Purification and characterization of bovine complement component C3 and its cleavage products.
OBJECTIVE
To purify complement component C3 from bovine serum, characterize and analyze NH2-terminal amino acid sequences from its various cleavage products, and do cross-species homology comparisons.


METHODS
2 healthy lactating Holstein cows, and 2 healthy adult female New Zealand White rabbits.


METHODS
Bovine C3 was isolated from serum, and was cleaved to C3b. The resulting protein was analyzed to determine apparent molecular mass of resulting protein segments. Bands were electroblotted onto a membrane and excised, then NH2-terminal amino acid sequences were determined.


RESULTS
The C3 preparation consisted of 6 segments, with molecular mass of 30, 40 (2 bands, a and b), 70, 75, and 115 kd. Via sequence comparisons, the 115-kd band was identified as the alpha chain; the 75-kd segment was determined to be the NH2-terminal portion of alpha chain; the 70-kd piece was identified as the intact beta chain; and the two 40-kd bands are believed to be located at the C-terminal portion of the alpha chain, at the cleavage site that yields C3f. The 30-kd band is the NH2-terminal portion of the alpha chain (minus the C3a segment). Sequence analysis of each band revealed a high degree of homology with human, rat, mouse, and horse C3. Polyclonal antibodies raised in rabbits yielded sera that reacted to the purified sample in manner similar to that of commercially available antibodies.


CONCLUSIONS
The purified preparation contained intact C3, C3b, and the degradation products iC3b and C3c, which had high sequence homology with those of other species. The C3a and C3d, and C3g segments of protein were not detected and may have been lost during purification, lyophilization, or transfer steps. Structure and cleavage characteristics of bovine C3 can be used to better understand immune responses to bacterial pathogens in the mammary gland.
Di Carlo
A L
AL
Department of Animal Sciences, University of Maryland, College Park 20742, USA.
Paape
M J
MJ
Hellman
J
J
Lilius
E M
EM
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D002417:Cattle; D003176:Complement C3 / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D005260:Female; D006736:Horses; D006801:Humans; D005779:Immunodiffusion / Q000662:veterinary; D051379:Mice; D008969:Molecular Sequence Data; D011817:Rabbits; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity
D000906:Antibodies; D003176:Complement C3