Molecular, biochemical and immunological analyses of porcine pancreatic DNase I

Deoxyribonuclease I(DNase I) was purified 26500-fold in 39% yield from porc ine pancreas to electrophoretic homogeneity using three-step column chromat ography. The purified enzyme was inhibited by an antibody specific to the p urified 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 a mplification of cDNA ends method, followed by sequencing. Mature porcine DN ase I protein was found to consist of 262 amino acids. Unlike all other mam malian DNase I enzymes that are inhibited by G-actin, porcine DNase I has H 65 and S114 instead of Y65 and A114, which presumably results in the lack o f inhibition. Porcine DNase I was more sensitive to low pH than rat or bovi ne enzymes. Compared with their primary structures. the amino acid at posit ion 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 enzy mes. (C) 2001 Elsevier Science B.V. All rights reserved.