Molecular, biochemical and immunological analyses of porcine pancreatic DNase I.
Deoxyribonuclease I (DNase I) was purified 26500-fold in 39% yield from porcine pancreas to electrophoretic homogeneity using three-step column chromatography. The purified enzyme was inhibited by an antibody specific to the purified enzyme but not by G-actin. A 1303 bp cDNA encoding porcine DNase I was constructed from total RNA from porcine small intestine using a rapid amplification of cDNA ends method, followed by sequencing. Mature porcine DNase I protein was found to consist of 262 amino acids. Unlike all other mammalian DNase I enzymes that are inhibited by G-actin, porcine DNase I has H65 and S114 instead of Y65 and A114, which presumably results in the lack of inhibition. Porcine DNase I was more sensitive to low pH than rat or bovine enzymes. Compared with their primary structures, the amino acid at position 110 was N in porcine enzyme, but S in rat and bovine enzymes. A porcine mutant enzyme in which N was substituted by S alone at position 110 (N110S) became resistant to low pH to a similar extent as the rat and bovine enzymes.
Mori
S
S
Department of Legal Medicine, Gunma University School of Medicine, Maebashi, Gunma, Japan.
Yasuda
T
T
Takeshita
H
H
Nakajima
T
T
Nakazato
E
E
Mogi
K
K
Kaneko
Y
Y
Kishi
K
K
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001483:Base Sequence; D019556:COS Cells; D003001:Cloning, Molecular; D003429:Cross Reactions; D018076:DNA, Complementary / Q000096:biosynthesis / Q000737:chemistry; D003850:Deoxyribonuclease I / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D007422:Intestines / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010179:Pancreas / Q000201:enzymology*; D010802:Phylogeny; D012313:RNA / Q000302:isolation & purification; D016415:Sequence Alignment; D013552:Swine
D018076:DNA, Complementary; D012313:RNA; D003850:Deoxyribonuclease I