Studies on sheep kidney nuclease

52

Biochimica et Biophysica Acta, 520 (1978) 52--60

© Elsevier/North-HollandBiomedicalPress

BBA 99230 STUDIES ON SHEEP KIDNEY NUCLEASE I. AN IMPROVED PURIFICATION METHOD AND SOME PROPERTIES

TAKAFUMIWATANABE * and KENICHI KASAI Department of Biological Chemistry, Faculty of Pharmaceutical Science, Hokkaido University, Sapporo (Japan)

(Received September 6th, 1977) (Revised manuscript received January 23rd, 1978)

Summary An improved purification method of the sheep kidney nuclease (nuclease SK) specific for single-strand nucleic acid, which includes extraction with 0.85% NaC1, treatment with DEAE-cellulose, fractionation with polyethylene glycol, phospho-cellulose chromatography, CM-Sephadex chromatography and phospho-cellulose rechromatography is described. The nuclease was purified approx. 390-fold. Identity was established by comparison with known properties. Molecular weight was estimated to be 52 000--53 000 by gel filtration on Sephadex G-100. The properties of the purified enzyme agreed well those reported previously. The purified enzyme hydrolyzed heat-denatured calf thymus DNA, yeast RNA and no hydrolytic activity for native calf thymus DNA, A2'-pA, A3'-pA, ADP, ATP, 5'-AMP and cyclic AMP.

Introduction The presence of an endonuclease in sheep kidney (nuclear SK) which hydrolyzed thermally denatured DNA in preference to native DNA has been reported by Healy et al. [1]. Kasai and Grunberg-Manago [2] partially purified this enzyme from sheep kidney acetone powder and studied some properties. The properties of this enzyme are as follows. * Present address: D e p a r t m e n t of Clinical Biochemistry, Tokyo College of Pharmacy, 1432-1 Horinouchi, Hachioji, Tokyo, Japan. Abbreviations: iPr2P-F, dilsopropylphosphofluoridate; TMD buffer, 2 • 10 -2 M Tris • HCI (pH 7.5)/2 • 1 0 --2 M 2 - m e r c a p t o e t h a n o l / 1 0 - 4 M i P r 2 P - F ; T M b u f f e r , 2 • 1 0 - 2 M "Iris • H C I ( p H 7 . 5 ) / 2 • 1 0 - 2 M 2 mercaptoethanol; bis-pNP, bis-p-nitrophenylphosphate; P C M B 0 p - c h l o r o m e r c u r i b e n z o a t e .

53 The optimum p H for R N A digestion is 7.5. This enzyme was most active in the presence of 5 • 10 -2 M MgCl2 and one of the endonucleases. If the nucleic acid used as substrate had ordered structure, it could not be hydrolyzed by this nuclease. The nuclease had no base specificity and the degradation products of the enzyme action were 5'-oligonucleotides. N o mononucleotide was produced and final products were 5'
Sheep kidney was provided from The Live-Stock Bureaw, Sapporo. Calf thymus DNA was obtained from the Sigma Co. DEAE-cellulose and phospho~cellulose was obtained from Brown Co. CM-Sephadex C50 was obtained from Pharmacia Fine Chemicals. Polyethylene glycol and yeast RNA were obtained from Nakarai Chemicals Co. iPr2P-F was purchased from Sumitomo Chemicals Co. 5'-AMP, 2'(3')-AMP, ADP and ATP were obtained from Kojin Co. A2'-pA and A3'-pA were a gift from Dr. Miura of this department. Assay of nuclease activity. To the reaction mixture containing 0.2 ml 0.2% thermally denatured calf thymus DNA, 0.05 ml 0.5 M Tris. HC1 (pH 7.5), 0.05 ml 1% bovine serum albumin and 0.05 ml 0.25 M 2-mercaptoethanol was added 0.05 ml of the enzyme solution. After the mixture was incubated for 15 rain at 37°C, a 0.2 ml aliquot was added to 0.8 ml of 10% HC104 and the mixture was chilled at 0°C for 15 rain. After centrifugation at 3000 rev./min for 15 min, the absorbance of the supernatant was measured at 260 rim. One unit of the enzyme activity corresponds to the amount of the enzyme that increases the absorbance of the supernatant by one unit after 1 h incubation. Assay of bis-pNP hydrolytic activity. To the reaction mixture containing 0.2 ml water, 0.2 ml 0.5 M Tris • HC1 (pH 7.5), 0.1 ml 1% bovine serum albumin, 0.1 ml 15 mM MgC12, 0.1 ml 0.25 M 2-mercaptoethanol and 0.2 ml 25 mM bis-pNP was added 0.1 ml of the enzyme solution; this was incubated

54

for 15 min at 37°C and a 0.2 ml aliquot taken o u t from the mixture was added to 0.8 ml 0.5% SDS {sodium dodecyl sulfate) in 0.5 M NH4C1/NH4OH (pH 9.5) and the absorbance of the mixture was measured at 420 nm. Purification o f nuclease SK. All purification procedures were carried out at 4°C and degassed water was used. Acetone powder of sheep kidney was prepared by the general m e t h o d [10]. Sheep kidney acetone p o w d e r (40 g) was homogenized in 200 ml 0.85% NaC1 containing 0.02 M 2-mercaptoethanol and 0.1 mM iPr2P-F and then allowed to stand overnight. After the centrifugation of this homogenate at 9000 rev./min for 90 min, the supernatant was diluted to 1000 ml with TMD buffer (crude extract). The crude extract was applied on a DEAE-cellulose column (2.5 X 40 cm) equilibrated with TMD buffer and the eluted fraction was collected (DEAE fraction). To 900 ml of this DEAE fraction, 50% {w/v) polyethylene glycol in TMD buffer was added at a final concentration of 25% and stirred for 30 min at 4°C. The precipitate produced was collected by centrifugation at 12 000 rev./min for 15 min and dissolved in 500 ml of TMD buffer {polyethylene glycol-1 fraction). The solution was applied on a phospho-cellulose column (2.5 X 41 cm) equilibrated with TMD buffer. The column was washed with 1000 ml of the same buffer and the elution was carried o u t with a linear gradient of NaC1 {0--0.5 M) in the same buffer. 20-ml fractions were collected at a flow rate of 50 ml/h. To the combined active fraction, 50% (w/v) polyethylene glycol was added to a final concentration of 20%; the mixture was allowed to stand for 30 min at 4 ° C. The precipitate produced was collected b y centrifugation at 12 000 rev./min for 15 min and dissolved in 25 ml of TM buffer {polyethylene glycol-2 fraction). After dialyzing against the same buffer, this fraction was applied on a CMSephadex C50 column {1.5 X 20 cm) equilibrated with TM buffer. After washing the column with the same buffer of 500 ml, the nuclease was eluted with a linear gradient of NaC1 (0--0.5 M) in the same buffer. 10-ml fractions were collected at a flow rate of 60 ml/h. The combined active fraction was dialyzed against TM buffer overnight. This fraction was applied to a phospho-cellulose column (1.5 X 20 cm) equilibrated with TM buffer. After washing the column with 500 ml of the same buffer, nuclease SK was eluted with a linear gradient of NaC1 (0--0.5 M) in the same buffer. 10-ml fractions were collected at a flow rate of 40 ml/h. The active fractions (purified nuclease SK) were combined and stored in 20% {w/v) glycerine at --20 ° C. Studies on substrate specificity. Relative activity for RNA, native DNA and thermally denatured D N A was compared by the increase in the absorbance at 260 nm of acid-soluble fraction obtained b y the standard assay method. Hydrolysis of A2'-pA and A3'-pA was studied b y paper electrophoresis as follows. To the reaction mixture containing 0.1 ml 6 . 6 . 1 0 - 4 M A2'-pA or A3'-pA, 0.05 ml 0.25 M 2-mercaptoethanol, 0.05 ml 0.5 M Tris • HCI {pH 7.5), 0.05 ml 15 mM MgCl2 and 0.05 ml 1% bovine serum albumin was added 0.02 ml of the enzyme solution {containing 8 units). The mixture was incubated at 37°C for 24 h, freezed and concentrated to 50 pl in vacuo. The concentrated hydrolyzate was applied to Whatman 3MM paper and electrophoresis was carried o u t with 0.05 M borate buffer (pH 9.5) at 12 V/cm, for 2 h.

55

Effect o f PCMB on nuclease activity. After dialysis of 4 ml of the purified nuclease preparation against 0.05 M Tris • HC1 (pH 7.5), 1 ml 10 -s or 10 -6 M PCMB was added and followed by standing for 30 min at 0°C. The remaining nuclease activity of the mixture was assayed by a standard assay method with use of thermally denatured calf thymus DNA as substrate in the presence or absence of 2-mercaptoethanol. Molecular weight determination o f nuclease SK. The molecular weight of nuclease was determined by the gel filtration method of Andrew [9]. Results

Purification o f nuclease SK The results of purification are summarized in Table I. The nuclease was purified 1.5-fold by the first DEAE-cellulose treatment of crude extract. The optimum condition for polyethylene glycol fractionation was examined in regard to temperature and the concentration of polyethylene glycol. The results showed that direct application of polyethylene glycol fractionation of the crude extract gave no good separation of the enzyme from other contaminants. Then, the fraction treated with DEAE-cellulose was subjected to polyethylene glycol fractionation at a final polyethylene glycol concentration at 20% at 4, 10 and 20 ° C, respectively. The results showed that at 10°C 100% of the activity and 20% of the protein were recovered in the precipitate. In this step, the purification was 5-fold. 10°C was the most preferable temperature. However, generally speaking, an experiment at 10°C was not practicable. Therefore, conditions were chosen as follows: temperature, 4°C and final concentration of polyethylene glycol, 20%. In this case, the nuclease was purified about 3-fold. Fig. 1 shows the result of phospho-cellulose chromatography. Nuclease activity determined with use of thermally denatured calf thymus DNA as substrate was eluted as a single peak at the position corresponding to a NaC1 concentration of 0.24 M. Previously, in sheep kidney some nucleases other than this nuclease have been found in our laboratory. These nucleases were eluted above 0.4 M NaC1. Fig. 2 shows the result of CM-Sephadex C50 chromatography. DNA hydrolytic activity was also eluted as a single peak at 0.15 M NaCl, but the peak of DNA hydrolytic activity and that of bis-pNP TABLE I SUMMARY OF PURIFICATION

Fraction

Protein (mg)

Activity (unit)

Specific activity

Purification

(units/mg

protein) Crude extract DEAE-cellulose Polyethylene glycol-fractionation ( I ) Phospho-cellulose Polyethylene glycol-fractionation (2) CM-Sephadex Phospho-cellulose

7600 3600 1200 260 96 17 1.9

52 35 34 18 16 7 5

400 000 000 000 000 000 200

6.9 9.7 27.6 69.7 166 407 2690

1 1.4 4.0 10 24 59 390

56 3000

l oL

I

ZOO0

E

Z

0.5

1000

0.5 0.4 0.3~ 0.2~ O.I

0 ~ 0

~

o

70 30 40 50 60 Fraction No. Fig. i . P h o s p h o - c e l l u l o s e c h r o m a t o g r a p h y o f n u c l e a s e S K . A m o l a r i t y g r a d i e n t ( 0 - - 0 . 5 ) o f NaCI in T M D b u f f e r w a s u s e d t o elute t h e e n z y m e . The solid curve i n d i c a t e d p r o t e i n c o n t e n t m e a s t t r e d as a b s o r b a n c e a t 2 8 0 n m . E n z y m e a c t i v i t y w a s a s s a y e d a c c o r d i n g to the standard assay m e t h o d described in the t e x t . o, p r o t e i n c o n t e n t ; e , the d e n a t u r e d D N A h y d r o l y t i c activity. 10

20

hydrolytic activity almost agreed. In order to separate bis-pNP hydrolytic activity from D N A hydrolytic activity this preparation was applied once more to a phospho-cellulose column {Fig. 3). D N A hydrolytic activity was eluted at a position corresponding to a NaC1 concentration of 0.22 M; bis-pNP hydrolytic activity eluted at 0.4 M NaC1, showing the separation of these t w o enzymes. To this purified nuclease SK solution, glycerine was added to 20% (w/v) and stored at --20°C. No loss of activity was observed for several months. A c t i o n o f purified nuclease S K on native and thermally denatured D N A

It is important to establish the identity between the properties of this preparation and that reported previourly, since this purification m e t h o d was extremely different from that reported. As indicated in Fig. 4, thermally denatured calf t h y m u s DNA was hydrolyzed, while native DNA was not, suggesting that no DNAase other than nuclease SK was contained in this preparation. Studies on substrate specificity

Although it is known that nuclease SK cleaves nucleic acid phosphodiester bonds, it has n o t been clear whether this nuclease hydrolyzes small nucleotide

57 700

0.7

t~

0.6

500 ~

0.5

~0.4

400

'~

~ ,~

I

U

00.3

300

ossoo 0.2

O. 5 2()0 )0

'1(

0.1

0.4 0.3~ E}.2~ 9.1

0 10

20 Fraction No.

30

F i g . 2. C M - S e p h a d e x C 5 0 c h r o m a t o g r a p h y

40

0

50

o f n u c l e a s e S K . A m o l a r l t y g r a d i e n t ( 0 - - 0 . 5 ) o f NaCI i n T M D

buffer waS u s e d t o elute the e n z y m e . S o l i d c u r v e indicated protein content m e a s u r e d as a b o s o r b a n c e a t 2 8 0 n m . o0 protein content; 0, denatured D N A h y d r o l y t i c a c t i v i t y ; $, b i s - p N P h y d r o l y t i c a c t i v i t y .

molecules. Therefore, the hydrolytic activity for small nucleotide was examined. After A2'-pA, A3'-pA, 5'-AMP, 2'(3').AMP, ADP, ATP and cyclic AMP were incubated, respectively, with nuclease SK at 37°C the products were examined by paper electrophoresis. In all cases, no other spot than starting material was observed. Considering these results, it may be concluded that in nuclease reaction it is essential for the substrate to be longer than a definite length. These results concerning the substrate specificity determination also suggest that the purified nuclease SK preparation contains no contaminating ribonuclease, phosphodiesterase and phosphatase. Effect o f heat treatment on nuclease S K Purified nuclease was inactivated rapidly by the heat treatment of 80°C for 10 min and the activity decreased to 50% of the original. This property also agreed with that already known. Effect o f PCMB on nuclease 8 K As shown in Table II, the activity of nuclease SK decrease from 96 to 21 units/ml in the presence of 10 -4 and 110-s M PCMB, respectively. But by addition of 2-mercaptoethanol to the assay mixture the activity was recovered. This result may suggest that nuclease SK is an SH-enzyme.

58 0.4

000

0.3

200

u

I000 G"

800

"~ O. 2

600

Z 0.5

400

0. I

0.4 0.3~ v

200

0.2 9 0.1

10

20 Froction No.

30

40

50

Fig. 3. P h o s p h o - c e l l u l o s e r e c h r o m a t o g r a p h y o f n u c l e a s e SK. A m o l a r i t y g r a d i e n t (0---0.5) of NaCI in TM b u f f e r w a s u s e d t o e l u t e t h e e n z y m e . T h e solid c u r v e i n d i c a t e s t h e p r o t e i n c o n t e n t m e a s u r e d as t h e a b s o r b a n c e a t 2 8 0 n m . E n z y m e a c t i v i t y w a s m e a s u r e d a c c o r d i n g t o t h e s t a n d a r d assay m e t h o d , o , p r o t e i n c o n tent; e, d e n a t u r e d D N A hydrolytic activity ; o, bis-pNP hydrolytic activity. 0.5

0.4

¢ 0.3

2o. 2

<~ 0 . 1

8 1

a 2

= 3

4

I n c u b a t i o n t i m e (hr) Fig. 4. A c t i o n o f n u c l e a s e S K o n t h e r t h e r m a l l y d e n a t u r e d a n d n a t i v e calf t h y m u s D N A . N a t i v e (o) a n d t h e r m a l l y d e n a t u r e d ( o ) D N A w a s u s e d as s u b s t r a t e in t h e s t a n d a r d a s s a y s y s t e m d e s c r i b e d in t h e t e x t . T h e r e a c t i o n m i x t u r e w a s i n c u b a t e d at 3 7 ° C a n d a f t e r a p p r o p r i a t e i n t e r v a l s , t h e i n c r e a s e o f t h e a b s o r b a n c e of t h e a c i d - s o l u b l e f r a c t i o n w a s m e a s u r e d .

59

TABLE EFFECT

II OF PCMB ON NUCLEASE

PCMB (M)

0 2 • I 0 -6 2 • 10 -5

SK ACTIVITY

Activity (units/ml) --2Mercaptoethanol

+2-Mercaptoethanol

84 21 5.1

98 96 90

Molecular weight of nuclease SK The molecular weight of nuclease SK was determined by gel filtration method with Sephadex G-100 column. The nuclease was eluted at the position correspond to a molecular weight of 52 000--53 000. Discussion The purification m e t h o d described in this report gave highly reproducible results. Although because on assaying this enzyme denatured DNA was used as a substrate instead of poly(A) used in previous report [2] the direct comparison of this procedure with that previously reported is difficult, the degree of purification appeared to increase from 120- to 390-fold. In the process of the fractionation with polyethylene glycol no decrease in the activity was shown. This purification step was simple and gave high reproducibility. In sheep kidney at least three nucleases other than nuclease SK were found. One of them was a ribonuclease specific for pyrimidine nucleotides as well as RNAase A. However, these nucleases were bound more tightly to the phosphocellulose column and could be eluted above 0.3 M NaC1. Therefore, it is suggested that the contamination of these nucleases in the nuclease SK preparation was negligible. In sheep kidney the hydrolytic activity for bis-pNP was also found in our laboratory. Initially this activity appeared to arise from nuclease SK in that bis-pNP hydrolytic activity was eluted at the same position as nuclease SK on gel filtration and was inhibited by PCMB. However, by heat treatment the activity was not affected and finally by phospho-cellulose rechromatography the separation of the activity from the preparation of nuclease SK was accomplished, suggesting bis-pNP hydrolytic activity to be derived from a diesterase other than nuclease SK. From the fact that this diesterase could hydrolyze NAD and FAD, it seems to be a NAD pyrophosphatase or a nucleotide pyrophosphatase [ 12]. As mentioned above, nuclease SK can hydrolyze RNA and denatured DNA only. Now, in recent years some nucleases specific for the single-strand nucleic acid such as nuclease SK have been found in various plants and fungi [13--18]. Laskowski isolated an endonuclease, optimum pH 5.0, from Mung bean sprouts and reported that the products of the enzyme action on DNA was almost mononucleotides. Under the ralatively low concentration of the enzyme the nuclease preferentially hydrolyzed single strand nucleic acid [ 13].

60 In the culture medium of Penicillium citrium Kuninaka [16] found a nuclease that hydrolyzed only RNA and denatured DNA. The enzyme was heat stable and inactivated by EDTA and the optimum pH was 5.0. Nuclease S1 obtained from Aspergillus oryzae [19], endonuclease IV from bacteriophage T4-infected Escherichia coli [20] were also single-strand specific nucleases. With the exception of endonuclease IV, in which the reaction products were not established, these nucleases produced mononucleotides as final products. Considering that the final products of the reaction of nuclease SK are di- and trinucleotides, it seems that the reaction mechanism of nuclease SK is different from single-strand specific nucleases obtained from plants and fungi. Furthermore, in the process of the enzyme reaction of nuclease SK, as the substrate is cleaved to short fragment, reaction hardly proceeds. Summarizing these properties of nuclease SK, this nuclease seems to be extremely useful for the limited digestion of the single strand DNA. From this point of view an application of this enzyme to nucleic acid research including limited digestion of ~X174 and the preparation of various oligodeoxynucleotides is being examined. Details will be reported in the following paper. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Healy, J.W., Stollar, M., Simon, M.I. and Levine, L. (1963) Arch. Biochem. Biophys. 1 0 3 , 4 6 1 - - 4 6 8 Kasal, K. and Grunberg-Manago, M. (1967) Eux. J. Biochem. 1 , 1 5 2 - - 1 6 3 Sanger, F. (1 972) Biochem. J. 124, 833--842 MinJov, W. and Fiers, W. (1972) Nature 237, 83---88 Ball, L. (1 969) Nature 224,967---969 Hindley, S. (1 969) Nature 2 2 4 , 9 6 4 - - 9 6 7 Bellemare, G,, J o r d a n , B.R. and Monier, R. (1972) J. Mol. Biol. 72,307---315 Spencer, M.F. and Walker, I.O. (1972) Eur. J. Biochem. 1 0 , 4 5 1 - - 4 5 6 Andrew, P. (1964) Biochem. J. 9 1 , 2 2 2 - - 2 2 3 Drysdale, G.T. and Lardy, H.A. (1953) J. Biol. Chem. 2 0 2 , 1 1 9 - - 1 3 6 J a c o b s o n , K.B. (1966) J. Biochem. BiophFs. Cytol. 3, 31---36 Anderson, B.M. (1966) Biochem. J. 1 0 7 , 3 9 2 - - 3 9 6 Jacobso n, P.H. an d Laskowski, M. (1970) J. Biol. Chem. 245, 891--898 Hanson. D.M. and Faixley, J.L. (1969) J. Biol. Chem. 244, 2 4 4 0 - - 2 4 4 9 Lerch, B. and Wolf, G. (1972) Biochim. Biophys. Acta 335, 208--218 K u n i n a k a , A. (1959) Bull. Agric. Chem. Soc. Jap. 123, 30---34 Hanamo rl, N. and Okazaki, R. (1973) Biochim. Biophys. Acta 335, 155--172 Sparks, Jr., B. (1972) Arch. Biochem. Biophys. 1 5 3 , 1 7 7 - - 1 7 9 Ando, T. (1966) Blochim. Biophys. Acta 114, 158--168 Sadowski, P.D. and Bakyta, I. (1972) J. Biol. Chem. 247,405---412