Agric. Biol. Chem., 46 (5), 1261 -1269, 1982 1261

Purification and Characterization of from Tea Leaves Hiroshi Imagawa, Hiromi Toryu, Tetsuo Ozawa and Yoshinori Takino* Institute of Applied Biochemistry, The University of Tsukuba, Ibaraki 305, Japan *Department of Agricultural Chemistry, Faculty of Agriculture, Tam'agawa University, Machida-shi, Tokyo 194, Japan Received October 23, 1981

Twoenzyme preparations having both and 3'- 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 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 enzymeswereshownto be (EC 3. 1.30.2) which liberated S'-mononucleotides and oligonucleotides from both RNAand DNAwith the following relative rate of hydrolysis: RNA>native DNA=heat-denatured DNA.

The present paper and three earlier publi- Tea, and stored at -20°C in a freezer. cations1 ~3) from our laboratory are concerned Chemicals. Yeast RNAused for assays was purchased with the isolation and characterization of the from Kohjin Co., E. coli ribosomal RNAfrom BDH involved in the enzymic changes of Chemicals Ltd., and yeast RNA(type XI), calf thymus nucleic acids and related compounds during DNA(type I), and 2'-, 3'- and 5'-nucelotides from Sigma the manufacture of black tea. In the previous Chemical Co. Heat-denatured DNAwas prepared by paper,1} the authors indicated the presence of heating a solution of native DNA(5mg/ml) at 100°C for 30 min, and then cooling it rapidly in ice. Good's buffers: enzymes which released 5/-mononucleotides 2-(7V-morpholino)ethane sulfonic acid, MES; piperazine- from RNA.In the course of purification, it A^,#/-bis(2-ethane sulfonic acid), PIPES; and tris(hy- was noted that two enzymefractions separated droxymethyl)methylglycine, Tricine, were the prod- by a DEAE-cellulose column were also active ucts of Dojindo Laboratories Co. Protein molecular on DNAand 3'-nucleotides. weight markers were obtained from Boehringer Nucleases having S'-nucleotidase activity Mannheim Yamanouchi Co. DEAE-and CM-cellulose (DE-52 and CM-52) were products ofWhatman Ltd., and were found in several kinds of higher Sephadex G-100, a product of Pharmacia Co. plants4~7) and fungi.8) A number of differ- ences have been detected amongplant nucle- Assay of nuclease activity. The usual reaction mixture ases with respect to specificities toward vari- (1.0ml), consisting of 0.5ml of 0.1 m MES-NaOHbuffer (pH 6.0), 0.25ml of RNA (lOmg/ml) or DNA (5mg/ml) ous substrates and other properties. solution and the enzyme solution, was incubated at 37°C This paper describes the partial purification for 1 hr. The reaction was stopped by addition of 1 ml of and characterization of the tea nucleases. 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 MATERIALS AND METHODS supernatant (0.5ml) was diluted 10-fold with water and the absorbance at 260nm was read against a blank Tea leaves. The first two leaves and a bud from fresh tea incubated without enzyme. One unit (u) of the nuclease shoots (Camellia sinensis, (L.) O. Kuntze, cv. Benihomare) activity was defined as the amount of enzyme which were picked in Mayat the National Research Institute of effected an increase in A260 of 1.0 in 1 min. 1262 H. Imagawa et al.

Assay of nucleotidase activity. The reaction mixture the same buffer for 24hr. After dialysis, the (2.0ml), consisting of 1.0ml of 0.1 m MES-NaOHbuffer precipitate was removed and discarded. (pH 6.0), 0.5ml of 10mM3'-AMP and enzyme solution, Step 3. First DEAE-cellulose column chro- was incubated at 37°C for 30 min. The amount of inorganic phosphate released was determined by matography. The dialyzed enzyme solution was Nakamura's method.9) One unit of the activity was defined applied to a DE-52 cellulose column. A typical as the amount ofenzyme that released 1 /xmol of inorganic elution pattern of enzymeactivities is shown in phosphate from 3'-AMP in 1 min. Fig. 1. Among the six peaks which showed Protein determination. Protein concentration was deter- RNAsplitting activity, two peaks (Ha and lib) mined by Lowry's procedure10) using bovine serum al- exhibited DNAand 3'-AMP splitting activity. bumin as the standard protein. For column eluates, only As it was assumed that these two peaks con- the absorbance at 280nmwas monitored. tained sugar non-specific nucleases, they are called nuclease A and B, respectively. Molecular weight determination. The molecular weight Fractions of both nuclease peaks were sep- of a nuclease was determined by gel filtration on Sephadex G-100 as described previously.3) arately pooled and concentrated to about 10ml with a membrane ultra filter (Amicon Identification of digestion products. Digestion products PM-10). were separated by two-dimensional paper chromatog- Step 4. Second DEAE-cellulose column chro- raphy on WhatmanNo. 1 filter paper in the following solvent systems: (1) isopropyl alcohol-28% aq. ammonia- matography. Each of the concentrated enzyme water (70: 5:25) in the first direction and (2) saturated solutions from step 3 was subjected to re- ammoniumsulfate-isopropyl alcohol-1 m sodium acetate chromatography on a DE-52 cellulose column (80:2: 18) or (3) isopropyl alcohol-cone.HCl-water (70:15: 15) in the second direction. The ultraviolet- absorbing spots were eluted with 0.01 m HC1and each eluate was identified from its Rfvalue and UVabsorption spectrum. The concentration of each nucleotide was deter- mined by measuring the absorption at 260nm. MC40à" 6 -20\ iJi RESULTS Purification of the enzyme The procedure is summarized in Table I. All a>20-ij -^4 ' >II å 10« operations were carried out in the cold at 4°C. § 1 A/V\f ^ j| .-o-0.4_ Step 1. Preparation of crude extract. Tea leaves (1.2kg) were homogenized in several separate batches with cold acetone for 1 min. The homogenate was filtered through filter 0 50 100 150 2OO (Fr.No.) paper with suction and the residue was washed 0.005Mt 0.05Mt O.IMt three times with a mixture of acetone and IJUIII l !p water (4: 1) to remove polyphenols. The ace- Acetate buffertpH 5.6) Fig. 1. First DEAE-Cellulose Column Chromatog- tone powderthus obtained was extracted for raphy. 2hr with 0.1 m sodium phosphate buffer (pH The dialyzed enzyme solution (160ml) was loaded on a 7.0), containing 1% of sodium ascorbate. DE-52 column (2.5 x 37 cm) equilibrated with 0.005m Step 2. Ammoniumsulfate fractionation. sodium acetate buffer, pH5.6, and the column waswashed Solid ammoniumsulfate was added slowly with 600ml of the same buffer. Elution was continued with stirring to the crude extract to 70% successively with 0.05 m, 0.1 m sodium acetate buffer (pH saturation. After standing for 30 min, the 5.6) and a linear gradient of zero to 0.25m NaCl in the same buffer. Fractions of 16ml were collected. Enzyme precipitate was collected by centrifugation and activities were measured with the following substrates: suspended in 0.005 m sodium acetate buffer, RNA (O O); denatured DNA (# #); 3 -AMP pH 5.6. The suspension was dialyzed against (A A)- , concentration of NaCl. Nucleases from Tea Leaves 1263 (1.6x39cm). This time, the column was Step 6. Sephadex G-100 column chromatog- equilibrated with 0.05 m sodium acetate buffer raphy. Each of the concentrated enzyme so- (pH 5.6) and elution was conducted with a lutions from step 5 was applied to a Sephadex linear gradient of 0.05m to 0.07m sodium G-100 column. As shown in Fig. 2, an identical acetate buffer (pH 5.6). Although each nucle- chromatographic behavior was exhibited by ase activity was found as a single peak, non- each of the four enzymatic activities, and the specific nucleotidase activity could not be re- small amountof non-specific nucleotidase ac- moved. Nuclease fractions from each column tivity which had remained in the enzymeso- were separately pooled and dialyzed against lution was removed. Nuclease A fractions (No. 0.01 m sodium acetate buffer (pH 5.6). 48 to 53) and nuclease B fractions (No. 49 to Step 5. CM-cellulose column chromatog- 54) were both free of 5'-nucleotidase, non- raphy. Each of the dialyzed enzyme solutions specific phosphomonoesterase and non- from step 4 was applied to a CM-52cellulose specific as described later. column (1.4 x 13cm) equilibrated with 0.01 m The results of a typical purification from sodium acetate buffer, pH 5.6. After washing 1.2kg of tea leaves are summarized in Table I. the column with 200ml of the same buffer, the The four enzymatic activities of each of the enzyme was eluted with a linear gradient of two nucleases were not separated, and the 0.01 mto 0.4m sodium acetate buffer (pH 5.6). ratios of their specific activities were nearly Nuclease activity appeared in the region of constant throughout the chromatographic pu- 0.09-0.12m buffer (tubes No. 35 to 40, in the rification steps. This suggests that each single case of the nuclease A fraction) and enzyme is responsible for these multiple 0.12-0.16m buffer (No. 32 to 36, the nuclease activities. B fraction), respectively. In both nuclease frac- tions, four enzymatic activities for RNA,na- Disc gel electrophoresis tive DNA, denatured DNAand 3'-AMP were Figure 3 shows the distribution of protein eluted together and most of the non-specific and enzymeactivities on a polyacrylamide gel nucleotidase activity was removed. Each of the (7.0%) after electrophoresis at pH 9. Although enzymefractions was concentrated to 1 ml each enzymepreparation at step 6 gave two or with an Amicon ultra filter. three protein bands, activities for RNA,DNA

Nuclease A Nuclease B o io- ft å å A -i.o

'^ '\l i\I \ i'^q o |5- 'A å å ' I\/\f« "a5l

ol .'å A-A-^-A-A^^q*. 8 . 1 I /.A^^y. ^. IQ 30 40 50 60 30 40 50 6 0 Fraction number Fig. 2. Sephadex G-100 Column Chromatography. Concentrated enzyme solution from step 5 was applied to a Sephadex G-100 column (1.5 x 91 cm) equilibrated with 0.04 msodium acetate buffer, pH5.6. Filtration was carried out with the same buffer and 2ml fractions werecollected. Enzymeactivities: RNA(OO);nativeDNA(à" #);denaturedDNA(à" à"); 3'- AMP(A A)- , absorbance at 280nm. 1264 H. Imagawa et al.

Table I. Purification of Nucleases from Tea Leaves

Specific activity (units/mg) for Protein ^ (mg) RNA ^ De"a'UAred 3-AMP DNA DNA Ammonium sulfate (70%) 1060 0.60 0.23 0. 18 - 2nd DEAE-cellulose Nuclease A 80 2.09 1.08 1.15 1.30 Nuclease B 140 1.07 0.51 0.68 0.56 CM-cellulose Nuclease A 19 7.86 4.06 3.50 3.81 Nuclease B 51 2.60 1.44 1.46 1.20 Sephadex G-100 Nuclease A 6.0 19.7 1 1.0 10.2 10.1 Nuclease B 12 10.8 5.50 5.86 4.90

Nuciease A Nuclease B <-)-» (±) (-)-> (+) 1 111 1 I II 1 1

> i.o- r~: ro j j

|0.5å J fJL H - I P h "å f= ~!i b» I!L r-( 0^ " 18 ' 36 0 18 36 Distance in millimeters Fig. 3. Distribution of Protein and Enzyme Activity in a Polyacrylamide Gel after Electrophoresis. Aliquots of each nuclease preparation of step 6 were applied on columns and subjected to electrophoresis at pH 9.0 and 3mAper column for 60 min at 4°C. Each gel was cut in half; one half was stained with amido black and the other half was cut into 3 mmsegments. The enzymewas eluted from each segment with 2 ml of0.025 m MES-NaOHbuffer, pH 6.0 and the activities were measured after incubating with each substrate for 30 min at 60°C. Enzyme activities for the following substrates: RNA( ), native DNA( ), 3 -AMP ( ).

and 3'-AMP were all associated with one about 35,000 and 33,000, respectively, by protein band. These results also suggest that Sephadex G-100 gel filtration. These values nuclease and 3 '-nucleotidase activities reside in were similar to those of nucleases from potato the same protein molecule. Nucleases A and B tubers (33,000),6) avena leaves (33,000)7) and showed almost the same mobility under the tobacco cell cultures (35,000).14) indicated conditions. Effect ofpH. Nucleases A and B showed the same pH-dependence toward various sub- Properties ofnucleases A and B strates (Fig. 4). The maximal activities for Molecular weight. The molecular weights of RNA,native and denatured DNAwere ob- nucleases A and B were determined to be served at around pH 6.0, 5.5 and 6.0, respec- Nucleases from Tea Leaves 1265

Nuclease A Nuclease 6

850";if à" B 85°-/ U\ -iliII s I h if I tt: ° NucleaseA à"ji?NucleaseB & *1\ 1 I 0«-à"-à"-à"-i-i-å' » ' å å » 4 5 6 7 8 9 4 5 6 7 8 9 °4 5 67 8 4 567 8 o PH pH pH pH Fig. 6. pH-Stability of Tea Nucleases. Fig. 4. pH-Activity Curves of Tea Nucleases. Each enzyme solution was kept at 4°C for 45 hr at various Enzymeactivities for each substrate were assayed under pHs (pH 4.0 to 6.0, sodium acetate buffer; pH 6.0 to 8.0, the standard conditions. The following buffers were used: PIPES-NaOH buffer; pH 8.0 to 9.0, Tricine-NaOH buf- 0.1u sodium acetate buffer (pH 4.5 to 6.0), 0.1m MES- fer). After adjustment of the pH to 6.0, the remaining NaOHbuffer (pH 5.5 to 7.0), 0.1 m PIPES-NaOH buffer activity was assayed. Enzyme activities: RNA(O O); (pH 7.5 to 8.0). Enzyme activities: RNA (O O); native DNA (# #); denatured DNA (à" à"); 3'- native DNA ( # -#); denatured DNA ( à" à"); 3'- AMP (A-A). AMP(A-A).

40- NucleaseA . NucleaseB

^3o" å A > / \ °50- Yjft å \ 12°- /\ å /\ tz NucleaseA \ NucleaseB l\*^^# *l0" /A\" / \ Lj L__I . Ljfi_l_l . I I l"'^ O 40 60 80 40 60 80 040 60 80 40 60 80 Temperature (°C) Temperature (°C) Fig. 7. Thermal Stability of Tea Nucleases. The enzyme solution in 0.066m MES-NaOHbuffer (pH Fig. 5. Temperature-Activity curves of Tea Nucleases. 6.0) was kept at various temperatures for 15 min, and the The enzyme solution was added to each substrate and remaining activity was assayed. Enzyme activities: RNA incubated for 30 min at various temperatures. Activity is (O O); native DNA (# *): denatured DNA expressed as the relative value to the activity at 37°C. (à" -à"); 3'-AMP(A-A). Enzyme activities: RNA (O O); denatured DNA (à" à"); 3'-AMP (A-A). activities for the three substrates were at around 70°C (nuclease A) and 60~70°C (nu- tively. The pH optima for S'-ribonucleotidase clease B). activity were observed at around pH 6.0 (for Stability of the enzyme. Enzymeactivities of GMPand CMP) and pH 6.5 (for AMPand nucleases A and B toward RNA, native DNA, UMP). denatured DNAand 3'-AMP showed similar Effect of temperature. The rate of hydrolysis stability. As shown in Fig. 6, both enzymes of RNA, denatured DNAand 3 -AMP by were unstable in acidic media, but stable in the nucleases Aand B was examined at various range of pH 6.0 to 8.0 at 4°C. They were stable temperatures. As shownin Fig. 5, the maximal at temperatures below 60°C, but over 80% 1266 H. Imagawa et al.

Table II. Effect of Various Substances on the Activity of Tea Nucleases The enzymeactivities were measured under the standard assay conditions, after incubating the enzyme solution with various metal ions or reagents (1.3nn) at 37°C for 30min. Nuclease A Nuclease B Relative activity (%) for Relative activity (%) for Substance - ' _XTA Native Denatured -, A.,D D1VTA Native Denatured A RNA DNA DNA 3"AMP RNA DNA DNA 3"AMP

None 100 100 100 100 100 100 100 100 NaF 107 120 107 1 12 130 95 106 99 BaCl2 116 104 107 102 114 81 106 101 CaCl2 97 96 102 99 100 88 96 95 FeSO4 45 76 66 37 90 108 1 1 1 93 CoCl2 42 76 57 86 46 84 61 82 CuSO4 0 10 0 0 6 15 18 4 MgCl2 72 73 83 98 67 76 67 95 MnCl2 71 85 61 89 64 84 50 85 Zn(CH3COO)2 1 6 76 1 9 1 3 30 100 40 30 FeCl3 23 64 54 54 43 74 64 5 1 EDTA 33 18 39 20 13 24 22 1 3 ICH2COOH 101 111 115 103 117 101 90 93 PCMB 79 31 67 30 52 28 26 4

Table III. Substrate Specificity of of the activities were lost on heating at 80°C Tea Nucleases for 15 min (Fig. 7). The two enzyme prepara- Nucleic acids were added at concentrations of 1.25 tions could be stored in 0.04m sodium acetate mg/ml and mononucleotides at 2.5niM. The standard buffer (pH 5.5) at 4°C for four months without assay conditions were used with 0.1u of nuclease or appreciable loss of the activities. 0.05 u of 3'-nucleotidase for each measurement. Effect of metal ions and inhibitors. The re- Relative activity (%) sults are summarized in Table II. The four Substrate ~ enzymatic activities of both enzymes were af- Nuclease A Nuclease B fected similarly by these substances. Both en- Yeast RNA 100 100 zymes were inhibited by Cu2+, Mg2+, Mn2+, E. coli rRNA 76 83 Co2+ and Fe3+, Cu2+ being the most effective Native DNA 56 51 among them. Zn2+ had little or no effect on Denatured DNA 52 54 the activity for native DNAbut considerably inhibited the other three activities. Significant 3 -AMP 100 100 3 -GMP 24 27 stimulation of activities could not be demon- 3 -UMP . 22 25 strated by any metal ion. Enzymatic activities 3 -CMP 12 ll 3 r-dAMP 1 1 of both nucleases were substantially inhibited 2'-AMP 0 0 by EDTAas for other plant nucleases. PCMB 5'-AMP 0 0 inhibited the enzyme activities, but monoiodo- 2/,3/-Cyclic AMP 0 0 acetate showedno effect. 3/,5/-Cyclic AMP 0 0 Substrate specificity. The two nuclease prep- PNPPa 0 0 arations showed similar substrate specificity BNPP" 0 0 under the standard assay conditions. Both /7-Nitrophenyl phosphate. enzymes hydrolyzed RNA, native DNAand Bis-/?-nitrophenyl phosphate. Activity was assayed denatured DNA to give 5/-phosphomono- by measuring the absorption at 420 nm, as described nucleotides and oligonucleotides as described previously. 1} Nucleases from Tea Leaves 1267 later. Although most known plant nucleases > 3-UMP > 3-CMP. Hydrolysis of 3'- hydrolyze denatured DNAfaster than native dAMPwas much slower than that of 3'-AMP. DNA,the enzymes in this work hydrolyzed Digestion of yeast RNAby tea nucleases. native DNAat a similar rate to the hydrolysis Yeast RNA(Sigma Type XI, 4mg) in 0.8 ml of of denatured DNA.Nucleases A and B also 0.1 m sodium acetate buffer (pH 5.5) was in- showed 3 '-nucleotidase activity but showed no cubated with 3.5 units of each nucleases at activity on the other substrates tested (Table 37°C. After incubation for various intervals, III). The relative rates ofhydrolysis of the four 40fA aliquots were taken and chromato- B'-ribonucleotides were: 3 -AMP > 3 -GMP graphed. The separated products were identi-

Table IV. Liberation of Mononucleotides from Yeast RNAby Tea Nucleases Each enzyme (3.5u, 0.8ml) was incubated at 37°C with 4mg of yeast RNA.

_ . å . Nucleotidesa Digestion time (hr) AMp CMp GMp UMp

1 78 -b 76 30 Nuclease A 2 87 1 1 87 37 6 93 31 90 46

1 57 - 46 15 Nuclease B 2 80 - 77 30 6 93 23 90 44

a Values indicate percentage liberation of the corresponding mononucleotides from the substrate. b -, not detected under UVlight.

Table V. Liberation of Mononucleotides from Native and Denatured DNAby Tea Nucleases Each 0.5 ml ofnuclease A (5.5 u) or nuclease B (4.6 u) was incubated at 37°C with 0.5 mg of native DNAor 1.0mg of denatured DNA. Substrate Digestion time (hr) Nucleotides" dAMP dCMP dGMP dTMP

Nuclease A Native DNA

Denatured DNA 43.2 50.5 59.7 35.1 37.2 13.1 35.9 45.6 53.2 56.7

25.0 30.2 38.3 23.5 34.3 48.6

21.4 24.7 32.4

Nuclease B Native DNA

Denatured DNA 22.7 23.8 48.4 ll.1

26.8 37.3 12.4 17.8 23.3 58.8 30.7

19.1 25.0 48.9 3.8 15.9 30.3

10.5 15.8 26.9

Values indicate percentage liberation of the corresponding 5'-mononucleotide from the substrate. -, not detected under UVlight. 1268 H. Imagawa et al. fied and determined. After 6hr digestion, (Table V). 5 -dCMPwas not detected until 8 hr four 5'~mononucleotides and oligonucle- digestion. otides were detected in the hydrolysate of Cleavage pattern of denatured DNA.Each yeast RNAwith both enzymes, but the rates enzyme preparation was incubated with dena- of appearance of these nucleotides were dif- tured DNA and the time courses for the ferent from each other, as shown in Table release of hydrolysis products soluble in tri- IV. It may be concluded that both the nu- chloroacetic acid (TCA) and also in uranyl cleases cleave all the phosphodiester bonds acetate-trichloroacetic acid (UA-TCA) were with an early release of 5'-AMP and -GMP, compared according to the method of Berry followed by release of 5 -UMP. 5 -CMP is and Campbell.12) It has been shown that an released after a considerable lag period. such as pancreatic DNase re- Digestion ofDNA. Native DNA(0.5 mg) or leases oligonucleotides, some of which are denatured DNA(1.0mg) in 0.5ml of 0.04m soluble in TCA but not in UA-TCA. sodium acetate buffer (pH 5.5) was incubated Therefore, plots of released products soluble in with 5.5 units (nuclease A) or 4.6 units (nu- each of the acid solutions represent different clease B) of each enzyme at 37°C. Liberated time course profiles. such as mononucleotides were characterized by the venomphosphodiesterase, on the other hand, same procedure as for RNAdigestion. Three release only 5/-mononucleotides which are so- deoxyribonucleoside-5'-phosphates were de- luble in both TCA solution and UA-TCA tected in all digestion products and the rate of solution, and give the same time course of appearance of these nucleotides from the sub- nucleotide liberation for both measurements. strate was in the order of dAMP> dGMP> Based on these facts, the profiles shown in Fig. dTMP. After 8 hr digestion, about 40-60% of 8 clearly indicate the two enzymes from tea adenine and guanine in the DNAwas released leaves to be endonucleases. Nuclease A Nuclease B "b / o DISCUSSION CD / S^

"o3 / I 6 J active on RNA,native DNA,denatured DNA O J / and 3'-nucleotides. The following results in 50.5- / - / this work suggest that a single enzyme is - I I responsible for these activities. 1) These ac- o / / tivities were not separated by several kinds of ?2 p y column chromatography and disc electro- ° I 2 3 1 2 3 phoresis. 2) The ratios of specific activities were nearly constant throughout the chroma- Time(hr) tographic purification procedure. 3) The four Fig. 8. Time Course of Release of Acid-soluble activities showed similar thermal stability and Products from Denatured DNA. pH stability. The effects of metal ions and A reaction mixture (14ml) containing 2.8 ml of denatured DNA (5mg/ml), 7ml of0.1 m MES-NaOH buffer, pH 6.0, EDTAon the activities were essentially the and the enzyme solution was incubated at 37°C for 3 hr. At same. the times indicated, two aliquots of the reaction mixture Sugar non-specific nucleases having 3'-nu- (1 ml) were taken and to each of them 0.5ml of either cold cleotidase activity have been isolated from 10% TCA or cold 0.5% UA in 10% TCA was added. various higher plants and their properties Suspensions were allowed to stand in ice water for 30 min, and then centrifuged for 10 min at 3,000 rpm. The characterized.4~7) Probably the primary supernatant was diluted 10-fold with water and the absor- difference among these nucleases is the ex- bance at 260nm was measured. O, soluble in TCA; #. tent of action on native DNA.The enzyme soluble in UA-TCA. preparations from mungbean4) and wheat5) Nucleases from Tea Leaves 1269 are much less active on native DNAunder bacco cell cultures14* and shiitake (Lentinus the usual assay conditions. Potato6) and edodes) was separated into two or three avena7) nucleases hydrolyzed both . components of different electrophoretic mo- However, the rate of hydrolysis was higher bilities. The physiological role of these on denatured DNA. In contrast, prepara- multiple forms of nucleases in plants re- tions from tea leaves were almost equally mains uncertain. active on both DNAs. According to the It can be assumed that the nucleases clarified Enzyme Nomenclature,16) enzymes active in this work also take part in releasing 5'- on both double- and single-stranded sub- nucleotides from nucleic acids during the strates are classified as an endonuclease process of manufacturing black tea. (EC 3.1.30.2). Therefore, the nucleases in this paper belong to this group and are dif- Acknowledgment. The authors wish to express their ferent from single-strand specific nucleases thanks to the National Research Institute of Tea for (EC 3.1.30.1) such as mung bean nuclease supplying tea leaves. and nuclease P1.8) REFERENCES The base specificity of a few plant nucleases has been investigated by determination of rel- 1) H. Imagawa, Y. Takino and M. Shimizu, Nippon Shohuhin Kogyo Gakkaishi, 23, 138 (1976). ative rates of released mononucleotides from 2) H. Imagawa, H. Toryu, M. Shimizu and Y. Takino, substrates. The enzyme from mungbean pre- Nippon Nogeikagaku Kaishi, 53, 1 17 (1979). feretially released S'-adenylic acid (pA) and 5'- 3) H. Imagawa, H. Yamano, K. Inoue and Y. Takino, deoxyadenylic acid (d-pA) from RNAand Agric. Biol Chem., 43, 2337 (1979). DNA,respectively. Tea nucleases also release 4) S. C. Sung and M. Laskowski, Sr., J. Biol. Chem., pA or d-pA rapidly. However, distinct differ- 237, 506 (1962). 5) D. M. Hanson andJ. L. Fairley, J. Biol. Chem., 244, ences were observed between mungbean and 2440 (1969). tea nucleases as to the relative amounts of 6) A. Nomura, M. Suno and Y. Mizuno, J. Biochem., mononucleotides released during hydrolysis. 70, 993 (1971). Mungbean nuclease released mononucleotides 7) N. V. Wyen, S. Erdei and G. L. Farkas, Biochim. at the following rates: pA>pU ^ pG>pC Biophys. Acta, 232, 472 (1971). 8) M. Fujimoto, A. Kuninaka and H. Yoshino, Agric. from RNA, and d-pA>d-pT>d-pC>d-pG Biol. Chem., 38, 777, 785, 1555, 2141 (1974). from DNA.Tea leaf nucleases showed the fol- 9) M. Nakamura, "Jikken Kagaku Koza," Vol. 23, lowing rates: pA=pG>pU>pCfrom RNA, Maruzen Co., Tokyo, 1957, p. 532. and d-pA>d-pG>d-pT>d-pC from DNA. 10) O. H. Lowry, N.J. Rosebrough,A. L. FarrandR.J. Randall, J. Biol. Chem., 193, 265 (1951). Both tea leaf nucleases have similar molec- ll) B. J. Davis, Ann. N. Y. Acad. Sci., 121, 404 (1964). ular weights, and their electrophoretic mobil- 12) S. A. Berry and J. N. Campbell, Biochim. Biophys. ities and other properties were nearly the Acta, 132, 84 (1967). same. They were separable only by DEAE- 13) C. M. Wilson, Plant Physiol, 48, 64 (1971). cellulose column chromatography. Therefore, 14) A. E. Oleson, A. M. Janski and E. T. Clark, Biochim. it was considered that the two nuclease Biophys. Acta, 366, 89 (1974). 15) K. Endo, Y. Umeyama, J. Nakajima and H. Kawai, preparations are the same type of enzyme Agric. Biol. Chem., 44, 1545 (1980). which have somewhat different physicochemi- 16) Nomenclature Committee of the International cal properties. Multiple forms have been de- Union of Biochemistry, "Enzyme Nomenclature," tected for several plant nucleases. Each of Academic Press Inc., New York, 1979, p. 274. the enzymes isolated from corn roots,13) to-