Characterization of a nuclease from malted barley roots

Journal of Cereal Science 5 (1987) 175-187

Characterization of a Nuclease from Malted Barley Roots* N. PRENTICE U.S. Department of Agriculture, Agricultural Research Service, Cereal Crops Research Unit, 501 N. Walnut Street, Madison, Wisconsin 53705 U.S.A. Received 25 April 1986 An exonuclease has been isolated from roots of malted barley by ammonium sulfate precipitation and chromatography on DEAE-trisacryl, Sephadex G 75, and Poly A Sepharose 4B. The enzyme is a Nuclease I (EC 3 . I . 30. 2) whose relative mol. wt (M r ) is approximately 30,000 by gel filtration and 35,000 by gel electrophoresis. It can be dissociated into two monomers of 18,000 M." Activation energies are II kcal/mole for RNase and 9 kcal/mole for DNase. Reaction products are 5' nUcleotides which after 2 h reaction represent about 37% of the RNA substrate. The optimum pH is 6·0 to 6'5 for both substrates. Activity loss was 30% after I"h at 40°C. The enzyme was present also in the remainder of the germinated barley kernel. The ratio of activities for RNA and DNA is 2. Reactions with various dinucleoside phosphates show that the uridylyl group on the 3' position of the ribose inhibits hydrolysis of this ester bond. Polyamines are strongly inhibitory.

Introduction There are two series of reactions that lead to the synthesis of nucleic acids. In one pathway, the de novo synthesis, the precursors are compounds such as amino acids, ethanoate, carbon dioxide and urea. The precursors in the other pathway are the products of nucleic acid catabolism, Le. nucleobases, nucleosides and nucleotides. The catabolic nucleic acid hydrolases are in the lytic compartments of cells where they are believed to degrade nucleic acids which are no longer functionaP.

Abbreviations used: RNA = ribonucleic acid; DNA = deoxyribonucleic acid; DEAE = diethyl aminoethyl; RNase = ribonuclease; DNase = deoxyribonuclease; poly A = polyadenylic acid; poly G = polyguanylic acid; poly U = polyuradylic acid; poly C = polycytidylic acid; Mr = relative molecular weight; t-RNA = transferribonucleicacid; CTP = cytidinetriphosphate ;CDP = cytidinediphosphate ; UTP = uridine triphosphate; ATP = adenosine triphosphate; ADP = adenosine diphosphate; GDP = guanosine diphosphate; AMP = adenosine monophosphate; CMP = cytidine monophosphate; GMP == guanosine monophosphate; UMP = uridine monophosphate; ApA = adenylyl adenosine; ApC = adenylyl cytidine; ApU = adenylyl uridine; ApG = adenylyl guanosine; OpA = guanylyl adenosine; OpC == guanylyl cytidine; GpU = guanylyl uridine; GpG = guanylyl guanosine; UpA = uridylyl adenosine; UpC = uridylyl cytidine; UpU uridylyl undine; UpG =uridylyl guanosine; CpC = cytidylyl cytidine; CpU = cytidylyl uridine; CpG = cytidylyl guanosine; PF = purification factor; U = units of RNase or DNase (units increase in absorption at 260 nm per min at 30°C); SA = specific activity, U/mg protein. * Mention ora trademark or proprietary product does not constitute a guarantee or warranty of the product by USDA and does not imply its approval to the exclusion of other products that may also be available.

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0733-5210/87/020175+ 13 $03.00/0

© 1987 Academic Press Inc. (London) Limited

176

N. PRENTICE

In industrial brewing the latter pathway appears to be important at the initial stage of fermentation after the wort has been inoculated with yeast. Although yeast (Saccharomyces carlsbergensis or Saccharomyces cerevisiae) can produce nucleic acids by de novo synthesis, a fermentation lag period is involved while adaptive enzymes are synthesized 2• This lag period, which is undesirable in a brewery functioning at capacity, is avoided if adequate (but as yet poorly defined) levels of nucleobases are supplied by the malt used in the mash. The nucleobases are the products of nucleic acid catabolism that has occurred during the malting of the barley and during the mashing of the malt. Nucleobases, nucleosides, and nucleotides are believed to contribute to the flavor of beer, but their effective levels are poorly defined 3 . Previous work4 has indicated that some malting barleys with good malting parameters, e.g. (X-amylase activity, diastatic power, and percent extract, may be marginal with respect to providing nucleobases in the mash. As expected, the activities of malt enzymes that are associated with nucleic acid degrll-dation correlate better with nucleobase levels in wort than with the conventional parameters. The work reported here is part of a continuing effort to characterize these enzymes so that their function may be accommodated in breeding programs for improved malting quality in barley. Materials and Methods

Preparation and extraction of tissues from germinating barley Barley seeds (cv. Morex) were germinated on filter paper in Petri dishes. Fifty seeds and water (4 ml) were used per dish. Germination proceeded at 15°C for 96 h, after which seedlings were separated into root, shoot, embryo and degermed caryopses tissues. The shoots and roots were cut as near as possible to the embryo but a small amount of root and shoot tissue remained with the embryo which therefore consisted of scutellum, median node and the traces of root and shoot tissues. The separated tissues were freeze-dried and then extracted as described previously5.

Malt roots Barley seeds (cv. Morex) were germinated for 96 h by the malting method described by Burger and Schroeder8 • The germinated barley was kilned with the thermal schedule: 9 hat 35 ee, 7 h at 45°C, 5 h at 55°C, 5 h at 65 ee, 2 h at 75°C and 2 h at 85°C. The roots were removed from the malt by agitation on a screen with apertures of 1·9 x 19 mm.

Extraction The roots were milled with a Udy Cyclone Mill (Udy Corp., Boulder, CO) and extracted with 0·1 M sodium ethanoate buffer pH 6·0 (7'5-10 ml/g roots) with stirring at 4 ec for 2 h. The suspension was filtered through cheese cloth and the filtrate was centrifuged at 20,000 g for 30 min at 4 ec.

Precipitation of enzyme with ammonium sulfate. Two hundred ml of supernatant were made 40% saturated with ammonium sulfate at 4°C. The precipitate was removed by centrifuging at 20,000 g and discarded. The supernatant was made 80% saturated with ammonium sulfate and the suspension was centrifuged as before. The

BARLEY NUCLEASE

precipitate was dissolved in 0'1 2 x 1300 ml of the buffer.

M

177

sodium ethanoate (115 ml), pH 6·0 and dialyzed twice against

DEAE-trisacryl chromatography DEAE-trisacryl (LKB Instruments Inc., Gaithersburg, MD), equilibrated with 0·01 M Tris chloride, pH 7·0 according to the manufacturer's directions, was poured as a slurry to fonn a column (2'5 x 30 em) when drained of excess buffer. Aliquots (8 m1) of the solution containing the material that precipitated between 40 and 80% saturation with ammonium sulfate (Table I) were dialyzed against 0·01 M Tris chloride pH 7 and applied to the column by upward flow at 16 mljh. The column was washed with the buffer for 3·5 hat 16 mljh. A linear gradient of sodium chloride was then introduced. A solution (150 g) of 0·0 I M Tris chloride pH 7 and O· 3 M NaCI was pumped into the stirred buffer (ISO g) which was pumped on the column at 16 mljh. The column eluate was collected at 15 min intervals (approximately 4 ml/fraction). Fractions were assayed for protein, RNase and DNase. Pooled fractions (fractions 57-62, Fig. I) (22 ml, Table I) were applied to the column again and the column was developed as above.

Gel filtration Sephadex G75 (pharmacia Fine Chemicals Inc., Piscataway, NJ) was equilibrated with 0·2 M sodium ethanoate, pH 6·5 in accordance with the manufacturer's instructions. The product from the DEAE-trisacryl was dialyzed against the buffer. To a column (2,5 x 80 em) 8 ml aliquots of the dialyzed solution were applied by upward displacement at 25 m1/h. The column was developed with the buffer at this rate of flow and four fractions per h were collected after 5 h until 60 fractions were obtained; these were assayed for protein, DNase and RNase.

Affinity chromatography with poly (A)-Sepharose 4B The ligand was poly A coupled with Sepharose 4B (Poly (A)-Sepharose 4B, Phannacia Fine Chemicals Inc.). A column (1 x 9 em) of this material was equilibrated with 0·025 M sodium ethanoate, pH 6·5. The solution (8 ml) from the second Sephadex G 75 treatment was dialyzed against the buffer and applied to the column by downward displacement at 17·2 ml/h. The column was washed with the buffer (10 ml) prior to the application of a linear NaCI gradient. For the gradient a solution (60 g) of 0·3 M NaCl in 0·025 M sodium ethanoate, pH 6·5 was pumped into the stirred buffer (60 g) which was pumped on the column. Fractions (2'9 m!) were collected as the gradient was applied at 17·2 ml/h, and assayed for enzyme activity and protein.

Enzyme assays RNase was measured by the method described previously7 and DNase by the method of Liao 8 • A unit of activity (U) in each case is a unit change in absorption at 260 nm per min. Specific activity is units per mg of protein.

Protein assay Except for the products from affinity chromatography, solutions were assayed for protein by the Warburg-Christian method 9• Solutions from the affinity column contained very low levels of protein the amounts of which were estimated by visual comparison with known amounts (0'1 Ilg to I Ilg) of bovine serum albumin after electrophoresis and then staining the gels by the silver stain method. CBR

5

178

N. PRENTICE

Determination of molecular weight by gel filtration A solution (8 ml) of RNase (35 U) and DNase (18 U) and a mixture of standards (Ribonuclease A, M r 13,700; cytochrome c, M r 12,500; chymotrypsin A, M r 25,000; apoferritin, Mr 48,000 and bovine serum albumin, Mr 66,000; Schwartz Mann Co., Orangeburg, NY) were separated by gel filtration on a column (2,5 x 89 cm) of Sephadex G 75 which was eluted with 0·2 M sodium ethanoate, pH 6· 5 at 25 mIJh. The M r of the enzyme was determined from a standard curve of logM r vs. Kavfor the standards where Kav = (VE - Vo)/(Vt - Vo)and V o = void vol. (ml); Vt = total column vol. (ml); VE = elution vol. (ml). Va was determined with blue dextran.

pH optimum The substrates RNA and DNA (0,1 mg/ml) were dissolved in 0·1 M sodium ethanoate at pH 4· 5, 5,0,5'5,6'0 and 6'5, and in 0·1 M Tris chloride at pH 6'5, 7·0 and 7·5. Activities were determined with the standard procedure with 5 ~I of enzyme (0'016 U RNase and 0·0112 U DNase).

pH stability The buffers used were those described for the pH optimum determination. The enzyme (5 ~l containing 0·016 U RNase and 0·0112 U DNase) was added to 0·1 ml of each buffer in quartz cuvettes and stored overnight at 4°C. The solutions were brought to 30°C for 45 min, substrate (0,9 ml) at 30°C was added to each and the reaction rate was measured by the standard procedure.

Activation energy Reactions with RNase (0,015 U) and DNase (0,009 U) were carried out under standard conditions except that temperatures of 23, 30, 35, 40, 45, 50 and 55°C were used. Log activity was plotted against the reciprocal of the absolute temperature, and activation energies (t;.Ha ) were determined from the Arrhenius equation.

Thermal stability The enzyme (initial activity 2·9 U RNase/ml, 1·8 U DNase/ml), in 0·025 M sodium ethanoate pH 6· 5 and 0·15 M NaCI, was incubated at 30, 40, 50, 60 and 70°C for I h. The percent of initial activity remaining after thermal treatments was calculated.

Reaction with polynucleotides Solutions (0,1 mg/ml in 0·1 M sodium ethanoate, pH 6'5) of poly A, poly G, poly C and poly U (Sigma Chemical Co.) were prepared. Amounts of enzyme added to substrate solutions (1 m1) were; to poly A, poly G and poly C; 50, 75 and 100 ~l of enzyme solution, respectively. The enzyme solution contained RNase (0,135 U/ml) and DNase (0,116 U/ml). The increase in absorbance at 260 nm was followed as for the standard RNase and DNase assays.

Reaction with RNA For electrophoretic studies the substrate solution was t-RNA (from yeast, Sigma Chemical Co.) in 0·1 M sodium ethanoate pH 6·5 (I mg/ml). The reaction system, consisting of substrate solution (0,5 ml) containing RNase (0,015 U), was incubated at 30°C for 90, 120, 150 and 180 min. At the end of each incubation period a sample (50 ~l) was removed and added to 50 ~l of formamide,

BARLEY NUCLEASE

179

pH 9·0 which contained 30% (w Iv) of sucrose. These aliquots were stored in a freezer until used in electrophoresis. For reactions in which products were to be examined by HPLC the substrate was yeast RNA, prepared by the method of Crestfield 1o . This RNA is a representative distribution of the RNA's in yeast and consists of 60 to 70 % of the total yeast RNA. The reaction mixtures consisted of 0·2 M sodium ethanoate, pH 6·5 (400 Ill) which contained RNA (1'0 mg) and RNase (0'034 U). Appropriate enzyme and substrate blanks were prepared. Blanks and the reaction mixtures were incubated at 30°C for 15, 30, 45, 60 and 120 min, after which they were placed in a water bath at 100°C for 5 min. Aliquots (10 Ill) were assayed for nucleobases, nucleosides and nucleotides by HPLCl1. Products were identified by elution times and by absorbance ratios, at selected wavelengths from 230 to 290 nm, which were compared to corresponding data for reference compounds.

Slab gel electrophoresis The electrophoretic equipment was the 16 cm BioRad Protean (BioRad Laboratories, Richmond, California). The non-denatured enzyme (0'1 to 1·5 U RNase) was electrophoresed by the procedure of the Sigma Chemical Co., Bulletin MKR-137, with the 8% polyacrylamide level. Gels were stained by the silver stain of the BioRad Laboratories (Bulletin 1089) and by the toluidin~ blue method of Wilson 12 • In the latter method the developed gels were soaked in RNA solution, washed, and then stained with the dye which adsorbs on the RNA in the gel. At the enzyme locations where RNA has been hydrolyzed a white spot appears on the blue background. For Mr determinations of non-denatured enzyme by slab gel electrophoresis the procedure of the Sigma Chemical Co., Bulletin MKR-137 was followed, in which the acrylamide levels in the gels were 6, 7, 8 and 9%. The standard proteins were ex-lactalbumin, carbonic anhydrase, chicken egg albumin and bovine serum albumin with Mr's of 14,200, 29,000, 45,000 and 66,000, respectively. These proteins were stained by the silver stain method. Enzyme preparations were electrophoresed on separate gels and stained with the RNA-toluidine blue method 12 • The molecular weight of the enzyme, denatured with sodium dodecyl sulfate, was determined by the method described in the Sigma Chemical Co. Bulletin MWS 877 L (February 1983) except that slab gels were used instead of tube gels. The protein standards were lysozyme, ~-lactoglobulin, carbonic anhydrase, egg albumin and bovine albumin with Mr's of 14,300,18,400,29,000,45,000 and 66,000, respectively. Polyacrylamide gels (11 %) were used to which 0·2 Ilg of each protein and enzyme were applied. After 15 rnA had been applied for 2 h, the gel slabs were stained with silver. For electrophoresis of RNA and its hydrolytic products, 10% polyacrylamide gels prepared in formamide were used 13 ; electrophoresis was carried out at 250 V and 18 rnA for 4 h. The gels were stained with toluidine blue to locate the RNA spots.

Reaction with 3'

--+

5' dinucleoside phosphates

Substrates (Table IV) were purchased from the Sigma Chemical Co. and prepared as 5 roM solutions in 0·1 Msodium ethanoate pH 6·5. To samples (50 Ill) ofeach substrate solution a portion (25 Ill) of enzyme in buffer solution (4 x 10- 3 U RNase) was added. The reaction proceeded for 2·5 h at 30°C and was then terminated at 100 °C for 5 min. Substrate blank consisted of substrate (50 Ill) and buffer (25111). Products were identified and measured by HPLCl1.

Reaction with nucleoside phosphates, di- and tri-phosphates, sugar phosphates and p-nitrophenyl phosphates Substrates were 10 mM solutions in 0·2 Msodium ethanoate, pH 6·5. Reaction mixtures consisted of substrate (50 Ill) and enzyme solution (25 Ill) which contained 3·5 x 10- 3 units of RNase. Substrate blanks consisted of substrate solution (50 Ill) and buffer (25 Ill). 8-2

N.PRENTICE

180

Blanks and reactions were incubated for 1 hat 40°C. Duplicate aliquots (35 Jll) of each reaction and blank were assayed for phosphorus1 4 , except for the p-nitrophenyl phosphates which were assayed for p-nitrophenoF6. The substrates used were 5/-CTP, 5'-CDP, 5'-UTP, 5'-ADP, 5'-ATP, 5'-GDP, 51 ·AMP, 3'-AMP, 5'-CMP, 3'-CMP, 5'-GMP, 3/-GMP. 51 -UMP, 3'-UMP, D-fructose-l phosphate, 2-glycerophosphate, o-ribose-5 phosphate, D-ribose-l phosphate, p.nitrophenyl phosphate and bis p-nitrophenyl phosphate.

Effect of inhibitors The standard assay procedures for RNase and DNase were used except that the substrate solutions contained 2'5 mM concentrations of the inhibitors shown in Table III. In some cases the DNase cofactor, MnH , was omitted. Results

Enzyme purification The purification and recovery achieved by each fractionation step are shown in Table I. The changing ratio of RNase to DNase was expected since nucleic acid hydrolases other than the one sought are removed by the treatments used. For example, the first treatment with DEAE-trisacryl (Fig. 1) removed a large amount of enzyme with high RNase and low DNase activity (fractions 64 to 74). The RNase to DNase ratio of 13 and R F of 0·35 upon electrophoresis (Fig. 2) indicate that this enzyme may be the major one from barley shoots as previously reported16 and which appears in substantial quantities in embryo and endosperm tissues, but is absent or at low levels in roots.

TABLE 1. Purification of the nuclease Fraction

Volume (ml) Protein (mg/ml) Recovered (%) RNase activity (U/ml) Specific activityB Recovered (%) Purification factor b DNase activity (U/ml) Specific activityB Recovered (%) Purification factor b

DEAE-trisacryl treatment 40-80% Crude Ammonium extract sulphate No.1 No.2

No.1

Poly A Sepharose No.2 4B

200 33 100 38·9 1·17 100 1 14·4 0·43 100 1

96 0·004 0·016 0·074 19 0·20 16·2 0·053 13 0·17 30

60 0·008 0·004 0·21 26 0·16 22 0'13 16 0·20 37

115 26·1 45 46·1 1·77 68 1'51 12·1 0·16 48 1-07

22 0·82 3·9 2·52 3·07 12·4 2·6 3·12 3-8 34 8·8

6·2 0·285 0'38 2·50 8·8 2'0 7·5 2·0 7·02 6'2 16'3

G75 Treatment

• Specific activity = units of activity per mg protein. Purification factor = specific activity of the fraction/specific activity of the crude extract.

b

12 2 x 10-· 3·6 x 10- 6 0·118 5·9 x 103 0·018 5 x 103 0·062 3·1 x 103 0·025 7 x 103

BARLEY NUCLEASE

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FIGURE 1. DEAE-trisacryl chromatography of the nuclease showing DNase activity (0-0), RNase activity (e-e), protein content (0-0) and NaCI (---). A sample (8 ml) of ammonium sulfate-precipitated fraction containing RNase (369 U) was applied and eluted with a NaCl gradient (0 to 0·3 M) in 0·01 M Tris chloride, pH 7·0.

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FIGURE 2. Gel electrophoresis of fractions from DEAE-trisacryl chromatography. Left to right: fractions 37-48, 50--56, 57-62 and 64-74. Spot 1, R F 0·35; spot 2, RF 0·55; spot 3, R F 0·70. The amounts of RNase activity electrophoresed were for pooled fractions 37-48, 0·4 units and for other pooled fractions, 0'75 U in each case. Electrophoresis conditions were: 8% polyacrylamide gels, 15mA, 250V for 2 h.

The enzyme with R F 0·55 (Fig. 2) is the one in fractions 57 to 62 (Fig. 1) and is also in embryo and endosperm extracts but present in only traces in shoot extracts. The enzyme with R F 0·70 is probably the RNase I from barley roots which we described previously5 and which is a major enzyme in extracts of embryo and endosperm tissues but a minor one in shoot tissue extracts.

N. PRENTICE

182

The second fractionation, made by applying fractions 57-62, (Fig. 1) to this ion exchange system, yielded only one enzyme peak, which showed R F O' 55 by electrophoresis and toluidine blue stain. Appreciable amounts of protein impurity were removed, and at this point a l6-fold RNase purification had been attained (Table I). Figure 3 shows the results of the first Sephadex G 75 treatment of the product from DEAE-trisacryl. A large peak of protein impurity was removed in the initial fraction. Similarly, a second treatment with Sephadex G 75 (Table I) removed additional inactive protein. At this point, purifications of RNase and DNase were 22-fold and 37-fold respectively.

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FIGURE 3. Sephadex G 75 chromatography of the nuclease. Symbols as for Fig. 1. A sample (8 ml; 20 U RNase) of product from second DEAE trisacryl treatment was applied and eluted with 0·2 M sodium ethanoate, pH 6·5.

The results of affinity chromatography with poly (A)-Sepharose 4B are shown in Fig. 4 and Table I. The product from this treatment (fractions 30-33, Fig. 4) had an extremely low protein level (Fig. 4) which was not measured accurately by absorption at 280 and 260 nm (the Warburg-Christian procedure). Therefore protein in the product was estimated by silver staining after electrophoresis. Only one spot was observed which displayed R F O' 55 on 8 % gels and which corresponded to the location of the enzyme by the same electrophoresis and the toluidine blue stain. High specific activities for RNase and DNase (Table I) had a ratio of approximately 2 in contrast to the ratio of 11 for the enzyme isolated from shoots 16 . Enzyme parameters

The pH optimum for both RNA and DNA substrates was pH 6·0 to 6·5. The enzyme showed optimum stability also over this pH range, although only about 40 % of the original activity remained after overnight storage at 4 dc. At pH's above and below this

183

BARLEY NUCLEASE 0,20

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FIGURE 4. Affinity (Poly A Sepharose 4B) chromatography of the nuclease. Symbols as for Fig. 1. A sample (8 ml; 1·7 U RNase) from the second G 75 treatment was applied and eluted with a gradient of NaCI (0 to 0·3 M) in 0·025 M sodium ethanoate, pH 6·5.

range, activity decreased markedly under conditions for both pH optimum and pH stability determinations. The temperature stability for the enzyme was not the same for both substrates. Relative to the activity of the control the activities remaining after 1 h were for RNase; 93 % at 30 DC; 70% at 40 DC; 49% at 50 DC; 18% at 60°C and 7% at 7 DC: for DNase, 85% at 30 and 40 DC; 11 % at 50 DC and 4% at 60 and 70 DC. Spatial alterations in the molecule may be more detrimental to the weak reaction with DNA than with the more efficient catalysis with RNA. When examined for temperature optimum the enzyme displayed activation energies (6.Ha ) of 11·0 kcallmole for RNase and 9·3 kcalfmole for DNase. The apparent M r of the active enzyme was 30,000 by gel filtration and 35,000 by gel electrophoresis. Treatment with sodium dodecylsulfate (4 %, wIv) and 1·35 M 2mercaptoethanol denatured the enzyme to form a product with M r 18,000 when measured by the electrophoretic procedure. The active enzyme appears to be two monomers, possibly joined by disulfide bonds. Reaction with RNA Gel electrophoresis of the t-RNA substrate before and after exposure to the enzyme showed the unreacted substrate with R F 0·30 and a single spot with R F 0·42 when examined over the incubation periods of 60 to 180 min. Similar results were obtained for the Crestfield RNA, but this RNA and its product did not form spots as discrete as did the t-RNA.

a Reactions system: Crestfield RNA (1 -0 mg) and enzyme (0,034 U) in 0·2 M ethanoate, pH 6· 5 (0·4 ml). Reaction at 30°C.

The products from the Crestfield RNA for several reaction times are shown in Table II. The reaction appeared to be essentially complete after 1 h with the formation mainly of 5'-AMP, 5'-GMP and 5'-UMP. The enzyme appears to hydrolyze the substrate until a limiting molecular size remains. No 2',3'-cyclic nucleotides were detected. The enzyme appears to be a Nuclease I (EC 3 . 1 .30 . 2).

Reaction with polynucleotides The specific activities (Ufmg protein) for poly A, poly C, poly U and poly G were 10, 5, 0·15 and 0 respectively. Because of the high affinity for poly A, poly A Sepharose was chosen for the affinity chromatography. TABLE III. Effect of inhibitors on nuclease activitya Inhibition (%) Inhibitor

RNase

DNase

BaC1 2 ZnC1 2 CaC1 2 MgC1 2 MnC1 2 Iodoacetamide Dithiothreitol Malemide Spermidine Spermine Putrescine

0 61 29 21 34 37 17

73 43

0 5

8 8 b

0 11 0

60 23

a Reaction system: Crestfield RNA substrate (0-1 mg/ml in 0·1 Methanoate pH 6'5), inhibitor (2'5 mM), and enzyme (0,03 U). Total volume 1 mI, IO min at 30 °C. b 5 mM MnCI, was routinely used in the DNase assay since it stimulates activity by 50%. C Precipitates in presence of Mn 2+.

BARLEY NUCLEASE

185

Effect of enzyme inhibitors

The results with inhibitors are shown in Table III. Activities with both RNA and DNA were sensitive to metal ions. Activity with DNA, in contrast to activity with RNA, was usually stimulated about 50% with 5 mM Mn H (see Methods). When this cation was present, inhibition was caused by the addition ofanother divalent cation. However, when Mn 2 + was omitted from the DNA substrate solution, BaH, Ca2+ and Mg2+ (but not Zn2+) stimulated activity by about 25%. The requirement for the divalent cation is apparently not restricted to Mn 2 + for this enzyme. The effects of the sulfhydryl-active reagents do not indicate the sulfuydryl group is involved in the enzyme's active site. The amines spermidine, spermine and putrescine characteristically inhibit RNase. Reaction with 3' ~ 5' dinucleoside phosphates

Activities with the 3' -+ 5' dinucleoside phosphates are shown in Table IV. It is apparent that if the uridylyl bond is on the 3' position of the ribose, the hydrolysis of the 3' ester bond is inhibited (UpA, UpC, UpU and UpG). Similarly, if guanosine is esterified on the 5' end of the dinucleoside phosphate, except for ApG, the reaction is inhibited (GpO, UpO and CpG). When the uridylyl bond is on the 3' end and the guanosine bond is on the 5' end (UpG) both nucleosides inhibit the reaction. The adenylyl bond on the 3' of the dinucleoside phosphate appears to be hydrolyzed fairly well (ApA, ApC, ApU and ApG). The guanylyl group of GPU is very susceptible to hydrolysis. TABLE IV. Extent of reaction of the nuclease with different 3' ~ 5' di-nucleoside phosphates R Substrate

Hydrolyzed (% )

Substrate

ApA ApC ApU ApG GpA GpC GpU GpG

13 24 30 17 30 26 81

UpA UpC UpU UpG CpC CpU CpG

Hydrolyzed

(% )

1 3 6 0 21 20 1

9

a Reaction system: Substrate (5 mM) in 0·1 M ethanoate, pH 6·5. Substrate solution (50 solution in buffer (25 fll, 4 x 10-3 U RNase) were incubated 2·5 h at 30°C.

~l)

and enzyme

Reaction with nucleoside mono-, di~ and triphosphates, sugar phosphates, 2-g/ycerophosphate and p-nitrophenyl phosphates

The enzyme did not catalyze the hydrolysis of the nucleoside 5' -mono, di-, or triphosphates, the p-nitrophenyl phosphates, the sugar phosphates, or the 2-glycerophosphate. It did catalyze the hydrolysis of the 3'-nucleoside monophosphates. As percent of substrate hydrolyzed the activities were: 3'-AMP, 11% ; 3'-CMP, 9% ; 3'-GMP, 1 % and 3'·UMP, 0·2%.

1&6

N.PRENTICE

Effect of storage and concentration on enzyme activity

Storage in the frozen state resulted in about 50% loss of both RNase and DNase within three weeks. Concentrating the enzyme by freeze drying or by dialysis against sucrose at 4 ac caused marked losses of activity. Discussion The nuclease described here is similar to the one isolated from barley shoots 16 in that their reaction products are the 5'-nucleotides. Both enzymes react with RNA and DNA and appear to have the characteristics of a Nuclease I (EC 3. 1.30.2) according to Wilson's classification!7. The apparent MrS are similar, 37,000 for the shoot nuclease and 30,000 to 35,000 for the root nuclease. Optimal pH's were also similar. However, the molecular charges were not the same since the electrophoretic RF's were 0·33 for the shoot enzyme and 0·55 for the root nuclease. Reactivity with RNA and DNA differed for the two nucleases. The shoot enzyme had an RNA: DNA activity ratio of II: 1 whereas the value for the root enzyme was 2: I (Table I). The nuclease from shoots was not reactive with 3' -+ 5' dinucleoside phosphates if the cytidylyl bond was on the 3' end of the molecule, whereas the root enzyme did not react well when the uridylyl group was esterified at the 3' position. Reactivities of the two nucleases with polynucleotides differed. The shoot enzyme showed the preference poly A > poly U > poly C> poly G, whereas for the root enzyme the sequence was poly A> poly C> poly U> poly G. The root nuclease appears to be less susceptible to the inhibitors to which it was subjected than was the shoot nuclease, particularly when exposed to the amines, spermidine, spermine and putrescine. Polyamines in plant tissue are thought to retard senescence by inhibiting RNase and protease18 , 19. A partially purified Nuclease I from barley malt, described by Sasakuma and Oleson 20 , is similar to the enzymes we report from roots and shoots, except that the RNase: DNase ratio is about I: 4, there is reaction with 2-glycerophosphate, and the optimum pH was 5 to 5,8, somewhat lower than the optimum pH we have found. Pietrzak et al. 21 have described a nuclease from barley seed which resembles, in some respects, the enzyme we describe from barley roots and the one from barley shoots16 • This nuclease has a M r of 37,000 daltons, pH optimum 6·8 and an RNase:DNase ratio of 3. Its retained activities in the presence of I mM MnCl 2 and I mM ZnS0 4 were 89 and 70% respectively, comparable to the behavior of the nuclease from roots (Table III) and the nuclease from shoots16 • However its preference for polynucleotides was poly U > poly A > poly C > poly G, which does not correspond to the sequence for either of the nucleases we have examined. Lia0 8 reported several DNase isozymes from barley malt. However, Ca ions were used in the purification to stabilize the DNase. No RNase activity remained in these preparations. RNase in our nuclease preparations was significantly inhibited by Ca2+ ions and may be largely inactivated over a prolonged exposure time. The isoelectric pH of nucleases in extracts of malt roots has been examined previously and found to be pH 4.5 5 • All nuclease activity (reaction with both DNA and RNA) in the extracts showed this isoelectric pH.

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