C5a fragment of bovine complement. Purification, bioassays, amino-acid sequence and other structural studies.
C5a and des-Arg-C5a have been purified from bovine serum in milligram amounts. The progress of the purification was followed by measuring the chemotactic activity of the complement fragments. The two polypeptides induce activation of neutrophil-oriented locomotion and secretion with very similar dose/response effects. After preparing a rabbit antiserum to bovine C5a/des-Arg-C5a, a competitive enzyme-linked immunosorbent assay (ELISA) was set up for the detection of C5a from 5 ng/mol to 1 microgram/ml. The complete primary structure of bovine C5a, which consists of 74 amino acids, has been determined by sequence analysis of the tryptic peptides, aligned by peptides derived from a chymotryptic digest, and by partially sequencing the intact molecule. Bovine C5a has a sequence homology of 78% and 70% with porcine and human C5a, respectively, reacts with an antiserum to porcine C5a and is recognized by cell surface receptors on human neutrophils. Finally, the secondary structure of bovine C5a was investigated by circular dicroic spectroscopy and predicted from the amino acid sequence. A comparison of the content and distribution of alpha-helical and/or hydropathic regions, suggests that the three-dimensional structure of C5a might be modeled from the known crystal structure of the homologous C3a molecule.
Gennaro
R
R
Simonic
T
T
Negri
A
A
Mottola
C
C
Secchi
C
C
Ronchi
S
S
Romeo
D
D
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002918:Chymotrypsin; D002942:Circular Dichroism; D003182:Complement C5 / Q000031:analogs & derivatives / Q000302:isolation & purification*; D015936:Complement C5a; D015937:Complement C5a, des-Arginine; D004588:Electrophoresis, Cellulose Acetate; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007120:Immunochemistry; D005779:Immunodiffusion; D009504:Neutrophils / Q000502:physiology; D010446:Peptide Fragments / Q000032:analysis; D014357:Trypsin
D003182:Complement C5; D015937:Complement C5a, des-Arginine; D010446:Peptide Fragments; D015936:Complement C5a; D002918:Chymotrypsin; D014357:Trypsin