Studies on Mode of Inhibition of Pancreatic DNases by Polyclonal Antibodies

• Main Authors: Liu,Yu-Fan, 劉玉凡
• Other Authors: Liao,Ta-Hsiu
• Format: Others
• Language: zh-TW
• Published: 1993
• Online Access: http://ndltd.ncl.edu.tw/handle/67233274422169912822

碩士 === 國立臺灣大學 === 生化學研究所 === 81 === Because a rabbit antiserum against bovine pancreatic(bp-) DNase had an inhibitory effect on bp-DNase butnot on porcine pancreatic (pp-) DNase, we raised an antiserum against pp-DNase and analyzed for its inhibitory effects.This anti-pp-DNase antiserum inhibited pp-DNase but not bp-DNase.We identified 15 peptides which account for over 90 % of the entire sequence of DNase. Four of the 15 peptides contain antigen determinat sites as determined with anti bpDNase polyclonal antibody and one of four peptides bp-DNase was positive using two monoclonal anti-bp-DNase antibodies. We found the inhibitery effect of the polyclonal antibody can be recovered by the Cu-iodoacetate inactivated DNase.But the effect can not be recovered by the three protease digests of DNase. We looked for a peptide that is 1] among the non-conservable protein sequence regions between bp-DNase and pp-DNase 2] antigenic regions 3] the regions is sensitive to hydrolysis by trypsin, chymotrypsin and thermolysin.This peptide is a 21 residue peptide corresponding to bp-DNase which was chemically synthesized and able to reverse the inhibitory effect. The antibody inhibitory effect depend on the metal ions used (Mn2+= Co2+= Ca2+ plus Mg2+> Mg2+). This order is the same as the order of metal ion for SiteI.Metal ions also have have effects on the mode of action of DNase. The DNase catalyzed pBR322 hydrolysis produced a significant amount of double strand cut with Mn2+, Co2+ or Mg2+ plus Ca2+ as activators and no double strand cuts were observed with only Mg2+as activator.The anti-bp- DNase antiserum inhibited the DNase action on double strand cut with Mn2+, Co2+ or Mg2+ plus Ca2+ as activators. The inhibitory effect of the antiserum is caused by the binding of an antibodyto Site I. The binding produce a reduced DNase hydrolysis rate and the inhibition of double cuts on duplex DNA.