Plant Cell Reports (1984) 3:81-84

Plant Cell Reports

© Springer-Verlag 1984

Polynucleotide phosphorylase from plant cells Eva Schumacher-Wittkopf, Gerhard Richter, and Sabine Schulze Institut ft~r Botanik der Universitfit Hannover, Herrenh/~user StraBe 2, D-3000 Hannover, Federal Republic of Germany Received July 8, 1983/Revised version received March 21, 1984 - Communicated by H. Kleinig

Abstract

Materials

T h e i s o l a t i o n of p o l y n u c l e o t i d e p h o s p h o r y l a s e (EC 2 . 7 • 7 . 8 ) f r o m s u s p e n s i o n c u l t u r e d p l a n t c e l l s of p a r s l e y ( P e t r o s e l i n u m sativum) a n d from tomato seedlings (Lycopersicon esculentum) is d e s c r i b e d . T h e p r o c e d u r e i n c l u d e s an ultracentrifugation step, a g l y c e r o l d e n s i t y g r a d i e n t c e n t r i f u g a t i o n a n d p r e p a r a t i v e gel electrophoresis under nondenaturing conditions. I s o e l e c t r i c f o c u s i n g g i v e s r i s e to a m a j o r c o m p o n e n t ( p I ~ 7.5) a n d to a m i n o r o n e (pI ~ 5). T h e e n z y m e c o n t a i n s f i v e s u b u n i t s w i t h a p p a r e n t M r v a l u e s of 160 000, 140 OO0, 70 OOO, 34 0 0 0 a n d 12 000, the 70 O O O - d a l t o n one being a glycoprotein.

Plant Material. Freely suspended callus cells o r i g i n a t i n g f r o m r o o t e x p l a n t s of P e t r o s e l i h u m s a t i v u m (parsley) w e r e g r o w n at 27oc u n d e r s t e r i l e c o n d i t i o n s in a s y n t h e t i c m e d i u m ( R i c h t e r a n d S e i t z 1970). The s u s p e n s i o n w a s a e r a t e d w i t h s t e r i l e air. T h e c e l l s w e r e h a r v e s t e d in the l o g a r i t h m i c g r o w t h p h a s e , f r o z e n i m m e d i a t e l y w i t h l i q u i d n i t r o g e n , and s t o r e d at -20oc. G r e e n s e e d l i n g s of t o m a t o (Lycopersicon esculentum "Moneymaker") were r a i s e d in a g r e e n h o u s e for 8-14 d at 1 8 - 2 5 o c in c o n s t a n t light.

Introduction Polynucleotide phosphorylase (polyribonucleotide ribonucleotidyl t r a n s f e r a s e , EC 2 . 7 . 7 • 8 ; P N P a s e ) c a t a l y z e s in v i t r o the p o l y m e r i z a t i o n of n u c l e o s i d e d i p h o s p h a t e s de n o v o or b y e l o n g a t i o n of a p r i m e r as w e l l as the p h o s p h o r o l y t i c c l e a v a g e of r i b o p o l y n u c l e o t i d e s . A l t h o u g h P N P a s e h a d b e e n d i s c o v e r e d 25 y e a r s ago, the p h y s i o l o g i c a l r o l e of this e n z y m e is still unknown. It is t y p i c a l l y f o u n d in p r o k a r y o t i c o r g a n i s m s , i.e. in b a c t e r i a ( G o d e f r o y - C o l b u r n and Grunberg-Manago 1972) a n d in c y a n o b a c t e r i a ( N o l d e n a n d R i c h t e r 1982). In c o n t r a s t to the w e a l t h of i n f o r m a t i o n on P N P a s e of t h e s e organismsjrelatively l i t t l e is k n o w n a b o u t the e x i s t e n c e a n d the p r o p e r t i e s of s u c h an enzyme in e u k a r y o t i c o r g a n i s m s . T h e r e h a v e b e e n a f e w r e p o r t s on b o t h s o l u b l e a n d m e m b r a n e b o u n d a c t i v i t i e s of P N P a s e in a n i m a l c e l l s (Godefroy-Colburn and Grunberg-Manago 1972) a n d in v a r i o u s p l a n t t i s s u e s ( B r u m m o n d et al. ]957; K e s s l e r a n d C h e n 1964; B r i s h a m m a r a n d J u n t t i 1974), b u t the e n z y m e has n e v e r b e e n p u r i f i e d a n d c h a r a c t e r i z e d on the m o l e c u l a r level. T h o s e a t t e m p t s w e r e h a m p e r e d b y the s m a l l q u a n t i t i e s of e n z y m e a c t i v i t y p r e s e n t a n d b y its i n s t a b i l i t y in d i l u t e s o l u t i o n s • T h e a i m of the p r e s e n t i n v e s t i g a t i o n s w a s to e s t a b l i s h the e x i s t e n c e of P N P a s e in h i g h e r p l a n t s a n d to e l u c i d a t e the m o l e c u l a r s t r u c t u r e of the e n z y m e .

and Methods

E n z y m e e x t r a c t i o n • 2 5 0 - g p o r t i o n s of f r o z e n p a r s l e y c e l l s w e r e s u s p e n d e d in an e q u a l v o l u m e of b u f f e r (0.2 M T r i s - H C l pH 8.5, 0 . 0 3 M M g C I 2, 0 . 0 6 M KC1, 0 . 2 M s u c r o s e , 0.14 mM phenylmethylsulfonylfluoride) and b l e n d e d for 3 x I m i n at full s p e e d in a Waring-type mixer. The resulting brei was g r o u n d in a m o t o r - d r i v e n t e f l o n - g l a s s h o m o g e n i z e r (3 x for 2 min) . T r i t o n X 100 w a s a d d e d to a f i n a l c o n c e n t r a t i o n of 2%. A f t e r i n c u b a t i o n for 15 m i n at O - 4 o c w i t h g e n t l e s t i r r i n g the s u s p e n s i o n w a s c e n t r i f u g e d at 30 0 O O • g for 5 m i n at OoC. T h e y e l l o w i s h - b r o w n sup e r n a t a n t w a s f i l t e r e d t h r o u g h two l a y e r s of 40 p m n y l o n cloth• A l i q u o t s of the f i l t r a t e (21 ml) w e r e a p p l i e d to the top of a 1.5 M s u c r o s e c u s h i o n (4 ml) f o r m e d in 0 . 0 4 M T r i s - H C l pH 8.5, O.01 M M g C I 2 , 0 . 0 2 M KC1. A f t e r c e n t r i f u g a t i o n for 150 m i n at 100 O O O g in a B e c k m a n 50.2 Ti r o t o r at O O C the p e l let w a s r e s u s p e n d e d in a m e d i u m c o n t a i n i n g 0 . 0 2 M T r i s - H C 1 p H 8.0, O.O1 M M g C I 2 , O.1 M KCI, 0 . 0 5 M E D T A , and d i a l y z e d o v e r n i g h t a g a i n s t 5 1 of T r i s - g l y c e r o l b u f f e r (TGb u f f e r ; 0 . 0 5 M T r i s - H C l pH 8.0, 0 . 0 5 M EDTA, 20% g l y c e r o l v/v) . T h e n T r i t o n X 100 w a s add e d to g i v e a t o t a l of O . 1 % v/v, a n d the l y s a t e c e n t r i f u g e d for 15 m i n a t 30 O O O • g a n d OoC. T h e s u p e r n a t a n t (= f r a c t i o n "PM") s e r v e d as s t a r t i n g m a t e r i a l for s u b s e q u e n t p u r i f i c a t i o n steps. F r o m t o m a t o s e e d l i n g s 150 - 200 g s h o o t tips were harvested, chilled, roughly chopped and h o m o g e n i z e d in p o r t i o n s as d e s c r i b e d for the c u l t u r e d cells, e x c e p t : b l e n d i n g w a s for 2 x 20 s at m e d i u m s p e e d f o l l o w e d b y 3 x 10 s at h i g h speed; the r e s u l t i n g b r e i w a s p a s s e d t h r o u g h two l a y e r s of 40 p m n y l o n c l o t h

82 before

addition

of T r i t o n

X 100.

E n z y m e assay. P N P a s e a c t i v i t y w a s a s s a y e d m e a s u r i n g the i n c o r p o r a t i o n of 1 4 C - l a b e l l e d ADP into ribopolynucleotide as d e s c r i b e d ( N o l d e n a n d R i c h t e r 1982). O n e u n i t of the e n z y m e c a t a l y z e s the i n c o r p o r a t i o n of I n m o l AMP into ribopolynucleotide at 37Oc in I min. P N P a s e e n r i c h e d in a p o l y a c r y l a m i d e g e l w a s l o c a t e d b y i n c u b a t i n g the l a t t e r in 2 m l of the t e s t m i x t u r e for at l e a s t 12 h at 37oc; the s u b s e q u e n t d e t e r m i n a t i o n of r a d i o a c t i v i t y i n c o r p o r a t e d has b e e n p u b l i s h e d e l s e w h e r e ( R i c h t e r 1973). A l t e r n a t i v e l y , the r i b o p o l y nucleotide formed was stained with acridine o r a n g e (30 p g / m l ) in d a r k n e s s . Protein determination. Protein content was d e t e r m i n e d b y a m o d i f i c a t i o n of the L o w r y p r o c e d u r e in a f i n a l v o l u m e of 0.5 m l ( S c h a c t e r l e a n d P o l l a c k ]973) a f t e r p r e c i p i t a t i o n w i t h 10% p e r c h l o r i c acid. Glycerol density gradient centrifugation. A l i q u o t s of 0.5 m l of f r a c t i o n "PM" c o n t a i n ing a b o u t 5 m g p r o t e i n w e r e l a y e r e d o n t o a 12 m l g r a d i e n t of 1 5 - 3 0 % (v/v) g l y c e r o l in 0 . 0 5 M T r i s - H C l pH 8.0 a n d 0.5 ~ 4 EDTA. C e n t r i f u g a t i o n w a s in a S p i n c o SW 40 r o t o r at 140 O O O • g for 4 h at 4oc. F r a c t i o n s of I m l w e r e c o l l e c t e d b y p u n c t u r i n g the b o t t o m of the tube, a n d e a c h o n e a s s a y e d for P N P a s e activity and protein, I s o e l e c t r i c f o c u s i n g w a s p e r f o r m e d on gel s l a b s of S e p h a d e x G 75 " s u p e r f i n e " w i t h L K B ampholines (pH 3.5-10) as r e c o m m e n d e d b y the m a n u f a c t u r e r s . F o r d e t e r m i n a t i o n of P N P a s e on the i s o e l e c t r o f o c u s i n g p l a t e s a p p r o p r i a t e s t r i p s w e r e s l i c e d i n t o s e g m e n t s of I c m w h i c h w e r e h o m o g e n i z e d in TG b u f f e r and c e n t r i f u g e d for 10 m i n at 5 O 0 0 • g; the a c t i v i ty w a s a s s a y e d in the s u p e r n a t a n t . 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 . C r u d e a n d purified enzyme preparations were analyzed electrophoretically in the p r e s e n c e of SDS as d e s c r i b e d b y L ~ m m l i (1970). P r o t e i n s w e r e stained with Coomassie brilliant blue R-250. S e p a r a t i o n b y d i s c e l e c t r o p h o r e s i s in n a t i v e c o n d i t i o n s w a s p e r f o r m e d in p o l y a c r y l a m i d e t u b e g e l s w i t h l a r g e p o r e size. C a r b o h y d r a t e s on the g e l s w e r e s t a i n e d b y e i t h e r the p e r i o d a t e - S e h ~ f f or the d a n s y l h y d r a z i n e - p e r i o d a t e m e t h o d ( E c k h a r d et al. 1976).

t o m a t o w h i c h c o n t a i n e d the b u l k of P N P a s e a c t i v i t y (= f r a c t i o n "PM"). F u r t h e r o u r i f i c a t i o n w a s a c h i e v e d b y c e n t r i f u g a t i o n of the s o l u b i l i z e d f r a c t i o n "PM" t h r o u g h a g l y c e r o l d e n s i t y g r a d i e n t . M o s t of the P N P a s e a c t i v i t y a c c u m u l a t e d n e a r the b o t t o m . I s o e l e c t r i c foc u s i n g of this m a t e r i a l (= f r a c t i o n "GG") g a v e r i s e to t w o p r o t e i n b a n d s w i t h P N P a s e a c t i v i t y r e p r e s e n t i n g a m i n o r c o m p o n e n t of pI W 5 a n d a m a j o r one of pI ~ 7 . 5 (Fig. I). A s u m m a r y s h e e t of P N P a s e p u r i f i c a t i o n f r o m t o m a t o s e e d l i n g s is g i v e n in T a b l e I. T h e data compare well with those obtained from p r e p a r a t i o n s of p a r s l e y cells. Table i. Purification of polynucleotide phosphorylase from tomato seedlings Fraction

Total protein (rag)

Crude extract

18 OOO

Polysome/ membrane lysate (fraction "PM")

Total activity (units) a

7O

Glycerol gradient 0.5 (fraction "GG") Isoelectric focusing pI ~ 5.0 pI ~7.5

O.21 O.18

43.6

2.4 x 10 -3

40.4

0.57

2.1

4.2

1.1 2.5

5.2 13.8

aNanomoles of AMP incorporated in ribopolynucleotide per min

-~ 18 0_ < E14

•

C~

.> U

Results P u r i f i c a t i o n of P N P a s e f r o m p a r s l e y c e l l c u l tures and tomato seedlings with conventional techniques which give positive results with the c o r r e s p o n d i n g e n z y m e f r o m p r o k a r y o t i c o r g a n i s m s , e.g. c y a n o b a c t e r i a (Nolden a n d R i c h t e r 1982) w a s u n s u c c e s s f u l d u e to the ins t a b i l i t y of the e n z y m e p r o t e i n . In the p r e s e n c e of ( N H 4 ) 2 S O 4 a m a r k e d loss in a c t i v i t y occurred; separation by column chromatography (OEAE-cellulose, poly (U)-Sepharose, Sephadex) g a v e r i s e to a v a r y i n g n u m b e r of a c t i vity peaks with patterns hardly reproducible. T h e s e f i n d i n g s c o n t r a d i c t the o b s e r v a t i o n s of the P N P a s e a c t i v i t y f r o m t o b a c c o l e a v e s (Brish a m m a r a n d J u n t t i 1974). F o r t h e s e r e a s o n s a l t e r n a t i v e p r o c e d u r e s h a d to be f o l l o w e d . As first step a polysome/membrane fraction was i s o l a t e d f r o m c e l l h o m o g e n a t e s of p a r s l e y a n d

Specific activity (units/mg Prot.

t~ 6

~2

./.

-.

~

C

~..../

/I

10 9 8 .7E -6:5 U .5~

~...."&"r i

m

4

5

1'0 1'5 Segment number

Fig. I. I s o e l e c t r i c f o c u s i n g of P N P a s e f r o m tomato seedlings partially purified by glycerol gradient centrifugation ( f r a c t i o n "GG"; 2 m g of p r o t e i n ) on a gel s l a b of S e p h a d e x G 75 a p p l y i n g a p H - g r a d i e n t of 3 . 5 - 1 0 . O . @ @, P N P a s e a c t i v i t y ; --- p H - g r a d i e n t . G e l s t r i p s w e r e s l i c e d i n t o 1-cm s e g m e n t s , treated with TG-buffer, and PNPase activity a s s a y e d in the e x t r a c t s .

83 F r o m the typical a c t i v i t i e s o u t l i n e d in Int r o d u c t i o n p u r i f i e d PNPase (fraction "GG") from both sources c a t a l y z e d the n u c l e o t i d e d i p h o s p h a t e p o l y m e r i s a t i o n de novo or by e l o n g a t i o n of an added p o l y n u c l e o t i d e primer as well as the r i b o p o l y n u c l e o t i d e p h o s p h o r o lysis (the n u c l e o s i d e d i p h o s p h a t e - o r t h o p h o s phate exchange was not assayed). During 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 of fraction "GG" from p a r s l e y cells in p o l y a c r y l a m i d e gels of large pore size a major a c t i v i t y band of PNPase ("I") and a m i n o r band ("II") were r e s o l v e d (Fig. 2, A and B) w h i c h c o i n c i d e d each with a p r o t e i n band (Fig. 2, C) . Band "I" stained p o s i t i v e l y for c a r b o h y d r a t e indicating its nature as a glycoprotein. Presum a b l y its p o s i t i o n in the gel does not reflect the true m o l e c u l a r mass since c h a r g e d groups of the c a r b o h y d r a t e m o i e t y may well have i n f l u e n c e d the migration. A t t e m p t s to analyze the p o l y p e p t i d e c o m p o s i t i o n of the two active bands by e l u t i n g them from the gel and s u b j e c t i n g each eluant to gel e l e c t r o p h o resis in the p r e s e n c e of sodium d o d e c y l s u l f a t e failed b e c a u s e of the low p r o t e i n c o n c e n t r a tions. E l e c t r o p h o r e s i s in large pore size gels of the two active c o m p o n e n t s of p I ~ 5 and p I ~ 7 . 5 (Fig. I) after r e c o v e r i n g them s e p a r a t e l y from the gel bed y i e l d e d one m a i n band of PNPase for each c o m p o n e n t a c c u m u l a t i n g in a p a r t i c u l a r r e y i o n of the gel (Fig. 3) . ~hese p o s i t i o n s compare well with those of the two PNPase bands r e g i s t e r e d after 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 under n o n d e n a t u r i n g c o n d i t i o n s (see above) of fraction "GG" (Fig. 2): the m i n o r c o m p o n e n t of pI 5 c o i n c i d e d in p o s i t i o n with band "II", that of pI 7.5 with band "I" thus i n d i c a t i n g their m u t u a l identity. A 1,2

A 1.! ¸

1.0 ¸

0.8

~

0.6,

15

~ 0.2

!

\ L,"

1

2

3

~,

B

c Fig. 2. G e l e l e c t r o p h o r e s i s of f r a c t i o n "GG" from p a r s l e y cells in a n o n d e n a t u r i n g polya c r y l a m i d e gel. 60-100 pg of p r o t e i n were a p p l i e d to a 3.75% (w/v) gel column (0.6 x 6 cm). S e p a r a t i o n was at 3 m A / g e l for about 2 h. PNPase a c z i v i t y was assayed as d e s c r i b e d under "Materials and Methods". A, [ 1 4 ~ A M P ~olymer s y n t h e s i z e d by g e l - b o u n d PNPase from ~-14~ADP; B, p a t t e r ~ of r i b o p o l y n u c l e o t i des formed from u n l a b e l l e d ADP and v i s u a l i z e d by staining with acridine orange; C, polypeptides after staining with C o o m a s s i e brilliant blue.

B1.2,

pl 5,0

5 6 1 Migration [cm}

1,0.

pl 7,5

1,0-

0,8-

0.8-

RO.6: u

u E



~5 2

3

~

5

6

Migrotion

7 {cm)

~

~

~

~

g

Migration

(cm)

Fig. 3. G e l e l e c t r o p h o r e s i s under n o n d e n a t u r i n g c o n d i t i o n s of the two components with PNPase a c t i v i t y o b t a i n e d by i s o e l e c t r i c focusing of f r a c t i o n "GG" from p a r s l e y cells. The two bands were eluted from the gel, c o n c e n t r a t e d and a p p l i e d to the gel column as d e s c r i b e d in Fig. 2.A, [14 0 AMP p o l y m e r s y n t h e s i z e d by the g e l - b o u n d active c o m p o n e n t of pI~5, and B, by the active c o m p o n e n t of pIm7.5.

84 From these results it is c o n c e i v a b l e that p a r s l e y PNPase exhibits two active forms w h i c h differ a p p a r e n t l y in m o l e c u l a r mass and / or charge. Since band II r e s p e c t i v e the c o m p o n e n t of pI~5 did not stain for carbohydrates, this form of PNPase o b v i o u s l y lacks the c o r r e & p o n ding structural c o m p o n e n t c h a r a c t e r i s t i c for band "I" r e s p e c t i v e the c o m p o n e n t of pI~7.5. This view is s u p p o r t e d by the results of the subunit analyses (s. Fig. 4). From e l e c t r o p h o r e s i s in S D S - p o l y a c r y l a m i d e gels (10%) it became evident that p o l y p e p t i des with apparent m o l e c u l a r masses of Mr 160 OOO, 140 O00, 70 OO0, 34 000 and 12 OOO were r e g u l a r l y p r e s e n t t h r o u g h o u t the purification up to active c o m p o n e n t of pI~7.5 obtained by isoelectric focusing (Fig. 4; lanes A, B, D). Since their amounts p a r a l l e l ed the activity of PNPase they are p r e s u m a b l y components of the enzyme. The subunit of M r~70 000 was i d e n t i f i e d as a g l y c o p r o t e i n (lane C). We have also examined the polypeptides c o n s t i t u t i n g the c o m p o n e n t of pI 5 (lane E). M o s t s t r i k i n g l y is the absence of t h e p o l y p e p t i d e s with Mr ~ 1 6 0 OOO and 140 OO0 from this active form of PNPase; moreover, from the g l y c o p r o t e i n of M r ~ 7 0 OOO only traces were detectable. A l t h o u g h a high degree of p u r i f i c a t i o n was a c h i e v e d we do not claim that these p r e p a r a t i o n s are homogeneous.

tion of c o n v e n t i o n a l column c h r o m a t o g r a p h y gave rise to a v a r y i n g number of active components w i t h d i f f e r e n t subunit p a t t e r n s indicating the i n s t a b i l i t y of PNPase as well as the influence of the c a r b o h y d r a t e m o i e t y w h i c h is p r o b a b l y charged. The plant PNPase, a p p a r e n t l y a h e t e r o o l i g o m e r of at least five subunits, is c l e a r l y disting u i s h a b l e from the "classical" p r o k a r y o t i c PNPase in respect to subunit c o m p o s i t i o n and m o l e c u l a r mass. These d i f f e r e n c e s together with the finding that the plant PNPase does not c r o s s - r e a c t with antisera raised a g a i n s t the PNPase of E s c h e r i c h i a coli seem to exclude an e v o l u t i o n a r y relationship. C o m m o n features, however, seem to be the a t t a c h m e n t of both to i n t r a c e l l u l a r membranes, and the complete activity i n h i b i t i o n by inorganic p h o s p h a t e (3 - 5 ~M) . Gel e l e c t r o p h o r e s i s under n o n d e n a t u r i n g conditions y i e l d e d two active formsof PNPase. Indications are that they are also a c c u m u l a ted into two active bands during i s o e l e c t r i c focusing of the p a r t i a l l y p u r i f i e d fraction "GG". The results from e l e c t r o p h o r e s i s under d e n a t u r i n g c o n d i t i o n s lend support to the c o n c l u s i o n that the PNPase form of low molecular mass - s e p a r a t e d as band "II" or comp o n e n t of pI 5 - lack the large subunits of M r ~ 160 000 and 140 0OO and p r o b a b l y also that of M r ~ 7 0 OOO. It is tempting to suggest that these subunits are c o n s t i t u t i v e for the h o l o e n z y m e but are of minor importance for the c a t a l y t i c activity, while the p o l y p e p t i des of M r ~ 12 000 and 34 O00 form the core enzyme c a t a l y z i n g the p o l y m e r i z a t i o n reaction. At p r e s e n t there is no answer to the q u e s t i o n w h e t h e r both forms of PNPase exist in vivo or o r i g i n a t e from a single enzyme by d e g r a d a t i o n during the p u r i f i c a t i o n procedure. Acknowledgement The authors are grateful to Dr. H. Soreq, W e i z m a n n Institute of Science, R e h o v o t / I s r a e l for s u p p l y i n g the antibody a g a i n s t p o l y n u c l e otide p h o s p h o r y l a s e of E.coli. References

Fig. 4. Sodium dodecyl sulfate gel electrophoresis in p o l y a c r y l a m i d e (5% stacking, 10% s e p a r a t i o n gel) of fractions r e s u l t i n g from various p u r i f i c a t i o n p r o c e d u r e s of PNPase from parsley cells. A l i q u o t s of the following fractions were applied to the slots of the gel: A, crude extract, B, p o l y s o m e / m e m b r a n e fraction, C,D, comp. of pI 7.5,E, of pI 5 ~ s o e l e c t r i c focusing of fraction "GG'~; C was stained with dansyl h y d r a z i n e - p e r i o d a t e for carbohydrate, A, B, D, E with C o o m a s s i e brilliant blue for protein. Discussion The results of the p r e s e n t study e s t a b l i s h the e x i s t e n c e of a PNPase in e u k a r y o t i c plant cells. The isolation and p u r i f i c a t i o n of this enzyme from both sources suffered from several shortcomings. In crude extracts and conc e n t r a t e d solutions the enzyme lost a c t i v i t y i r r e v e r s i b l y w i t h i n hours; the specific activity was quite low when compared to PNPase from p r o k a r y o t i e o r g a n i s m s (Soreq and L i t t a u er 1977; N o l d e n and Richter 1982). A p p l i c a -

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