Purification and characterization of subcomponent C1q of the first component of bovine complement

Abstract

Bovine Clq, a subcomponent of the first component of complement, was purified in high yield by a combination of euglobulin precipitation, and ion-exchange and molecularsieve chromatography on CM-cellulose and Ultrogel AcA 34. Approx. 12-16mg can be isolated from 1 litre of serum, representing a yield of 13-18%. The molecular weight of undissociated subcomponent Clq, as determined by equilibrium sedimentation, is 430000. On sodium dodecyl sulphate/polyacrylamide gels under non-reducing conditions, subcomponent Clq was shown to consist of two subunits of mol.wts. 69000 and 62000 in a molar ratio of 2: 1. On reduction, the 69000-mol.wt. subunit gave chains of mol.wts. 30000 and 25000 in equimolar ratio, and the 62000-mol.wt. subunit decreased to 25000. The amino acid composition, with a high value for glycine, and the presence of hydroxyproline and hydroxylysine, suggests that there is a region of collagen-like sequence in the molecule. This is supported by the loss of haemolytic activity and the degradation of the polypeptide chains of subcomponent Clq when digested by collagenase. All of these molecular characteristics support the structure of six subunits, each containing three different polypeptide chains, with globular heads connected by collagen triple helices as proposed by Reid & Porter (1976) (Biochem. J. 155,(19)(20)(21)(22)(23) for human subcomponent Clq. Subcomponent Clq contains approx. 9% carbohydrate; analysis of the degree of substitution of the hydroxylysine residues revealed that 91% are modified by the addition of the disaccharide unit Gal-Glc. Bovine subcomponent Clq generates full Cl haemolytic activity when assayed with human subcomponents Clr and Cls.

Purification and characterization of subcomponent C1q of the first component of mouse complement

“…The overall amino acid composition of mouse C lq is very similar to that of human C lq (Table 2) and those reported for the rabbit (Reid et al, 1972) and bovine Clq (Campbell et al, 1979). Notable features of the amino acid composition are the high glycine content and the presence of hydroxyproline and hydroxylysine, suggestive of a collagen-like sequence in mouse C lq also.…”

“…It is well known that collagen molecules are widely distributed among the members of the Animal Kingdom, including invertebrates, and that the complement system is probably present in invertebrates and has been conserved throughout evolution (Gigli & Austen, 1971). Not surprisingly, therefore, interest in the biochemistry or structure on C lq of various animals besides man has increased greatly, and purification and partial characterization of rabbit (Reid et al, 1972;Volanakis & Stroud, 1972;Lowe & Reid, 1974), bovine (Campbell et al, 1979), equine (McDonald & Burger, 1979), rat (Hiffken et al, 1978) and frog Clq (Alexander & Steiner, 1980) have been reported. Each has been shown to have an unusual amino acid composition (hydroxyproline, hydroxylysine and a high percentage of glycine) and to be composed of essentially the similar subunit structure.…”

A general method for affinity purification of complement component C3b using factor H-sepharose

“…Immunological methods. Immunodiffusion analysis was carried out by the method of Ouchterlony (1958), as described by Campbell et al, (1979). Rocket immunoelectrophoresis was carried out by a modification to the method of Laurell (1966) described by Weeke (1973).…”

Use of Clq enzyme‐linked immunosorbent assay to detect plant viruses and their serologically different strains

“…The precipitate was then dissolved with 32 ml of buffer B, centrifuged at 5000 g for 5 min and the supernatant dialysed against 4 litres of buffer C for 4 h. The resultant precipitate was sedimented and washed with buffer C and then dissolved with 32 ml of buffer D and centrifuged at 5000 g for 5 min. The supernatant was dialysed against 4 litres of buffer E for 5 h, and the resultant precipitate sedimented and washed with buffer E. Finally the precipitate was dissolved in 10ml of buffer F. The absorbance at 280 nm was measured and the preparation stored at -18 "C in small amounts 0.5-1 ml (c. 200 pg assuming = 0.72; Campbell, Booth & Fothergill, 1979).…”