Two enzyme preparations having both nuclease and 3'-nucleotidase activities were partially purified from an extract of tea leaves. They resemble each other in most enzymatic properties, but are separated by DEAE-cellulose column chromatography. The enzyme activities for RNA, native DNA, heat-denatured DNAand 3'-AMP of each preparation showed a high degree of similarity with respect to the following properties: pH stability, thermal stability and response to EDTA. Both enzymeswere shownto be endonucleases (EC 3. 1.30.2) which liberated S'-mononucleotides and oligonucleotides from both RNA and DNAwith the following relative rate of hydrolysis: RNA>native DNA=heat-denatured DNA.The present paper and three earlier publications1~3) from our laboratory are concerned with the isolation and characterization of the enzymes involved in the enzymic changes of nucleic acids and related compounds during the manufacture of black tea. In the previous paper,1} the authors indicated the presence of enzymes which released 5/-mononucleotides from RNA.In the course of purification, it was noted that two enzymefractions separated by a DEAE-cellulose column were also active on DNAand 3'-nucleotides. Chemicals. Yeast RNAused for assays was purchased from Kohjin Co., E. coli ribosomal RNAfrom BDH Chemicals Ltd., and yeast RNA(type XI), calf thymus DNA(type I), and 2'-, 3'-and 5'-nucelotides from Sigma Chemical Co. Heat-denatured DNAwas prepared by heating a solution of native DNA(5mg/ml) at 100°C for 30 min, and then cooling it rapidly in ice. Good's buffers: 2-(7V-morpholino)ethane sulfonic acid, MES; piperazine-A^,#/-bis(2-ethane sulfonic acid), PIPES; and tris(hydroxymethyl)methylglycine, Tricine, were the products of Dojindo Laboratories Co. Protein molecular weight markers were obtained from Boehringer Mannheim Yamanouchi Co. DEAE-and CM-cellulose (DE-52 and CM-52) were products ofWhatman Ltd., and Sephadex G-100, a product of Pharmacia Co.Assay of nuclease activity. The usual reaction mixture (1.0ml), consisting of 0.5ml of 0.1 m MES-NaOH buffer (pH 6.0), 0.25ml of RNA (lOmg/ml) or DNA (5mg/ml) solution and the enzyme solution, was incubated at 37°C for 1 hr. The reaction was stopped by addition of 1 ml of uranyl reagent (0.25% uranyl acetate in 0.2m perchloric acid) and allowing to stand in the cold for 30 min. The precipitate formed was removed by centrifugation. The supernatant (0.5ml) was diluted 10-fold with water and the absorbance at 260nm was read against a blank incubated without enzyme. One unit (u) of the nuclease activity was defined as the amount of enzyme which effected an increase in A260 of 1.0 in 1 min.

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