ionic control of enzymic degradation of double-stranded rna

40

Biochimica et Biophysica Acta, 609 (1980) 40--52 © Elsevier /Nor th-Hol land Biomedical Press

BBA 99706

IONIC CONTROL OF ENZYMIC DEGRADATION OF DOUBLE-STRANDED RNA

S A L V A T O R E S O R R E N T I N O a, A N T O N E L L A C A R S A N A a, A D R I A N A F U R I A a, JIl~f DOSKO(~IL b and MASSIMO LIBONATI a , ,

a Institute of Organic and Biological Chemistry, Faculty of Sciences, University of Naples, Via Mezzocannone 16, 1-80134 Napoli (Italy) and b Institute of Molecular Genetics, Czechoslovak Academy of Sciences, Flemingovo ndm$sti 2, 16610 Praha 6 (Czechoslovakia)

(Received November 20th , 1979)

Key words: Double-stranded RNA; RNAase A; RNA degradation; (Bovine, Pike whale)

Summary

The pattern of the degradation of various double-stranded polyribonucleotides by several ribonucleases (bovine RNAase A and its cross-linked dimer, bovine seminal RNAase, and pike-whale pancreatic RNAase) has been studied as a function of ionic strength and pH.

It appears that (1) there is no direct correlation between the secondary structure of double-stranded RNA and its resistance against enzymatic breakdown, i.e., the stability of the secondary structure of double-helical RNA is not the main variable in the process. (2)The activity responses of the enzymes examined to changes of ionic strength and pH suggest that enzymic degradation of double-stranded RNA is mainly controlled by ion concentration, and that the process may fall within the phenomena interpreted by the theory of the ionic control of biochemical reactions advanced by Douzou and Maurel (Douzou, P. and Maurel, P. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 1013-- 1015). (3) The activity curves of the enzyme studied show, at a given pH, a shift toward higher ionic strengths as a function of the basicity of the enzyme protein. This finding explains the already observed correlation between number and/or density of positive charges of a ribonuclease molecule and its ability to attack double-stranded RNA in 0.15M sodium chloride/0.015 M sodium citrate (SSC). (4) A careful analysis of the influence of ionic strength and pH on the reaction appears to be necessary in order to characterize a ribonuclease

* T o w h o m c o r r e s p o n d e n c e shou ld be addressed. Abbrev ia t ions : Poly(A), poly(C), poly(U), polyadenylate, polycytidylate, and polyuridylate; poly(A)" poly(U), the c o m p l e x f o r m e d b e t w e e n p o l y a d e n y l a t e and polyurldylate; SSC, 0.15 M sodium chloride/ 0.015 M sodium citrate; n X SSC, n-fold concentrated SSC.

41

which shows activity towards double-stranded RNAs, and to allow a meaningful comparison between different enzymes capable of attacking these substrates.

Introduction

Single-strand-specific nucleases, such as nuclease $1 from Aspergillus oryzae [1], are widely used as tools for detection of hybrid nucleic acids. For this purpose a maximal differentiation between degradation of single- and double- stranded forms of a nucleic acid is necessary.

Bovine pancreatic ribonuclease A (EC 3.1.4.22) is considered an enzyme very useful to identify and characterize double-stranded RNAs [2,3]. However, a satisfactory differential measurement of the double- and single-stranded forms of RNA with this enzyme is limited to rather strict assay conditions. Resistance of double-stranded RNA to ribonuclease attack is high, but not absolute, at salt concentrations of about 0.15 M NaC1, at pH 7 (where single- stranded RNA is quickly and completely degraded) [3]. It becomes almost complete at 0.2--0.3 M NaC1 [4](where also single-stranded RNA is degraded at a slower rate [5,6], declining, however, dramatically with the lowering of salt concentration [2--4,7,8].

These facts are usually ascribed to the variable stability of the secondary structure of double-stranded RNA as a function of counterion concentration.

Our aim in this work was ( i ) to ascertain the role of the stability of the secondary structure of the substrate, whose importance as the only variable in the process of enzymic degradation of double-stranded RNA was already questioned by some observations [8]; and (ii)to investigate what influence other variables, such as ionic strength and pH, have on the enzyme involved in the process and/or its interaction with the double-helical substrate.

Two completely different types of double-stranded polyribonucleotides were used as substrates of various ribonuclease species: viral double-stranded RNA and the poly(A) • poly(U) complex, on one side; the acidic forms of poly(A) and poly(C), on the other. They differ in their structure and at least in one important characteristic: the stability of the double-helical secondary structure of double-stranded RNAs decreases by lowering the ionic strength of the medium [2]; that of acidic poly(A) and poly(C) definitely increases under the same conditions [9--13]. The system used is suitable for differentiating the effects of the ionic environment on the substrate on one hand, and the enzymes (and/or the enzyme-substrate interaction) on the other.

Materials and Methods

Nucleic acids. [8-~H]Poly(A) and [3H]poly(C) (spec. act., 45.1 Ci/mol P, and 49 Ci/mol P, respectively), and the corresponding non radioactive polymers were Miles products. [5-3H]Poly(U) (spec. act., 1.63 Ci/g) was purcb, ased from the Radiochemical Centre, Amersham. Non radioactive poly(U) was from Sigma Chem. Co. Labelled and unlabelled polymers were mixed to give the following final solutions. Poly(A), 3.2 mg/ml, spec. act. 1 .3.106 cpm/mg;

42

poly(C), 4.05 mg/ml, spec. act. 6 • l 0 s cpm/mg; poly(U), 5 mg/ml, spec. act. 3.5 • 106 cpm/mg. Poly(A) • poly(U) ($20 = 15; hyperchromicity, 43%) was a Miles product.

Viral double-stranded RNA was obtained by infecting non-permissive (su-) bacteria, Escherichia coli F ÷, with bacteriophage f2 sus 11, which carries a non- polar amber mutat ion in the coat protein cistron [14].

Using the RNAase III-deficient strain AB 105 [15] kindly donated by Dr. P.H. Hofschneider, Miinchen, double-stranded RNA was stable in the cells and could be obtained in intact form by extraction with sodium dodecyl sulphate [16] and purification [3]. Its mean sedimentation coefficient in SSC was 10.6 s. Electron micrographs indicated the presence of bihelical linear mole- cules of several classes of length, the value of 0.9 gm occurring most frequently.

Enzymes. Four ribonucleases have been used in this work. They are considered specific for single-stranded RNAs. However, three of them are also capable of degrading double-stranded polyribonucleotides, with different efficiencies, under conditions where bovine pancreatic RNAase A is almost inactive (see Table I). Bovine RNAase A [17] ( type XII-A), specific activity, 100 Kunitz units/mg [18], was purchased from Sigma Chem. Co. The dimers of RNAase A, obtained by cross-linkage with dimethyl suberimidate [19], were in part a generous gift of Professor Stanford Moore, the Rockefeller University, New York, in part prepared according to the procedure outlined by Wang et al. [19]. They have a higher density of basic charges (like the aggregated dimers [20,21]) than native, monomeric RNAaseA. Bovine seminal RNAase was purified as described [22]. It is a dimeric protein (molecular weight, 27 400), homologous to pancreatic RNAase A [22--24]. The ribonuclease from the pancreas of pike-whale (Balaenoptera acutorostrata), purified as described [25,26], was a generous gift of Dr. Jaap J. Beintema, the University of Groningen. The enzyme, a single polypept ide chain homologous to bovine RNAase A, is quite active on double-stranded RNAs in SSC [27,28]. These enzymes differ in their charge characteristics (see Table I), bovine RNAase A being the least basic enzyme protein.

T A B L E I

C H A R G E C H A R A C T E R I S T I C S A N D A C T I V I T Y V A L U E S ON S I N G L E - A N D D O U B L E - S T R A N D E D

R N A s O F T H E R I B O N U C L E A S E U S E D

A c t i v i t y o n s ingle-s tranded R N A are e xp r e s se d as K u n i t z u n i t s p e r m g o f prote in . Ac t iv i ty v a l u e s o n

double - s t randed R N A ( d e t e r m i n e d i n S S C ) are r e l a t i v e t o that o f b o v i n e R N A a s e A se t e q u a l t o 1 [ 2 8 ] .

E n z y m e G l u + As p A r g + L y s His A c t i v i t y o n

single- double- stranded stranded

RNA RNA

B o v i n e p a n c r e a t i c R N A a s e A 1 0 1 4

Cross- l inked d imers o f b o v i n e R N A a s e A 1 0 * 1 4 *

B o v i n e s e m i n a l R N A a s e 1 0 * 1 8 *

P i k e - w h a l e p a n c r e a t i c R N A a s e 1 0 17

4 1 0 0 1 4 * 6 8 8 .5 4 * 4 0 1 0 - - 1 7

5 2 5 - - 3 3 3 8

* p e r s u b u n i t .

43

Assays. Enzyme activity with po ly (A) , poly(U) was assayed as described [29], by following the increase in absorbance at 260 nm as a function of time on a recording Zeiss PM6 spectrophotometer equipped with a linear scale expander. Specific activity, deduced from the slope of the linear part of the recordings, is expressed as AA260/min per mg of protein, with 50 ~g of poly(A) • poly(U) per ml, at 25°C. With viral double-stranded RNA as a substrate, the enzymic activity was determined with a spectrophotometr ic procedure similar to that used with poly(A) • poly(U). The absorbance of a buffered solution (containing various amounts of NaC1; see legend to Fig. 1 for details) of double-stranded RNA was controlled at 260 nm for at least 5 min at 25°C before adding the enzyme. The reaction mixtures were also checked at 340 nm to ascertain that absorbance was small or absent. The increase in A260 produced by enzyme activity was linear over at least 5--10 min, and proportional to enzyme concentration. The assays were carried out either with a Zeiss PM6 or with a Cary, Model 118, spectrophotometer . Specific activity, calculated from the slope of the linear part of the recordings, wherein 1--1.5% of the substrate was degraded per min, is expressed as AA260/min per mg of protein, with 20/~g of double-stranded RNA/ml, at 25°C.

Enzymic activity with radioactive poly(A), poly(C) and poly(U) as substrates was determined as described [29], with minor modifications. The reaction, lasting from 5 to 60 min with poly(A), and up to 60 s with poly(C) or poly(U) was s topped by adding cold, concentrated trichloroacetic acid and NaC1 solutions to obtain a final concentration of 6% (w/v) of the former, and 0.15 M of the latter. The precipitates (in duplicate) were filtered through MiUipore mem- branes (HAWP00010, Millipore Filter Co.), and washed with chilled 6% trichloroacetic acid. The acid-insoluble radioactivity retained on the filters was counted in a Beckman LS-133 liquid scintillation spectrometer. Specific activ- i ty is expressed as /~g of homopolymer solubilized per min per mg of protein, at 37°C with poly(A), at 20°C with poly(C) or poly(U). It was calculated from the linear part of the curve, wherein 30 to 50% of the substrate was degraded.

Thermal transition profiles of the double-stranded RNAs o r of poly(A), poly(C) and poly(U) were determined in stoppered cuvettes (Starna Ltd., London, type 29) with a Zeiss PM6 spectrophotometer equipped with a thermostatically controlled water bath.

Results

Experiments with viral double-stranded RNA Activity of several ribonucleases under various ionic strength and pH condi-

tions. As a preliminary point, the stability of the secondary structure of the viral double-stranded. RNA at the lowest concentrations of counterions~ and at various pH values, used in the experiments which follow was checked by determining the thermal transition profiles of the nucleic acid.

The melting curves were of a cooperative type, and Tm values (se~ Table II) indicate that the stability of the structure was very high, and comparable with that of double-stranded f2 sus 11 or MS2 RNAs in 0.1 × SSC, pH 7 (Tin values, 89°C and 87°C, respectively), and not so far from that of double-stranded MS2

44

T A B L E II

T m A N D H Y P E R C H R O M I C I T Y V A L U E S OF V I R A L 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 IONIC A N D p H C O N D I T I O N S

T h e r m a l t r ans i t ion prof i les were d e t e r m i n e d as descr ibed in Materials an d Methods . C o n c e n t r a t i o n of double - s t randed R N A , 14 .5 ~ g /ml Ini t ia l A 2 6 0 , 0.3.

Condi t ions T m H y p e r c h r o m i c i t y (°C) (%)

NaC1 Buffer p H (raM) (10 raM)

5.0 Tris/HC1 8.7 86 .0 27 .6 7.5 Tris/HC1 7.7 87 .0 35 .0 7.5 Imidazo le /HC1 7.0 89 .0 32 .4 7.5 SSC 5.0 85.0 36 .0

RNA in SSC, pH 7 (Tin, 103°C) [2,8]. The results obtained by investigating the influence of ionic strength on the

action of various enzymes on viral double-stranded RNA under different pH conditions, appear in Fig. 1.

From a comprehensive view of the results, it appears that the patterns of double-stranded RNA degradation can be essentially distinguished in two types: bell-shaped curves showing a maximum of activity at a certain Na ÷ concentration at a given pH, and curves of activity which appear to increase indefinitely by lowering the Na ÷ concentrat ion of the reaction mixture. The curves, either bell-shaped or not, of each enzyme tested, are shifted towards lower Na ÷ concentrations by increasing the pH of the reaction mixture. More- over, it appears that at each pH tested, the more basic enzyme proteins show the tendency to shift their curves of activity vs. ionic strength towards higher values. This results in a definitely higher degrading capacity of these enzymes under high salt concentrations.

From a more detailed inspection of the activity curves of three enzymes at pH 7, the following facts emerge. Whereas of 0.15 M Na ÷ the action of bovine RNAase A is very modest (spec. act., 0.013; Fig. la) , under identical conditions bovine seminal RNAase shows a higher activity (spec. act., 0.08; Fig. lc) , and whale RNAase is definitely more active (spec. act., 2.85; Fig. ld ) than bovine RNAase A and seminal RNAase, in agreement with what pointed out above. This pattern, however, changes dramatically when assays are carried out under lower Na ÷ concentrations. For instance, at 0.02 M Na ÷, whereas the specific activity of bovine RNAase A increases to about 16 (Fig. la) , those of seminal RNAase (Fig. lc ) and of whale RNAase (Fig. ld ) are 0.47 and 13.5, respectively.

Experiments with the poly(A ). poly(U) complex The secondary structure of the poly(A) • poly(U) complex is stabilized by

salt [13]. However, we found that under certain conditions, i.e., when NaC1 and MgC12 are present in certain proportions, the stability of the secondary structure of this nucleic acid may decrease by increasing the concentrat ion of NaC1. This fact has also been reported to occur with DNA [30].

45

4 8 4 8

d 4 0 ¢0

3 2 32

2 4 24

16 16

8 8

0 0 ._ 0 0.05 0.1 0.15 0 0.05 0.1 0.15 o

In 0.5 C

16

0 . 3

8

0

0 . . . . . . . . . . . . . ' • 0 0 0 5 0.1 0.15 0 0 .05 0.1 0. ,5

[ N , ' ]

Fig. I . D e g r a d a t i o n o f v i ra l d o u b l e - s t r a n d e d R N A b y va r i ous r i b o n u c l e a s e s u n d e r d i f f e r e n t i on ic a n d p H c o n d i t i o n s . (a) A c t i v i t y o f b o v i n e p a n c r e a t i c R N A a s e A. R e a c t i o n m i x t u r e s : bu f f e r s , 1 0 m M s o d i u m c i t r a t e / c i t r i c a c id f o r p H va lues 5 (A ~) a n d 6 (o a ) ; 1 0 m M i m i d a z o l e / H C l f o r p H 7 (o o) ; 1 0 m M Tr i s /HCI f o r p H va lues 7 .7 ( a A) a n d 8 .7 (¢ --); NaCl , as i n d i c a t e d o n t h e

absc i s sa ; d o u b l e - s t r a n d e d R N A , 2 0 ~ g / m l ; e n z y m e , 0 . 1 - - 2 0 Dg/ml . (b) A c t i v i t y o f c ro s s - l i nked d i m e r s o f b o v i n e R N A a s e A. R e a c t i o n m i x t u r e s : b u f f e r s f o r p H values 5 (~ ~), 7 (o o) a n d 8 .7 (e - ') , as d e s c r i b e d u n d e r (a) ; NaCI, as i n d i c a t e d o n t h e absc issa ; d o u b l e - s t r a n d e d R N A , 2 0 ~ g / m l ; e n z y m e , 0 . 1 - - 2 0 / ~ g / m l . (c) A c t i v i t y o f b o v i n e s e m i n a l R N A a s e . R e a c t i o n m i x t u r e s : b u f f e r s f o r p H va lues 5 (~ ~), 7 (o o) , 7 .7 (A Jr) a n d 8 .7 ( - - ') , as d e s c r i b e d u n d e r (a) ; NaCI, as i n d i c a t e d o n t h e absc issa ; d o u b l e - s t r a n d e d R N A , 2 0 Dg/ml ; e n z y m e , 5---30 Dg/ml . (d) A c t i v i t y o f p i k e - w h a l e p a n c r e - a t i c R N A a s e . R e a c t i o n m i x t u r e s : b u f f e r s f o r p H va lues 5 (L A), 7 (o o) a n d 8 .7 ( - - ' ) , as d e s c r i b e d u n d e r (a) ; NaCI, as i n d i c a t e d o n t h e absissa; d o u b l e - s t r a n d e d R N A , 2 0 Dg/ml ; e n z y m e , 0 . 0 5 - - 5 .9 Dg/ml . In all e x p e r i m e n t s t he v o l u m e o f i n c u b a t i o n was 1 m l ; t e m p e r a t u r e , 2 5 ° C . Assays we re p e r f o r m e d as d e s c r i b e d u n d e r M e t h o d s .

Table III shows Tm and hyperchromicity values of poly(A) • poly(U) in the presence (section B) or absence (section A) of 2 mM MgCl=. While in the absence of MgCI= Tm values increased regularly by increasing the concentration of NaC1, in the presence of Mg 2+ the stability of the double-helical structure of poly(A) • poly(U) decreased, as indicated by its Tm values, by increasing NaC1 concentrations.

The results o f an experiment carried out with bovine RNAase A and poly(A) • poly(U) as a substrate under the conditions of Table III, section B, are shown in Fig. 2. As it appears, enzyme efficiencies at degrading the double- stranded nucleic acid in the presence of Mg 2÷ and o f 0 .15 M (Tm, 70°C), 0 .05 M (Tm, 72.5°C) and 0.02 M (Tm, 75°C) NaC1 are in the ratio of 1 : 22 : 54, respectively. In other words, degradation of p o l y ( A ) , poly(U) occurs at definitely higher rates under low ionic strength conditions, notwithstanding a higher stability of its secondary structure.

46

T A B L E I I I

T m A N D H Y P E R C H R O M I C I T Y V A L U E S O F T H E P O L Y ( A ) • P O L Y ( U ) C O M P L E X U N D E R V A R I O U S

I O N I C C O N D I T I O N S

T h e r m a l t r a n s i t i o n p rof i l es , d e t e r m i n e d as desc r ibed in Mater ia l s a n d Me thods . C o n c e n t r a t i o n o f t he

p o l y ( A ) • p o l y ( U ) , 50 ~ g / m l . In i t ia l a b s o r h a n e e at 260 n m , 1.0.

C o n d i U o n s T m H y p e r c h r o m i c i t y

(°C) (%) Tris-HC1, NaC1 MgC12 p H 7.3 ( m M ) ( m M )

( raM)

A 10 20 n o n e 49 .0 51.5

10 50 n o n e 54.0 51.7 10 150 n o n e 62 .0 50.7

B 10 20 2 75 .0 51.3

10 50 2 72.5 51.7 10 150 2 70.0 51.8

Experiments with synthetic homopolyribonucleotides A. Activity of ribonucleases on poly(A ). The actual conformation of poly(A)

in the range of ionic strengths and pH values used in the experiments which follow was controlled by determining its thermal transition profiles at acidic and neutral pH.

8

6 >

o

o 4

u o

2

o ~ ° o o . o 5 o . ,o o.15

IN.'] Fig. 2. D e g r a d a t i o n o f p o l y ( A ) • p o l y ( U ) by b o v i n e p a n c r e a t i c R N A a s e A u n d e r va r i ous ion ic c o n d i t i o n s , a t p H 7.3 . R e a c t i n d m i x t u r e s : 1 m l o f 10 m M T I ~ / H C I . p H 7.3, c o n t u s i n g 2 m M MgC12, a n d d i f f e r e n t

concentrations "of NaCI as indicated on the abscissa; poly(A) - poly(U), 50 #g; RNAase A, 1.4--10 #g.

T e m p e r a t u r e , 25°C. Assays were p e r f o r m e d as d e s c r i b e d u n d e r Me thods . E a c h p o i n t was o b t a i n e d in

t r ip l i ca te .

47

Melting curves at pH 5 always showed a cooperative pattern, typical of a double stranded structure [11,13,31,32]; those at pH 7 were, as expected, uncooperative (data not shown), since the homopolymer is essentially single- stranded at neutral or alkaline pH [10,11,13]. The data obtained (Table IV) confirmed that the stability of the secondary structure of acidic poly(A) increases by decreasing the concentration of salt [9,10], at least to 0.003 X SSC. At salt concentrations above 0.3 X SSC poly(A) probably occurs as a supercoiled structure. Alternatively, it might aggregate [33]. This can be argued from the hyperchromicity values, which are 161% and 256% at 0.5X and 1 X SSC, respectively, and from the corresponding low values of initial absorbances (Table IV).

The activity curves of monomeric and dimeric RNAase A, and of seminal RNAase, determined as a function of salt concentration under different pHs, appear in Fig. 3. Both at pH 8.1 (Fig. 3a) and 5 (Fig. 3c) they appear at lower ionic strengths than at pH 7 (Fig. 3b).

Moreover, the activity maxima of the three enzymes at a given pH value appear to be gradually and significantly shifted towards higher ionic strengths as a function of the basicity of the enzyme proteins.

B. Activity o f ribonucleases on poly(C). Like poly(A), poly(C) undergoes definite conformational changes under the ionic strength and pH conditions of the experiments described above. At acidic pH poly(C) has a double-stranded structure, which is stabilized by the lowering of ionic strength [11,13,32] (Table IV). Thermal transition profiles determined at pH 5 were of a cooperative type, while at pH 7 melting curves were uncooperative (data not shown), indicating that poly(C) is essentially single-stranded at (and above) neutral pH [10,13].

The pattern of poly(C) degradation by the three enzyme species is shown in Fig. 4 (a and b). Unlike poly(A), poly(C) is an optimal substrate for a ribo-

T A B L E IV

T m A N D H Y P E R C H R O M I C I T Y V A L U E S OF P O L Y ( A ) A N D P O L Y ( C ) D E T E R M I N E D A T V A R I O U S SSC C O N C E N T R A T I O N S , A T p H 5

T h e r m a l trans i t ion prof i l es w e r e d e t e r m i n e d as descr ibed in the Materials and Methods . Initial absorbance were : for p o l y ( A ) , a t 260 n m , 0 ,200 f r o m 0 . 0 0 0 3 X up to 0.1 X SSC; 0 .1 6 4 a t 0.3 X SSC; 0 .201 and 0 . 1 3 8 at 0.SX and 1 X SSC, respect ive ly . These last t w o values were ob t a ined by doub l ing the a m o u n t of p o l y ( A ) in so lu t ion (see also t e x t ) . Initial absorbance for poly(C) , a t 274 n m , 0.270---0.328.

Poly(A) Poly(C)

Salt T m Hype r - Salt T m Hyper - c o n c e n t r a t i o n s ( °C) c h r o m i c i t y c o n c e n t r a t i o n s (OC) c h r o m i c i t y (n X SSC) (%) (n X SSC) (%)

0 .0003 80.3 53 0 .001 69 .5 23 0.001 81.5 49 0.01 74 .0 22 0 .003 89.5 44 0.1 71 .3 20 0.01 85 .5 47 0 .2 71 .0 0.1 80 .5 54 0.3 67 .0 16 0.3 66 .0 65 0 .5 65 .0 14 0.5 63 .0 161 1.0 62 .0 14 1 .0 59 .0 256 3.0 60 .5 11

48

1 2 0

2 8 0

u I

4O u Q

0 , , , , 0 0.1 0,3 0.5

120

80

4 0

0 0

b

0.2 0,4 0.6 0.8

n x S S C

6 0

20

0 0

C

0.2 0.4 0,6 0.8

Fig. 3. Degradation of poly(A) by monomeric and dimeric RNAase A, and by bovine seminal RNAase,

under different ionic and pH conditions. (a) pH 8.1; (b) pH 7; (c) pH 5. Reaction mixtures: 0.5 ml of n-fold concentrated SSC (brought to pH with small amounts of NaOH in (a), or HCI in (b) and (c)) con- t a ined p o l y ( A ) , 2.6 /~g ( 3 4 0 0 c p m ) , and m o n o m e r i c (¢ "-) or d imer ic (© ©) R N A a s e A, and semina l R N A a s e (A ~), 2 .3 - -8 .8 pg each. T e m p e r a t u r e , 37°C. Assays p e r f o r m e d as desc r ibed u n d e r Methods .

>

u m

u

u

¢t U)

x 10 .3 x 10 .3

8 0 0

6 0 0 ~

40C

2 0 0

a

/

0 0.2 0.4 0.6 0.8 1 2

2 o

st

0 0.2 0.4 0.6 0.8 1

. x S S C

o

Fig. 4. D e g r a d a t i o n of po ly (C) by m o n o m e r i c a nd d imer ic R N A a s e A, and by bovine semina l RNAase , u n d e r d i f f e r en t ionic and p H condi t ions . (a) p H 7; (b) p H 5. R e a c t i o n mix tu re s : (a), 0 .5 ml of n-fold c o n c e n t r a t e d SSC c o n t a i n e d po ly(C) , 7.5 /~g (4500 c p m ) an d m o n o m e r l c (¢ -') or d imer ic (o o) RNAase A, or seminal RNAase (A ~), 22--80 ng each; (b), 0.5 ml of n-fold concentrated SSC (brought to pH with small amounts of HCI) contained poly(C), 7.5/~g (4500 cpm), and monomeric (e e) or d imer ic (o o) R N A a s e A, or semina l R N A a s e (~ ~), 0 .5 - -2 .3 /~g each. T e m p e r a - tu re , 20 ° C. Assays p e r f o r m e d as descr ibed u n d e r Me thods

49

nuclease of the bovine pancreatic type, as it appears by comparing to each other the specific activities determined with poly(A) and poly(C) as substrates at neutral and acidic pHs.

However, the general behaviour of the three enzyme species at degrading poly(C) at pH 7 or 5 did not significantly differ from that shown with poly(A) as substrate, the range of ionic strengths at which the phenomena occur being only shifted toward higher values. In fact, bell-shaped activity curves were obtained, which show a general tendency to be displaced toward lower ionic strengths by increasing the pH of the reaction mixture from 5 to 7. The results of some experiments carried out at pH 8.1 (not shown) indicate that optimal activity of monomeric RNAase A occurs at ionic strength (0.1 × SSC) even lower than that determined at pH 7.

As with poly(A), the activity curves are shifted, at a given pH, towards higher SSC concentrations as a function of the basicity of the enzyme mole- cules. The phenomenon may be rather easily observed with dimeric RNAase A; less clearly with seminal RNAase.

C. Activity o f ribonucleases on poly(U). Poly(U), unlike poly(A) and poly(C), does not undergo conformational changes in dependance on ionic strength and pH. A secondary structure may only occur at low temperature [ 13]. Thermal transition profiles confirmed these assertions (data not shown).

Experiments on poly(U) digestion by monomeric and dimeric RNAase A, and .by seminal RNAase, were possible only at acidic pH (Fig. 5). At neutral or alkaline pHs the assays gave, both for the control and the digested samples, non dependable results, which were not improved by adding to the incubation mixtures bovine serum albumin, up to 200 ~g/ml.

Bell-shaped curves were also obtained with poly(U) as a substrate. Between 0.3× and 0.7 × SSC, the activity of monomeric RNAase A shows a dramatic

100

• - 80 i

u

a 60 u

4¢ e

20 / ~ . ~" - " -~

=

0 0 ,2 0 .4 0 .6 0 .8 1 .0

n x S S C

Fig. 5. A c t i o n of m o n o m e c i c a n d d i m e r i c R N A a s e A, a n d s e m i n a l R N A a s e , o n p o l y ( U ) at pH 5, as a f u n c - t i o n of SSC c o n c e n t r a t i o n . React ion mixtures: 0 . 5 m l o f n - f o l d c o n c e n t r a t e d SSC (brought to pH w l t h sma l l a m o u n t s o f HCI) c o n t a i n e d p o l y ( U ) , 1 0 # g ( 3 5 0 0 e p m ) , a n d m o n o m e r l c (e e ) o r dimerlc (o o) R N A a s e A, a n d s e m i n a l R N A a s e (4 ~), 7 6 - - 3 8 0 n g each. Temperature , 20°C. Assays per formed as described u n d e x M e t h o d s .

50

increase, while both the dimeric form of the enzyme, and seminal RNAase, are significantly less efficient, their activity being quite similar to each other.

Discussion

From the experiments presented in this work several points can be deduced. (i) Degradation of viral double-stranded RNA, of the poly(A) • poly(U) com-

plex and of the acidic forms of poly(A) and poly(C) by ribonucleases which are known to be active toward single-stranded RNAs showed, in all cases, similar patterns as a function of ionic strength and pH. These patterns are also similar to those obtained with single-stranded polyribonucleotides as substrates [ 5].

The fastest enzymic breakdown of the acidic, double-stranded forms of poly(A) and poly(C) as substrates does not occur under ionic conditions where the double-helical structure of the polymers shows its lowest stability. The same is true with the poly(A) • poly(U) complex and with viral double-stranded RNA: the susceptibility (which can be very high) of these double-helical nucleic acids to ribonuclease at tack is not necessarily correlated with a low stability of their structure. Therefore, the stability of the secondary structure seems not to be the only factor which controls the process.

(ii) The main variable which regulates enzymic degradation of double-helical RNAs, at a given pH, instead appears to be ion concentration. Therefore, this process seems to fall within the phenomena described and interpreted by Douzou and Maurel in their theory of ionic regulation of biochemical reactions [5,34,35].

The patterns of enzymic degradation of double-stranded polyribonucleotides obtained in this work may well fit this theory. At any pH, and with all substrates used, the activity curves of the enzymes tested are of the two types (bell-shaped or monotonic) described by Douzou and Maurel [5,34,35]. Both show a shift toward low ionic strengths by increasing the pH of the reaction mixture, as predicted by the theory. An exception is represented by the activity curves obtained with poly(A) at pH 5 (Fig. 3c), which show their maxima at salt concentrations similar to those at which the corresponding curves at higher pH were determined. However, in the light of the theory, this discrepancy may be explained by the reduction of the electrostatic potential of the polyanion in consequence of the salt link present in the acidic form of poly(A) between the positively charged NI of an adenine and the phosphate of the opposite strand [31].

(iii) An additional fact emerging from the results obtained is that the charge characteristics of the enzyme protein have a definite influence on its activity response to ionic strength and pH. With any polynucleot ide as substrate activity curves of enzymes endowed with a higher number and/or density or basic charges than bovine RNAase A are displaced, at a given pH, towards higher ionic strength values. This fact may explain previous observations, i.e., the greater efficiency in SSC, relative to that of RNAase A, of ribonucleases more basic than bovine pancreatic RNAase towards double-stranded RNA [27], DNA-RNA hybrids [36,37], and poly(A) [21,27], as well as the inversion of this effect occurring under low ionic strength conditions [8,38,39]. The results presented in this work show, in fact, that one can obtain very different activity

51

ratios between the various enzymes depending on the choice of the ionic strength and pH conditions of the assay. Therefore, activity values determined for different enzymes at only a certain salt concentration (at a given pH) represent just single points of quite differently shaped curves. As a practical consequence, a careful analysis of enzyme activity response to ionic strength and pH appears to be necessary in order to characterize a ribonuclease activity towards double-stranded RNA and to allow a meaningful comparison between different enzyme activities. Otherwise, the assertion that a ribonuclease (non specific for double-stranded RNA) is, under certain conditions (usually rela- tively high salt concentrations), more active or less active than another on double-stranded RNA may lose much of its significance.

Acknowledgements

We are grateful to Dr. Patrick Maurel for his advice and stimulating criticism, to Professor Martin A. BiUeter for his kind advice and careful critical reading of the manuscript, and to Professor Charles Weissmann for suggestions and criticism. We also acknowledge the help of Dr. Jaap J. Beintema, Professor Stanford Moore, Dr. Dalton Wang and Dr. Glynn Wilson in preparing the manuscript. This work has been supported by C.N.R. grant No. CT 77.01388.04.

References

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ionic control of enzymic degradation of double-stranded rna

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