Specificity of the natural rat liver ribonuclease inhibitor towards nuclease activities of subcellular fractions

323

Biochimica et Biophysica Acta, 353 (1974) 323--333

Q Elsevier Scientific Publishing Company, Amsterdam .-. Printed in The Netherlands BBA 98036 SPECIFICITY OF T H E N A T U R A L RAT L I V E R RIBONUCLEASE INHIBIT O R TOWARDS NUCLEASE ACTIVITIES OF S U B C E L L U L A R FRACTIONS

c. GAGNON, G. LALONDE and G. de LAMIRANDE lnstitu! du Cancer de Montreal, tl6pital Notre-Dame, and Departemen! de Biochimie, Universit~ de Montrdal. Monlrdal (Canada)

(Received December 13th, 1973) (Revised manuscript received March 18th, 1974 ) Summary Th e specificity of the natural rat liver RNAase inhibitor towards various nucleolytic activities of subcellular fractions is reported. The inhibitor acts not only on the supernatant alkaline RNAase but also on ribosomal, mitochondrial and nuclear RNAases. T he o p t i m u m pH values of mitochondrial and nuclear RNAases inhibited by the RNAase inhibitor are 8.0 and 6.5. N ot m ore than 25--30% o f acid RNAase activity at pH 5.8 is inhibited. The purified inhibitor does n o t affect DNAase, poly(A) hydrolase and phosphodiesterases of the rat liver. All ribonucleases which can be inactivated by the RNAase inhibitor are stable in H2 SO4 at a final c o n c e n t r a t i o n of 0.09 M.

Introduction The presence of RNAase inhibitor in rat liver has been report ed by m any investigators [ 1--4] and various m et hods for its purification have been published [ 3 - 8 ] . Some of its physical properties were also reported [ 2 , 3 , 9 - - 1 3 ] . However, the physiological role of the RNAase inhibitor is not y e t understood. An increase o f the ratio RNAase inhibitor/alkaline RNAase was observed in tissues characterized by a high rate of RNA synthesis and cell division [14 1 9 ] . On the ot her hand, a decrease of this ratio was noted in tissues showing a low rate of protein synthesis or an increase in their catabolic activities [20 2 9 ] . In neoplastic tissues, reported results were c o n t r a d i c t o r y , some showing an increase and others a decrease o f RNAase inhibitor [ 3 0 - - 3 3 ] . The interpretation of data is very difficult due to the lack of knowledge of the specificity of the inhibitor. It has been reported that rat liver supernatant stabilizes polysomes [34-3 6 ] , polyribosome-like particles [ 3 7 ] , mRNA [38] and rapidly-labelled nuclear RNA [39] in vitro. Acid RNAase is slightly or n o t inhibited in vitro [ 2 , 4 0 ] .

32-1 Alkaline RNAase was shown to be in a latent form in the cell probat)ly complexed with RNAase inhibitor [ 2, 3, 41] . Most of these studies were carried out with the supernatant fraction or crude preparation of the inhibitor. In order to better understand the role of the inhibitor, the effect of a highly purified preparation on the various nucleolytic activities of the subcellular fractions of rat liver has been studied. Materials and Methods

Chemicals l lighly polymerized yeast RNA, salmon sperm DNA, polyadenylic acid, p-nitrophenyl-thymidine-5'-phosphate and p-nitrophenyl-thymidine-3'-phosphate were purchased from Calbiochem; pancreatic RNAase A t ype X11-A. c y t o c h r o m e c from horse heart (type 111), RNAase free sucrose and bovine serum albumin from Sigma Chem. Corp. All other chemicals were of reagent grade from J.T. Baker. Crude subcellular fractions Wistar male albino rats of an average weight of 200 g were starved for 40 h before being sacrificed by decapitation. The livers were perfused with 200 ml of ice-cold 0.25 M sucrose. The livers were then excised, chilled in cold sucrose solution and minced in a cold Plexiglass squeezer. The liver pulp was homogenized in a Potter-Elvehjem homogenizer (4 min) to give a 25% hom ogenat e in 0.25 M sucrose. Crude subcellular fractions were obtained by the m e t h o d of De Duve et al. [42[ and resuspended in distilled water. Purified subcellular fractions Rats were starved for 40 h and the livers were perfused with 0.25 M sucrose. Nuclei were prepared by the m e t h o d of Blobel and Potter [43] slightly modified. The nuclei were sedimented at 7 5 0 0 0 X g average during 50 min in a Spinco SW25.2 rotor. Mitochondria were isolated by the m e t h o d of Vignais and Nachbaur [44] 4 days after intraperitoneal injection of 170 mg of Triton WR1339 (Ruger Chem. Co. Inc.). Lysosomes were prepared by the m e t h o d of Sawant et al. [45] and ribosomes by the modified m e t h o d of Tashiro and Siekevitz (see refs 46 and 47). The subcellular fractions were resuspended in distilled water. The supernatant was obtained by a 1 0 5 0 0 0 X g (2 h) centrifugallon of the homog,'mate. Enzymatic determinations The various enzymatic activities have been determined as described below. Controls with o u t substrate or w i t hout e n z y m e were run simultaneously. Acid phosphatase. The activity was measured as described previously [48] e x c e p t that the c o n c e n t r a t i o n of ~-glycerophosphate used was 0.02 M. Glucose-6-phosphatase. This enzymatic activity was determined as reported previously 148]. Cytochrome c oxidase. The reaction mixture contained: 2.0 ml of 0.15 M potassium phosphate buffer pH 7.5, 0.8 ml of a suitable dilution of subcellular fractions and 0.2 ml of 0.625 mM reduced e y t o c h r o m e c. '['he activity was

325 measured at room t e m p e r a t u r e by the decrease in absorbance, at 550 nm using an extinction coefficient for the reaction of 18.7 • 103 1 • m o k f I • cm -~ Nuclease activities o f nuclear fractions. DNAase, poly(A)hydrolase and RNAase activities were determined in the following incubation mixture: 0.5 ml o f 0.1 M sodium phosphate buffer pH 7.0, 0.1 ml of 0.2 M MgC12, 0.45 ml of nuclear fraction, 0.2 ml of 0.001 M potassium phosphate buffer pH 6.0 with or w i t h o u t RNAase inhibitor and 0.25 ml of substrate (denatured DNA, 1 mg/ml; poly(A), 2 mg/ml and RNA, 4 mg/ml). After an incubation of 30 rain at 37"C, the reaction was stopped by addition of ice-cold ttC104 to give a final concentration of 3.33%. The reaction mixture was centrifuged at 3 0 0 0 0 X g, 10 min and the absorbance at 260 nm was used as a measure of p o l y m e r breakdown. 5'-phosphodiesterase. The activity was measured I)y a previously described m e t h o d [48] except that the pit was brought down to 8.5 and that 0.2 ml of 0.001 M potassium phosphate buffer pH 6.0 with or w i t hout inhibitor was added. Nuclease activities o f mitochondrial fractions. 1.0 ml of mitochondrial fraction was incubated with 0.5 ml of 0.1 M Tris--HCl buffer pH 7.5 containing 0.8 mM MgC12 and 0.056 M K2 H P O 4 , 0 . 5 ml of 0.001 M potassium phosphate buffer pH 6.0 with or without RNAase inhibitor and 0.5 ml of suhstrate. The same concentrations of DNA, poly(A) and RNA were used as with the nuclear fraction. After 30 min of incubation at 37°C, the reaction was stopped and treated as m e n t i o n e d above. 3'-phosphodiesterase. The incubation m i xt ure contained: 0.5 ml of 0.1 M a m m o n i u m acetate buffer pH 5.7. 0.1 ml of 0.1 mM EDTA pH 7.0, 0.9 ml of cell fraction, 0.5 ml of 0.001 M potassium phosphate buffer pH 6.0 with or w i t h o u t RNAase inhibitor and 0.5 ml of a solution of p-nitrophenyl-thymidine3'-phosphate (1 mg/ml}. The reaction m i xt ure was incubated and treated as for 5'-phosphodiesterase. Acid RNAase. This activity was measured according to the m e t h o d of Kraft and Shortman [ 2 5 ] , except that the substrate c o n c e n t r a t i o n used was 4 mg/ml and that freezing-thawing was omitted. After the incubation, the reaction mixture was treated as above. Ribosomal RNAase. The ribosomal RNAase was determined by the method of Gagnon and de Lamirande [ 4 9 ] . The incubation mixture was com posed of 0.5 ml of 0.1 M Tris--HCl buffer pH 8.0 containing 0.1 M EDTA, 0.50 mt of 0.001 M potassium phosphate buffer with or w i t hout RNAase inhibitor and 0.25 ml of ribosomal fraction. When ribosomal RNAase was measured against exogenous yeast RNA. the ribosomal fraction was incubated in the presence of 1 mg o f RNA. Alkaline RNAase. This activity was measured as previously reported [8] except that 0.001 M potassium phoshate buffer pH 6.0 was used to dilute the RNAase inhibitor. pH curves. The reaction m i xt ur e contained: 1.0 ml of 0.05 M Tris- HC1-succinate buffer of various pH, 0.02 ml of 0.1 M EDTA pH 7.0, 0.2 ml of cell fraction, 0.5 ml o f RNA solution (4 mg/ml) and distilled water to 2.5 ml. tt2SO~ treatment. H2SO, was added to a cell fraction suspension to a final c o n c e n t r a t i o n of 0.09 M. After 2 rain the mixture was neutralized by the addition of NaOH to a final pH of 7.0.

326

Isolation of RNAase inhibitor. The RNAase inhibitor was purified 1 0 0 0 fold by a two-step procedure "already described [ 8 ] . Units of RNAase inhibitor. Units of RNAase inhibitor were determined by the inhibition of pancreatic RNAase in the same conditions as those used for each different enzymatic assay. One unit of RNAase inhibitor was taken as the a m o u n t of RNAase inhibitor necessary to inhibit the activity of 1 ng of pancreatic RNAase A by 50%. Protein and DNA determination. Proteins were determined by the biuret reaction [ 5 0 ] , using bovine serum albumin as standard. DNA was measured by the m e t h o d of Schneider [51] with highly polymerized salmon sperm DNA as standard. Results

Supernatan t fraction Alkaline RNAase is not present in a free state in normal liver and incubation of the 105 000 X g supernatant with RNA, in presence of EDTA, does not produce any acid-soluble material. Indirect proof such as the effect of PCMB which re-established the RNAase activity w i t h o u t dissociating the e n z y m e - inhibitor complex suggested that the e n z y m e was inhibited in the supernatant fraction. To show if this was really the case and if the purified inhibitor could inhibit alkaline RNAase, the supernatant was treated with H: SO4 at a final concentration of 0.09 M. This treatment dissociates the enzyme--inhibitor complex and inactivates the inhibitor w i t h o u t destroying the e n z y m e [ 4 1 ] . The treated supernatant was incubated in the presence and absence of purified RNAase inhibitor at three different concentrations; the second and third being 4 and 16 times greater than the first. Table I shows that the purified inhibitor

TABLE I INHIBITION OF ALKALINE RIBONUCLEASE ACTIVITIES FROM VARIOUS T I O N S BY R A T L I V E R S U P E R N A T A N q " R N A a s e I N H I B I T O R , T h e a m o u n t o f s u b c e l l u l a r f r a c t i o n p r o t e i n s in t h e i n c u b a t i o n m i x t u r e for the supernatant, ribosome, mitochondria and nuclei respectively.

Inhibit(Jr (units)

Fraction alone CA )

Fract: )n + inhibitor (A )

Inhibition (%)

Supernatant 0.240 0.960 3.840

was 1.21,0.38,0.48

Inhibitor (units)

Fraction alone 0.11 . . . . . . . . . . . . . .

FRAC-

and 1.25mg

Fraction lnhibi+ inhibitor tion (A) (c;) . . . . . . . .

Ribosome 0.295 0.299 0.297

(I.217 0.044 0.017

27 85 94

Mitochondria 0.160 0.640 2.560

SUBCELLULAR

0.355 1 .-120 5.680

0.175 0.1 (; 1 0.153

0.146 0.056 0.037

17 (i 5 76

Nuclei . . . . . . . . . . . . . . . . . . . . . . . . 0.562 0.547 0.545

0.310 0.220 0.1 RO

45 60 67

0.100 0,,I00 1.600 . . . . . . . . .

0.1 }}9 0.202 0. 2 0 7 . . . . . . . .

O. 1 5 5 0.030 0.000 . . . .

22 85 100

327 decreases the supernatant alkaline RNAase activity by 27, 85 and 94% depending on its concentration.

Ribosomal fraction Table I shows also that the auto-degradation of ribosomes isolated by the modified method of Tashiro and Siekevitz (see refs 46 and 47) is also affected by the purified inhibitor. This enzymatic activity is decreased by 17% in the presence of 0.355 unit of RNAase inhibitor. The inhibition is increased to 65 and 76% at the two other concentrations used. The same level of inhibition was obtained when the ribosomal preparation was incubated in the presence of yeast RNA as substrate.

Mitochondrial fraction Mitochondrial fraction isolated by the m e t h o d of De Duve et al. [42] shows DNAase, poly(A)hydrolase and RNAase activities. The action of the inhibitor was tested against these enzymes. DNAase and poly(A)hydrolase activities were not affected by the inhibitor. RNAase activity, however, was inhibited by 67% as shown in Table I. It was impossible with the isolated mitochondrial fraction to obtain a greater inhibition. Since poly(A)hydrolase activity can degrade RNA [52], the fraction was treated with H2SO4 at a final concentration of 0.09 M. This treatment destroys poly(A)hydrolase and DNAase activities along with any possible complexed inhibitor. RNAase itself is only mildly affected by such treatment showing a loss of one third of its activity. Fig. 1 shows that the RNAase activity of the treated mitochondrial fraction is totally inhibited by the inhibitor. The optimal pH of this RNAase activity was determined by incubating, at different pH values, the mitochondrial fraction treated by H2 SO4 with and without inhibitor. Two peaks of RNAase activity were observed, one at pH 6.5 and the other at pH 8.0. The ratio of activities at 6.5 and 8.0 was 1.5. The inhibitor (16.5 units) decreased the activity at pH 6.5 by 42% and at pH 8.0 by 83%. A more purified mitochondrial fraction was prepared by the method of Vignais and Nachbaur [44] to eliminate as much as possible the activity observed at acid pH which might be due to a lysosomal contamination. This method gives a yield of 30% as compared to 45% by that of De Duve et al. [42]. The

0

6o

|0 100 0

08

,

,

16

24

Inhibitor

• 32

40

(units)

Fig. 1. I n h i b i t i o n o f m i t o e h o n d r i a l R N A a s e b y t h e p u r i f i e d rat l i v e r R N A a s e i n h i b i t o r . A m i t o c h o n d r i a l f r a c t i o n w a s t r e a t e d b y H 2 S O 4 a t a f i n a l c o n c e n t r a t i o n o f 0 . 0 9 M. n e u t r a l i s e d t o p H 7 . 0 a n d i n c u b a t e d with increasing amount of i n h i b i t o r . The incubation mixture contained 0.6 m g o f mitochondrial protein.

:/28

E

I

0

.

50

.

.

.

.

60

.

.

~

~

IO

~ 4 . 80

~ . 90

,

. . . .

100

pH

F i g . 2. R N A a s e a c t i v i t y a t v a r i o u s p H v a l u e s o f a p u r i f i e d m i t o c h o n d r i a l Mitochondrial fraction (0.2 mg) incubated with (0 ...... --o) and without

f r a c t i o n t r e a t e d by H 2 S O 4. (u . . . . a ) a l a r g e e x c e s s o f

RNAase inhibitor (16.5 units).

yield is calculated from the marker e n z y m e activity ( c y t o c h r o m e c oxidase) of the fraction and of the homogenate. This fraction is cont am i nat ed by only 1.7 and 0.5% of lysosomes and of microsomes as compared to 17 and 0.8% by the m e t h o d of De Duve et al. [ 421 • The contaminations were calculated from acid phosphatase and glucose-6-phosphatase activities used as markers. The ptl curve of the purified mitochondrial fraction treated with H2 SO4 is shown in Fig. 2. T w o peaks of activity are still observed, but the ratio of the activity at acid and alkaline pit, which was 1.5 as m ent i oned above, is now 0.8. This reflects the loss of activity at acid pH due to the better elimination of lysosomes. In the presence of a large excess of inhibitor the enzymatic activity at alkaline pH, which is optimal at 8.0, is totally inhibited. A low RNAase inhibitor activity at acid pH might be due to a weak capacity of inhibition at these pH values. To examine this possibility further, the inhibition capacity against pancreatic RNAase A has been studied in function of the pH. The results summarized in Fig. 3 show that the inhibition is optimal between pFl 7.0 and 8.0. However, the inhibitory activity is still 50 and 72% o f the o p t i m u m at pH 5.5 and 6.0 when 0.28 units of inhibitor are used. Consequently, 16.5 units of inhibitor were used for the determination of the pH--inhibition curve of the various cellular fractions studied. The inhibitor, in

I00

8o r

60 40

2O 0 5O

60

70

80

90

100

F i g . 3. p H c u r v e o f t h e i n h i b i t i o n o f 0 . 2 n g ()f p a n c r e a t i c inhibitor.

RNAase A by 0.28 units of the purified RNAase

329 such large excess, should probably inhibit any enzyme susceptible to its action from pH 5.5 to 9.5.

Nuclear [faction Purified nuclei were isolated by the method of Blobel and Potter [43] with a yield of 30% based on DNA content. The contamination by other cell constituants was very low; 0.07% of mitochondria, 0.44% of microsomes and no detectable presence of lysosomes as determined from the activities of the marker enzymes mentioned above. The nuclear fraction was incubated, in the presence and absence of the RNAase inhibitor, with the following substrates: DNA, poly(A), RNA and p-nitrophenyl-thymidine-5'-phosphate. The inhibitor was without effect on the DNAase, poly(A)hydrolase, RNAase and 5'-phosphodiesterase. The purified nuclear fraction was then treated with H2 SO4 as described above. This treatment destroyed completely the DNAase, poly(A)hydrolase and 5'-phosphodiesterase but showed little effect on the RNAase activity. The treated nuclear fraction was then incubated in the presence and absence of the inhibitor. Table ! shows that 0.1 unit of inhibitor caused a 22% decrease of the RNAase activity, and a total inhibition is observed in presence of 1.6 units. In order to find the optimal pH of the inhibited enzymatic activity, the pH curve was established. The upper curve, Fig. 4, shows very high activities at pH 5.5 and 9.5 for the purified nuclear fraction. After treatment with H2 SO4 (middle curve) the activity at pH 5.5 is still relatively high whereas that at pH 9.5 is greatly decreased. In the presence of 16.5 units of inhibitor (lower curve) the enzymatic activity is totally inhibited between ptI 7.0 and 9.5 whereas only traces of activity still remain at pH 6.0 and 6.5. The pH curve of the inhibited RNAase activity is shown in Fig. 5. This curve has been obtained

E ©

60

70

80

9o

pH Fig. 4. R N A a s e a c t i v i t y a t v a r i o u s p H v a l u e s o f a p u r i f i e d n u c l e a r f r a c t i o n . N u c l e a r f r a c t i o n ( 0 . 9 2 m g ) w i t h o u t H 2 S O 4 t r e a t m e n t (A . . . . A). w i t h H 2 S O 4 t r e a t m e n t i n a b s e n c e (m m) a n d in p r e s e n c e (o o) of a large excess of RNAase inhibitor (16.5 units).

330

E

10 j

/

05 1

ol 50

60

70

80

90

100

pH

Fig. 5. p H c u r v e o f n u c l e a r R N A a s e i n h i b i t e d b y t h e p u r i f i e d R N A a s e i n h i b i t o r . T h i s c u r v e w a s o b t a i n e d b y s u b t r a c t i n g t h e v a l u e s o f t h e l o w e r c u r v e f r o m t h a t o f t h e m i d d l e c u r v e o f t h e Fig. 4.

by subtracting the values of the lower curve from those of the middle curve in Fig. 4. The optimal pH for the RNAase activity of the purified nuclear fraction inhibited by the RNAase inhibitor is 6.5.

Lysosomal fraction The lysosomal fraction isolated by the method of De Duve et al. [42] has also been studied for the action of the inhibitor on its 3'-phosphodiesterase and acid RNAase activities. Table II shows that the 3'-phosphodiesterase is not affected whereas the acid RNAase activity is decreased by 22% in presence of 2.18 units of inhibitor. It was never possible to obtain an inhibition greater than 25--30%. The same results were obtained with a purified lysosomal fraction prepared by the method of Sawant et al. [45]. Discussion Ribosomal RNAase is inhibited by the inhibitor isolated from rat liver supernatant. This may explain the observation reported by many investigators that the supernatant fraction stabilizes polysomes [34--36]. They are also

T A B L E II EFFECT OF RAT I,IVER SUPERNATANT LYSOSOMES

RNAase INHIBITOR

T h e a m o u n t o f l y s o s o m a l p r o t e i n s in t h e i n c u b a t i o n 3'-phosphodiesterase deter~nination.

Inhibitor (units)

Fraction alone (A)

Acid rit)onuclease . . . . . . . 0.136 0.54-1 2.180 . . . . . . . . .

.

.

l"raction + inhibitor (A)

.

.

0.771 0.758 0.791 . . . . . . . .

.

.

.

.

.

.

.

ACTIVITIES

m i x t u r e s w a s 0 . 0 8 a n d 0.,lO m g for a c i d R N A a s e

Inhibition (%)

.

ON NUCLEOLYTIC

Inhibitor (units)

.

.

0.696 10 0.636 16 0.618 22 . . . . . . . . . . . . . . .

Fraction alone (A)

3'-Phosphodiesterase . . . . . . . . 0.300 1.200 4.800 . . . . . .

0.379 0.374 0.382

Fraction + inhibitor (A)

.

.

.

.

0.376 0.383 0.384

.

.

OF

and

Inhibition (%)

.

. 0 O 0

331

TABLE

III

INHIBITION

EACTOR

OF THE VARIOUS

RNAase

ACTIVITIES

T h e i n h i b i t i o n f a c t o r is t h e r a t i o o f t h e a m o u n t s o f i n h i b i t o r n e c e s s a r y t o d e c r e a s e b y 5 0 % t h e e n z y m e s t u d i e d a n d p a n c r e a t i c R N A a s c a c t i v i t i e s m e a s u r e d in t h e i n c u b a t i o n c o n d i t i o n s o f tile e n z y m e s t u d i e d . Fraction

Factor

Mitochondrial Nuclear Ril)osomal Supernatant

0.16 1.20 1.81 2.20

p r o t e c t e d against the s u p e r n a t a n t alkaline R N A a s e since this e n z y m e is c o m p l e tely inhibited b y t h e inhibitor. T h e a c t i o n o f the i n h i b i t o r on the m i t o c h o n d r i a l and n u c l e a r f r a c t i o n R N A a s e a c t i v i t y c o u l d n o t really be d e t e r m i n e d d u e to the p r e s e n c e o f enz y m e s in these f r a c t i o n s able to d e g r a d e R N A a n d insensitive to t h e inhibitor. H o w e v e r , a f t e r t r e a t m e n t o f the f r a c t i o n s with H2 SO4, which d e s t r o y s c o m p l e tely these e n z y m e s , a t o t a l i n h i b i t i o n o f the acid-resistant R N A a s e activity o f m i t o c h o n d r i a l and n u c l e a r f r a c t i o n s was o b t a i n e d . A c o m p a r i s o n was m a d e b e t w e e n the various inhibited R N A a s e s and an i n h i b i t i o n f a c t o r was c a l c u l a t e d f o r each o f t h e m . This f a c t o r is the ratio o f the a m o u n t s o f i n h i b i t o r necessary to decrease b y 50% the e n z y m e studied and p a n c r e a t i c R N A a s e activities m e a s u r e d in identical c o n d i t i o n s . T a b l e IIl s h o w s t h a t this f a c t o r varies f r o m 0.16 f o r the m i t o c h o n d r i a l f r a c t i o n R N A a s e to 2.20 for s u p e r n a t a n t alkaline R N A a s e . Values o f 1.20 and 1.81 w e r e o b t a i n e d for n u c l e a r and r i b o s o m a l RNAascs. H o w e v e r , o n e has to k e e p in m i n d t h a t these ratios have been o b t a i n e d with subcellular f r a c t i o n s as sources o f various enz y m a t i c activities studied. This p r o v i d e s c o m p l e x s y s t e m s in which various f a c t o r s m i g h t be involved such as i n t e r a c t i o n b e t w e e n s u b s t a n c e s p r e s e n t in the f r a c t i o n and e n z y m e or inhibitor. Nevertheless, this f a c t o r m a y give a g o o d i n d i c a t i o n o f w h a t is h a p p e n i n g in the cell organelles or in the cell as a whole. It has b e e n s h o w n in a similar s y s t e m ( D N A a s e - - D N A a s e inhibitor) t h a t the c o m p l e x f o r m e d was in the p r o p o r t i o n o f o n e m o l e c u l e o f i n h i b i t o r to o n e m o l e c u l e o f e n z y m e [ 5 3 ] . A s s u m i n g a similar s i t u a t i o n f o r the R N A a s e - R N A a s e i n h i b i t o r s y s t e m , it w o u l d thus seem t h a t it takes 2.20 times m o r e alkaline R N A a s e m o l e c u l e s to p r o d u c e the s a m e activity as o n e m o l e c u l e o f p a n c r e a t i c R N A a s e u n d e r identical c o n d i t i o n s . T h e m o s t active e n z y m e w o u l d t h e n be m i t o c h o n d r i a l R N A a s e with a f a c t o r o f 0.16. H o w e v e r , the i n h i b i t o r f a c t o r m a y also m e a n t h a t it takes 2.20 times m o r e i n h i b i t o r m o l e c u l e s to inhibit alkaline R N A a s e t h a n p a n c r e a t i c R N A a s e . If such is t h e ease the m i t o e h o n d r i a l R N A a s e w o u l d require less i n h i b i t o r m o l e c u l e s to be a f f e c t e d . B o t h i n t e r p r e t a t i o n s lead to the c o n c l u s i o n t h a t the various R N A a s e activities sensitive to the i n h i b i t o r are m o s t p r o b a b l y d i f f e r e n t e n z y m e s . T h e results o f G o t o and M i z u n o [ 4 1 ] suggest t h a t the c o m p l e x e n z y m e - i n h i b i t o r is c o m p o s e d o f o n e m o l e o f R N A a s e a n d o n e m o l e o f R N A a s e inhibitor. T h e first e x p l a n a t i o n w o u l d thus s e e m m o r e p r o b a b l e .

332 T h e p r e s e n t s t u d y has revealed a certain specificity o f the liver supern a t a n t RNAase inhibitor. This i n h i b i t o r seems to e x e r t its action on RNAases present in all cell fractions even t h o u g h its e f f e c t is r a t h e r limited on the acid RNAase activity o f the lysosomes. T h e i n h i b i t o r is specific for RNAases since it has no e f f e c t on DNAase, p o l y ( A ) h y d r o l a s e or phosphodiesterases, it seems to he especially effective on alkaline RNAases with the e x c e p t i o n of the nuclear fraction RNAase which has an o p t i m a l pII o f activity at 6.5. However, this RNAase, like the alkaline ones, is resistant to the t r e a t m e n t with If2 SO4.

Acknowledgements This investigation was s u p p o r t e d by grants from T h e National Cancer Institute o f Canada, le Ministbre des Affaircs sociales du Qudbec, La F o n d a t i o n J.H. Biermans and Les F o n d a t i o n s J. Rh4aume. C. Gagn~on is Fellow of the Medical Research Council o f Canada, G. L a l o n d e is a S u m m e r Medical S t u d e n t Research Fellow o f Le Minist~re des Affaires Sociales du Qu6bec and G. de L a m i r a n d e is Research associate of T h e National Cancer Institute o f Canada.

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