RESUMO
With a new ektacytometry, we studied the relation between the microstructure of red blood cell (RBC) membrane and the rheological properties of RBCs in a shear flow field of low viscosity. The main contributions of this paper are as follows: 1. The hemorheological meanings of the orientation index (DI)or and the small deformation index (DI)d were explored. (DI)or is an overall rheological index depending on the deformability and morphology of RBCs. The better the physiological shape of RBCs is maintained, the greater the (DI)or is. (DI)d can be used to describe the lipid fluidity of RBC membrane. Such an explanation for the meaning of (DI)d has been forcefully supported by our experiments using electron spin resonance (ESR) and fluorescence polarization. 2. The influence of wheat germ agglutinin (WGA) of different concentrations on the lipid fluidity of membrane is different from that of concanavalin A (ConA). The lipid fluidity of membrane changes with WGA concentration treating RBCs and there is a maximum value for the membrane fluidity at a specific concentration of WGA. However, the deformability of membrane described by the integrate deformation index (IDI) monotonically decreased with the increase in WGA concentration treating RBCs. 3. It is concluded that the increase in the lipid fluidity of red cell membrane is not necessarily associated with the improvement of RBC deformability.
Assuntos
Concanavalina A/farmacologia , Deformação Eritrocítica/efeitos dos fármacos , Membrana Eritrocítica/metabolismo , Receptores de Concanavalina A/efeitos dos fármacos , Receptores Mitogênicos/efeitos dos fármacos , Aglutininas do Germe de Trigo/farmacologia , Animais , Concanavalina A/sangue , Lipídeos de Membrana/sangue , Coelhos , Receptores de Concanavalina A/sangue , Receptores Mitogênicos/sangue , Aglutininas do Germe de Trigo/sangueRESUMO
A high-performance concanavalin A (Con A) affinity column Gelpack GL-L55C (Hitachi Kasei Industries) was successfully used for the fractionation of human serum Con A-binding proteins. Serum proteins that have strong affinity to Con A (ca. 11% of the recovered proteins) could be fractionated within 80 min. By analysing the eluates from the column by micro two-dimensional electrophoresis, followed by blotting and Con A staining, the specificity of the column was effectively visualized. Although the protein-binding capacity of the column gradually decreased during repeated loading of serum or tissue extracts, the specificity of the column to Con A-binding proteins did not change. Serum lipoproteins have been eluted from the column with 6 M urea, suggesting that the capacity decrease is caused by the binding of lipids or lipoproteins to the column.
Assuntos
Receptores de Concanavalina A/sangue , Cromatografia Líquida de Alta Pressão , Colódio , Eletroforese em Gel de Poliacrilamida , Humanos , Imunoquímica , Técnicas Imunoenzimáticas , Lectinas/isolamento & purificaçãoRESUMO
The concanavalin A receptor from human erythrocyte membranes has been isolated by affinity chromatography using the mild, readily-dialyzable detergent dodecyltrimethylammonium bromide. The purified protein has been reincorporated into large unilamellar phospholipid vesicles using a detergent dialysis technique. The mean diameter of these vesicles increases as the lipid: protein ratio decreases. Binding of succinyl-concanavalin A to these vesicles was quantitated using 125I-labelled lectin in a filtration assay. The concanavalin A receptor in lipid bilayer vesicles provides specific high affinity binding sites for succinyl-concanavalin A with an association constant of 2.13 . 10(6) M-1. Scatchard plots indicate positive cooperativity of binding at very low lectin concentrations, a characteristic also seen in concanavalin A binding to intact human erythrocytes. The presence of bovine serum albumin has little effect on lectin binding and is not required for expression of cooperativity. Concanavalin A effectively competes with succinyl-concanavalin A for binding to the vesicles with an association constant of 4.83 . 10(6) M-1. Receptor-bearing vesicles are readily agglutinated by concanavalin A but not by its succinylated derivative. The kinetics of vesicle agglutination are biphasic, with an initial rapid phase followed by a pseudo-first order process. We suggest that studies on reassembled receptor proteins in lipid bilayers can provide valuable insight into receptor involvement in transmembrane signalling events and the factors involved in cell membrane behaviour and cell agglutination.
