Carp hepatopancreatic DNase I: biochemical, molecular, and immunological properties.
A survey of DNase I in nine different carp tissues showed that the hepatopancreas has the highest levels of both DNase I enzyme activity and gene expression. Carp hepatopancreatic DNase I was purified 17,000-fold, with a yield of 29%, to electrophoretic homogeneity using three-step column chromatography. The purified enzyme activity was inhibited completely by 20 mM EDTA and a specific anti-carp DNase I antibody and slightly by G-actin. Histochemical analysis using this antibody revealed the strongest immunoreactivity in the cytoplasm of pancreatic tissue, but not in that of hepatic tissue in the carp hepatopancreas. A 995-bp cDNA encoding carp DNase I was constructed from total RNA from carp hepatopancreas. The mature carp DNase I protein comprises 260 amino acids, the same number as the human enzyme, however, the carp enzyme has an insertion of Ser59 and a deletion of Ala225 in comparison with the human enzyme. These alterations have no influence on the enzyme activity and stability. Three amino acid residues, Tyr65, Val67, and Ala114, of human DNase I are involved in actin binding, whereas those of carp DNase I are shifted to Tyr66, Val68, and Phe115, respectively, by the insertion of Ser59: the decrease in affinity to actin is due to one amino acid substitution, Ala114Phe. The results of our phylogenetic and immunological analyses indicate that carp DNase I is not closely related to the mammalian, avian or amphibian enzymes, and forms a relatively tight piscine cluster with the tilapia enzyme.
Mogi
Kouichi
K
Department of Legal Medicine, Gunma University School of Medicine, Maebashi, Gunma 371-8511, Japan.
Takeshita
Haruo
H
Yasuda
Toshihiro
T
Nakajima
Tamiko
T
Nakazato
Emiko
E
Kaneko
Yasushi
Y
Itoi
Masako
M
Kishi
Koichiro
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002347:Carps; D003850:Deoxyribonuclease I / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D029941:Fish Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D015971:Gene Expression Regulation, Enzymologic; D008099:Liver / Q000201:enzymology* / Q000276:immunology; D008969:Molecular Sequence Data; D010179:Pancreas / Q000201:enzymology* / Q000276:immunology; D010802:Phylogeny
D029941:Fish Proteins; D003850:Deoxyribonuclease I