Purification, specificity, and other properties of a ribonuclease from Octopus vulgaris

ARCHIVES

OF BIOCHEMISTIgY

AND

:BIOPHYSICS

146, 327-336 (1971)

Purification, Specificity, and Other Properties of a Ribonuc!ease from Octopus vulgaris ~ FRANCESCO

D E L O R E N Z O , G U I D O MOLEA, AND M A R I O M O L I N A R O

Institute of Biochemistry (I and II Chair), Medical School, University of Naples, Naples, Italy, and Institute of Histology and General Embryology, Medical School, University of Rome, Rome, Italy Received December 15, 1970; accepted June 8, 1971 A ribonuclease from the hepatopanereas of Octopus vulgaris has been purified 900-fold. The purified enzyme shows one band upon electrophoresis in polyacrylamide gels and is homogeneous with respect to size as shown by analytical ultracentrifugation. The molecular weight of the enzyme is approximately 34,000 and its sedimentation coefficient s20.,~is 3.2. The enzyme activity has an optimum at pH 5.5 and is inhibited by Zn 2+, Cu 2+, Fe 2+, PMB, and iodoacetate. The purified enzyme has neither deoxyribonuelease nor phosphomonoesterase activity; it exhibits endonuelease activity and specifically cleaves phosphodiester bonds next to purine bases, preferentially adenine, with the formation of oligonucleotides terminating in 3~-purine nucleotides, via cyclic phosphate intermediates. Ribonucleases (RNases) are widely distributed in nature and there have been m a n y reports on their specificity and purification from biological sources (reviewed in 1, 2). RNases with strict and well defined base specificity, such as the bovine pancreatic enzyme (cleaving only next to pyrimidine nueleotides), and takadiastase T1 as well as the UI enzyme from Ustilago sphaerogena (specifically hydrolyzing phosphodiester bonds between guanosine 3'-phosphate and other nucleotides), have proved to be extremely useful biochemical tools for studies of the p r i m a r y structure of R N A . For such studies, an enzyme having high specificity for the hydrolysis of the phosphodiester bonds between adenosine and other nucleosides in ribonucleic acid could provide useful information. Only R N a s e T2 from Aspergil1 This investigation has been supported in part by grants from the National Institutes of Health, U. S. Public Health Service (TW 00258-2 to F.D.L. and ROI-EC 00086 to M.M.) and in part from a grant of Consiglio Nazionale delle Ricerche, Roma, Italy.

lus oryzae has been shown to have preferential specificity for hydrolysis at the phosphodiester bonds next to adenylic acid during the early stages of hydrolysis (3). Furthermore, several RNases specific for bonds involving pyrimidines have been isolated from bacteria and animM tissues (1); RNases specific for purines have been isolated in large number only from bacteria (1), while no animal R N a s e having preferential specificity for purines has been described so far. I n a search for RNases from animal organisms we have examined extracts from the hepatopancreas of Octopus vulgaris, since this organ contains high level of enzymes involved in the degradation of both R N A (4) and D N A (5). I n the course of these investigations we have isolated an R N a s e highly specific for purines, with a higher selectivity for the hydrolysis of the phosphodiester bonds between adenosine 3'-phosphate and other nucleotides, via a 2 ' , 3 ' cyclic phosphate intermediate. I n this paper we describe the purification of this ribonuclease and some of the properties of the homogeneous enzyme. 327

328

D E L O R E N Z O , MOLEA, A N D M O L I N A R O MATERIALS AND METHODS

Materials Calcium d i - p - n i t r o p h e n y l phosphate, p - n i t r o p h e n y l phosphate, yeast R N A (type II), calf t h y m u s D N A (type I), and Dowex 1-X8 were p u r c h a s e d from Sigma Chemical Co. CM-Sephadex, D E A E - S e p h a d e x A-25, A-50, a n d Sephadex G-75 were purchased from P h a r m a c i a (Uppsala). P o l y ( U ) , poly(A), and poly(C) were o b t a i n e d from Miles Chemical Co. Escherichia coli alkaline p h o s p h a t a s e was o b t a i n e d from W o r t h i n g t o n Biochemical Co.

Assay for RNase Activity R N a s e a c t i v i t y was assayed by m e a s u r i n g t h e r a t e of digestion of y e a s t R N A at pH 5 (6). T h e r e a c t i o n mixture was i n c u b a t e d at 30 ~ for 25 m i n and c o n t a i n e d in 2.0 ml t h e following components : 0.5 ml of 0.4% yeast RNA, 0.1 M sodium acetate buffer p H 5.0, and an appropriate a m o u n t of enzyme solution. To stop the e n z y m a t i c r e a c t i o n 0.5 ml of 0.75% u r a n y l acetate in 25% perchloric acid was added. The p r e c i p i t a t e was removed b y centrifugation. An aliquot (0.2 ml) of t h e s u p e r n a r a n t fluid was diluted to 3 ml w i t h distilled w a t e r and t h e absorbance at 260 m~ was d e t e r m i n e d in a B e c k m a n D U s p e c t r o p h o t o m e t e r . T h e values were corrected for zero time blanks. A u n i t of R N a s e a c t i v i t y is defined as t h e a m o u n t of enzyme t h a t causes an increase in a b s o r b a n c e of 1.00 at 260 mt~ u n d e r t h e above conditions.

Phosphalase and Phosphodiesterase Assays P h o s p h a t a s e a c t i v i t y was assayed b y incub a t i n g 0.7 ml of 1 mM p - n i t r o p h e n y l p h o s p h a t e , 0.2 ml of 0.5 M sodium acetate buffer pH 5.0, 0.1 ml of an a p p r o p r i a t e dilution of t h e enzyme, a n d w a t e r to 1.5 ml. After 30 rain of i n c u b a t i o n a t 37 ~ 1.5 ml of 0.1 M NaO}{ was added, a n d t h e absorbance was measured at 440 mt*. An identical m e t h o d was employed for assaying phosphodiesterase a c t i v i t y except t h a t calcium di-p-nitrop h e n y l p h o s p h a t e was used as s u b s t r a t e .

Esterification of Leucine with tRNA Acceptor a c t i v i t y was measured as described (7), using as source of a m i n o - a c y l - t R N A s y n t h e tase t h e s u p e r n a t a n t at 100,000g from b a k e r ' s y e a s t cells. Purified b a k e r ' s yeast t R N A was prep a r e d as previously reported (8).

R N a s e (1 ml, final volume); a control incubation, w i t h o u t t h e enzyme, was also performed. T h e i n c u b a t i o n was carried out in dialysis bags placed into small flasks containing 10 ml of a m m o n i u m acetate buffer and shaken at 37 ~ After 30 rain of i n c u b a t i o n t h e dialysis bags were removed and the dialyzed m a t e r i a l was dried in a flash evaporator. To remove traces of salts t h e flash e v a p o r a t i o n was repeated twice. The m a t e r i a l obtained was analyzed for t h e presence of cyclic nucleotides b y paper c h r o m a t o g r a p h y (9).

Ultracentrifuge Studies The u l t r a c e n t r i f u g e studies were carried out on a Spinco Model E a n a l y t i c a l ultracentrifuge equipped w i t h the s t a n d a r d schlieren and R a y leigh interference optical systems. T e m p e r a t u r e was controlled at 20 ~ on all runs w i t h the R T I C control u n i t . S e d i m e n t a t i o n values were determined at various protein c o n c e n t r a t i o n s for purified RNase. T h e buffer s y s t e m consisted of 20 mM p o t a s s i u m p h o s p h a t e , p i t 7.0. T h e s e d i m e n t a t i o n coefficient was calculated as described b y Schachm a n (10) and corrected to w a t e r at 20 ~. The diffusion coefficient D:0,w was calculated from t h e areas of t h e schlieren diagrams in the sedimentation experiments and corrected for radial dilution and for t h e m o v e m e n t of t h e b o u n d a r y in t h e centrifugal field according to Elias (11). The molecular weight was o b t a i n e d on t h e basis of the relationship: M = RTs/D(1 -- ~).

Polyacrylamide Gel Electrophoresis Electrophoresis in polyacrylamide gels was employed to m o n i t o r the n u m b e r a n d characteristics of c o n t a m i n a n t s t h r o u g h o u t t h e purification procedure. Electrophoresis was performed in 7.5% s t a n d a r d gels, at pH 4.5, at 4 ~ and at 3.0 m A per t u b e as described by O r n s t e i n and Davis (12). RESULTS

Enzyme Purification All the operations were carried out at 0-4 ~ unless otherwise indicated. The recove r y of p r o t e i n a n d t h e specific a c t i v i t y achieved at each step are shown in Table I. Source of RNase. H e p a t o p a n c r e a s , e x c i s e d f r o m 1 5 0 0 - 2 0 0 0 - g s p e c i m e n s of 0 . vulgaris, w a s u s e d as t h e s o u r c e of e n z y m e . T h e g l a n d s w e r e f r e e d of t h e c a p s u l e a n d p l a c e d in a freezer at --18 ~ until processed.

Identification of 2' ,3' Cyclic Phosphate Nucleotides

Steps 1 and 2: Preparation of homogenate and of acetone powder. T h e f r o z e n g l a n d s

T r a n s f e r R N A (200 OD) in 50 mM a m n m n i u m a c e t a t e was digested w i t h 12 and 24 tLg of purified

(2.2 k g ) w e r e a l l o w e d t o t h a w a n d w e r e homogenized for 2 rain in a Waring Blendor

RIBONUCLEASE FROM Octopus vulgaris

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TABLE I t)URIFICATION OF A I~IBONUCLEASE FROM HEPATOPANCREAS OF O c t o p ~ t s a Step

1 2 3 4 5 6 7 8 9

Fraction

Homogenate Acetone powder Ammonimn sulfate CM-Sephadex Heat step SephadexG-75 DEAE-Sephadex 2ndHeat step DEAE-Sephadex

Total volume (ml)

3030 2430 1270 535 20 15 13 2 5.5

Protein (g)b

89.25 58.5 19.3 1.480 0.819 0.117 0.053 0.029 0.005

Specific activity (units/mg)

Total activity (units)

Purification (-fold)

0.7 2.1 5.1 24.6 31.1 79.5 117 215 564

68,000 120,000 97,000 37,000 26,000 9,750 6,200

1 3 7.3 35.2 44.5 114.0 167 307 905

6,200 2,530

Ribonuclease activity was determined by measuring the rate of digestion of yeast RNA (see MateriMs and Methods). b Protein concentration was measured by the Lowry method (13). (4 liters capacity) with twice their weight of 10 m ~ Tris-tIC1, p H 7.8, containing 0.25 sucrose. The insoluble material was removed by centrifuging the suspension for 15 rain at 2000g. The supernatant solution was chilled to 0 ~ and 20 vol of acetone, preeooled to 0 ~ was added under constant stirring. The precipitate was washed several times with cold acetone, collected by filtration, and dried under vacuum.

Step 3: Ammonium sulfate precipitation. The acetone powder was suspended in cold 0.1M sodium acetate buffer p H 5.5 (final vol 2430 ml) and stirred for 2 hr. After centrifugation at 20,000g for 15 rain, to the supernatant fluid solid a m m o n i u m sulfate was slowly added to 35 % saturation, and the inactive precipitate was removed by centrifugation. To the supernatant fraction, solid a m m o n i u m sulfate was again added to 80 % saturation. The precipitate was allowed to stand for 2 hr and then collected by centrifugation and dissolved in 0.1 ~ acetate buffer, p H 5.5, and dialyzed against the same buffer.

each chamber. The p a t t e r n obtained in a typical experiment is shown in Fig. 1. Step 5: Heat treatment. The active fractions from all four runs of the CM-Sephadex step were pooled and dialyzed against 20 m ~ potassium phosphate, p H 6.2. Tile total volume was 535 ml and the protein concentration approximately 3 m g / m l . The enzyme preparation was divided into two aliquots, and each aliquot was heated separately at 60 ~ for 7 rain. The precipitate resulting h'om heat t r e a t m e n t was centrifuged (20,000g for 10 rain) and the supernatant was concentrated b y v a c u u m dialysis to a volume of 20 ml and a protein concentration of 40 m g / m l .

Step 6: Gel filtration on Sephadex G-75. A

Step 4: Ion-exchange chromatography on CM-Sepha~ex. After dialysis the volume was

column (2.5 X 150 era) of Sephadex G-75 equilibrated with 20 m ~ potassium phosphate, p H 6.2, was used for gel filtration of the material obtained in Step 5 (6.5 ml at a time in each of the three runs). The p a t t e r n obtained in a typical run is shown in Fig. 2. The active fractions were pooled, dialyzed against potassium phosphate buffer, 40 m~r, p H 6.8, and concentrated by v a c u u m dialysis.

1270 ml and the protein concentration approximately 15 mg/ml. A column (3 X 25 era) of CM-Sephadex C-50 equilibrated with 0.1 M N a acetate buffer, p H 5.5, was used for chromatography (300 ml at a time in each of four runs). Elution was accomplished with a linear gradient of N a acetate buffer, p H 5.5 (0.1-1.0-~.~), with 600 ml in

To the enzyme solution (15 rot) obtained from Step 6, 0.4 g of DEAE-Sephadex A-50 (previously washed and equilL brated with potassium phosphate buffer, 40 m~, p H 6.8) was added and mixed gently for 30 rain at 4% The DEAE-Sephadex was removed b y centrifugation and washed twice

Step 7: Treatment with DEAE-Sephadex.

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FIG. 1. Pattern obtained in Step 4 of the purification procedure. The material from the ammonium sulfate step (1270 ml) was adsorbed (300 ml in each run) onto a column (3 X 25 em) of CM-Sephadex equilibrated with 0.1 M Na acetate buffer, pH 5.5. Elution was accomplished with a linear gradient of Na acetate buffer pH 5.5 (0.1-1.0 M) with 600 ml in each chamber. The flow rate was 50 ml/hr; fractions of 15 ml were collected. O O, absorbance at 280 mtL of the eluate; 9 9 salt gradient; 9 9 enzymic activity (in arbitrary units). Fractions 39-52 were pooled for the next step in the purification procedure. .

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with the same buffer; the combined supern a t a n t s contained most of the R N a s e activity. Step 8: Second heat treatment. T h e e n z y m e solution f r o m the previous step (13 ml) was subjected again to heating at 65 ~ for 7 min. T h e inactive precipitate was r e m o v e d b y eentrifugation and the s u p e r n a t a n t fluid c o n c e n t r a t e d b y v a c u u m dialysis.

Step 9: Chromatography on DEAE-Sepha~ dex. T h e concentrated solution (2 ml) was applied to a D E A E - S e p h a d e x A-50 column (0.8 • 4 era) equilibrated with 4 m ~ potassium p h o s p h a t e buffer, p H 6.8, and eluted with the same buffer. T h e p a t t e r n obtained is shown in Fig. 3.

Properties of the RNase T h e purest enzyme preparations were free of phosphodiesterase (when calcium di-p-

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Fro. 2. Pattern obtained in Step 6 of the p u r l fieation procedure. The material from the heat treatment step (15 ml) was applied (6.5 ml in each run) to a column (2.5 X 150 era) of Sephadex G-75 equilibrated with 20 mu potassium phosphate, pH 6.2. The flow rate was 16 ml/hr. Each fraction contained 5 ml. O O, absorbance at 280 m~ of the effluent fractions; 9 .... 9 enzymic activity (in arbitrary units). Fractions 60-78 were pooled for the next step in the purification procedure. nitrophenyl p h o s p h a t e was used as substrate), deoxyribonuelease, and phosphomonoesterase activity. E n z y m e solutions in the range of p H 5.0-8.0 were stable in the frozen state for several months. Electrophoresis in polyacrylamide gel. T h e most purified R N a s e preparation was examined b y electrophoresis in 7.5 % polyacrylamide gel. A single distinct protein b a n d was obtained w h e n 80 ug of the e n z y m e was subjected to eleetrophoresis at p H 4.5 (Fig. 4) and 8.3. Ultracentrifuge studies. Figure 5 represents the sehlieren p a t t e r n s of the purified e n z y m e at a concentration of 4 m g / m l . T h e S2o,w extrapolated to zero protein concentration was calculated to be 3.2. The diffusion coefSeient D2o,w was calculated to be 7.5 • 10 -7 em 2 see -1 as described u n d e r Materials and M e t h o d s (10). The molecular weight for the R N a s e was calculated to be 34,200. T h e partial specific volume of the protein was assumed to have the value of 0.696

~/g.

Effect of pH on the activity of RNase. T h e purified R N a s e is active at p H between 5

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FIG. 3. Pattern obtained in Step 7 of the purification procedure. The material from the second heat treatment step was applied to a DEAESephadex A-50 column (0.8 X 4 cm) equilibrated with 4 II1Mpotassium phosphate buffer ptI 6.8 and eluted with the same buffer. The flow rate was 9 ml/hr. Fractions of 3 ml were collected. 9 9 absorbanee at 280 mtt of the effluent fr~ctions; 9 .... O, enzymic activity (in arbitrary units). Fractions 2-4 were pooled.

and 6, with maximum activity occurring at pit 5.5. Effects of metal ions on the activity of RNase. Table II shows the effect of various metal ions on RNase activity. It was found that the enzyme activity was strongly inhibited by Zn2+, Cu2+, and Fe ~+, but was not affected by Ca2+ and only partially by Mg 2+. Effect of inhibitors on the activity of RNase. The results of studies on the inhibitory effects of a number of substances tested are summarized in Table III. p-Hydroxy-mercuribenzoate (PMB) appears to be a good inhibitor of ~RNase activity. Some inhibition was also produced by iodoacetate, while no effect was observed with EDTA, mercaptoethanol, or NaF. Endonuclease activity. Figure 6 shows the rate of formation of acid-soluble products during digestion of tRNA by the enzyme. The comparison between extinction at 2t~

Fro. 4. Eleetrophoretie pattern on polyaerylamide gel of the purified I~Nase. The eleetrophoresis is from the anode to the cathode at p t l

4.5. m~ of the material soluble in perchloric a d d and the extinction of the material soluble in perehlorie acid plus uranyl acetate gives information about the chain length of the products of hydrolysis, since oligonucleotides are partially soluble in perchlorie acid, whereas only mononucleotides are soluble when uranyl acetate is added. Figure 6 shows that the A2c0 of the acid-soluble fraction decreases by about 30-40 % in the presence of 0.25% uranyl acetate. This result indicates that the products of digestion are heterogeneous with regard to chain length. This mechanism of hydrolysis involving the production of fragments of different length is peculiar to an enzyme having endonuclease activity. This mechanism is also confirmed by the rate of inactivation of tRNA T M ~eceptor activity during

332

DE LORENZO, MOLEA, A N D MOLINAlZO

FI~. 5. Sehlieren pattern obtained during velocity centrifugation of the l~Nase (4 mg/ml) at 56,100 rpm ~nd 20~ The photographs were taken at an angle of 60 ~ at 16, 32, 48, 64, and 80 min, respectively, after reaching full speed of 56,100 rpm. Sedimentation from right to left. TABLE

II

TABLE

EFFECT OF METAL IONS ON I{NASE ACTIVITY a Metal ion

Concentration (na~)

Relative(%)activity

-1 5 1 5 0.75 1.5 1.5 1.5

100 54 33 19 16 69 51 1O0 85

Inhibitor

None Zn 2+ Cu ~+ Fe ~+ Ca ~+ Mg ~+

The reaction was carried out at 30 ~ for 25 rain in the presence of 100 mlvtN a acetate (pH 5.0) and the metal ions tested in a total volume of i ml. The assay was performed as described in Materials and Methods. digestion. I n f a c t 98 % i n a c t i v a t i o n of leucine a c c e p t o r a c t i v i t y is p r o d u c e d b y 5 % d i g e s t i o n of t h e t R N A molecules. T h i s effect is c o n s i s t e n t w i t h a n e n d o n u c l e a s e a c t i v i t y of t h e e n z y m e . A n exonuclease a c t i v i t y w o u l d cause less d r a m a t i c effects b y c l e a v i n g off n u c l e o t i d e s a t t h e 5' e n d (14). Average chain length of the product. T h e f o r m a t i o n of a h e t e r o g e n o u s p o p u l a t i o n of oligonucleotides d u r i n g e n z y m i c d i g e s t i o n w a s i n v e s t i g a t e d b y c h r o m a t o g T a p h y of t h e d i g e s t on a D E A E - S e p h a d e x A-25 column, e l u t e d w i t h a linear s a l t g r a d i e n t c o n t a i n i n g 7 ~ urea. This chromatographic procedure resolves t h e oligonucleotides on t h e basis of t h e n u m b e r of p h o s p h o d i e s t e r b o n d s . T h e chloride c o n c e n t r a t i o n is used as a m e a s u r e of t h e c h a i n l e n g t h of t h e v a r i o u s oligon u c l e o t i d e s (7). F i g u r e 7 shows a chro-

III

EFFECT OF VARIOUS INHIBITORS ON RN.
None PMB Mercaptoethanol EDTA Iodoacetate N aF

Concentration (m~)

Relative activity (%)

-0.05 0.15 10 1.5 0.5 5 1.5

100 79 22 100 100 100 70 100

Assay mixtures contained 2 mg of RNA in 100 m~ N a acetate buffer (pH 5.0), an aliquot of diluted enzyme and the compound tested as indicated in a total volume of 1 ml. Incubation at 30 ~ for 25 min. The assay was performed as described in Materials and Methods. m a t o g r a p h i e p a t t e r n of t h e oligonucleotides p r o d u c e d b y d i g e s t i o n of t R N A w i t h t h e e n z y m e : oligonueleotides u p to h e x a n u c l e o tides a n d l a r g e r u n r e s o l v e d ones were observed. Specificity of hydrolysis. F i g u r e 8 shows a t i m e curve of d i g e s t i o n of s e v e r n p o l y n u c l e o t i d e h o m o p o l y m e r s . A f t e r 90 rain of i n c u b a t i o n p o l y a d e n y l i e acid was c o m p l e t e l y h y d r o l y z e d , while p o l y u r i d y l i c acid was u n a f f e c t e d a n d p o l y c y t i d y l i c acid w a s c l e a v e d to a small e x t e n t ( a b o u t 2 0 % ) . W h e n t h e i n c u b a t i o n was carl~ed o u t for longer periods, t h e specificity of h y d r o l y s i s g r a d u a l l y d e c r e a s e d a n d after o v e r n i g h t incubation both polyuridylie and polyeytidylic a c i d were h y d r o l y z e d extensively. T h e e n z y m e was also i n c u b a t e d w i t h t R N A a n d tile m o n o n u c l e o t i d e s p r o d u c e d were chro-

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FIG. 6. Time curve of hydrolysis of tRNA. The reaction mixture contained in 1 ml : purified enzyme 0.75 tLg, purified baker's yeast tRNA (8) 200 A~60 units, and 10 ~,moles of potassium acetate, pit 5.0. The incubation was performed at 30~ Two aliquots of 100 ~1 were taken at 10, 20, 40, and 90 min of incubation. To one aliquot was rapidly added 0.5 ml of cold 7% perchloric acid and to the other 0.5 ml of 7% perchloric acid containing 0.25% uranyl acetate. The absorbance of the acid-soluble material was read at 260 mr* after suitable dilution. The percentage of digestion is calculated taking as 100% the A260 of the soluble material in 7% perchlorie acid after 3 hr incubation. O----O, fraction soluble in 7% perchloric acid. 9 .... 9 fraction soluble in 7% perchloric acid containing 0.25% uranyl acetate. m a t o g r a p h e d on a Dowex 1-X8 formate column. The mononucleotides produced b y digestion under these conditions are derived from sequences of at least two bases preferentially recognized b y the enzyme. As shown in Table I V almost 90 % of the mononueleotides produced after 90 min of digestion are purine nucleotides.

Position of the phosphomonoester bonds in the products of digestion. Transfer R N A was incubated with the enzyme for 3 hr at 30 ~ The mononucleotides produced during enzymic digestion were removed from the oligonueleotides by chromatography on D E A E - S e p h a d e x column. The oligonueleotides were then hydrolyzed with alkali; this procedure yields nueleosides only if the oligonueleotides do not bear a terminal 2'- or W-phosphate group. The presence of nucleosides after alkaline hydrolysis was checked

b y c h r o m a t o g r a p h y on a DEAE-cellulose column (7). This procedure showed that only mononucleotides were present in the alkaline digest, suggesting the presence of terminal 2'- or 3'-phosphate groups. When the oligonucleotides were pretreated with phosphomonoesterase, which removes the terminal 2'- or 3'-phosphate group, alkaline hydrolysis gave rise to nucleosides from terminal positions. The hydrolysis of the R N A b y the enzyme goes via 2',3'-cyclic nucleotides. Adenosine and guanosine 2', 3'cyclic phosphate were identified b y paper c h r o m a t o g r a p h y as early p r o d u c t s of the enzymic digestion, following the procedure described in Materials and Methods. The relative amounts of the two cyclic intermediates were determined b y spectrophotometric measurements and were found to be

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Fro. 7. D e t e r m i n a t i o n of t h e l e n g t h of t h e oligonucleotides produced b y complete digestion. T r a n s f e r R N A was i n c u b a t e d for 3 hr, as described in Fig. 6. A t t h e end of digestion the p H was raised to 8.0 w i t h c o n c e n t r a t e d N a O H a n d t h e reaction m i x t u r e was i n c u b a t e d for 2 h r in the presence of alkaline p h o s p h o m o n o e s t e r a s e (0.5 u n i t s per one A~60 u n i t of t R N A ) and t h e n applied to a D E A E - S e p h a d e x A-25 column (0.7 X 50 cm) e q u i l i b r a t e d w i t h 20 m g Tris-HC1, p H 7.6, c o n t a i n i n g 7.0 M urea. E l u t i o n was accomplished w i t h a linear g r a d i e n t of 500 ml of NaC1 (0-0.3 M) in 20 m ~ Tris buffer, p H 7.6, containing 7 ~ urea. F r a c t i o n s of 10 ml were collected at a flow r a t e of 150 m l / h r and b o t h A260 and t h e chloride g r a d i e n t were determined. - - , absorbance at 260 m~ of t h e effluent fractions; ..... , salt gradient.

67 % cyclic adenylic acid and 33 % cyclic guanylic acid. DISCUSSION The RNase isolated from hepatopancreas of Octopus vulgaris and purified by the methods outlined above appears homogeneous when examined by disc-gel electrophoresis and by sedimentation analysis. The sedimentation coefficient and molecular weight have been calculated to be 3.2S and 34,000, respectively. Among the properties of the RNase, the inhibition by divalent metal ions such as Zn2+ and Cu 2+ was similar to the inhibitory effect of these cations on the known RNases (15, 16). The strong inhibitory effect of PMB might imply the presence of a sulfhydryl group(s) at the catalytic site. The Octopus RNase shows heat stability, as most RNases do (2), while its pH optimum between 5 a n d 6 is characteristic for RNases from animal-~ssues (2).

It has been shown that this enzyme is an endonuclease, since the products obtained by hydrolysis of RNA consist of several classes of oligonucleotides of different size, most of them smaller than hepta or octa nucleotides. This conclusion is supported by the differential solubility of the digestion products in perchloric acid and in perchloric acid-containing uranyl acetate and by the chromatographic pattern of cleaved tRNA on DEAE-Sephadex A-25. The rapid loss of leucine acceptor activity of tRNA as a function of digestion also supports an endonuclease mechanism of hydrolysis. In fact, an exonuclease which begins hydrolysis from the 5' end, such as spleen exonuclease, should affect the acceptor activity only when the digestion has involved about 30 % of the molecule (14). An exonuclease starting from the 3t terminus, such as venom exonuclease, rapidly inactivates the tRNA acceptor activity, but should produce nucleotide d-phosphates.

RIBONUCLEASE FROM Octopus vulgaris 100-

335 o

~ o

J

80re

o

oJ 6O

oL_

40-

0.

20-

~

0

30

-~

~

60

~

~

90

Period of incubQtion (min) FIG. 8. Time curve of digestion of polynucleotide homopolymers. The incubation was carried out in 1 ml of 0.1 M sodium acetate, pH 5, containing 2 mg of homopolymers and 0.05 mg of enzyme. Aliquots of 0.2 ml were withdrawn at several intervals of time up to 90 rain, mixed with 1 ml of a cold solution of 7% perchloric acid, and centrifuged. The absorbance of the supernatant was read at 260 mu after dilution, using as blank a zero time sample. An additional sample for each homopolymer was completely digested with alkali ~nd the absorbance at 260 mt~ of its acid-soluble fraction was considered to represent 100% digestion. The rate of digestion of polyuridilie acid, because of its solubility in acid, was followed by measuring the absorbance at 260 m~ of the supernal, ant after addition of 7% perchloric acid containing 0.25% uranyl acetate. Digestion rate of O O poly A; A .... A poly C; @----@ poly U. TABLE IV SPECIFICITY OF I~NASE FI~OM Octopus vulgaris ~ Mononucleotides produced by enzymatic digestion

Moles %

Adcnylic acid Uridylic acid Guanylic acid Cytidylic acid

50.0 7.5 37.1 5.3

Transfer RNA (600 A260 units) was incubated with 1.5 ttg of enzyme for 90 rain at 30 ~ in 1 ml of 0.1 M sodium acetate buffer, pH 5.0. The mononucleotides produced were separated by ion-exchange chromatography on a Dowex l-X8 formate column (1 X 10 cm), using the procedure described by 5Jeelon et al. (7). T h e specificity of t h e e n z y m e h a s b e e n e s t a b l i s h e d using b o t h p o l y n u c l e o t i d e h o m o p o l y m e r s a n d t R N A . I n t h e presence of b o t h s u b s t r a t e s t h e e n z y m e is h i g h l y specific

for t h e p h o s p h o d i e s t e r b o n d b e t w e e n p u r i n e nucleoside 3 ' - p h o s p h a t e a n d the 5 ' - h y d r o x y l g r o u p s of t h e a d j a c e n t nucleotides, y i e l d i n g oligonueleotides t e r m i n a t i n g in p u r i n e n u eleotides a n d m o n o n u c l e o t i d e s (preferentially adenine nueleotides). The hydrolysis p r o c e e d s t h r o u g h t h e f o r m a t i o n of cyclic 2 ' , 3 ' - p u r i n e n u e l e o t i d e s t h a t h a v e been identiffed i n the e a r l y s t a g e of digestion. U n d e r these conditions no p y r i m i d i n e cyclic n u cleotides are d e t e c t a b l e . F o l l o w i n g long p e r i o d s of i n c u b a t i o n t h e specificity of c l e a v a g e g r a d u a l l y decreases a n d p y r i m i d i n e n u e l e o t i d e s are also p r o d u c e d . W h e t h e r this i n d i c a t e s a r e s i d u a l c o n t a m i n a t i o n of t h e R N a s e i s o l a t e d in t h e p r e s e n t studies w i t h o t h e r nucleases or t h e l a c k of a b s o l u t e specificity r e m a i n s to be clarified. T h e high specificity e x h i b i t e d b y t h e

336

DE LORENZO, MOLEA, AND MOLINARO

RNase at the beginning of the digestion process can be very useful in sequencing RNA. ACKNOWLEDGMENTS We thank the Director and the staff of the Zoological Station, Naples, Italy, for the large supply of Octopuz vulgaris, used for the studies reported here. We are grateful to Dr. C. B. Anfinsen, Dr. G. L. Cantoni, a**d Prof. F. Salvatore, for the critical reading of the manuscript. P~EFEP~ENCES 1. BARNARD, E. A., Ann. Rev. Biochem. 38, 677

(1969). 2. ANFINSEN, C. B., .!kND WHITE, F. H. JR., Enzymes 5, 95 (1961). 3. }l,usmzKY, G. W., Am) SOBEa, I-I. A., J. Biol. Chem. 238, 371 (1963). 4. FARINA,B., G. Bioehim. 13, 77 (1964). 5. ANTANOGLOU, O., AND GEORGATSOS, J. G., Arch. Biochem. Biophys. 127, 813 (1968). 6. ANFINSEN, C. B., REDFIELD, R. R., CI-IOATE, W. L., PAGE, J., ANDCARROL,W . R., J. Biol. Chem. 207,201 (1954).

7. NEELON, F. A., MOLINARO,M., ISHIKIJRA,H., SHEIMER, L. B., AND CANTONI, G. L., J. Biol. Chem. 242, 4515 (1967). 8. CANTONI, G. L., AND t:~ICItARDS,]71. H., Procedures Nucl. Acid Res. p. 624 (1966). 9. M~RI~HAM,R., Methods Enzymol. 3,804 (1957). 10. SCHACHMAN, I-t K., Methods Enzymol. 4, 32 (1957). 11. ELIAS, H. G., "Ultrazentrifugen Methoden." Beckman Inst., GmbH, Munich, Germany, 1961. 12. OmNSTEIN, L., A~) DAVIS, B. J., in "Disc Etectrophoresis," preprinted by Distillation Products Industries, Rochester, New York, 1962. 13. LowRY, O. H., ROSEBROUGH, •. J., ]~ARR, A. L., AND I~:kNDALL,t~. J., d. Biol. Chem. 193, 265 (1951). 14. STULBERG, M. P., AND ISHAM, K. R-, Proc. Nat. Acad. Sci. U. S. A. 57, 1310 (1967). 15. McDoN.r M. R. Methods Enzymol. 2, 427 (1955). 16. EG,VMI, F., T:X~.AHASHL K., AND UCI~IIDA,T., Progr. Nucl. Acid Res. Mol. Biol. 3, 59

(1964).