exogenous phosphatidylserine incorporated into their membrane outer monolayer, by prior inhibition of the aminophospholipid transporter, stimulate protein kinase C significantly more than red cells in which exogenous phosphatidylserine is allowed to translocate to the inner membrane monolayer. Kinase activation is comparable for normal cells and cells not exposed to lipid in which the aminophospholipid transporter is inhibited with sulfhydryl reagents (diamide or N-ethylmaleimide). However, Ca(2+)-loading results in an increase in activation of protein kinase C over control cells, consistent with previous reports that Ca2+ induces the exposure of erythrocyte and platelet phosphatidylserine. By reference to protein kinase C activation by phosphatidylserine in model systems, the quantity of phosphatidylserine on the cell surface may be estimated. Thus, protein kinase C activation affords a sensitive and specific measure of phosphatidylserine in the outer monolayer of biological membranes.
Daleke
D L
DL
Department of Chemistry, Indiana University, Bloomington 47405.
Huestis
W H
WH
Newton
A C
AC
D000328:Adult; D000818:Animals; D002118:Calcium / Q000097:blood / Q000494:pharmacology; D002417:Cattle; D004789:Enzyme Activation; D004910:Erythrocyte Membrane / Q000737:chemistry* / Q000502:physiology; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry*; D008081:Liposomes; D010713:Phosphatidylcholines / Q000494:pharmacology; D010718:Phosphatidylserines / Q000097:blood* / Q000737:chemistry / Q000235:genetics; D010743:Phospholipids / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D012680:Sensitivity and Specificity; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D014178:Translocation, Genetic / Q000187:drug effects
D008051:Lipid Bilayers; D008081:Liposomes; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D010743:Phospholipids; D013438:Sulfhydryl Compounds; D011493:Protein Kinase C; D002118:Calcium