Physicochemical characterization of fluid phase (SC5b-9) and membrane derived (MC5b-9) attack complexes of human complement purified by immunoadsorbent affinity chromatography or selective detergent extraction |
| |
Authors: | Carl F. Ware Rick A. Wetsel William P. Kolb |
| |
Affiliation: | Department of Biochemistry, University of Texas Health Science Center, San Antonio, TX 78284, U.S.A. |
| |
Abstract: | Immunoadsorbent affinity chromatography, employing a monospecific caprine anti-human C5 (IgG)-agarose bead matrix, was applied to the purification of complement attack complexes from insulin activated normal human serum (SC5b-9). Gel filtration chromatography (Biogel A-15M) of the SC5b-9 complexes eluted from the anti-C5 column by guanidine-HCl was utilized as a second step to obtain a highly purified SC5b-9 preparation. Sodium dodecyl sulfate-polyacrylamide slab gel electrophoretic (SDS-PAGE) analysis of SC5b-9 isolated by immunoadsorbent chromatography revealed the appropriate subunit composition. Complement attack complexes extracted from complement lysed erythrocyte membranes (MC5b-9) upon solubilization with either Triton X-100 (TX-100) or deoxycholate (DOC) were also purified by anti-C5 immunoadsorbent and gel filtration chromatography, but with somewhat less than satisfactory results. Thus, an alternative purification procedure was developed based upon the ability of zwitterionic detergents to selectively extract MC5b-9 from erythrocyte membranes. Zwitterionic detergent solubilized MC5b-9 complexes were isolated in high yield in a one-step purification procedure by gel filtration column chromatography (Biogel A-15M). SDS-PAGE analysis of MC5b-9 isolated in this manner revealed a subunit composition which was similar to SC5b-9 except for the complete absence of S protein.Sucrose density gradient ultracentrifugal analysis of MC5b-9 complexes solubilized by TX-100, DOC, or zwitterionic detergents revealed similar sedimentation profiles with the major portion of MC5b-9 expressing a sedimentation coefficient of29S with minor peaks of 23S and 34S or greater. Thus MC5b-9 complexes solubilized from biological membranes by nonionic, anionic, or zwitterionic detergents form a heterogeneous population of high molecular weight, ordered, oligomeric structures with the 23S form representing MC5b-9 monomer, 29S the MC5b-9 dimer and 34S the MC5b-9 trimer or possibly tetrameric structure. These results clearly suggest a possible relationship between the observed MC5b-9 complex physical heterogeneity and the heterogeneity in the effective size of functional complement lesions. |
| |
Keywords: | BEP pH 8.8 EA antibody coated sheep erythrocytes EAC complement lysed EA NHS normal human serum PBS phosphate buffered saline PBS-E PMSF phenylmethyl sulfonyl fluoride EDTA SDS sodium dodecyl sulfate TX-100 triton X-100 DOC deoxycholate TW-85 Tween 85 SC5b-9 C5b-9 complex, containing S protein, assembled upon complement activation of NHS MC5b-9 C5b-9 complex assembled on EAC membranes |
本文献已被 ScienceDirect 等数据库收录! |
|