Purification of the 11- and 5-kDa antibacterial polypeptides from guinea pig neutrophils.
It has been known that neutrophils contain various antimicrobial components in the granules, which contribute to the oxygen-independent host defense mechanism. In this study, we have isolated the two antimicrobial polypeptides from guinea pig neutrophil granules. Urea-SDS-PAGE analysis revealed that the molecular masses of the polypeptides were 11 and 5 kDa under nonreducing conditions. Under reducing conditions, the molecular mass of the 5-kDa polypeptide did not change, whereas the molecular mass of the 11-kDa polypeptide changed to about 5 kDa, suggesting that the 11-kDa polypeptide is a dimer composed of 5-kDa subunits joined with a disulfide bond. The amino acid composition and sequence data indicated that the 5-kDa subunit of the 11-kDa polypeptide contained 9 lysine, 8 arginine, and 1 cysteine residues and that the 11-kDa polypeptide was a homodimer of G1LRKKFRKTRKRIQKLGRKIGKTGRKVWKAWREYGQIPYPCRI43 (4599 Da) joined with one disulfide bond. Amino acid sequence of the 11-kDa polypeptide showed partial homology (19-30%) to the active peptides of rabbit and human cationic antimicrobial proteins of 18 kDa (CAP18), suggesting the 11-kDa polypeptide might be a homologue of CAP18. In contrast, the amino acid analysis of the 5-kDa antibacterial polypeptide revealed that the polypeptide was composed of 41 amino acids (5007 Da) containing 7 lysine, 10 arginine, and 2 cystine residues. However, sequence analysis indicated that the N-terminus of the 5-kDa polypeptide was likely blocked. The 11- and 5-kDa polypeptides showed almost the same antibacterial activities; ED50 values were 30-35 nM against Escherichia coli and 90-120 nM against Staphylococcus aureus, which were 4- to 20-fold lower than those of defensins. Furthermore, the 11- and 5-kDa polypeptide retained the antibacterial activities even at the physiological concentration of NaCl (0.15 M), although the antibacterial activity of defensin was completely lost in the presence of NaCl.
Yomogida
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Department of Biochemistry, Juntendo University School of Medicine, Tokyo, Japan.
Nagaoka
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Yamashita
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D000595:Amino Acid Sequence; D000818:Animals; D001770:Blood Bactericidal Activity; D002851:Chromatography, High Pressure Liquid; D003594:Cytoplasmic Granules / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000187:drug effects; D006168:Guinea Pigs; D006801:Humans; D046911:Macromolecular Substances; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009504:Neutrophils / Q000502:physiology*; D010455:Peptides / Q000097:blood* / Q000737:chemistry / Q000494:pharmacology; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D013211:Staphylococcus aureus / Q000187:drug effects
D046911:Macromolecular Substances; D010455:Peptides