Distribution, properties and specificity of polynucleotide phosphorylase in wheat roots

MICA ET BIOPHYSICA ACTA

ROPERTIES AND SPECIFIC )HOSPHORYLASE IN WHEA KESSLER

AND

r

D. CHEN

t

Plant Physiology and Biochemistry, ity In~Iitute o] Agriculture, Rehovoth ( (Received October i l t h , 1963)

SUMMARY

presence of polynucleotide phosphorylase, which cat~ catalyses the tion of nucleoside diphosphates and the 3~P-ADP exchange, was dem~ wheat :s. This enzyme was found to be active in nuclear, mitochondr mi al and ble subcellular fractions. The enzymatic activities oJ of these fr ~red in r Km values, p H and temperature relations and in their tl stab! se and ~ethylAase (EC 3.1.4.5) had no effect on poly-A formation ; (2-chlor nonium chloride and 2,4-dichlorobenzyl-tributylphosp)honium c related polymerization up to 5o %. The enzyme synthesized homopol :opolys of adenosine, guanosine, cytidine and uridine monophosphates m, from correading diphosphates. The rates of formation of poly-A, poly-G, poly-C and poly-U cell as the co composition of the copotymers were specific for J each subcellular fraction. as well

~1 ~ . . . . . . .

rD

INTRODUCTION

dies in this laboratory on the resistance of plants to, dehydration indicated close Stud: .tionships between the ability to withstand environrr vironmental stress conditions and relationshi" the metabolism of RNA 1,~. Under drought conditions a rnet synthesis of "abnormal" n c o u n t e r e d in o l i v e lleaves e a v e ~ 2 and a n d seedlings seedlin~,~ s. s_ In I n the course of this work we ,live RNAA wn~ was eencountered olive obtained indications that ulpon gentle dehydration RNA changes parallel with: the activity of polynucleotide phospho )hosphorylase. )horl No information on this group of enzymes is available for higher plants•• This paper presents evidence for the existence of polynucleotide phosphorylase inn plants and some of its properties. A second paper will discuss relations between this his enzyme and environmental influences. MATERIALS AND METHODS

Fractionation o/ the roots Wheat seedlings were g~ grown in sand in the dark. Approx. IO days after germination the roots were detached ~ed for fractionation. For dry matter determinations root samples were dried at 65 ° for 48 h. Abbreviations used: CCC, (2-chloroethyl)-trimethylammonium chloride; phosphon, 2,4-dichlorobenzyltributylphosl~phonium chloride. * Contribution from Thee National and University I n s t i t u t e of Agriculture, R e h o v o t h , Israel. 1963 Series, No. 565-E.

Biochim. Biophys. Acta, 8o (1964) 533-541

D. K E S S L E R ,

D. C H E N

ttion steps were carried out at ten cedure was a modification of the Ir roots were suspended in 700 m l , Ls gently squeezed and then again s was repeated 7 times. were first slowly stirred for 2o mi and o.ooI M CaCl~ and then with e roots were finally homogenized ooo rev./min). The cell debris was 1 ~le-coated cheese cloth. The residuq fibers and with a protein-N content of less than 2 ~o of ' the tot ,ts, was discarded. Nuclear [raction: The filtrate was now centrifuged ed fc for 15 rain inco Model L ultracentrifuge (Step I). The supernata )ernatant was r atment. The pellet was suspended in 5o ml Tris buffer buffei (0.2 M, M sucrose and centrifuged for IO min at 34 ooo ×g. This " proc :e more. The precipitate was then washed twice with Tris but t finally suspended in IO ml of the same buffer. This fraction fr wa nuclear material as determined microscopically (ace! acetocarminining) and contained about 8o % of the total D N A ('(Table I)

)etween ibed b y C1. Tile 7oo ml ml Tris mposed ~¢ith an passing mainly of the g in the further Ltaining epeated pH 7.4) mainly flet-KI ed with

TABLE I rRIBUTION OF I ~ N A , D N A AND PROTEIN IN VARIOUS SUBCE SUBCELLVLARFgACTIOSS (AS FRESH TISSUE)

Subcellular traction

Nucleus Microsomes Soluble cytoplasm

RNA

DNA

Protein

2.4 88.6 9.o

81.5

5.3 2. 7 92.0

I8. 5

o

% OF g

henylamine e. l lae main c o n t a m i n a n t s of this nuclear nucleai fraction were starch grain., diphen~ ins which could not be separa,ted with the aid of other methods eitheff. I n comparable studies;s of polynucleotide phosphorylase activity (as follows ) ions obtained b y the m e t h o d of JOHSSTON et al. 7 and th~ between the nuclear fractions the above outlined procedure the latter gave a considerably higher 3~P-ADP exchang~e activity. ted the solubilization of polynucleotide phosphorylase b y3 Various authors reported t( an ultrasonic t r e a t m e n t s . F oorr this purpose the nuclear fraction was agitated for up to mic oscillator (MSE). No obvious increase in 8~P-ADt DP 30 min in a 20 kcycles somc re exchange activity was obtaained and nearly 60 ~o of the initial e n z y m e activity reauclear pellets. This t r e a t m e n t was, therelore, omitted mained in the insoluble nuclear mitted. aln Mitochondrial [raaion:~: The s u p e r n a t a n t of Step I was centrifuged for IZ mii at 41 ooo × g (Step 2). The.' precipitate was suspended in Tris buffer (0.2 M, p H 7-4 7-4) nd recentrifuged for 12 min at 80 ooo x g. The mitochon ~choncontaining o.2 M sucrose and washed twice and resuspended finally in IO ml Tris buffel drial precipitate was now washed !fer (0.2 M, p H 7-4). B i o c h i m . B i o p h y s . Aura, 8o (1964) 5 3 3 - 5 4 .I

E PHOSPHORYLASE IN WHEAT RO0

toplasmatic [raction: The supern~ too ooo ×g (Step 3). The microsoII Tris buffer (0.2 M, pH 7.4)- The ~toplasmatic fraction. DNA and proteins between the The small amount of DNA found m a slight contamination of nuclei

Step 2 te was t from :ellular Jsomal

Aiter prehminary methodologacal logical studies the following follow~ meth~ opted: A D P exchange with 3,p: This procedure followed the th~ original GRUNG-MANAGO et al. 3. The standard incubation mixture contained c o.Iml buffer (0.2 M, p H 7.8), o.I ml ADP (4.1o -3 M), o.02 ml MgC1 ~.o5 ml in potassium phosphate buffer (pI4 7.8), o.I ml enzy~'me soluti ; made to volume. After incubation for 2o min in a metabolic shaker the th~ tubes w~ in an NGER lo bath. The following assay steps were adopted from ]N I E L S E N nodified by AVRONn. ~BERG 8 Polymerization o[ [8-14C]ADP: The procedure of ILITTAUER slightly modified as follows: the incubation mixture of 0.2 m] ~sed of • ml Tris buffer (o.I M, pH 7.6), 0.03 ml ADP (o.< o.o3M), o. ;]ADP )o counts/min per reaction mixture; specific activity activit 25.8/* ~.oi ml ;13 (o.oi M), and o.I ml enzyme solution. After incubation incu] for so min in a mettic shaker at 3 °0 , the reaction was stopped byr ice-cold ice-col IO % trichloroacetic acid abolic and 0.5 ml casein (2 %) was added as carrier. The mixture mixt was left in the cold for nin after which it was centrifuged and the precipita 15 mln ntate washed twice with cold s%, perchloric acid. The precipitate was dissolved in o.5 ml KOH (0.05 M). o.I-ml aliq uots were dried on planchets and counted in a gasas-flow counter. Unit enzyme activit" vity is expressed as mmoles incorporated per ml enzyme el solution per second. Aliquots of the acid-insoluble precipitates of t h e various fractions were hydrol[ysed with o.3 N KOI-I for 2o h at 37 °. The hydrolysa~ ,sate was chromatographed on ~l~'mcln No. ~'~'t TI ~paper ' ~ O r ~n~t - h r n m a f n o ~ - ~ r n scanned for radioactivity. Whatman and t'ho the ~towolnnocl developed tchromatograms Polymerization o/A DP,~, GDP, CDP and UDP: For polymerization experiments the following radioactive riboside fiboside diphosphates were employed (Schwarz Bio-Research, Inc.): [8-14C]GDP (sp. ;p. activity 19/~C/mmole), [2-14C]CDP (sp. activity 24/zC/ mmole), [8JaC]ADP (sp. activity •tivity 28. 7/,C/mmole), [2-14C]UDP (sp. activity 16.5 #C/ mmole). The reaction mixture xture contained: 0.04 ml Tris buffer (o.oi M, p H 7.6), 0.02 ml [14C]nucleoside di"phosphate (4/~M), o.oi mlMgC1, (o.i M) and o.i ml enzyme solution. The reaction ion mixture was brought up to volume with Tris buffer. In the case of copolymer synthesis 1thesis the reaction solution contained 0.06 ml of a mixture of the above four nucleoside ;ide diphosphates at equimolar concentrations of 4 # M each. All preparations were~carried out in an ice bath. The reactions were continued for so min in a shaking incubator ubator at 37 °. After incubation the tubes were immersed in an ice bath, adding 7 % IKC104 and casein as carrier. The mixture was now centrifuged for 20 min at 5000 rev. rev./min. The acid-insoluble precipitate was washed twice with 1 % HC104 and then dissob dissolved in 0.05 N KOH. Finally aliquots were dried on planchets and counted in a• gas-flow counter. Biochim. Biophys. Acta, 80 (1964) 533-541

B. K E S S L E R ,

D. C H E N

the polymer composition the ak vernight with o.3 N K O H at 37 °. n precipitated with o.I ml HC104 was adjusted to p H 7. Aliquots oi ?. I paper with isopropanol-HC1 After development the chromatogn ith o.I N HCl and counted in the

soluble arts of d after e chro,panol:anned, ounter.

RESULTS

Distribution o/polynucleotide phosphorylase: All proh protoplasmatJ gnucleotide phosphorylase activity, as illustrated in ir Table I :tion exhibited highest specific ADP-polymerization activity

~howed osomal soluble

TABLE II 'RIBUTION

BETWEEN

cellular location

leus :osome Soluble cytoplasm

SUBCELLULAR F R A C T I O N S O F A D P - P , OLYMERIZI PHOSPHORYLASE ACTIVITY

Micro-units en~ytne (m/~M ADP[ rot/see)

Total microunits per g fresh matter

Distribution o/micro-units (% o/total)

Specilic activity (units/g protein)

113 50 6I

38 83 2o 3

11.8 25.6 62.6

0. 7 3.7 0. 3

LEOTIDE

4.0 4.0 1.112

e present in the roots. cyto~plasm contained over 62 % of the total micro-units enzyme rmerization products and their chroHydrolysis of the acid-insoluble ADP-polymerizatic to the RF of the corr( matcographic analysis showed one radioactive spot corresponding monomer. re~nlt~ were wf:re oobtained aSP-ADP exchange ex )btained for the a*P-ADP which also showed Parallel results

TABLE szP-ADP

nI

GE ACTIVITY OF VARIOUS SUBCELLULAR FRACTIONS EXCHANGE

Fraction

Specilic activity

Nucleus )chondria Mitoc Microsome tble cytoplasm Soluble

15o 420

I75 4°

a similar distribution bet~veen the various protoplasmatic fractions (Table I I I ) .

~bstrate concentrations: The concentrations of the enzyme Effect o/enzyme and substrate solution in the reaction m ixtures were in the linear range of substrate-enzyme Biochim.

B i o p h y s . Acre, 8 0 (1964) 5 3 3 - 5 4 1

)E P H O S P H O R Y L A S E

IN W H E A T

RO

or ADP :osomal

rage and ADP incorporation. The. 3r the nuclear fraction, 4.o.Io -3 t oluble I'raction. temperature optima ol both the (Fig. I). On the other hand, the mi num but its activity increased.,

I

s'~

/.~,'

I

/

I/

',

I

1

microenzyme to 7 °0

I

X

*

,,oo b-

eV

* ....... %--

. . . . . .

-o

\ 1

Fig.• i. T e m p e r a t u r e

relations

I

I

i

I

I

of n u c l e a r a n d m i c r o s o m a l

I:

I

I

p o l y nnucleotide phosphorylase activitlY

(sgP-ADP exchange).

1

I

I

I

I

I

E

~00

6OO I

I

I I I I J rEM~f'~ro~lr Fig. 2. Temperature relations of mitochondrial polynucleotide phosphorylase activity (ssP-ADI sP-ADP exchange). BiocMra. Biophys. Acta, 80 (i964) 533-541 533-54]

B. KESSLER, D. CHEN

mitochondrial fractions were pre respectively (Fig. 3). Both fracti( atment. Preheating the nuclear heating periods became inhibitor 2 ed linearly to the preheating peri ts do not agree with other report., ven short preheating period s.

ffuuu

0

I

I

70° for ed dif5 min .st, the entire ctivity

I

NUCLEAR

~oo~ i -

~000

--

i

2000

.. ~ "

Li [

/,D'MITOCHONDRIAL FRACTION

I

1

10 20 HINUTES

I 30

ntervals upon the Fig. 3. The effect of preheating at 7°° for various time iinterval )f the nuclear and mitochondrial fractions. activity of

32P-ADP exchange

lear, microsomal and mitochondrial fractions was assayed The activity of the nuclear after storage for 4 weeks at --15 °. The nuclear fraction remained stable and exhibited it the end of 4 weeks storage. Under the same conditions 93 % of its initial activity at the microsomal fraction retained rained 76 % and the mitochondrial fraction only 55 % of their initial activities. pH: pI-I optima of the nuclear and microsomal fractions ranged between 6 and 7 (Fig. 4) above which the activity decreased markedly. Again, the mitochondrial enzyme behaved differently; y; its maximum 3~p exchange activity was attained at pH 6.8 (Fig. 5), levelling off If to a plateau. E//ect o/RNAase, DNAase, !ase, CCC and phosphon: RNAase and DNAase (EC 3-1.4.5 ) had no effect upon the pol)ymerization of ADP, while CCC and phosphon were activating (Table IV). lular fractions of wheat roots are able to polymerize Speci/icity: All subcellular Biochira.

Biophys.

Acta,

8o (1964) 533-541

)E PHOSPHORYLASE IN WHEAT ROq

I

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I

~,'I

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\ - - .

--4

..,~ • 4. T h e e t f e c t o f p H

]

upon

I

I

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$

6

?

#

the 82P-ADP

exchange

I 9

activity

pU of the nu

I

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:rosomal

fractions.

~ , ~ o o -'-+"

~I00

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--

--~,

]

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4;

5

6

7

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9

~. The T h e eeffect f f e c t of o f pH nI-[ upon ur~on the t h e s:P-ADP s2P-ADP e x e h a n ~ , e activity actlvit'~ of the mitochondrial fraction Fig.• 5exchange n

TABLE IV THE EFFECT OF : R N A A s E , D N A AASE, s E , CCC AND P~OSP~ON ON TaE YORMATIONOF POLY-A CATALYSEr TALYSED BY THE NUCLEAR FRACTION

Treatment

Counts/rain

Control l%NAase DNAase CCC Phosphon

198 186 I9o 278

308

Change ]rora control

(%)

6.1 -- 4.o +40.5 +55-5 --

( T a b l e V) h o m o p o l y m e r s a n d c o p o l y m e r s of A D P , G D P , U D P a n d C D P . T h e rrat~ ate of s y n t h e s i s of t h e c o p o l y mh e r A G U C c o n t a i n i n g t h e f o u r species of m o n o n u c lHeotide eotid~ u n i t s is c o n s i d e r a b l y s l o w e,'r r t h a n t h e r a t e s of f o r m a t i o n of h o m o p o l y m e r s , as foun~ found Biochim. Biophys. Acta, 8o (1964) 533-54] 533-541

B. K E S S L E R ,

D. C H E N

,l. 9 and OCHOA AND MIIlZ This difJ larkedly slows down the synthesis ( rizing reaction and its relative spe~ The difference between the subc TABLE

S

AND

r in the :ions is

V

COPOLYMERS

CLEAR,

be due FS9,12,13.

MICROSOMAL

FROM AND

EQUIMOLAR SOLUBLE

P0

)F

RIBO-

E

PHOS-

PHORYLASE

ate incorporation E,zyme localion

per

reaction

G

A

C

U

373 400 189

12o 4 581 507

422 94 176

206 345 286

35 39 41

12o 118 98

I3O 88 115

55 42 5°

mixtu 2 -

n.

*e

line

~opolymers:

[ucleus licrosome oluble fraction

3 D

4

~olyrners :

rucleus I_icrosome oluble fraction

I I o

3 I

4

purine/pyrimid copolyst pronounced in the ratios of AU/GC and of purine s while rs these ratios are similar in the case of the nuclear and solu (Table V). The purine )urine/ se of the microsomal fraction are considerably higher higl fraction. The poly-7-imidine ratio for homopolymers differs for each subcellular sut md the relatively high rate of o merization rization of GDP is frequently rather restricted 14 and primers 19 in the wheal heat ~P incor incorporation might be related to the presence of suitable su GDP root enzyme preparations. DISCUSSION

~side exchange and ribonucleosid~ Both th assay methods employed in this work, i.e., 3zp e: dstence )itates, demonstrated the existence hosphate incorporation into acid-insoluble precipitates diphos t nhn~nhnrvl~e netivitie~ in the nuclear, microsomal, mitosphorylase activities mito of nnlvrihnmmlo~ticl~ polyribonucleotide phosphory md ~cellular fractions of wheat roots. Other authors foun( chondrial and soluble subcelh solsimilar enzyme activities in the nucleus 15, ribosomes ~6,~7, cell membranes ~8 and sol lisuble portion of the celP *, but no studies were reported on the total subcellular dis Ir tribution of polynucleotidee phosphorylase activities, which still is uncertain 19. In ,luble lear enzyme activity seems to be located in the insolubh the present study the nuclear anot be released b y ultrasonic treatment. 62.6 % of the th~ fraction from which it cannot ile ~horylase activity was located in the soluble fraction whih total polynucleotide phosphor~ lso the highest specific activit:y was exhibited by the microsomes. The latter were als( OSdistinguished by a relativehly high purine/pyrimidine ratio of ribonucleoside diphos COphate incorporation with regard to the formation of both homopolymers and co polymers. tots :ure optima of the enzyme preparations from wheat root, The p H and temperature )m orted for polynucleotide phosphorylase preparations frorr were lower than those reported aich es. It is as yet premature to comment upon this fact whict bacterial and animal sources. might be closely related to the different natural environment of plant life. Biochim.

B i o p h y s . A c t a , 8o (1964) 5 3 3 - 5 4 .1

)E PHOSPHORYLASE IN WHEAT RO vet c o n c l u d e w h e t h e r t h e d i f f e r e n t iphosphates are due to different e t i o n s , t o d i f f e r e n t e n z y m e specific l i b i t o r s ~° w h i c h m a y b e p r e s e n t ii l o w e r v a l u e of t h e a p p a r e n t M i c h a m p e r a t u r e a n d p H r e l a t i o n s of the all o t h e r f r a c t i o n s .

lymerillations ifferent rations. t of t h e rial en-

ACKNOWLEDGEMENT

r t h a n k s a u d u e t o Mr. I. KIMHI for v adl u a b l e sugg, e s t i o n s i r *P-incorporation assays.

)P and

REFERENCES

18

1~ 18 19

3. KESSLER, Advan. But., 2 (1961) 1153. 3. KESSLER AND JUDITH FRANK-TISHEL, Nature, 196 (1962) 542 • 2. H. WEST, Plant Physiol., 37 (1962) 565 . ?. O. P. Tso AND C. S. SATO, Exptl. Cell Res., 17 (1959) 227?. O. P. Tso AND C. S. SATO, Exptl. Cell Res., 17 (1959) 237. ~. DISCHE, Mikrochemie, 8 (193o) 4. ?. B. JOHNSTON, G'. SETTERFIELD AND H. STERN, J. Bioph) >hys. Bioche~ 959) 53. vl. Z. LITTAUER AND A. KORNBERG, J. Biol. Chem., 226 (I9~ 1957) lO77. ~. GRUNBERG-MANAGO, P. J. ORTIZ AND S. OCHOA, Biochi~ Biochim. Biophys. 56 ) 269. ;. O. NIELSEN AND A. L. LEHNINGER, J. Biol. Chem., 2I 5 (ic (1958) 558. ~I. AVRON, Biochim. Biophys. Acta, 4 ° (196o) 257. 3. OCHOA AND S. MII, J. Biol. Chem., 236 (1961) 3303 . [. T. TILDON AND J. SZULMAJSTER, Biochim. Biophys. Acta, 47 (1961) 199. ~I. F. SINGER, L. A. HEPPEL AND 1R. J. HILMOE, J. Biol. Chem Chem., 235 (I96O) 738. R. J. HILMOE AND L. A. HEPPEL, J. Am. Chem. Soc., 79 (1957) (1957 4810G~r. . E. STRASDINE, L. A. HOGG AND J. J. R. CAMPBELL, Biochi' Biochim. Biophys. Acta, 55 (1962) 231I. H7I. . EE. . WADE AND S. LOWETT, Biochem. J., 8z (1961) 319. p•.. MC~AMARA AND A. ABRAMS, Federation. Proc., 20 (1961 1961) 362. MVl. . GRUNBERG-MANAGO, Ann. Rev. Biochem., 31 (1962) 3Ol. ~.. F. STEINER AND R. F. BEERS, Jr., Polynucleotides, Elsevier,', Amsterdam, I96O, p. 404 .

Biochim. Bi~ Biophys. Acta, 8o (1964) 533-541 533-54