Poly(C9), the tubular 27 S complex forming the transmembrane channel of the membrane attack complex of complement, was purified to homogeneity by gel filtration and sucrose density gradient ultracentrifugation. The molecular weight of poly(C9) was determined by two independent methods in addition to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. First, sedimentation equilibrium analysis using 0.2% SDS for 1% deoxycholate containing buffer as solvents yielded a point weight average molecular weight exclusive of bound detergent of 0.9 to 1.3 x 106 and a weight average molecular weight of all poly(C9) complexes of 1,050,000 ± 40,000 (S.D.). SDS and deoxycholate binding of poly(C9) was measured in an air-driven ultracentrifuge and was determined to be 0.53 ± 0.065 (S.D.) g of SDS and 0.26 ± 0.015 (S.D.) g of deoxycholate/g of poly(C9), respectively. Second, the mass of 27 S poly(C9) devoid of bound detergent was determined by electron scattering of unstained specimens in the scanning transmission electron microscope. The molecular weight obtained by this method was 1,078,000 ± 194,000. The inner diameter of poly(C9) tubules imaged in top view projections by negative staining electron microscopy varied between 9 and 12 mm. The accumulated data suggest a true heterogeneity of the molecular weight of poly(C9) due to polymers with varying protomer numbers. Using a mean value of 73,500 for the molecular weight of monomeric C9, the protomer number of poly(C9) tubules appear to vary between 12 and 18 C9 subunits. Approximately 50-75% of the tubules have 14 to 16 subunits as deduced from the mass distribution determined by electron scattering and from ring size measurements. It is suggested that poly(C9) tubules with various protomer numbers may arise due to limited flexibility in the C9-C9 interaction.
|Original language||English (US)|
|Number of pages||7|
|Journal||Journal of Biological Chemistry|
|State||Published - 1984|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology