419

Biochimica et Biophysica Acta, 517 (1978) 419--428 © Elsevier/North-Holland Biomedical Press

BBA 99124

PURIFICATION OF RNAase II BY PREPARATIVE POLYACRYLAMIDE GEL ELECTROPHORESIS

MICHAEL

LEINEWEBER

and G E O R G

R. PHILIPPS *

Botanisches Institut and Physiologisch-Chemisches Institut, University of Bonn, Bonn (G.F.R.) (Received April 1st, 1977) (Revised manuscript received August 8th, 1977)

Summary Purification of RNAase II to electrophoretic homogeneity is described. The exonuclease~is activated by K ÷ and Mg2÷ and hydrolyses poly(A) to 5'-AMP, exclusively as described by Nossal and Singer (1968, J. Biol. Chem. 243, 913-922). To separate RNAase II from ribosomes, DEAE-cellulose chromatography was used. Two additional chromatographic steps give a preparation that yields 10 bands after analytical polyacrylamide gel electrophoresis. Preparative polyacrylamide gel electrophoresis resulted in a final preparation which on analytical polyacr;ylamide gels gives a single band. A molecular weight of 76 000 -+ 4000 was obtained from Sephadex G-200 chromatography, with three bands from sodium dodecyl sulfate (SDS) denaturation and SDS gel electrophoresis. The subunits have a molecular weight of 40 000 -+ 2000, 33 000 -+ 2000, and 26 000 -+ 1000. The enzyme thus appears to consist of three dissimilar subunits.

Introduction E s c h e r i c b i a c o li RNAase II is an exonuclease, activated by K ÷ and Mg2÷ [ 1], which cat~yses the degradation of single-stranded polynucleotides to nucleoside 5-monophosphates starting at the 3'-hydroxyl end [1,2]. RNA chains with a highly ordered structure are poor substrates for the enzyme [3]. A given enzyme molecule tends to hydrolyse a single RNA chain by sequentially liberating mononucleotides from the polynucleotide chain until its 5'-hydroxyl end is released as a small oligonucleotide; it is only then that the enzyme molecule attacks another RNA chain. This mode of action has been called processive degradation [3]. The physiological role of the ubiquitous exonuclease is not * T o w h o m c o r r e s p o n d e n c e s h o u l d be directed at the present address: N e u r o l o g i s c h e Klinik, University of W/irzburg, Josef-Schneiderstxasse 11, 8700 Wlirzburg, G .F .R.

420 known. An involvement of RNAase II in the degradation of m R N A has been discussed (for review, see ref. 4). It was earlier implicated in the conversion of precursors to ribosomal RNA [5] and, more recently, in the processing of t R N A precursors [6]. The enzyme has been partially purified [1,7,8] and here we report complete purification of RNAase II and its characterization by analytical polyacrylamide gel electrophoresis. Materials and Methods Frozen cells of E. coli strain B, grown to stationary phase, where obtained from Merck, Darmstadt, G.F.R. DEAE-cellulose DE-52 was from Whatman Biochemicals Ltd., Maidenstone, England. Hydroxyapatite SC {Lot No 27 035), acrylamide, N,N'-methylenebisacrylamide, and N,N,N',N'-tetramethylethylenediamine were from Serva, Feinbiochemica, Heidelberg, G.F.R. Polyethyleneimine-impregnated cellulose plastic sheet (F 1440, LS 254) came from Schleicher and Schfill, Dassel, G.F.R. 3H-labelled poly(A) was obtained from New England Nuclear Corp., Boston, Mass. U.S.A., and dissolved in water; it was diluted to a specific activity of 1 pCi per 18.75 #mol phosphorus with unlabelled poly(A) from Boehringer, Mannheim, G.F.R. DNAase I (EC 3.1.4.5, from bovine pancreas) was from Worthington Biochemical Corp., Freehold, N.J. RNAase II activity was determined in a volume of 100 gl containing 0.187 pmol poly(A), 6.25 #mol Tris. HC1 (pH 7.0), 12 pmol KC1, 0.5 pmol magnesium acetate, 60 pl bovine serum albumin (Serva Feinbiochemica), and various amounts of enzyme. Incubation was for 2 and 5 min at 37°C and the reaction was stopped with 250 #l of ice-cold 2.5% HC104. The samples were filtered immediately through Whatman GF/C glass fiber filters. The filters were washed with a total of 750 #l HC104.1 ml of the filtrate was added to 5 ml Unisolve 1 scintillator (Koch, Ltd, Colnbrook Brucks, England) and counted in a Packard Liquid Scintillation Counter. The efficiency was determined in separate experiments in which 5 gl 3H-labelled poly(A) and 1 ml 2.5% HC104 were added to 5 ml scintillation fluid. Under these conditions, 1 #mol 3H-labelled poly(A) gave 29 000 cpm. One enzyme unit was defined as that a m o u n t of protein which released 1 pmol of AMP from poly(A) per min at 37°C. Contamination by other nucleases was excluded in two ways. For preliminary checks, incubations were made in the presence and absence of K ÷. Since RNAase II is activated more than RNAase I by K + [ 1], the ratio of nucleotides solubilized in the presence and absence of KC1 was used to estimate contamination by RNAase I. More accurate results were obtained by thin-layer chromatography. For this purpose, enzyme fractions were incubated with 3H-labelled poly(A) for various times up to 2 h, as described above. The reaction mixture was deproteinized [9] and 5 #1 of filtrate were applied to a polyethyleneimineimpregnated cellulose plastic sheet which was developed in 50 mM Na2B40~ (pH 6.8)/0.6 M LiC1 [10] together with markers for 2',3'-AMP, 5'-AMP, ADP, and adenosine. Since RNAase II yields only 5'-AMP while RNAase I yields 2',3'AMP and polynucleotide phosphorylase yields ADP, the absence of the latter nucleotides was taken as evidence that the samples were n o t contaminated by either of these enzymes. Also, the absence of free adenosine was taken as evidence that 5'-nucleotidase was n o t present.

421 Protein concentrations were determined by the method of Lowry et al. [11] using bovine serum albumin as standard. The protein concentration of purified RNAase II was estimated from analytical polyacrylamide gels by comparison of stained gels after separation of fractions IV and V (Table I). The gels were stained with Coomassie Brilliant Blue G 250 and, after destaining, the intensity of the retained stain in the different bands was monitored in a Gilford ultraviolet spectrophotometer at 550 nm. The intensity of the stain in the RNAase II band was then compared (after integration) with that of the stain in all protein bands. A b o u t 1/30th of the proteins in fraction IV constituted RNAase II. Preparative gel electrophoresis was performed in an Ultraphor apparatus (Colora Messtechnik, Lorch, Germany) as described by the manufacturer. Buffers used were: (1) for separating gels: 0.37 M Tris • HC1 (pH 8.5)/0.01 M magnesium acetate; (2) for stacking gels 0.06 M Tris • HC1 (pH 7.5)/0.01 M magnesium acetate/10% glycerol; (3) in electrode vessels: 0.05 M Tris/0.4 M glycine (pH 8.4}/0.01 M magnesium acetate; (4) for elution of proteins: 0.12 M Tris • HC1 (pH 8.5)/0.01 M magnesium acetate/5 mM ~-mercaptoethanol/10% glycerol; (5) for the concentration of proteins: 0.36 M Tris • HC1 (pH 8.5)/0.03 M magnesium acetate/5 mM ~-mercaptoethanol/10% glycerol. The stacking gel was 2.5% acrylamide/0.6% N,N'-methylenebisacrylamide; the separating gel contained 5.5% acrylamide/0.14% N,N!-methytenebisacrylamide. For the estimation of the molecular weight of the enzyme, Sephadex chromatography on G-200 was used. The buffer was 0.1 M potassium phosphate (pH 6.9)/0.1 M NaC1/5 mM magnesium acetate and the column was operated b y upward flow. Markers were from Serva Feinbiochemica, Heidelberg (MS II collection). For analytical gels, the same buffer system and the same acrylamide concentration was used as for preparative gel electrophoresis. Analytical gel electrophoresis was performed either in the analytical chamber of the Ultraphor apparatus or by the technique in ref. 12. Gels were stained with Coomassie Brilliant Blue in 7% acetic acid. 5 and 10% gels were used as in ref. 13. Protein markers were ~-globulin (Miles-Seravac), horse myoglobin (Serva), and c y t o c h r o m e c (Serva). Results

Purification of RNAase II All operations were performed at 4 ° C. Frozen cells were allowed to thaw in buffer I {0.02 M Tris-HC1 (pH 7.6)/12 mM magnesium acetate/10 mM EDTA/5 mM ~-mercaptoethanol/10% glycerol). They were passed through a , French Pressure Cell at 60--80 kP/cm 2. To the highly viscous solution were added 100 pg of DNAase and the mixture was stirred for 20 min. It was then centrifuged in a Sorvall rotor SS 34 for 10 min at 15 000 rev./min. The supernatant was decanted and dialysed overnight against buffer I. This solution was applied to a first DE-52 column equilibrated with buffer II (0.02 M Tris • HCI (pH 7.6)/10 mM magnesium acetate/5 mM ~-mercaptoethanol/10% glycerol). The column was eluted stepwise with buffer II containing 0, 0.1, and 0.2 M NaC1. Ribosomes and some proteins were eluted in the first step; proteins containing RNAase II activity in 0.1 M NaC1, and other non-nucleolytic proteins in 0.2 M. Fractions containing RNAase activity were concentrated by overnight

422

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-8

120

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12

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40

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20

30

40

50

60

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F'roction number

F i g . 1. C h r o r a a t o g r a p h y o n D E A E - c e l l u l o s e . T h e c o l u m n ( 2 0 × 2.5 c r a ) e q u i l i b r a t e d w i t h b u f f e r II, w a s l o a d e d w i t h 7 0 0 r a g o f fTaction I I ( T a b l e I) in 1 2 0 m l . T h e f l o w r a t e w a s 1,5 r a l / r a i n a n d 7-ral f r a c t i o n s w e r e c o l l e c t e d . T h e c o l u m n w a s w a s h e d till f r a c t i o n 23 w i t h b u f f e r I I . T h e b u f f e r w a s t h e n a d j u s t e d t o 0.1 M NaC1 a n d , s t a r t i n g w i t h f r a c t i o n N o . 51 w i t h 0.2 M NaC1. E n z y m a t i c a c t i v i t y w a s d e t e r m i n e d o n 50-pl a l i q u o t s . F r a c t i o n s 3 3 - - 4 9 w e r e c o l l e c t e d ( 1 2 0 ral) as i n d i c a t e d b y t h e b a r . o o, a h s o r b a n c e at 280 nra; a A e n z y m a t i c a c t i v i t y in u n i t s × 10 -3.

dialysis against buffer II containing 30% polyethylene glycol type 20 000. This fraction contained nucleases other than RNAase II, since the release of AMP from poly(A) increased only by a factor of two in the presence of KC1, whereas with our most purified fractions this increase was four times under these conditions. A second DE-52 column equilibrated with buffer II was loaded with this sample. After washing the column with buffer II, the enzyme was eluted with 0.1 M NaC1 as above. As shown in Fig. 1, all RNAase II activity was eluted with this buffer while another protein fraction was retained on the column and could only be eluted with higher salt concentrations. Fractions indicated in Fig. 1 were pooled and tested for activity with and w i t h o u t KCI. The results indicated that the sample did not contain appreciable amounts of other nucleases. This sample was then concentrated in the presence of 30% polyethylene glycol. Three-quarters of this concentrate was applied to a hydroxyapatite column used as supplied by the manufacturer in a b o u t 1 mM phosphate buffer (pH 6.8). The column was washed with 5 mM potassium phosphate (pH 6.8)/5 mM fl-mercaptoethanol/10% glycerol. The proteins were then eluted in a stepwise manner as indicated in Fig. 2 with increasing concentrations of potassium phosphate (pH 6.8). Most of the RNAase II activity was eluted with 75 mM phosphate buffer and fractions 39--46 were collected. The material was concentrated in buffer III (0.01 M Tris • HC1 (pH 8.5)/5 mM magnesium acetate/5 mM fl-mercaptoethanol/10% glycerol) containing 30% polyethylene glycol. Aliquots of 15--20 mg protein in buffer III were used for preparative polyacrylamide gel electrophoresis. Immediately after the proteins were eluted from the gel, they

423

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Fig. 2. C h r o m a t o g r a p h y o n h y d r o x y a p a t i t e . T h e c o l u m n w a s 18 X 2.5 c m a n d n o t e q u i l i b r a t e d b e f o r e use. I t w a s l o a d e d w i t h 3 0 6 m g (45 m l ) of c o n c e n t r a t e d f r a c t i o n I I I ( T a b l e I). A f t e r all p r o t e i n h a d b e e n a b s o r b e d ( t u b e N o . 7), t h e c o l u m n w a s w a s h e d w i t h 1 2 5 m l of 5 m M p o t a s s i u m p h o s p h a t e ( p H 6.8)15 m M ~ - m e r c a p t o e t h a n o l / 1 0 % g l y c e r o l . F l o w r a t e w a s 1 m l l m i n a n d 1 4 - m i f r a c t i o n s w e r e c o l l e c t e d a n d imm e d i a t e l y analyssed f o r e n z y m a t i c a c t i v i t y . T h e c o n c e n t r a t i o n o f t h e p h o s p h a t e b u f f e r w a s raised w h e n e v e r n o e n z y m a t i c a c t i v i t y w a s e l u t e d . H e r e , R N A a s e I I w a s e l u t e d w i t h 75 m M p h o s p h a t e , in a n o t h e r e x p e r i m e n t w i t h a d i f f e r e n t b a t c h o f h y d r o x y a p a t i t e it w a s e l u t e d w i t h 50 m M p h o s p h a t e [ 1 7 ] . F r a c t i o n s 3 9 - - 4 6 w e r e c o l l e c t e d ( 1 2 4 m l ) as i n d i c a t e d b y t h e h o r i z o n t a l bar. o o, a b s o r b a n c e a t 2 8 0 n m ; ~, e n z y m a t i c a c t i v i t y in u n i t s × 1 0 -3.

6

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Fig. 3. P r e p a r a t i v e p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s . T h e p r o c e d u r e a n d c o n d i t i o n s h a v e b e e n d e s c r i b e d in t h e t e x t . T h e c h a m b e r w a s l o a d e d w i t h 6 m l o f c o n c e n t r a t e d f r a c t i o n I V ( T a b l e I), a b o u t 2 0 absorb a n c e u n i t s a t 2 8 0 n m . T h e e f f l u e n t o f t h e e l u t i o n c h a m b e r w a s c o l l e c t e d in 1 . 7 5 - m i f r a c t i o n s a t a p u m p r a t e o f 0 . 4 m l l m i n . A l i q u o t s o f 5 0 pl w e r e u s e d f o r d e t e r m i n a t i o n of t h e e n z y m a t i c a c t i v i t y (4 A) a n d a b s o r b a n c e a t 2 8 0 n m w a s r e c o r d e d (o o). O n l y f r a c t i o n s a f t e r m o s t o f t h e t r a c k i n g d y e h a d b e e n e l u t e d w e r e r e c o r d e d ( t h e high a b s o r b a n c e in t h e v e r y first f r a c t i o n s r e p r e s e n t t h e rest o f t h e d y e ) .

424 TABLE I PURIFICATION

O F R N A a s e II

Fraction

Total protein (mg)

Total activity (units)

Specific activity ( u n i t s × 10-3~

I II III IV V

8000 700 425 80 3

50.8 164.0 66.9 46 14

6.35 234.9 157.4 * 580.0 4670.0

H o m o g e n a t e afte~ c e n t r i f u g a t i o n 1st D E A E c h r o m a t o g r a p h y 2nd DEAE chromatography Hydroxyapatite chromatography P r e p a r a t i v e gel electrophoresis

* O n l y a f t e r e l u t i o n f r o m t h e 2 n d D E A E c o l u m n is e x o n u c l e a s e a c t i v i t y m a x i m a l l y a c t i v a t e d b y KC1.

were concentrated in the elution chamber. The eluate was collected in small fractions. Fig. 3 shows the absorbance profile and the distribution of enzymatic activity in the eluate. Since the protein concentration was very low, 100 pg of serum albumin in 40 pl buffer were usually added to the tubes to stabilize the enzyme. Table I summarizes the purification. Since RNAase II is rather unstable at very low concentrations, we experienced a poor increase of the specific activity at the very last step when no serum albumin was added as in Table I. In another experiment, where bovine serum albumin was added beforehand, the increase of the specific activity after preparative gel electrophoresis was 15-fold. As with other nucleases (e.g. ref. 14), the extent and yield of the purification cannot be determined properly since a number of nucleases are present in less purified fractions. The increase of the specific activity after the first chromatography was probably due to the release of latent nucleases from ribonucleoprotein particles. RNAase I [15] and about two-thirds of RNAase II [1] sediment with ribosomes upon ultracentrifugation in the presence of Mg 2÷. We have avoided this step and used 12 mM Mg 2÷ plus 10 mM EDTA after observing that under these conditions chromatography on DEAE-cellulose released the majority of bound RNAase II but only some RNAase I. An exact examination of the distribution of either enzyme between the ribosome fraction (not bound to DEAEcellulose) and the soluble proteins released with 0.1 M NaC1 was n o t undertaken since this would have been beyond the scope of this research. We also made no a t t e m p t to strip ribosomes completely of all RNAase II activity because it is easier to start with a larger a m o u n t of cells than to separate RNAase II from other nucleases. Thus, some RNAase II might have been lost in the second step. In Fig. 4 the analytical gel electrophoresis of RNAase II purified by preparative gel electrophoresis is shown. Only one band is seen which could be stained with protein dyes. The sample is compared with RNAase II-containing material which was derived from the h y d r o x y a p a t i t e column and was chromatographed on Sephadex G-200 (see below) after addition o f bovine serum albumin. This sample showed at least seven bands in addition to the fast migrating serum albumin. The original h y d r o x y a p a t i t e fraction showed 10 bands. When fractions from the beginning and the end of the gel electrophoresis peak in Fig. 3 were analysed, the same electrophoretic mobility was observed.

425

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Fig. 4. A n a l y t i c a l p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s of R N A a s e II. T h e e x p e r i m e n t w a s p e r f o r m e d in t h e a n a l y t i c a l c h a m b e r o f t h e U l t r a p h o r a p p a r a t u s . T h e gel c o n t a i n e d 5.5% a c r y l a m i d e a n d a b o u t 4 0 !ug p r o tein w a s a n a l y s e d . L e f t : p u r i f i e d R N A a s e I I ( f r a c t i o n V); right: f r a c t i o n IV a f t e r c h r o m a t o g r a p h y o n S e p h a d e x G - 2 0 0 c o n t a i n i n g b o v i n e s e r u m a l b u m i n . M i g r a t i o n w a s f r o m t o p to b o t t o m .

Physical characterization o f RNAase H Fig. 5 shows an analysis of the hydroxyapatite fraction on Sephadex G-200. To retain full activity, serum albumin was added beforehand (0.5 mg/ml). The exonuclease was eluted in a single symmetrical peak. Purified proteins were used as molecular weight markers. An apparent molecular weight of 76 000 -+ 4000 was determined for RNAase II. RNAase II was also investigated after denaturation by sodium dodecyl sulfate (SDS) gel electrophoresis. After RNAase II (about 40 #g) had been incubated with SDS and/~-mercaptoethanol for 2 h at 37°C, analysis of the gel pattern by chromoscan gave three peaks in a ratio of 1 : 1 : 1 (Fig. 6a). From three such experiments and by comparison with 7-globulin, ovalbumin, chymotrypsinogen, and myoglobin, molecular weights of 40 000 + 200.0, 33 000 + 2000, and 26 000 + 1000 were determined. For u n k n o w n reasons, some samples from another preparation gave also three bands on SDS-polyacrylamide gel electrophoresis, but in a ratio of 1 : 2 : 1 (Fig. 6b). The molecular weights of these polypeptide chains were 30 000 + 2000, 22 000 + 1000, and 18 000 + 1000. We cannot y e t explain these results, b u t under certain conditions the molecular weight unit of 40 000 may dissociate into two chains of 22 000 and 18 000.

426

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60

FPoction number

F i g . 5. C h r o m a t o g r a p h y on S e p b a d e x 6 - 2 0 0 . T h e c o l u m n w a s 95 X 2.5 c m a n d 15 m l o f c o n c e n t r a t e d f r a c t i o n I V ( 2 0 r a g ) c o n t a i n i n g a b o u t 7.5 m g b o v i n e s e r u m a l b u m i n w e r e a p p l i e d . F l o w r a t e w a s 1 m l / m i n a n d 6 . 5 - m l f r a c t i o n s w e r e c o l l e c t e d . R N A a s e II w a s e l u t e d b e t w e e n f r a c t i o n 44 a n d 50 ( p e a k f r a c t i o n 5 0 ) . The same column was used for the protein markers. The insert shows the semi-log plot for estimation of the m o l e c u l a r weight, using (1) aldolase (147 000), (2) and (3) bovine serum a l b u m i n ( d i m e r : 134 000; m o n o m e r : 67 0 0 0 ) , ( 4 ) o v a l b u m i n ( 4 5 0 0 0 ) , ( 5 ) c h y m o t r y p s i n o g e n ( 2 5 5 0 0 ) , ( 6 ) m y o g l o b i n ( 1 7 8 0 0 ) .

w o~

A Migration in dises

Migration

A in dises

Fig. 6. Densitometry tracing of SDS-denatured R N A a s e II a f t e r p o l y a e r y l a m i d e gel electrophoresis. Approx. 40 pg of RNAase II w e r e d e n a t u r e d as d e s c r i b e d in ref. 1 3 a n d e l e c t r o p h o r e t i c s e p a r a t i o n w a s as d e s c r i b e d i n r e f . 1 2 . S c a n s o f t w o d i f f e r e n t p r e p a r a t i o n s are s h o w n ; i n (a) a 1 0 % g e l a n d i n ( b ) a 5% gel was used. For details see text.

427 Biochemical characterization The product of hydrolysis of poly(A) by RNAase II was 5'-AMP, exclusively. No other nucleotides could be detected b y thin-layer chromatography. At 0.0625 M KC1, optimal activity was observed at a Mg 2÷ concentration of 1 mM. When assayed for stimulation by KC1 in the presence of 5 mM magnesium acetate, an optimal concentration of 0.04 M KC1 was found. With 1.87 #mol of poly(A) and 5 #g of RNAase II in the assay, the rate of hydrolysis of poly(A) at 37°C remained constant for 15 min and slowly decreased for the next 10 min until no substrate remained.

Discussion The aim of this study was to completely purify RNAase II. The final product of the preparation consists of a single band on polyacrylamide gel electrophoresis. The enzyme is activated by K ÷ and Mg 2÷ as described [1,2] and the product of hydrolysis of poly(A) is 5'-AMP, exclusively. It is very unlikely that the enzyme which was eluted after preparative gel electrophoresis as a single symmetrical peak is contaminated by other nucleases, because as we have observed even small changes in the protein structure of an enzyme can be visualized by gel electrophoresis (unpublished data). In view of recent reports (e.g. ref. 16) that E. coli contains many nucleases, it appears extremely important to obtain the enzymes free of other nucleases if one wishes to study the effect of a particular enzyme on a distinct polynucleotide chain such as precursor tRNA. The m e t h o d described is gentle and up to fraction IV does n o t require specialised apparatus. It is very important not only to avoid extreme ionic conditions [17] b u t also to achieve purification in the minimum time. Compartmentation in the cell results in a delicate steady state which is destroyed after homogenization. Our method requires, at the most, 3 days up to step IV. Limitations are imposed at the last step, b u t fraction IV can be stored frozen after concentration without loss of activity. Approx. 0.15--0.25 mg of protein (1.2-2.0 units) were obtained by each gel electrophoresis step when 15--20 mg of fraction IV were used. We were unable to purify more enzyme in one run since loading the chamber with more protein gave a rather poor separation (possibly by clogging up the pores of the gel). A disadvantage is the low protein concentration of the final product. Usually the enzyme was eluted with a protein concentration of a b o u t 15/~g/ml. The loss of activity could partially be avoided b y addition of albumin. Experiments are under way to concentrate the enzyme b y one of the various means available. The yield of enzyme might be higher in other partially purified preparations [1,7,8] b u t since the activity critically depends on the substrate used [18], the results cannot be compared properly. No gel electrophoretic analyses were performed in refs. 1 and 7. The final product of ref. 8 contained at least eight bands on analytical gel electrophoresis. We also tried to obtain the pure enzyme by the combination of various chromatographic methods b u t were unsuccessful. Thus, preparative polyacrylamide gel electrophoresis as final step is a valuable alternative where complete purification cannot be achieved otherwise. The molecular weight of native RNAase II was estimated b y Sephadex chro-

428 matography to be 76 000 + 4000; in ref. 8 it was 73 000 determined by the same method. Nossal and Singer [2] gave a value of 65 000 obtained by sucrose density gradient centrifugation. It appears that the native enzyme consists of several dissimilar subunits [8]; however, it is not clear h o w large they are. Using the established m e t h o d of SDS-mercaptoethanol denaturation [13], we obtained three polypeptides in all preparations but the molecular weights differed under donditions where other polypeptides gave identical results. We do not know y e t if this is due to an impeded dissociation o f subunits or to proteolytic or otherwise caused nicking of polypeptide chains during purification. The latter explanation seems to us rather unlikely since it would presume some unc o m m o n features in the primary structure of RNAase II not only because tRNA-nucleotidyltransferase obtained from the same organism under similar conditions (unpublished) did always give one polypeptide chain. Certainly, further studies are required to clarify this point. Acknowledgements This research was supported by the Deutsche Forschungsgemeinschaft. The studies were part of the dissertation of M.L. submitted to the Math.-Naturwissenschaftliche Fakult~it der Universit~it Bonn and supported b y a predoctoral fellowship. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

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