Purification and properties of extracellular nuclease from tobacco pollen

Proteins diffusing from tobacco pollen grains exhibit different phosphohydrolytic activities. Molecular sieving produces nuclease fractionation into forms I, II and III with apparent molecular masses ≥ 60 × 10 3 , 32.9 × 10 3 and 24.6 × 10 3 , respectively, and separation of principal forms II and III from phosphatase and major part of 5′- and 3′-nucleotidase activities. These forms did not differ in the mode of substrate attack and were combined for further enzyme characterization. The preparation had 3′-nueleotidase activity even after further purification by DEAE-cellulose chromatography. The enzyme is an endonuclease with preference for single stranded molecules. The endolytical cleavage of native DNA occurs simultaneously in both strands and generates limit products of about 58 pairs of nucleotides. DNA duplex polymers are also cleaved by a terminally-directed, exonuclease-like process. The products of DNA degradation are oligonucleotides and 5′-mononucleotides. In the presence of NaCl, both endolytical and exonucleaselike activities on bihelical DNA are inhibited and the proportion of mono-to oligonucleotides produced increases. The enzyme can rapidly convert superhelical plasmid DNA to a nicked open circular form, and then to a unit-length linear molecule. On the basis of these properties and of those found earlier (sugar-unspecificity, acidic pH optimum, activation by Zn 2+ ions), the extracellular nuclease of tobacco pollen can be classified as plant nuclease I (EC 3.1.30.x).