Abstract
The eighth component of human complement (C8) has been purified in high yield from Cohn Fraction III and characterized with regard to its physicochemical properties and subunit structure. The purified product was found to be similar to functional C8 isolated from plasma or serum. Human C8 possesses a molecular weight of 151,000 and is composed of a 1:1:1 ratio of three nonidentical subunits: alpha (Mr = 64,000), beta (Mr = 64,000), and gamma (Mr = 22,000). These subunits occur as a covalently linked alpha-gamma dimer which is noncovalently associated with beta. After purification and characterization of alpha, beta, and gamma, each was found to possess different amino acid compositions and NH2-terminal sequences. Both alpha and beta subunits contain similar but exceptionally high percentages of hydrophobic aromatic amino acids. As measured by circular dichroism, the secondary structure of C8 contains 12% alpha-helix, 24% beta structure, and 64% unordered structure, values typical of globular proteins. Complete secondary structure, as well as hemolytic activity, can be recovered after exposure to 6 M guanidinium hydrochloride or 8 M urea. The alpha-gamma and beta subunits were dissociated and isolated in the presence of sodium dodecyl sulfate and after removal of detergent, neither was found to possess independent hemolytic activity. Significantly, activity equivalent to that of native C8 was generated when alpha-gamma and beta were recombined in an equimolar ratio. These results indicate that C8 is an atypical serum protein with regard to both its subunit structure and denaturation characteristics.
Highlights
Amphiphilic macromolecular complex of C5b-9 which, unlike has been purified inhigh yield from Cohn FractionI11 its individual constituents,exhibits ahighaffinity for cell and characterized with regard to its physicochemical membranes, liposomes, phospholipid, and certain detergents properties and subunit structure
When assembled on a cell surface, C5b-9 effectively was found to be similar to functional CS isolated from disrupts membrane organization without degradation of coplasmaorserum.Human C8 possesses amolecular valent structures in the lipid bilayer and, proweight of 151,000 and is composed of a 1:l:l ratio of duces membranelysis and cell death [11,12,13,14,15]. three nonidentical subunits: a (Mr = 64,000), /3 (Mr = Theproteins of C5b-9 areunique in thateachhasthe 64,000), and y (Mr = 22,000)
As well as he- may correspond to extended sequenceosf hydrophobic amino molytic activity, can be recovered after exposure to 6 acids similar to those found in integral membrane proteins
Summary
Becausethea-chain molecularweightwassignificantly fraction 3 where maximum activity isobserved It is important lower than 77,000 reported for the same subuninitC8 isolated to note thagtel profiles inFig. 3 show a component in fractions from serum [20], we considered the possibility that our puri- 1and 2 which co-migrates with the, subunit on nonreduced fied material was proteolytically degraded. T o examine this further, we isolated C8 under conditions where the potentiaflor proteolytic degradation is a t a minimum.Intheseexperiments,freshfrozen plasma supplemented with proteolytic inhibitors was subjected to the same purification procedures used for Cohn mately M , = 72,000 on reducedgels It isemphasized that this component has coincidental electrophoremticobility but does not contribute to C8 activity.This is particularlyclear in fraction 1 which contains predominantly this component and has a low specific activity.Becausethisimpurityextends significantly far into the C8 region of the elution profile, a.
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