PURIFICATION AND CHARACTERIZATION OF TILAPIA (OREOCHROMIS-MOSSAMBICUS) DEOXYRIBONUCLEASE-I - PRIMARY STRUCTURE AND CDNA SEQUENCE

DNase I of tilapia (Oreochromis mossambicus) was purified to homogenei ty. Tilapia DNase I is most active at pH 8.5 with Mg2+ as activator. T he Ca2+/Mg2+ pair has a synergistic effect on activation, The enzyme i s readily inactivated by heating above 55 degrees C, but is not inacti vated by trypsin or 2-mercapto-ethanol under alkaline conditions, with or without CaCl2. Its isoelectric point is 6.0. The 258-amino-acid se quence of tilapia DNase I was derived from overlapping sequences of tr yptic, chymotryptic and CNBr peptides, The purified enzyme has two var iants differing by a single Lys-->Arg mutation at position 125. The po lypeptide chain has one disulfide bridge and one carbohydrate side cha in. By mass spectrometry, the purified enzyme shows many molecular mas s forms differing by Lys/Arg substitution and sugar-chain length, The major form has a molecular mass of 30914 Da, A 1061-bp nucleotide sequ ence for the cDNA of tilapia DNase I, obtained by gene cloning and DNA sequencing, contains an ORF coding for a putative 26-residue transmem brane peptide and the mature DNase I polypeptide.