PURIFICATION AND CHARACTERIZATION OF BOVINE COMPLEMENT COMPONENT C3 AND ITS CLEAVAGE PRODUCTS

Objective-To purify complement component C3 from bovine serum, charact erize and analyze NH2-terminal amino acid sequences from its various c leavage products, and do cross-species homology comparisons. Animals-2 healthy lactating Holstein cows, and 2 healthy adult female New Zeala nd White rabbits. Procedure-Bovine C3 was isolated from serum, and was cleaved to C3b. The resulting protein was analyzed to determine appar ent molecular mass of resulting protein segments, Bands were electrobl otted onto a membrane and excised, then NH2-terminal amino acid sequen ces were determined. Results-The C3 preparation consisted of 6 segment s, with molecular mass of 30, 40 (2 bands, a and b), 70, 75, and 115 k d. Via sequence comparisons, the 115-kd band was identified as the alp ha chain; the 75-kd segment was determined to be the NH2-terminal port ion 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-ter minal 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 degr ee of homology with human, rat, mouse, and horse C3, Polyclonal antibo dies raised in rabbits yielded sera that reacted to the purified sampl e in manner similar to that of commercially available antibodies. Conc lusions-The purified preparation contained intact C3, C3b, and the deg radation products iC3b and C3c, which had high sequence homology with those of other species, The C3a and C3d, and C3g segments of the prote in were not detected and may have been lost during the purification, l yophilization, or transfer steps. Structure and cleavage characteristi cs of bovine C3 can be used to better understand immune responses to b acterial pathogens in the mammary gland.