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Molecular weight of poly(C9). 12 to 18 C9 molecules form the transmembrane channel of complement
Insights
The study purified poly(C9), a key component of the complement membrane attack complex, revealing its molecular weight heterogeneity. This heterogeneity arises from variations in the number of C9 subunits, impacting the structure of the transmembrane channel.
Area of Science:
- Biochemistry
- Immunology
- Structural Biology
Background:
- The membrane attack complex (MAC) is crucial for complement-mediated cell lysis.
- Poly(C9) forms the transmembrane pore of the MAC.
- Understanding poly(C9) structure is vital for deciphering complement system function.
Purpose of the Study:
- To determine the molecular weight and structural heterogeneity of purified poly(C9).
- To investigate the number of C9 protomers in poly(C9) tubules.
- To elucidate the relationship between poly(C9) structure and its function in the MAC.
Main Methods:
- Purification of poly(C9) using gel filtration and sucrose density gradient ultracentrifugation.
- Molecular weight determination via sedimentation equilibrium analysis and scanning transmission electron microscopy (STEM).
- Analysis of detergent binding and electron microscopy for structural insights.
Main Results:
- Poly(C9) was purified to homogeneity, exhibiting a molecular weight of approximately 1.05-1.08 million Daltons.
- Two independent methods confirmed the molecular weight, accounting for bound detergent.
- Electron microscopy revealed inner diameters of 9-12 nm and suggested varying protomer numbers (12-18 C9 subunits per tubule).
Conclusions:
- Poly(C9) exhibits significant molecular weight heterogeneity due to variations in the number of C9 subunits.
- Most poly(C9) tubules consist of 14-16 C9 subunits.
- This structural variability may stem from flexibility in C9-C9 interactions, influencing pore formation.
Abstract:
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 or 1% deoxycholate containing buffer as solvents yielded a point weight average molecular weight exclusive of bound detergent of 0.9 to 1.3 X 10(6) and a weight average molecular weight of all poly(C9) complexes of 1,050,000 +/- 40,000 (S.D.). SDS and deoxycholate binding to 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.