H. Takeshita et al., DEOXYRIBONUCLEASE-I FROM RAT URINE - AFFINITY PURIFICATION, CHARACTERIZATION, AND IMMUNOCHEMICAL STUDIES, Journal of Biochemistry, 118(5), 1995, pp. 932-938
Deoxyribonuclease I (DNase I) from rat urine was purified about 3,000-
fold to apparent homogeneity with a 14% yield by affinity chromatograp
hy utilizing polyguanylic acid-agarose and DNA-cellulose. The purified
enzyme preparation was found to contain no other detectable nucleases
. Isoelectric focusing electrophoresis revealed that all six isoelectr
ic forms of the enzyme had been purified, and the resulting bands all
contained DNase I activity. Quantitative amino acid analysis and N-ter
minal amino acid sequencing were performed on the purified DNase I. Th
e N-terminal sequence up to the 15th residue of the enzyme was identic
al to that of rat parotid DNase I. The enzyme was found to be a glycop
rotein, containing 1 fucose, 10 galactose, 17 mannose, 12 glucosamine,
and at least 3 sialic acid residues per molecule, The isoelectric mul
tiplicity of the enzyme was partly due to differences in the sialic ac
id content of the isoforms, Gel filtration on Superose 12 and electrop
horesis on sodium dodecyl sulfate polyacrylamide gels indicated an app
roximate molecular mass for DNase I of 32 kDa. The enzyme had an optim
um pH of 6.5 and required divalent cations such as Ca2+ for its activi
ty, Its activity was inhibited by 1 mM EDTA and EGTA, but not G-actin,
An antibody against the purified enzyme was found to be monospecific
against rat urine and the pure antigen, and completely blocked the act
ivity of the purified enzyme.