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How much is secondary structure responsible for resistance of double-stranded RNA to pancreatic ribonuclease A?
277
Biochimica et Biophysica Acta, 5 1 8 ( 1 9 7 8 ) 2 7 7 - - 2 8 9 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 99140
HOW MUCH IS SECONDARY STRUCTURE RESPONSIBLE FOR RESISTANCE OF DOUBLE-STRANDED RNA TO PANCREATIC RIBONUCLEASE A?
MASSIMO
L I B O N A T I and M A R T A
PALMIERI
Istituto di Chimica Organica e Biologica, Facoltd di Scienze, Universitd di Napoli, Via Mezzocannone, 16, 80134 Napoli (Italy)
(Received July 28th, 1977)
Summary 1. Double-stranded f2 susll or Q/~ RNAs, resistant to bovine pancreatic RNAase A in 0.15 M NaC1/0.015 M sodium citrate (SSC), are quickly and completely degraded at 10-fold lower ionic strength (0.1 X SSC) under otherwise similar conditions. At this ionic strength the secondary structure of doublestranded RNA is maintained, as judged by the following: (a) the unchanged resistance of double-stranded RNA and DNA, under similar low ionic strength conditions, to nuclease S, from Aspergillus oryzae, in contrast with the sensitivity of the corresponding denatured nucleic acids to this enzyme, specific for single-stranded RNA and DNA; (b) the co-operative pattern of the thermaltransition profile of double-stranded RNA (with a Tm of 89°C) in 0.1 X SSC. 2. Whereas in SSC bovine seminal RNAase (RNAase BS-1) and whale pancreatic RNAase show an activity on double-stranded RNA significantly higher than that of RNAase A, in 0.1 X SSC the activity of the latter enzyme on this substrate becomes distinctly higher than that of RNAase BS-1, and similar to that of whale RNAase. 3. From these results it is deduced that the secondary structure is probably not the only nor the most important variable in determining the susceptibility of double-stranded RNA to ribonuclease. Other factors, such as the effect of ionic strength on the enzyme and/or the binding of enzyme to nucleic acids, may play an important role in the process of double-strnaded RNA degradation by ribonucleases specific for singlestranded RNA.
Abbreviation: SSC, standard saline-citrate solution, 0.15 M sodium chloride/0.015 M s o d i u m citrate, pH 7.
278 Introduction
It is well established that double-stranded RNA is highly resistant to digestion by bovine pancreatic ribonuclease A in 0.15--0.2 M NaC1, while singlestranded RNA is rapidly degraded under these conditions [1--4]. On the other hand, it is also well known that at lower salt concentrations double-stranded RNA becomes highly sensitive to RNAase A [1,3,4]. In this work evidence is presented that double-stranded RNA may be quickly and completely degraded by pancreatic RNAase A under salt conditions where the double-helical structure of the RNA is maintained, as judged by: (i) the co-operative pattern of its thermal-transition profile, and (ii) the resistance of the nucleic acid to nuclease $1 from Aspergillus oryzae, an enzyme specific for single-stranded RNA and DNA [ 5--7 ]. Materials and Methods
Substrates. Double-stranded [3H]RNA, prepared as described [8,9] from non-perpermissive Escherichia coli K-38 cells infected with f2 sus11 bacteriophage, was a generous gift of Dr. Jirf Doskocil, Czechoslovak Academy of Science, Praha. The concentration of double-stranded RNA was determined assuming an E260 value of 210 dl" g-1. cm-1 [1]. Its specific activity was approx. 5500 cpm per pg. Single-stranded [3H]RNA was obtained by heat denaturation of the double-stranded viral RNA under low salt conditions [10]. 32P-Labelled double-stranded and single-stranded Qfl RNAs were a generous gift of Dr. Tadatsugu Taniguchi and Professor Charles Weissmann, The University of Zurich, Switzerland. Double-stranded [14C]DNA was prepared as described [11,12], with minor modifications, from E. coli K-12 cells, with [~4C]thymidine (spec. act. 62 Ci/mol; The Radiochemical Centre, Amersham) as a radioactive precursor. Purified DNA had a spec. act. of 1800 cpm/pg. Radioactive single-stranded DNA was obtained by heat denaturation of native E. coli DNA in 0.007 M NaC1, pH 7. 3H-Labelled poly(A) and poly(C) and the corresponding non-radioactive homopolymers were purchased from Miles. They were mixed to obtain a solution of 3.2 pg/ml, with a spec. act. of approx. 1300 cpm/ gg. Cytidine 2',3'-cyclic phosphate was purchased from Sigma Chemical Co. Enzymes. Bovine pancreatic RNAase (type XII-A) was purchased from Sigma. Bovine seminal ribonuclease (RNAase BS-1) was purified as described [13]. Its specific activity was 46 Kunitz units [14] per mg. Pancreatic RNAase from the lesser rorqual or pike-whale, Balaenoptera acutorostrata, purified as described [15,16], was a kind gift of Dr. Jaap J. Beintema, The University of Groningen, The Netherlands. Nuclease S~ was purified from crude a-amylase powder (catalog No. A 6630) from Aspergillus oryzae, purchased from Sigma, according to the procedure outlined by Vogt [ 7]. However, the chromatography on DEAE-cellulose was repeated twice, without going through the SulfoSephadex and Sephadex G-100 steps. The purified enzyme was completely inactive towards double-stranded DNA under the assay conditions outlined by
Vogt [7]. Methods. Incubations with bovine RNAase A and RNAase BS-1 and with whale pancreatic RNAase were carried out in SSC or in various dilutions of it,
279 usually at pH 7. When necessary, the pH was adjusted to 5 with small amounts of 0.1 M HC1. E n z y m e activity was determined as described [10,12], by measuring the acid-insoluble radioactivity retained on Millipore membranes (HAWP00010, Millipore Filter Co.) after precipitating samples with trichloroacetic acid (final concentration 6%) at 0°C, and filtering and washing with chilled trichloroacetic acid. Details are given in the legends to the figures and to Table II. Assays with nuclease $1 were performed essentially according to Vogt [7], at 37 or 45°C, using radioactive DNA or RNA as substrates. In the case of RNA concentrations of nuclease $1 7--10 times higher than with DNA were used, because of the correspondingly lower activity of the enzyme on RNA [ 7]. Moreover, in the control assays under low salt concentrations (see Table I, buffer B), with either denatured DNA or RNA as substrate, the concentration of $1 nuclease was doubled since 0.1 M NaC1 is r e c o m m e n d e d [7] for optimal enzymic activity. Enzyme activity was measured by determining the acidinsoluble radioactivity retained on Millipore filters as described above for the ribonuclease assays. Details are given in the legend to Table I. Activity on p o l y ( A ) a n d poly(C) was also assayed as described above, by measuring the acid-insoluble radioactivity retained on Millipore filters. Specific activity of RNAase A with poly(A) as substrate is given as pg of polymer degraded/rain per mg protein, at 37 ° C. E n z y m e assays with cytidine 2' : 3'-cyclic phosphate as a substrate were performed essentially as described [13,17]. Details are given in the legend to Table III. All assays were performed in duplicate. Protein concentration was determined either spectrophotometrically (for RNAase A and RNAase BS-1) as described [18], or by the procedure of Lowry et al. [19]. Thermal-transition profiles of double-stranded RNA were determined at 260 nm using stoppered cuvettes (type 29, Starna Ltd., London), with a Zeiss PM QII spectrophotometer equipped with a thermostatically controlled water bath. Results
Susceptibility o f double-stranded R N A to R N A a s e A at different salt concentrations Fig. l a shows the results of an incubation of double-stranded RNA with 16 pg of pancreatic RNAase A per ml at 25°C under various ionic strength conditions. The resistance of the nucleic acid to the action of the enzyme is high in SSC, but decreases dramatically already by halving the SSC concentration, becomining almost nil when incubation is carried out in 10-fold diluted SSC. In particular (Fig. lb), under these conditions, at 20°C, only 30 and 18% of the RNA remains acid-precipitable after 40 and 90 s of incubation respectively, with as little as 0.94/~g of RNAase A per ml. When single-stranded RNA is used as a substrate, the rate at which the nucleic acid is degraded by RNAase A in 10-fold diluted SSC is only about twice that measured in standard SSC (Figs. 4 and 6). Moreover, the activities of
280
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Fig. 1. A c t i o n o f p a n c r e a t i c R N A a s e A o n d o u b l e - s t r a n d e d R N A u n d e r v a r i o u s i o n i c s t r e n g t h c o n d i t i o n s , a. A s s a y m i x t u r e s o f 0 . 5 m l c o n t a i n e d 8.1 /~g o f R N A a s e A a n d 0 . 3 6 # g ( a p p r o x . 2 0 0 0 c p m ) o f d o u b l e - s t r a n d e d [ 3 H ] R N A d i s s o l v e d i n SSC ( A ) , 2 - f o l d d i l u t e d SSC (B), 5 - f o l d d i l u t e d SSC (C), a n d 1 0 - f o l d d i l u t e d SSC (D). T e m p e r a t t L r e , 2 5 ° C . b . S a l t c o n d i t i o n s as d e s c r i b e d u n d e r D a b o v e . R N A a s e A c o n c e n t r a t i o n , 0 . 9 4 / ~ g p e r m L T e m p e r a t u r e , 2 0 ° C . In a a n d b i n c u b a t i o n s w e r e s t o p p e d b y a d j u s t i n g , w h e r e n e c e s s a r y , t h e salt c o n c e n t r a t i o n t o S S C , a n d a d d i n g a t 0°C t r i c h l o r o a c e t i c a c i d (final c o n c e n t r a t i o n 6%). P r e c i p i t a t e s w e r e f i l t e r e d t h r o u g h M i l l i p o r e m e m b r a n e s (see M a t e r i a l s a n d M e t h o d s ) , a n d r a d i o a c t i v i t y w a s c o u n t e d as d e s c r i b e d [ 1 0 , 1 2 ] .
the enzyme on double- and single-stranded R N A in 0.1 X SSC are essentially of the same order of magnitude (Table II). The findings concerning double-stranded RNA, which are n o t novel [1,3,4], could be attributed to partial denaturation of the R N A double helix at low ionic strength. However, with the experiments described below we offer evidence that the different susceptibility of double-stranded R N A to enzymic attack at high and low ionic strength is probably n o t due to a change in its secondary structure. Thermal-transition profile o f double-stranded R N A at low ionic strength The pattern of the thermal denaturation of double-stranded f2 sus11 R N A under low salt conditions is shown in Fig. 2. Absorbancy increase as a function of temperature in 0.1 X SSC starts at approx. 73.5°C, following a typical cooperative pattern, with a Tm value around 89 ° C. The hyperchromicity is 34%, a value similar to that f o u n d in SSC (31%), where the absorbancy increase starts at approx. 87°C. At r o o m temperature the absolute absorbance of double-stranded f2 sus11 R N A is the same in SSC and in 0.1 X SSC. These data indicate that there is no substantial difference in the degree of secondary structure o f f2 sus11 R N A in SSC or in 0.1 × SSC. The Tm value determined for double-helical f2 sus11 R N A in 0.1 X SSC is similar to the corresponding value reported for double-stranded MS2 RNA, the Tm values of which are 103°C in SSC, 87°C in 0.1 X SSC, and approx. 80°C in 0.01 × SSC [1]. The difference between these Tra values is certainly n o t large. Therefore, it seems that, at least at the temperature of the RNAase assay (20--25°C), the stability of the secondary structure o f double-stranded R N A should not be very different whether it is dissolved in SSC, where it is highly
281 resistant to RNAase A digestion, or in 0.1 X SSC, where it is highly sensitive to the enzyme.
Action of nuclease Sl from Aspergillus oryzae on double-stranded RNA at different ionic strengths The following experiments were devised to ascertain whether the ionic strength-dependent susceptibility of double-stranded RNA to enzymic attack might occur also with a different nuclease, thus indicating it could be a more general phenomenon, or whether it is characteristic for digestion by pancreatic RNAase A. The results (Table I) show that nuclease $1 is almost completely inactive toward native double-stranded RNA and DNA under high (buffer A) and low (buffer B) salt conditions, similar to those used in the experiments with pancreatic RNAase A. Moreover, the resistance of double-stranded RNA remains unchanged under the significantly lower ionic strength conditions of buffer C. On the other hand, under the same salt conditions, where native double-stranded RNA or DNA were resistant, the corresponding denatured (i.e., single-stranded) nucleic acids were highly sensitive to the $1 nuclease (Table I). Thus, the secondary structure of double~tranded RNA at low TABLE I ACTION OF NUCLEASE S 1 FROM ASPERGILLUS ORYZAE ON NATIVE OR DENATURED BLE-STRANDED RNA AND DNA UNDER VARIOUS CONDITIONS
DOU-
B u f f e r A : 0 . 0 5 M s o d i u m a c e t a t e , p H 5 / 0 . 1 2 M s o d i u m c h l o r i d e / 0 . 0 0 1 3 M Z n S O 4. B u f f e r B: 0 . 0 1 5 M s o d i u m a c e t a t e , p H 5 / 0 . 0 0 1 3 M Z n S O 4. B u f f e r C: 0 . 0 0 5 M s o d i u m a c e t a t e , p H 5 / 0 . 0 0 1 3 M Z n S O 4. Spec i f i c a c t i v i t i e s o f t h e r a d i o a c t i v e n u c l e i c acids: d o u b l e - s t r a n d e d [ 3 H ] R N A , 5 5 0 0 c p m / / ~ g ; [ 1 4 C ] D N A , 1 8 0 0 c p m / p g . I n c u b a t i o n s w e r e carried o u t in 1 m l f o r 30 r a i n at 37°C. T u b e s w e r e t h e n t r a n s f e r e d in a n i c e - b a t h , salt c o n c e n t r a t i o n w a s adjusted t o 0 . 1 5 M N a C I , a n d 1 / 1 0 vol. o f c o l d 6 0 % t r i c h l o r o a c e t i c a c i d w a s added. A f t e r 1 0 - - 3 0 rain a t 0°C, p r e c i p i t a t e s w e r e filtered t h r o u g h Millipore m e m b r a n e s and w a s h e d 5 - 6 t i m e s w i t h chilled 6% t r i c h l o r o a c e t i c acid. A c i d - i n s o l u b l e r a d i o a c t i v i t y r e t a i n e d o n filters w a s c o u n t e d as d e s c r i b e d u n d e r Materials and M e t h o d s . B a c k ~ o u n d values have b e e n s u b t r a c t e d f r o m figuzes appearing in t h e t a b l e .
Substrate
Buffer A
Buffer B
cpm a. D o u b l e - s t r a n d e d R N A 1. N o e n z y m e 2. 7 # g S1 3.10 #g S 1
3805 . 3706
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100 .
. 97.4
Buffer C
cpm
%
cpm
%
3461 . 3288
100
736 699 --
100 95 --
680 . 0
100
95
b. D e n a t t t r e d d o u b l e - s t r a n d e d R N A 1. N o e n z y m e 2221 2. 10 # g S 1 534 3.20 ~g S 1 --
100 24 --
2610 -55
100 -2.1
c. D o u b l e - s t r a n d e d D N A 1. N o e n z y m e 2. 1 ~ g S 1 3. 10 /~g S 1
100 99.2 99.7
1097 1012 --
100 92.2 --
100 10.6
693 . 9
1048 1040 1045
d. D e n a t u r e d d o u b l e - s t r a n d e d D N A 1. N o e n z y m e 669 2. 1 ~zg S 1 71 3 . 2 pg S 1 --
--
100 .
. 1.3
0
282
salt concentrations is retained, at least to the extent o f conferring resistance to an enzyme such as nuclease $1, which is absolutely specific for singlestranded R N A or D N A [ 5--7 ]. The main variable between the experiments with RNAase A and nuclease $1 being the different pH of the incubation mixtures (optimal activity of S1 nuclease is at acidic pH) and the presence of ZnSO4, an experiment was performed to check any possible influence of the acidic pH and of ZnSO4 on the ribonuclease reaction. Double-stranded R N A was incubated with RNAase A in the same buffers used in the experiments with nuclease $1 (buffers A and B of Table I). At pH 5, 75 and 92% of the double-stranded R N A were degraded after incubation for 25 min at 25°C with 16 #g of RNAase A/ml under the high and low salt conditions, respectively. Moreover, a sensitivity of doublestranded RNA to RNAase A significantly higher than at pH 7 has been also observed in SSC at pH 5.
Susceptibility of double- and single-stranded RNA to RNAase A, RNAase BS-1 and whale pancreatic RNAase under different salt conditions It has been established that bovine seminal ribonuclease (RNAase BS-1), a
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Fig. 2. Thermal-transition profile o f d o u b l e - s t r a n d e d f2 s u s l 1 R N A . C o n c e n t r a t i o n o f d o u b l e - s t r a n d e d R N A , 1 8 . 4 / ~ g per m l in 0.1 X SSC. A b s o r b a n c e at 2 6 0 n m w a s d e t e r m i n e d as described u n d e r Materials and M e t h o d s . Fig. 3. C o m p a r i s o n o f R N A a s e A and R N A a s e BS-1 activities o n d o u b l e - s t r a n d e d R N A at different ionic strengths. C o n d i t i o n s : a, i n c u b a t i o n in SSC; d o u b l e - s t r a n d e d [ 3 H ] R N A ( e , o). 1 . 3 4 / ~ g / m l , or d o u b l e stranded [ 3 2 p ] R N A ( A A), 5 . 6 / ~ g / m l ( a p p r o x . 18 1 0 0 cpm//~g). R N A a s e A ( a e) or R N A a s e BS-1 ( A o), 1 8 . 5 #g/m1, or 2 7 . 3 / ~ g / m l w i t h the 3H-labelled or t h e 32p-labelled substrate, respectively, b. I n c u b a t i o n in 0.1 X SSC; d o u b l e - s t r a n d e d [ 3 H ] R N A ( e , o ) , 1 . 3 4 / ~ g / m l , or double-stranded [ 3 2 p ] R N A ( a z~). 2 . 8 5 #g/m1 a p p r o x . 1 6 4 0 0 cpm#~g). R N A a s e A ( A e) or R N A a s e BS-1 (o, A), 1.85/~g/rnl o r 2 . 7 3 / ~ g / m l w i t h the 3H-labelled or the 32p-labelled substrate, respectively. A s s a y m i x t u r e s v o l u m e s , 0 . 5 m l w i t h d o u b l e - s t r a n d e d [ 3 H ] R N A or 0 . 2 m l w i t h d o u b l e - s t r a n d e d [ 3 2 p ] R N A . T e m p e r a t u r e , 20°C. A t the t i m e s indicated t h e r e a c t i o n w a s s t o p p e d and s a m p l e s w e r e treated as described under Fig. 1.
283 well characterized, dimeric enzyme [13,17,20], and whale pancreatic RNAase [15,16], both of which are proteins more basic than RNAase A, are definitely more efficient than the latter enzyme at degrading double-stranded polyribonucleotides in SSC [18]. Figs. 3a and b show the results of experiments carried o u t in SSC and in 0.1 × SSC, respectively, with RNAase A and RNAase BS-1, using doublestranded RNA as a substrate. It is quite clear that while at the higher salt concentration RNAase A is less active than RNAase BS-1 (Fig. 3a), the opposite is true in 0.1 X 88C (Fig. 3b). Actually, as shown in Table II, where a comparison on a quantitative basis is possible, the activity of both enzymes towards double-stranded RNA is increased at low ionic strength, but, whereas RNAase A in 0.1 × 8SC becomes more than 2000 times as active as in SSC, the activity of RNAase BS-1 at low salt concentration is only 80 times higher than in 88C. This accounts for the reversal in the relative activities of bovine RNAase A and RNAase BS-1. Fig. 4 shows the effect of a 10-fold difference in ionic strength on the susceptibility of single-stranded Qfl RNA to bovine RNAase A and RNAase BS-1. Whereas the difference in salt concentration slightly affects the action of RNAase A, which at low ionic strength is a b o u t twice as active as in 88C, it has no influence on the action of RNAase B8-1 (see also Table II). The behaviour of whale pancreatic RNAase is shown in Fig. 5 and in Table II. Whereas, as already reported [18], the enzyme is significantly more efficient than RNAase A (and RNAase BS-1) at degrading double-stranded RNA in SSC, its action appears only a b o u t twice that of RNAase A in 0.1 × SSC (Figs. 5a and b). Under these conditions, in fact, the increase in activity of whale RNAase is lower than that of bovine RNAase A, the ratio of whale RNAase activities in 0.1 × 8SC and SSC being a b o u t 5 times lower than that of the corresponding RNAase A activities (Table II). If single-stranded R N A is 100
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Fig. 4. R N A a s e A a n d R N A a s e B S - I activities o n single-stranded R N A u n d e r d i f f e r e n t salt c o n d i t i o n s . Single-stranded Q~ R N A l a b e l l e d w i t h 3 2 p , 5 . 3 / ~ g / m l ( a p p r o x . 1 2 4 0 0 c p m / # g ) . R N A a s e A or R N A a s e BS-I, 2.73 ~g/per ml. o o or • ~, R N A a s e A a c t i v i t y in SSC or in 0 . I X SSC, r e s p e c t i v e l y . ~ or • A R N A a s e BS-1 a c t i v i t y in SSC or in 0.1 X SSC, r e s p e c t i v e l y . A s s a y m i x t u r e s v o l u m e , 0 . 2 ml. T e m p e r a t u r e , 2 0 ° C . A t t h e t i m e s i n d i c a t e d t h e r e a c t i o n w a s s t o p p e d a n d s a m p l e s w e r e t r e a t e d as d e s c r i b e d u n d e r Fig. 1.
RNA UNDER DIFFERENT
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A B B
R N A a s e BS-1
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1968 3093 4590
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A c t i v i t y in Activity in 0.1 × SSC
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D o u b l e - s t x a n d e d R N A as s u b s t r a t e
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1.00 0.95 1.40
1.97 1.96 2.23
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E n z y m e a c t i v i t y is g i v e n as /~g o f R N A d e g r a d e d / m i n p e r m g p r o t e i n , a t 2 0 ° C . E x p t . A: d o u b l e - s t r a n d e d [ 3 2 p ] R N A as s u b s t r a t e . C o n d i t i o n s d e s c r i b e d u n d e r F i g s . 3 a a n d b . E x p t . B: d o u b l e - s t r a n d e d [ 3 H ] R N A , 4 . 3 /~g/ml; R N A a s e A o r R N A a s e B S - 1 , 2 0 /~g/ml; w h a l e R N A a s e , 1 4 . 8 ~ g / m l . A s s a y m i x t u z e s , 0 . 5 m l ; t e m p e r a t u r e , 2 0 ° C . E x p t . C: d o u b l e - s t m a n d e d [ 3 H ] R N A , 1 ~ g ~ n l ; R N A a s e A o r R N A a s e B S - I , 1 . 8 6 ~ g / m I ; w h a l e R N A a s e , 0 . 6 /~g/m]. A s s a y m i x t u r e s , 0 . 5 m l ; t e m p e r a t u r e , 2 0 ° C . E x p t . D: i n S S C o r i n 0 . 1 X S S C , s i n g l e - s t r a n d e d [ 3 2 p ] R N A , 3 . 1 5 /~g/ml; R N A a s e A o r R N A a s e B S - 1 , 2 . 6 5 /~g/ml. A s s a y m i x t u r e s , 0 . 2 m l ; t e m p e r a t u r e , 2 0 ° C . E x p t . E: c o n d i t i o n s d e s c r i b e d u n d e r Fig. 4. E x p t . F: c o n d i t i o n s d e s c r i b e d u n d e r Fig. 6.
C O M P A R I S O N O F R N A a s e A , R N A a s c BS-1 A N D W H A L E R N A a s e A C T I V I T I E S O N D O U B L E - A N D S I N G L E - S T R A N D E D STRENGTH CONDITIONS
T A B L E II
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Fig. 5. Comparison of R N A a s e A and whale R N A a s e activitieson double-stranded R N A at different ionic strengths, a. In SSC, double-stranded [ 3 H ] R N A , 2.6 #g/ml. R N A a s e A (e), 5.47 #g/ml, whale R N A a s e ( o ) , 4 . 7 5 p g / m l . A s s a y m i x t u r e s v o l u m e , 0 . 2 m l . T e m p e r a t u r e , 2 0 ° C . b . In 0.1 X SSC0 d o u b l e ~ t r a n d e d [ 3 H ] R N A , 0 . 5 2 / ~ g / m l . R N A a s e A ( e ) or w h a l e R N A a s e ( o ) , 0 . I # g / m l . A s s a y m i x t u r e s v o l u m e , 1 m l . T e m p e r a t u r e , 2 0 ° C . A t t h e t i m e s i n d i c a t e d t h e r e a c t i o n w a s s t o p p e d a n d s a m p l e s w e r e t r e a t e d as d e s c r i b e d u n d e r Fig. 1.
used as substrate, both whale and ox pancreatic RNAases show a slight, and essentially similar, increase in their activity at the lower salt concentration, as compared with that measured in SSC (Fig. 6 and Table II).
Action of RNAase A, RNAase BS-1 and whale RNAase on cytidine 2',3'-cyclic phosphate at different ionic strength The effect of different salt concentrations on the action of bovine RNAase 100
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1.5
mif*,.
Fig. 6. R N A a s e A a n d w h a l e R N A a s e a c t i v i t i e s t o w a r d s i n g l e - s t r a n d e d R N A a t d i f f e r e n t i o n i c s t r e n g t h s . S i n g l e - s t r a n d e d Q~ R N A , l a b e l l e d w i t h 3 2 p , 5 . 3 # g / m l ( a p p r o x . 1 0 7 0 0 ¢ p m / ~ g ) . R N A a r e A o r w h a l e RNAase, 1.5 #g/m L o o or ¢ ~-, R N A a s e A a c t i v i t i e s i n S S C o r in 0 . 1 X SSC, r e s p e c t i v e l y . A ~ or • • , w h a l e R N A a s e a c t i v i t i e s i n S S C o r i n 0 . I X SSC, r e s p e c t i v e l y . A s s a y m i x t u r e s volume, 0.2 ml. Temperature, 20°C. At the times indicated the reaction was stopped, and samples were t r e a t e d as d e s c r i b e d u n d e r F i g . 1.
286 TABLE III A C T I O N O F R N A a s e A , R N A a s e BS-1 A N D W H A L E P A N C R E A T I C R N A a s e O N C Y T I D I N E 2' : 3 ' - C Y CLIC PHOSPHATE UNDER DIFFERENT IONIC STRENGTH CONDITIONS T o 1 m l of 0.2 m M c y t i d i n e 2 ' : 3 ' - c y c l i c p h o s p h a t e dissolved in 0.01 M imidazole-HC1 b u f f e r c o n t a i n i n g v a r i o u s c o n c e n t r a t i o n s o f NaCl, 0 . 9 - - 1 . 8 p g of e n z y m e w e r e added, and change in a b s o r b a n c e at 2 8 7 n m w a s r e c o r d e d w i t h a C a r y m o d e l 1 1 8 s p e c t r o p h o t o m e t e r , a t 24°C. Specific activities are e x p r e s s e d as change in a b s o r b a n c e / m i n p e r m g p r o t e i n . Imidazole-HC1 buffer, pH 6.92
Salt c o n d i t i o n s (M NaCD
Specific a c t i v i t i e s o f RNAase A
R N A a s e BS-1
(M) 0.01 0.01 0.01 0.01
Whale
RNAase 0 0.05 0.10 0.15
3,50 2.73 2.28 2.06
3.42 3.78 3.85 3.15
1.28 1.13 0.95 0.84
A and RNAase BS-1, and of whale pancreatic RNAase toward cytidine 2',3'cyclic phosphate is shown in Table I I I . The results of this experiment essentially agree with those obtairied using single-stranded RNA as a substrate. While in the case of RNAase BS-1 the activity values may be considered as randomly scattered around an average specific activity of 3.5 the activity of RNAase A, and, to a lower extent, t h a t of whale RNAase regularly decrease by increasing the ionic strength of the assay mixture. Discussion Pancreatic RNAase A has a modest activity on double-stranded RNA under high salt conditions, the remarkable resistance of this species o f RNA to the enzyme being usually attributed to the presence and the maintainance of the secondary structure in the nucleic acid. Other well characterized ribonucleases, such as, for instance, seminal RNAase BS-1 and whale pancreatic RNAase, are definitely more efficient than RNAase A at degrading double-helical RNA under identical ionic strength conditions. To explain the activity on double-stranded polyribonucleotides by ribonucleases specific for single-stranded RNA, we proposed, as a tentative mechanism of action, a destabilization of the RNA double helix, similar to t h a t shown on DNA by RNAase A [21] or, more efficiently, by RNAase BS-1 [22], resulting from the interaction of a ribonuclease molecule with double-stranded RNA. This destabilization would then allow the enzymic attack on the nucleic acid [10,18,23]. On the other hand it seems improbable t h a t a RNA double helix can fit the active site of a ribonuclease w i t h o u t a simultaneous unwinding of the double-stranded nucleic acid to yield hydrolyzable single-stranded segments. The efficiency of this labilizing activity was correlated with the number, or the density, of basic charges present on the enzyme molecule [18]. In fact, bovine RNAase BS-I, whale RNAase and dimers or higher aggregates of RNAase A, enzyme species distinctly more efficient than monomeric RNAase A at
287 degrading double-stranded RNA in SSC [12,18,24,25], are proteins more basic (or with a higher density of basic charges) than the bovine pancreatic enzyme. From the experiments presented in this work it appears that in 0.1 × SSC double-stranded RNA is degraded by RNAase A, RNAase BS-1 and whale RNAase at a much higher rate than in SSC. However, the activity of RNAase A increases dramatically in comparison with that of the other two enzymes, becoming higher than the activity of RNAase BS-1 and close to that of whale RNAase. On the other hand, in 0.1 X SSC the secondary structure of doublestranded RNA is maintained, as judged by the absorbance, the thermal-transition profile and the unchanged resistance of the nucleic acid to nuclease $1 under these salt conditions. Therefore, the data obtained at low ionic strength on one hand indicate that the secondary structure of the RNA may not be the most important variable in the process of enzymic degradation of doublestranded RNA; on the other hand, they seem to contradict the correlation between enzyme basicity and activity on the RNA double helix (with or without previous destablization), observed at high salt concentration [18]. Why is the pattern of double-stranded RNA degradation by the three enzymes not maintained at low ionic strength? While a trivial explanation of the results obtained, i.e., a uniform inhibition of all RNAase activities by salt, may be ruled out by the experiments performed in parallel on double- and single,stranded RNA, the data presented above suggest that other factors may play a role in the phenomena observed. For instance, the ionic strength, the importance of which for RNAase A has been already shown by Kalnitsky et al. [26], could differently affect different enzyme proteins, their interaction with the double-stranded nucleic acid, and therefore their turnover numbers. By examining the data of Table II it appears that the activity of RNAase A on double-stranded RNA in 0.1 X SSC is close to that on single-stranded RNA determined under identical salt conditions, the activity on the latter substrate being only about twice that on double-stranded RNA. The same is true with whale RNAase. The activity of this enzyme toward single-stranded RNA in 0.1 X SSC is also about twice that measured on double-stranded RNA at the same ionic strength. In other words, the fact that RNA is in double- or singlestranded form makes no great difference for the action of RNAase A and whale RNAase, provided the ionic strength is low. RNAase BS-1, however, in 0.1 X SSC is about 9 times less active on double-stranded than on single-stranded RNA. Moreover, the rate at which this enzyme degrades single-stranded RNA appears unchanged at high or low ionic strength (see also Figs. 4 and 6). These facts could be probably ascribed to a different salt effect on the tertiary structure of the various proteins considered. Whereas whale RNAase is a monomeric protein, homologous to RNAase A [16,27], RNAase BS-1 is a dimer, containing two identical 124-amino acid peptide chains (homologous to RNAase A [28]) linked through two interchain disulfide bridges [29,30]. Both whale and BS-1 RNAases have a higher number of basic amino acids than RNAase A [16,28]. It might be reasonable to assume that the conformation of different enzyme proteins, which can be influenced by the number of charges present on their surface and/or by their quaternary structure, could be dif-
288 ferently affected b y changes in ionic strength conditions. On the other hand, the results presented in this work are reminiscent of what was observed with poly(A) as a substrate, although this polymer cannot be considered a "normal" substrate for a ribonuclease. Polyadenylic acid was also more efficiently degraded b y RNAase BS-1 [31] and dimeric RNAase A [10] than by monomeric RNAase A under high salt conditions, whereas the opposite occurred (and with much smaller amounts of enzyme) under low salt conditions. This behaviour can be observed both at pH 5 and 7. Moreover, at pH 5 RNAase A is distinctly more active on poly(A) in 0.1 × SSC than in standard SSC, the corresponding specific activity values being 20.2 and 1.2, respectively. Similar results have been obtained with poly(C) as substrate (Sorrentino, Palmieri and Libonati, unpublished results). Since at pH 5 poly(A) and poly(C) have a well-characterized secondary structure which is stabilized by low salt concentrations [32], the presence of the secondary structure in these polymers does not seem to be an important variable in their susceptibility to the action of RNAase A, in contrast with what was originally thought for poly(A) [10]. In conclusion, provided the ionic strength is 0.1 that of SSC, always the same effect by RNAase A is observed, i.e., a more efficient degradation of a double-stranded polyribonucleotide, either double-helical RNA, which is less stable, or the acidic double-stranded forms of poly(A) and poly(C), which are more stable at low salt concentrations. These arguments could strengthen the possibility that the secondary structure of a polyribonucleotide, although being a key factor, is n o t the only one in determining the resistance of double-stranded R N A to pancreatic RNAase A. An advancement in the understanding o f the facts presented could be probably achieved only by using different experimental approaches, as, for instance, NMR analyses of the various ribonucleases considered under different conditions. Moreover, the results presented might also be considered and further studied in the light of the hypothesis of the catalytic implications of electrostatic potentials, advanced by D o u z o u and Maurel [33,34]. Finally, it is probably worth considering that physiological ionic strength values in vivo are close to that of SSC. Therefore, the significance of the various ribonucleases reported here and elsewhere [35] to be also active on doublestranded RNA, and the physiological implications of this action, should be referred to enzyme activities studied at an ionic strength similar to that of SSC. Acknowledgements We are very grateful to Professor Stanford Moore, Professor Charles Weissmann and Dr. Glynn Wilson for important suggestions and criticism. References 1 Billeter, M.A., Weissmann, C. and Warner, R.C. (1966) J. Mol. Biol. 17, 145--173 9 Weissmann, C., Billeter, M.A., Vinuela~ E. and Libonati, M. (1966) in Viruses of Plants (Beemster, A.B.R. and Dijkstra, J., ed.), pp. 249--274, North-Holland Publ. Co., A m s t e r d a m
289 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
Iqbal, Z.M. and K o h n , K.W. (1975) Arch. Biochem. Biophys. 166, 5 1 8 - 5 2 5 Edy, V.G., Szekely, M., Loving, T. and Dreyer, C. (1976) Ettr. J. Biochem. 6 1 , 5 6 3 - 5 7 2 A n d o , T. (1966) Biochim. Biophys. Aeta 114, 158--168 Sutton, W.D. (1971) Biochim. Biophys. Acta 240, 5 2 2 - 5 3 1 Vogt, V.M. (1973) Eur. J. Bioehem. 33, 1 9 2 - - 2 0 0 Paces, V., Doskocfl, J. and Sorm, F. (1968) Biochtm. hiophys. Aeta 1 6 1 , 3 5 2 - - 3 6 0 D'Aless~o, G., Doskocfl, J. and Libonat], M. (1974) Biochem. J. 1 4 1 , 3 1 7 - - 3 2 0 Palmieri, M. and Libonati, M. (1977) Bioehtm. Biophys. Acta 474, 456--466 Habieh, A., W e ~ m n , C., IAbonati~ M. and Warner, R.C. (1966) J. Mol. Biol. 21, 255--264 Libonati, M. and Florldi, A. (1969) Ettr. J. Biochem. 8, 61--87 D'Alessio, G., Floridi, A., De Prisco, R , Plgnero, A. and Leone, E. (1972) Eur. J. Biochem. 26, 1 5 3 - 161 Kunitz, M. (1946) J. Biol. Chem. 164, 563--566 Wierenga, R.K., Huizinga, J.D., Gaastra, W., Welling, G.W. and Beintema, J j . (1973) FEBS Lett. 31, 181--185 E m m e n s , M., Wenln~, G.W. and Beintema, J j . (1976) Bioehem. J. 157, 317--323 Florldi, A., D'Alessio, G. and Leone, E. (1972) Eur. J. Biochem. 26, 162--167 Lihonat], M., Furia, A. and Beintema, J J . (1976) Eur. J. Bioehem. 69, 445--451 Lowry, O.H., Rosehrough, N.J., Farr, A.L. and Randall, R J . (1951) J. Biol. Chem. 193, 265--275 D'Alessto, G., Parch,e, A., Guida, C. and Lenone, E. (1972) FEBS Let,. 27, 285--268 Felsenfeld, G., Sandeen, G. and von Hippel, P.H. (1963) Proe. Nat]. Acad. ScL U.S. 50, 644--651 IAbonati, M. and Beintema, J~l. (1977) Biochem. Soc. Trans. 5, 470--474 Libonat], M. Sorrentino, S., Galli, R., La Montagnal R. and Di Donato, A. (1975) Biochtm. Biophys. Acta 4 0 7 , 2 9 2 - - 2 9 8 Libonati, M. (1971) Biochim. Biophys. Aeta 2 2 8 , 4 4 0 - - 4 4 5 Wang, D., Wilson, G. and Moore, S. (1976) Biochemistry 15, 660----665 Kalnitsky, G., H u m m e l , J.P. and Dierks, C. (1959) J. Biol. Chem. 234, 1 5 1 2 - - 1 5 1 6 Beintcma, J.J., Graastra, W., Lenstra, J.A., Welling, G.W. and Fitch, W.M. (1977) J. Mol. Evol. 10, 49--71 Barker, W.C. and Dayhoff, M.O. (1976) in Atlas of Protein Sequence and Structure (Dayhoff, M.O., ed.), Vol. 5, Suppl. 2, pp. 77--103, National Biomedical Research F o u n d a t i o n , Washington, D.C. Di Donato, A. and D'Alessio, G. (1973) Biochem. Biophys. Res. C o m m u n . 55, 919--928 D'Alesslo, G., Malorni, M.C. and Parente, A. (1975) Biochemistry, 14, 1 1 1 6 - - 1 1 2 2 Pahnierl, M. and Libonati, M. (1976) Boll. Soc. It. Biol. Sper, 52, 80--84 Fekenfeld, G. and Miles, H.T. (1967) A n n u . Rev. Biochem. 36, 407--446 D o u z o u , P. and Maurel, P. (1977) Proc. Natl. Acad. Sci. U.S. 74, 1013--1015 D o u z o u , P. a n d Maurel, P. (1977) Trends Bioehem. Sei. 2, 14--17 BaxdSn, A., Sierekowska, H. and Shugar, D. (1976) Bio~hlm~ Biophys. Acta 438, 461--473
Biochimica et Biophysica Acta, 5 1 8 ( 1 9 7 8 ) 2 7 7 - - 2 8 9 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
BBA 99140
HOW MUCH IS SECONDARY STRUCTURE RESPONSIBLE FOR RESISTANCE OF DOUBLE-STRANDED RNA TO PANCREATIC RIBONUCLEASE A?
MASSIMO
L I B O N A T I and M A R T A
PALMIERI
Istituto di Chimica Organica e Biologica, Facoltd di Scienze, Universitd di Napoli, Via Mezzocannone, 16, 80134 Napoli (Italy)
(Received July 28th, 1977)
Summary 1. Double-stranded f2 susll or Q/~ RNAs, resistant to bovine pancreatic RNAase A in 0.15 M NaC1/0.015 M sodium citrate (SSC), are quickly and completely degraded at 10-fold lower ionic strength (0.1 X SSC) under otherwise similar conditions. At this ionic strength the secondary structure of doublestranded RNA is maintained, as judged by the following: (a) the unchanged resistance of double-stranded RNA and DNA, under similar low ionic strength conditions, to nuclease S, from Aspergillus oryzae, in contrast with the sensitivity of the corresponding denatured nucleic acids to this enzyme, specific for single-stranded RNA and DNA; (b) the co-operative pattern of the thermaltransition profile of double-stranded RNA (with a Tm of 89°C) in 0.1 X SSC. 2. Whereas in SSC bovine seminal RNAase (RNAase BS-1) and whale pancreatic RNAase show an activity on double-stranded RNA significantly higher than that of RNAase A, in 0.1 X SSC the activity of the latter enzyme on this substrate becomes distinctly higher than that of RNAase BS-1, and similar to that of whale RNAase. 3. From these results it is deduced that the secondary structure is probably not the only nor the most important variable in determining the susceptibility of double-stranded RNA to ribonuclease. Other factors, such as the effect of ionic strength on the enzyme and/or the binding of enzyme to nucleic acids, may play an important role in the process of double-strnaded RNA degradation by ribonucleases specific for singlestranded RNA.
Abbreviation: SSC, standard saline-citrate solution, 0.15 M sodium chloride/0.015 M s o d i u m citrate, pH 7.
278 Introduction
It is well established that double-stranded RNA is highly resistant to digestion by bovine pancreatic ribonuclease A in 0.15--0.2 M NaC1, while singlestranded RNA is rapidly degraded under these conditions [1--4]. On the other hand, it is also well known that at lower salt concentrations double-stranded RNA becomes highly sensitive to RNAase A [1,3,4]. In this work evidence is presented that double-stranded RNA may be quickly and completely degraded by pancreatic RNAase A under salt conditions where the double-helical structure of the RNA is maintained, as judged by: (i) the co-operative pattern of its thermal-transition profile, and (ii) the resistance of the nucleic acid to nuclease $1 from Aspergillus oryzae, an enzyme specific for single-stranded RNA and DNA [ 5--7 ]. Materials and Methods
Substrates. Double-stranded [3H]RNA, prepared as described [8,9] from non-perpermissive Escherichia coli K-38 cells infected with f2 sus11 bacteriophage, was a generous gift of Dr. Jirf Doskocil, Czechoslovak Academy of Science, Praha. The concentration of double-stranded RNA was determined assuming an E260 value of 210 dl" g-1. cm-1 [1]. Its specific activity was approx. 5500 cpm per pg. Single-stranded [3H]RNA was obtained by heat denaturation of the double-stranded viral RNA under low salt conditions [10]. 32P-Labelled double-stranded and single-stranded Qfl RNAs were a generous gift of Dr. Tadatsugu Taniguchi and Professor Charles Weissmann, The University of Zurich, Switzerland. Double-stranded [14C]DNA was prepared as described [11,12], with minor modifications, from E. coli K-12 cells, with [~4C]thymidine (spec. act. 62 Ci/mol; The Radiochemical Centre, Amersham) as a radioactive precursor. Purified DNA had a spec. act. of 1800 cpm/pg. Radioactive single-stranded DNA was obtained by heat denaturation of native E. coli DNA in 0.007 M NaC1, pH 7. 3H-Labelled poly(A) and poly(C) and the corresponding non-radioactive homopolymers were purchased from Miles. They were mixed to obtain a solution of 3.2 pg/ml, with a spec. act. of approx. 1300 cpm/ gg. Cytidine 2',3'-cyclic phosphate was purchased from Sigma Chemical Co. Enzymes. Bovine pancreatic RNAase (type XII-A) was purchased from Sigma. Bovine seminal ribonuclease (RNAase BS-1) was purified as described [13]. Its specific activity was 46 Kunitz units [14] per mg. Pancreatic RNAase from the lesser rorqual or pike-whale, Balaenoptera acutorostrata, purified as described [15,16], was a kind gift of Dr. Jaap J. Beintema, The University of Groningen, The Netherlands. Nuclease S~ was purified from crude a-amylase powder (catalog No. A 6630) from Aspergillus oryzae, purchased from Sigma, according to the procedure outlined by Vogt [ 7]. However, the chromatography on DEAE-cellulose was repeated twice, without going through the SulfoSephadex and Sephadex G-100 steps. The purified enzyme was completely inactive towards double-stranded DNA under the assay conditions outlined by
Vogt [7]. Methods. Incubations with bovine RNAase A and RNAase BS-1 and with whale pancreatic RNAase were carried out in SSC or in various dilutions of it,
279 usually at pH 7. When necessary, the pH was adjusted to 5 with small amounts of 0.1 M HC1. E n z y m e activity was determined as described [10,12], by measuring the acid-insoluble radioactivity retained on Millipore membranes (HAWP00010, Millipore Filter Co.) after precipitating samples with trichloroacetic acid (final concentration 6%) at 0°C, and filtering and washing with chilled trichloroacetic acid. Details are given in the legends to the figures and to Table II. Assays with nuclease $1 were performed essentially according to Vogt [7], at 37 or 45°C, using radioactive DNA or RNA as substrates. In the case of RNA concentrations of nuclease $1 7--10 times higher than with DNA were used, because of the correspondingly lower activity of the enzyme on RNA [ 7]. Moreover, in the control assays under low salt concentrations (see Table I, buffer B), with either denatured DNA or RNA as substrate, the concentration of $1 nuclease was doubled since 0.1 M NaC1 is r e c o m m e n d e d [7] for optimal enzymic activity. Enzyme activity was measured by determining the acidinsoluble radioactivity retained on Millipore filters as described above for the ribonuclease assays. Details are given in the legend to Table I. Activity on p o l y ( A ) a n d poly(C) was also assayed as described above, by measuring the acid-insoluble radioactivity retained on Millipore filters. Specific activity of RNAase A with poly(A) as substrate is given as pg of polymer degraded/rain per mg protein, at 37 ° C. E n z y m e assays with cytidine 2' : 3'-cyclic phosphate as a substrate were performed essentially as described [13,17]. Details are given in the legend to Table III. All assays were performed in duplicate. Protein concentration was determined either spectrophotometrically (for RNAase A and RNAase BS-1) as described [18], or by the procedure of Lowry et al. [19]. Thermal-transition profiles of double-stranded RNA were determined at 260 nm using stoppered cuvettes (type 29, Starna Ltd., London), with a Zeiss PM QII spectrophotometer equipped with a thermostatically controlled water bath. Results
Susceptibility o f double-stranded R N A to R N A a s e A at different salt concentrations Fig. l a shows the results of an incubation of double-stranded RNA with 16 pg of pancreatic RNAase A per ml at 25°C under various ionic strength conditions. The resistance of the nucleic acid to the action of the enzyme is high in SSC, but decreases dramatically already by halving the SSC concentration, becomining almost nil when incubation is carried out in 10-fold diluted SSC. In particular (Fig. lb), under these conditions, at 20°C, only 30 and 18% of the RNA remains acid-precipitable after 40 and 90 s of incubation respectively, with as little as 0.94/~g of RNAase A per ml. When single-stranded RNA is used as a substrate, the rate at which the nucleic acid is degraded by RNAase A in 10-fold diluted SSC is only about twice that measured in standard SSC (Figs. 4 and 6). Moreover, the activities of
280
~_1 0 0 ~
100
o
%
o '
'o,%'
rain,
Fig. 1. A c t i o n o f p a n c r e a t i c R N A a s e A o n d o u b l e - s t r a n d e d R N A u n d e r v a r i o u s i o n i c s t r e n g t h c o n d i t i o n s , a. A s s a y m i x t u r e s o f 0 . 5 m l c o n t a i n e d 8.1 /~g o f R N A a s e A a n d 0 . 3 6 # g ( a p p r o x . 2 0 0 0 c p m ) o f d o u b l e - s t r a n d e d [ 3 H ] R N A d i s s o l v e d i n SSC ( A ) , 2 - f o l d d i l u t e d SSC (B), 5 - f o l d d i l u t e d SSC (C), a n d 1 0 - f o l d d i l u t e d SSC (D). T e m p e r a t t L r e , 2 5 ° C . b . S a l t c o n d i t i o n s as d e s c r i b e d u n d e r D a b o v e . R N A a s e A c o n c e n t r a t i o n , 0 . 9 4 / ~ g p e r m L T e m p e r a t u r e , 2 0 ° C . In a a n d b i n c u b a t i o n s w e r e s t o p p e d b y a d j u s t i n g , w h e r e n e c e s s a r y , t h e salt c o n c e n t r a t i o n t o S S C , a n d a d d i n g a t 0°C t r i c h l o r o a c e t i c a c i d (final c o n c e n t r a t i o n 6%). P r e c i p i t a t e s w e r e f i l t e r e d t h r o u g h M i l l i p o r e m e m b r a n e s (see M a t e r i a l s a n d M e t h o d s ) , a n d r a d i o a c t i v i t y w a s c o u n t e d as d e s c r i b e d [ 1 0 , 1 2 ] .
the enzyme on double- and single-stranded R N A in 0.1 X SSC are essentially of the same order of magnitude (Table II). The findings concerning double-stranded RNA, which are n o t novel [1,3,4], could be attributed to partial denaturation of the R N A double helix at low ionic strength. However, with the experiments described below we offer evidence that the different susceptibility of double-stranded R N A to enzymic attack at high and low ionic strength is probably n o t due to a change in its secondary structure. Thermal-transition profile o f double-stranded R N A at low ionic strength The pattern of the thermal denaturation of double-stranded f2 sus11 R N A under low salt conditions is shown in Fig. 2. Absorbancy increase as a function of temperature in 0.1 X SSC starts at approx. 73.5°C, following a typical cooperative pattern, with a Tm value around 89 ° C. The hyperchromicity is 34%, a value similar to that f o u n d in SSC (31%), where the absorbancy increase starts at approx. 87°C. At r o o m temperature the absolute absorbance of double-stranded f2 sus11 R N A is the same in SSC and in 0.1 X SSC. These data indicate that there is no substantial difference in the degree of secondary structure o f f2 sus11 R N A in SSC or in 0.1 × SSC. The Tm value determined for double-helical f2 sus11 R N A in 0.1 X SSC is similar to the corresponding value reported for double-stranded MS2 RNA, the Tm values of which are 103°C in SSC, 87°C in 0.1 X SSC, and approx. 80°C in 0.01 × SSC [1]. The difference between these Tra values is certainly n o t large. Therefore, it seems that, at least at the temperature of the RNAase assay (20--25°C), the stability of the secondary structure o f double-stranded R N A should not be very different whether it is dissolved in SSC, where it is highly
281 resistant to RNAase A digestion, or in 0.1 X SSC, where it is highly sensitive to the enzyme.
Action of nuclease Sl from Aspergillus oryzae on double-stranded RNA at different ionic strengths The following experiments were devised to ascertain whether the ionic strength-dependent susceptibility of double-stranded RNA to enzymic attack might occur also with a different nuclease, thus indicating it could be a more general phenomenon, or whether it is characteristic for digestion by pancreatic RNAase A. The results (Table I) show that nuclease $1 is almost completely inactive toward native double-stranded RNA and DNA under high (buffer A) and low (buffer B) salt conditions, similar to those used in the experiments with pancreatic RNAase A. Moreover, the resistance of double-stranded RNA remains unchanged under the significantly lower ionic strength conditions of buffer C. On the other hand, under the same salt conditions, where native double-stranded RNA or DNA were resistant, the corresponding denatured (i.e., single-stranded) nucleic acids were highly sensitive to the $1 nuclease (Table I). Thus, the secondary structure of double~tranded RNA at low TABLE I ACTION OF NUCLEASE S 1 FROM ASPERGILLUS ORYZAE ON NATIVE OR DENATURED BLE-STRANDED RNA AND DNA UNDER VARIOUS CONDITIONS
DOU-
B u f f e r A : 0 . 0 5 M s o d i u m a c e t a t e , p H 5 / 0 . 1 2 M s o d i u m c h l o r i d e / 0 . 0 0 1 3 M Z n S O 4. B u f f e r B: 0 . 0 1 5 M s o d i u m a c e t a t e , p H 5 / 0 . 0 0 1 3 M Z n S O 4. B u f f e r C: 0 . 0 0 5 M s o d i u m a c e t a t e , p H 5 / 0 . 0 0 1 3 M Z n S O 4. Spec i f i c a c t i v i t i e s o f t h e r a d i o a c t i v e n u c l e i c acids: d o u b l e - s t r a n d e d [ 3 H ] R N A , 5 5 0 0 c p m / / ~ g ; [ 1 4 C ] D N A , 1 8 0 0 c p m / p g . I n c u b a t i o n s w e r e carried o u t in 1 m l f o r 30 r a i n at 37°C. T u b e s w e r e t h e n t r a n s f e r e d in a n i c e - b a t h , salt c o n c e n t r a t i o n w a s adjusted t o 0 . 1 5 M N a C I , a n d 1 / 1 0 vol. o f c o l d 6 0 % t r i c h l o r o a c e t i c a c i d w a s added. A f t e r 1 0 - - 3 0 rain a t 0°C, p r e c i p i t a t e s w e r e filtered t h r o u g h Millipore m e m b r a n e s and w a s h e d 5 - 6 t i m e s w i t h chilled 6% t r i c h l o r o a c e t i c acid. A c i d - i n s o l u b l e r a d i o a c t i v i t y r e t a i n e d o n filters w a s c o u n t e d as d e s c r i b e d u n d e r Materials and M e t h o d s . B a c k ~ o u n d values have b e e n s u b t r a c t e d f r o m figuzes appearing in t h e t a b l e .
Substrate
Buffer A
Buffer B
cpm a. D o u b l e - s t r a n d e d R N A 1. N o e n z y m e 2. 7 # g S1 3.10 #g S 1
3805 . 3706
%
100 .
. 97.4
Buffer C
cpm
%
cpm
%
3461 . 3288
100
736 699 --
100 95 --
680 . 0
100
95
b. D e n a t t t r e d d o u b l e - s t r a n d e d R N A 1. N o e n z y m e 2221 2. 10 # g S 1 534 3.20 ~g S 1 --
100 24 --
2610 -55
100 -2.1
c. D o u b l e - s t r a n d e d D N A 1. N o e n z y m e 2. 1 ~ g S 1 3. 10 /~g S 1
100 99.2 99.7
1097 1012 --
100 92.2 --
100 10.6
693 . 9
1048 1040 1045
d. D e n a t u r e d d o u b l e - s t r a n d e d D N A 1. N o e n z y m e 669 2. 1 ~zg S 1 71 3 . 2 pg S 1 --
--
100 .
. 1.3
0
282
salt concentrations is retained, at least to the extent o f conferring resistance to an enzyme such as nuclease $1, which is absolutely specific for singlestranded R N A or D N A [ 5--7 ]. The main variable between the experiments with RNAase A and nuclease $1 being the different pH of the incubation mixtures (optimal activity of S1 nuclease is at acidic pH) and the presence of ZnSO4, an experiment was performed to check any possible influence of the acidic pH and of ZnSO4 on the ribonuclease reaction. Double-stranded R N A was incubated with RNAase A in the same buffers used in the experiments with nuclease $1 (buffers A and B of Table I). At pH 5, 75 and 92% of the double-stranded R N A were degraded after incubation for 25 min at 25°C with 16 #g of RNAase A/ml under the high and low salt conditions, respectively. Moreover, a sensitivity of doublestranded RNA to RNAase A significantly higher than at pH 7 has been also observed in SSC at pH 5.
Susceptibility of double- and single-stranded RNA to RNAase A, RNAase BS-1 and whale pancreatic RNAase under different salt conditions It has been established that bovine seminal ribonuclease (RNAase BS-1), a
0.510
0.49( '~ 0.47(
100
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~ 0.410
0.390
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Temperature (°C)
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,
.
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Fig. 2. Thermal-transition profile o f d o u b l e - s t r a n d e d f2 s u s l 1 R N A . C o n c e n t r a t i o n o f d o u b l e - s t r a n d e d R N A , 1 8 . 4 / ~ g per m l in 0.1 X SSC. A b s o r b a n c e at 2 6 0 n m w a s d e t e r m i n e d as described u n d e r Materials and M e t h o d s . Fig. 3. C o m p a r i s o n o f R N A a s e A and R N A a s e BS-1 activities o n d o u b l e - s t r a n d e d R N A at different ionic strengths. C o n d i t i o n s : a, i n c u b a t i o n in SSC; d o u b l e - s t r a n d e d [ 3 H ] R N A ( e , o). 1 . 3 4 / ~ g / m l , or d o u b l e stranded [ 3 2 p ] R N A ( A A), 5 . 6 / ~ g / m l ( a p p r o x . 18 1 0 0 cpm//~g). R N A a s e A ( a e) or R N A a s e BS-1 ( A o), 1 8 . 5 #g/m1, or 2 7 . 3 / ~ g / m l w i t h the 3H-labelled or t h e 32p-labelled substrate, respectively, b. I n c u b a t i o n in 0.1 X SSC; d o u b l e - s t r a n d e d [ 3 H ] R N A ( e , o ) , 1 . 3 4 / ~ g / m l , or double-stranded [ 3 2 p ] R N A ( a z~). 2 . 8 5 #g/m1 a p p r o x . 1 6 4 0 0 cpm#~g). R N A a s e A ( A e) or R N A a s e BS-1 (o, A), 1.85/~g/rnl o r 2 . 7 3 / ~ g / m l w i t h the 3H-labelled or the 32p-labelled substrate, respectively. A s s a y m i x t u r e s v o l u m e s , 0 . 5 m l w i t h d o u b l e - s t r a n d e d [ 3 H ] R N A or 0 . 2 m l w i t h d o u b l e - s t r a n d e d [ 3 2 p ] R N A . T e m p e r a t u r e , 20°C. A t the t i m e s indicated t h e r e a c t i o n w a s s t o p p e d and s a m p l e s w e r e treated as described under Fig. 1.
283 well characterized, dimeric enzyme [13,17,20], and whale pancreatic RNAase [15,16], both of which are proteins more basic than RNAase A, are definitely more efficient than the latter enzyme at degrading double-stranded polyribonucleotides in SSC [18]. Figs. 3a and b show the results of experiments carried o u t in SSC and in 0.1 × SSC, respectively, with RNAase A and RNAase BS-1, using doublestranded RNA as a substrate. It is quite clear that while at the higher salt concentration RNAase A is less active than RNAase BS-1 (Fig. 3a), the opposite is true in 0.1 X 88C (Fig. 3b). Actually, as shown in Table II, where a comparison on a quantitative basis is possible, the activity of both enzymes towards double-stranded RNA is increased at low ionic strength, but, whereas RNAase A in 0.1 × 8SC becomes more than 2000 times as active as in SSC, the activity of RNAase BS-1 at low salt concentration is only 80 times higher than in 88C. This accounts for the reversal in the relative activities of bovine RNAase A and RNAase BS-1. Fig. 4 shows the effect of a 10-fold difference in ionic strength on the susceptibility of single-stranded Qfl RNA to bovine RNAase A and RNAase BS-1. Whereas the difference in salt concentration slightly affects the action of RNAase A, which at low ionic strength is a b o u t twice as active as in 88C, it has no influence on the action of RNAase B8-1 (see also Table II). The behaviour of whale pancreatic RNAase is shown in Fig. 5 and in Table II. Whereas, as already reported [18], the enzyme is significantly more efficient than RNAase A (and RNAase BS-1) at degrading double-stranded RNA in SSC, its action appears only a b o u t twice that of RNAase A in 0.1 × SSC (Figs. 5a and b). Under these conditions, in fact, the increase in activity of whale RNAase is lower than that of bovine RNAase A, the ratio of whale RNAase activities in 0.1 × 8SC and SSC being a b o u t 5 times lower than that of the corresponding RNAase A activities (Table II). If single-stranded R N A is 100
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Fig. 4. R N A a s e A a n d R N A a s e B S - I activities o n single-stranded R N A u n d e r d i f f e r e n t salt c o n d i t i o n s . Single-stranded Q~ R N A l a b e l l e d w i t h 3 2 p , 5 . 3 / ~ g / m l ( a p p r o x . 1 2 4 0 0 c p m / # g ) . R N A a s e A or R N A a s e BS-I, 2.73 ~g/per ml. o o or • ~, R N A a s e A a c t i v i t y in SSC or in 0 . I X SSC, r e s p e c t i v e l y . ~ or • A R N A a s e BS-1 a c t i v i t y in SSC or in 0.1 X SSC, r e s p e c t i v e l y . A s s a y m i x t u r e s v o l u m e , 0 . 2 ml. T e m p e r a t u r e , 2 0 ° C . A t t h e t i m e s i n d i c a t e d t h e r e a c t i o n w a s s t o p p e d a n d s a m p l e s w e r e t r e a t e d as d e s c r i b e d u n d e r Fig. 1.
RNA UNDER DIFFERENT
IONIC
A B B
R N A a s e BS-1
Whale RNAase
A B
Expt.
2.5 2.4 7.0
0.6 0.5
SSC
A C C
A C
Expt.
197 201 2867
1327 1246
0.1 × S S C
80 84 409
2211 2492
Activity in SSC
D E F
D E F
Expt.
1405 2150 3900
1000 1579 2055
SSC
D E F
D E F
Expt.
1405 2051 5478
1968 3093 4590
0.1 × SSC
Activity in
A c t i v i t y in Activity in 0.1 × SSC
S i n g l e - s t r a n d e d R N A as s u b s t r a t e
D o u b l e - s t x a n d e d R N A as s u b s t r a t e
RNAasca
Enzyme
1.00 0.95 1.40
1.97 1.96 2.23
A c t i v i t y in S S C
Activity in 0.1 X SSC
E n z y m e a c t i v i t y is g i v e n as /~g o f R N A d e g r a d e d / m i n p e r m g p r o t e i n , a t 2 0 ° C . E x p t . A: d o u b l e - s t r a n d e d [ 3 2 p ] R N A as s u b s t r a t e . C o n d i t i o n s d e s c r i b e d u n d e r F i g s . 3 a a n d b . E x p t . B: d o u b l e - s t r a n d e d [ 3 H ] R N A , 4 . 3 /~g/ml; R N A a s e A o r R N A a s e B S - 1 , 2 0 /~g/ml; w h a l e R N A a s e , 1 4 . 8 ~ g / m l . A s s a y m i x t u z e s , 0 . 5 m l ; t e m p e r a t u r e , 2 0 ° C . E x p t . C: d o u b l e - s t m a n d e d [ 3 H ] R N A , 1 ~ g ~ n l ; R N A a s e A o r R N A a s e B S - I , 1 . 8 6 ~ g / m I ; w h a l e R N A a s e , 0 . 6 /~g/m]. A s s a y m i x t u r e s , 0 . 5 m l ; t e m p e r a t u r e , 2 0 ° C . E x p t . D: i n S S C o r i n 0 . 1 X S S C , s i n g l e - s t r a n d e d [ 3 2 p ] R N A , 3 . 1 5 /~g/ml; R N A a s e A o r R N A a s e B S - 1 , 2 . 6 5 /~g/ml. A s s a y m i x t u r e s , 0 . 2 m l ; t e m p e r a t u r e , 2 0 ° C . E x p t . E: c o n d i t i o n s d e s c r i b e d u n d e r Fig. 4. E x p t . F: c o n d i t i o n s d e s c r i b e d u n d e r Fig. 6.
C O M P A R I S O N O F R N A a s e A , R N A a s c BS-1 A N D W H A L E R N A a s e A C T I V I T I E S O N D O U B L E - A N D S I N G L E - S T R A N D E D STRENGTH CONDITIONS
T A B L E II
to 00
285 100
100
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610
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Fig. 5. Comparison of R N A a s e A and whale R N A a s e activitieson double-stranded R N A at different ionic strengths, a. In SSC, double-stranded [ 3 H ] R N A , 2.6 #g/ml. R N A a s e A (e), 5.47 #g/ml, whale R N A a s e ( o ) , 4 . 7 5 p g / m l . A s s a y m i x t u r e s v o l u m e , 0 . 2 m l . T e m p e r a t u r e , 2 0 ° C . b . In 0.1 X SSC0 d o u b l e ~ t r a n d e d [ 3 H ] R N A , 0 . 5 2 / ~ g / m l . R N A a s e A ( e ) or w h a l e R N A a s e ( o ) , 0 . I # g / m l . A s s a y m i x t u r e s v o l u m e , 1 m l . T e m p e r a t u r e , 2 0 ° C . A t t h e t i m e s i n d i c a t e d t h e r e a c t i o n w a s s t o p p e d a n d s a m p l e s w e r e t r e a t e d as d e s c r i b e d u n d e r Fig. 1.
used as substrate, both whale and ox pancreatic RNAases show a slight, and essentially similar, increase in their activity at the lower salt concentration, as compared with that measured in SSC (Fig. 6 and Table II).
Action of RNAase A, RNAase BS-1 and whale RNAase on cytidine 2',3'-cyclic phosphate at different ionic strength The effect of different salt concentrations on the action of bovine RNAase 100
15
# ~
£
5c
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0
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Fig. 6. R N A a s e A a n d w h a l e R N A a s e a c t i v i t i e s t o w a r d s i n g l e - s t r a n d e d R N A a t d i f f e r e n t i o n i c s t r e n g t h s . S i n g l e - s t r a n d e d Q~ R N A , l a b e l l e d w i t h 3 2 p , 5 . 3 # g / m l ( a p p r o x . 1 0 7 0 0 ¢ p m / ~ g ) . R N A a r e A o r w h a l e RNAase, 1.5 #g/m L o o or ¢ ~-, R N A a s e A a c t i v i t i e s i n S S C o r in 0 . 1 X SSC, r e s p e c t i v e l y . A ~ or • • , w h a l e R N A a s e a c t i v i t i e s i n S S C o r i n 0 . I X SSC, r e s p e c t i v e l y . A s s a y m i x t u r e s volume, 0.2 ml. Temperature, 20°C. At the times indicated the reaction was stopped, and samples were t r e a t e d as d e s c r i b e d u n d e r F i g . 1.
286 TABLE III A C T I O N O F R N A a s e A , R N A a s e BS-1 A N D W H A L E P A N C R E A T I C R N A a s e O N C Y T I D I N E 2' : 3 ' - C Y CLIC PHOSPHATE UNDER DIFFERENT IONIC STRENGTH CONDITIONS T o 1 m l of 0.2 m M c y t i d i n e 2 ' : 3 ' - c y c l i c p h o s p h a t e dissolved in 0.01 M imidazole-HC1 b u f f e r c o n t a i n i n g v a r i o u s c o n c e n t r a t i o n s o f NaCl, 0 . 9 - - 1 . 8 p g of e n z y m e w e r e added, and change in a b s o r b a n c e at 2 8 7 n m w a s r e c o r d e d w i t h a C a r y m o d e l 1 1 8 s p e c t r o p h o t o m e t e r , a t 24°C. Specific activities are e x p r e s s e d as change in a b s o r b a n c e / m i n p e r m g p r o t e i n . Imidazole-HC1 buffer, pH 6.92
Salt c o n d i t i o n s (M NaCD
Specific a c t i v i t i e s o f RNAase A
R N A a s e BS-1
(M) 0.01 0.01 0.01 0.01
Whale
RNAase 0 0.05 0.10 0.15
3,50 2.73 2.28 2.06
3.42 3.78 3.85 3.15
1.28 1.13 0.95 0.84
A and RNAase BS-1, and of whale pancreatic RNAase toward cytidine 2',3'cyclic phosphate is shown in Table I I I . The results of this experiment essentially agree with those obtairied using single-stranded RNA as a substrate. While in the case of RNAase BS-1 the activity values may be considered as randomly scattered around an average specific activity of 3.5 the activity of RNAase A, and, to a lower extent, t h a t of whale RNAase regularly decrease by increasing the ionic strength of the assay mixture. Discussion Pancreatic RNAase A has a modest activity on double-stranded RNA under high salt conditions, the remarkable resistance of this species o f RNA to the enzyme being usually attributed to the presence and the maintainance of the secondary structure in the nucleic acid. Other well characterized ribonucleases, such as, for instance, seminal RNAase BS-1 and whale pancreatic RNAase, are definitely more efficient than RNAase A at degrading double-helical RNA under identical ionic strength conditions. To explain the activity on double-stranded polyribonucleotides by ribonucleases specific for single-stranded RNA, we proposed, as a tentative mechanism of action, a destabilization of the RNA double helix, similar to t h a t shown on DNA by RNAase A [21] or, more efficiently, by RNAase BS-1 [22], resulting from the interaction of a ribonuclease molecule with double-stranded RNA. This destabilization would then allow the enzymic attack on the nucleic acid [10,18,23]. On the other hand it seems improbable t h a t a RNA double helix can fit the active site of a ribonuclease w i t h o u t a simultaneous unwinding of the double-stranded nucleic acid to yield hydrolyzable single-stranded segments. The efficiency of this labilizing activity was correlated with the number, or the density, of basic charges present on the enzyme molecule [18]. In fact, bovine RNAase BS-I, whale RNAase and dimers or higher aggregates of RNAase A, enzyme species distinctly more efficient than monomeric RNAase A at
287 degrading double-stranded RNA in SSC [12,18,24,25], are proteins more basic (or with a higher density of basic charges) than the bovine pancreatic enzyme. From the experiments presented in this work it appears that in 0.1 × SSC double-stranded RNA is degraded by RNAase A, RNAase BS-1 and whale RNAase at a much higher rate than in SSC. However, the activity of RNAase A increases dramatically in comparison with that of the other two enzymes, becoming higher than the activity of RNAase BS-1 and close to that of whale RNAase. On the other hand, in 0.1 X SSC the secondary structure of doublestranded RNA is maintained, as judged by the absorbance, the thermal-transition profile and the unchanged resistance of the nucleic acid to nuclease $1 under these salt conditions. Therefore, the data obtained at low ionic strength on one hand indicate that the secondary structure of the RNA may not be the most important variable in the process of enzymic degradation of doublestranded RNA; on the other hand, they seem to contradict the correlation between enzyme basicity and activity on the RNA double helix (with or without previous destablization), observed at high salt concentration [18]. Why is the pattern of double-stranded RNA degradation by the three enzymes not maintained at low ionic strength? While a trivial explanation of the results obtained, i.e., a uniform inhibition of all RNAase activities by salt, may be ruled out by the experiments performed in parallel on double- and single,stranded RNA, the data presented above suggest that other factors may play a role in the phenomena observed. For instance, the ionic strength, the importance of which for RNAase A has been already shown by Kalnitsky et al. [26], could differently affect different enzyme proteins, their interaction with the double-stranded nucleic acid, and therefore their turnover numbers. By examining the data of Table II it appears that the activity of RNAase A on double-stranded RNA in 0.1 X SSC is close to that on single-stranded RNA determined under identical salt conditions, the activity on the latter substrate being only about twice that on double-stranded RNA. The same is true with whale RNAase. The activity of this enzyme toward single-stranded RNA in 0.1 X SSC is also about twice that measured on double-stranded RNA at the same ionic strength. In other words, the fact that RNA is in double- or singlestranded form makes no great difference for the action of RNAase A and whale RNAase, provided the ionic strength is low. RNAase BS-1, however, in 0.1 X SSC is about 9 times less active on double-stranded than on single-stranded RNA. Moreover, the rate at which this enzyme degrades single-stranded RNA appears unchanged at high or low ionic strength (see also Figs. 4 and 6). These facts could be probably ascribed to a different salt effect on the tertiary structure of the various proteins considered. Whereas whale RNAase is a monomeric protein, homologous to RNAase A [16,27], RNAase BS-1 is a dimer, containing two identical 124-amino acid peptide chains (homologous to RNAase A [28]) linked through two interchain disulfide bridges [29,30]. Both whale and BS-1 RNAases have a higher number of basic amino acids than RNAase A [16,28]. It might be reasonable to assume that the conformation of different enzyme proteins, which can be influenced by the number of charges present on their surface and/or by their quaternary structure, could be dif-
288 ferently affected b y changes in ionic strength conditions. On the other hand, the results presented in this work are reminiscent of what was observed with poly(A) as a substrate, although this polymer cannot be considered a "normal" substrate for a ribonuclease. Polyadenylic acid was also more efficiently degraded b y RNAase BS-1 [31] and dimeric RNAase A [10] than by monomeric RNAase A under high salt conditions, whereas the opposite occurred (and with much smaller amounts of enzyme) under low salt conditions. This behaviour can be observed both at pH 5 and 7. Moreover, at pH 5 RNAase A is distinctly more active on poly(A) in 0.1 × SSC than in standard SSC, the corresponding specific activity values being 20.2 and 1.2, respectively. Similar results have been obtained with poly(C) as substrate (Sorrentino, Palmieri and Libonati, unpublished results). Since at pH 5 poly(A) and poly(C) have a well-characterized secondary structure which is stabilized by low salt concentrations [32], the presence of the secondary structure in these polymers does not seem to be an important variable in their susceptibility to the action of RNAase A, in contrast with what was originally thought for poly(A) [10]. In conclusion, provided the ionic strength is 0.1 that of SSC, always the same effect by RNAase A is observed, i.e., a more efficient degradation of a double-stranded polyribonucleotide, either double-helical RNA, which is less stable, or the acidic double-stranded forms of poly(A) and poly(C), which are more stable at low salt concentrations. These arguments could strengthen the possibility that the secondary structure of a polyribonucleotide, although being a key factor, is n o t the only one in determining the resistance of double-stranded R N A to pancreatic RNAase A. An advancement in the understanding o f the facts presented could be probably achieved only by using different experimental approaches, as, for instance, NMR analyses of the various ribonucleases considered under different conditions. Moreover, the results presented might also be considered and further studied in the light of the hypothesis of the catalytic implications of electrostatic potentials, advanced by D o u z o u and Maurel [33,34]. Finally, it is probably worth considering that physiological ionic strength values in vivo are close to that of SSC. Therefore, the significance of the various ribonucleases reported here and elsewhere [35] to be also active on doublestranded RNA, and the physiological implications of this action, should be referred to enzyme activities studied at an ionic strength similar to that of SSC. Acknowledgements We are very grateful to Professor Stanford Moore, Professor Charles Weissmann and Dr. Glynn Wilson for important suggestions and criticism. References 1 Billeter, M.A., Weissmann, C. and Warner, R.C. (1966) J. Mol. Biol. 17, 145--173 9 Weissmann, C., Billeter, M.A., Vinuela~ E. and Libonati, M. (1966) in Viruses of Plants (Beemster, A.B.R. and Dijkstra, J., ed.), pp. 249--274, North-Holland Publ. Co., A m s t e r d a m
289 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
Iqbal, Z.M. and K o h n , K.W. (1975) Arch. Biochem. Biophys. 166, 5 1 8 - 5 2 5 Edy, V.G., Szekely, M., Loving, T. and Dreyer, C. (1976) Ettr. J. Biochem. 6 1 , 5 6 3 - 5 7 2 A n d o , T. (1966) Biochim. Biophys. Aeta 114, 158--168 Sutton, W.D. (1971) Biochim. Biophys. Acta 240, 5 2 2 - 5 3 1 Vogt, V.M. (1973) Eur. J. Bioehem. 33, 1 9 2 - - 2 0 0 Paces, V., Doskocfl, J. and Sorm, F. (1968) Biochtm. hiophys. Aeta 1 6 1 , 3 5 2 - - 3 6 0 D'Aless~o, G., Doskocfl, J. and Libonat], M. (1974) Biochem. J. 1 4 1 , 3 1 7 - - 3 2 0 Palmieri, M. and Libonati, M. (1977) Bioehtm. Biophys. Acta 474, 456--466 Habieh, A., W e ~ m n , C., IAbonati~ M. and Warner, R.C. (1966) J. Mol. Biol. 21, 255--264 Libonati, M. and Florldi, A. (1969) Ettr. J. Biochem. 8, 61--87 D'Alessio, G., Floridi, A., De Prisco, R , Plgnero, A. and Leone, E. (1972) Eur. J. Biochem. 26, 1 5 3 - 161 Kunitz, M. (1946) J. Biol. Chem. 164, 563--566 Wierenga, R.K., Huizinga, J.D., Gaastra, W., Welling, G.W. and Beintema, J j . (1973) FEBS Lett. 31, 181--185 E m m e n s , M., Wenln~, G.W. and Beintema, J j . (1976) Bioehem. J. 157, 317--323 Florldi, A., D'Alessio, G. and Leone, E. (1972) Eur. J. Biochem. 26, 162--167 Lihonat], M., Furia, A. and Beintema, J J . (1976) Eur. J. Bioehem. 69, 445--451 Lowry, O.H., Rosehrough, N.J., Farr, A.L. and Randall, R J . (1951) J. Biol. Chem. 193, 265--275 D'Alessto, G., Parch,e, A., Guida, C. and Lenone, E. (1972) FEBS Let,. 27, 285--268 Felsenfeld, G., Sandeen, G. and von Hippel, P.H. (1963) Proe. Nat]. Acad. ScL U.S. 50, 644--651 IAbonati, M. and Beintema, J~l. (1977) Biochem. Soc. Trans. 5, 470--474 Libonat], M. Sorrentino, S., Galli, R., La Montagnal R. and Di Donato, A. (1975) Biochtm. Biophys. Acta 4 0 7 , 2 9 2 - - 2 9 8 Libonati, M. (1971) Biochim. Biophys. Aeta 2 2 8 , 4 4 0 - - 4 4 5 Wang, D., Wilson, G. and Moore, S. (1976) Biochemistry 15, 660----665 Kalnitsky, G., H u m m e l , J.P. and Dierks, C. (1959) J. Biol. Chem. 234, 1 5 1 2 - - 1 5 1 6 Beintcma, J.J., Graastra, W., Lenstra, J.A., Welling, G.W. and Fitch, W.M. (1977) J. Mol. Evol. 10, 49--71 Barker, W.C. and Dayhoff, M.O. (1976) in Atlas of Protein Sequence and Structure (Dayhoff, M.O., ed.), Vol. 5, Suppl. 2, pp. 77--103, National Biomedical Research F o u n d a t i o n , Washington, D.C. Di Donato, A. and D'Alessio, G. (1973) Biochem. Biophys. Res. C o m m u n . 55, 919--928 D'Alesslo, G., Malorni, M.C. and Parente, A. (1975) Biochemistry, 14, 1 1 1 6 - - 1 1 2 2 Pahnierl, M. and Libonati, M. (1976) Boll. Soc. It. Biol. Sper, 52, 80--84 Fekenfeld, G. and Miles, H.T. (1967) A n n u . Rev. Biochem. 36, 407--446 D o u z o u , P. and Maurel, P. (1977) Proc. Natl. Acad. Sci. U.S. 74, 1013--1015 D o u z o u , P. a n d Maurel, P. (1977) Trends Bioehem. Sei. 2, 14--17 BaxdSn, A., Sierekowska, H. and Shugar, D. (1976) Bio~hlm~ Biophys. Acta 438, 461--473