JOURNAL OF BACTERIOLOGY, May 1977, p. 869-876Copyright C 1977 American Society for Microbiology

Vol. 130, No. 2Printed in U.S.A.

Isolation and Characterization of a PolynucleotidePhosphorylase from Bacillus amyloliquefaciens

ROBERT J. ERICKSON* AND JOSEPHINE C. GROSCHMolecular Biology Department, Miles Laboratories, Inc., Elkhart, Indiana 46514

Received for publication 8 December 1976

Bacillus amyloliquefaciens BaM-2 produces large amounts of extracellularenzymes, and the synthesis of these proteins appears to be dependent upon

abnormal ribonucleic acid metabolism. A polynucleotide phosphorylase (nucleo-side diphosphate:polynucleotide nucleotidyl transferase) was identified, puri-fied, and characterized from this strain. The purification scheme involved celldisruption, phase partitioning, differential (NH4)2SO4 solubilities, agarose gelfiltration, and diethylaminoethyl-Sephadex chromatography. The purified en-

zyme demonstrated the reactions characteristic of polynucleotide phosphorylase:polymerization, phosphorolysis, and inorganic phosphate exchange with the 3-

phosphate of a nucleotide diphosphate. The enzyme was apparently primerindependent and required a divalent cation. The reactions for the synthesis ofthe homopolyribonucleotides, (A),, and (G),, were optimized with respect to pHand divalent cation concentration. The enzyme is sensitive to inhibition byphosphate ion and heparin and is partially inhibited by rifamycin SV andsynthetic polynucleotides.

The enzyme polynucleotide phosphorylase(PNPase) has been isolated from a variety ofmicroorganisms (7,21) and catalyzes the bio-chemical reaction summarized in the followingreaction scheme: NDP + (NMP),, f (NMP), +1+ Pi, where NDP is nucleoside 5'-diphosphate,NMP is nucleoside monophosphate, and Pi isinorganic pyrophosphate. The forward reactionresults in the polymerization of ribonucleoside-5'-diphosphates and has been important in thesupply of synthetic polyribonucleic acids for bi-ological research. Some forms of PNPase re-quire that n be greater than a certain criticalvalue (primer-dependent PNPase), whereasother forms will initiate synthesis from thecomponent mononucleotides (10). Primer-de-pendent syntheses have been utilized to modifynaturally occurring ribonucleic acid (RNA) byaddition of a defined sequence to the 3'-terminiof the molecule (17). The reverse reaction re-sults in the processive degradation of polynu-cleotide chains in the 3' to 5' direction (5,11),and in this capacity the enzyme has been uti-lized to remove (adenylic acid),, [(A)j] segmentsfrom eucaryotic messenger RNA (mRNA) mole-cules (20,23).The apparent ubiquity of the enzyme in mi-

croorganisms suggests an important role in cellphysiology; however, an unequivocal demon-stration of its function in cell metabolism isabsent from the literature. Two divergent, con-tradictory views have been presented to explain

the role of PNPase. One view suggests thatPNPase participates in RNA metabolism byincreasing the turnover rate of ribonucleotidesthrough the processive degradative pathway(15,18). The opposing viewpoint suggests thatthe polymerization reaction is the physiologi-cally important function and that the enzymemay modify the 3'-hydroxyl end ofmRNA mol-ecules so as to increase the half-life of the mes-sage (16). The recent demonstration of (A),, seg-ments on the 3'-termini of some mRNA mole-cules in Escherichia coli (12) may add supportto this latter viewpoint and may indicate thatonly certain classes of mRNA's are modified.Bacillus amyloliquefaciens produces large

quantities of extracellular proteins and themRNA molecules for these products have anapparently long half-life (4, 6). We have initi-ated a study ofRNA metabolism in B. amyloli-quefaciens and, in this report, describe the pu-rification and characterization of one of twoPNPase activities found in cell extracts.

MATERIALS AND METHODS

Bacterial cells. B. amyloliquefaciens BaM-2 is as-porogenic and was isolated by E. W. Boyer. Largequantities of the strain were grown and concen-trated for us by G. Mercer of the Marschall ResearchLaboratory, Miles Laboratories.

Chemicals. Isotopically labeled and unlabeledsynthetic polynucleotides are products of Miles Re-search Products Division. Tritiated NDPs and inor-ganic 32p were purchased from Schwarz/Mann, and

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870 ERICKSON & GROSCH

heparin, actinomycin D, rifamycin SV, N-ethylmal-eimide, and dithiothreitol (DTT) were obtained fromCalbiochem. The nonionic detergent Nonidet P-40(NP-40) was supplied by Shell Chemical Co. Allreagents used in the gel electrophoresis were prod-ucts of Canalco. Phenylmethylsulfonyl fluoride(PMSF) was a product of Sigma, polyethylene glycol6000 (PEG) was from Matheson, and (NH4)2SO4 (en-zyme grade) was from Schwarz/Mann. Bio-Gel A 1.5-m (200 to 400 mesh) was purchased from Bio-Rad,and the dextran T-500 and diethylaminoethyl(DEAE)-Sephadex A-50 were from Pharmacia.

Standard polymerization assay. A standard reac-

tion was carried out in a volume of 50 ul at 370 C in a

mixture that contained the following: adenosine 5'-diphosphate (ADP), 6.8 nmol; ethylenediaminetet-raacetic acid (EDTA), 68 nmol, NH4Cl, 1.0 ,imol;MgCl2, 0.45 Amol; DTT, 45 pmol; NP-40, 0.03%; so-

dium cacodylate (pH 5.0), 1.1 ,umol; and [3H]ADPwas added to a specific activity of 20 cpm/pmol ofADP in our systems of quantitation. After 20 min ofincubation, the reaction was stopped by the additionof a mixture of trichloroacetic acid (100%)-saturatedNa4P207-saturated NaH2PO4 in a volume ratio of1:1:1 at 4°C. The insoluble product was collected andwashed on nitrocellulose filters. The filters were

dried, placed in a toluene-based scintillation fluid,and assayed in a liquid scintillation spectrometer.

Standard phosphorolysis assay. The phosphoroly-tic activity of the preparations was assessed in 50 Alof reaction mixture containing: P042, 90 nmol;MgCl2, 0.45 ,Lmol; EDTA, 68 nmol; NH4Cl, 1.0 ,umol;DTT, 45 pmol; tris-(hydroxymethyl)aminomethane(Tris)-hydrochloride (pH 7.5), 2.5 ,umol; NP-40,0.03%; and [3H](A)0, 100 ng (specific activity of 87cpm/ng). The reaction was incubated at 37°C for 20min and assayed for remaining acid-insoluble poly-nucleotide as described for the polymerase assay.

Assay for inorganic [32P]ADP exchange reaction.The exchange reaction between the S-phosphate ofADP and inorganic [32p] was carried out as previ-ously described (16). E. coli PNPase was obtainedfrom J. Colbourn of Miles Research Products De-partment and used as a positive control.Enzyme purification. (i) Cell disruption. Fifty

grams of cells frozen in 8% dimethyl sulfoxide were

suspended in 200 ml of buffer I (0.01 M Tris-hydro-chloride, pH 8.4, 1 mM EDTA, pH 7, 0.01 M MgCl2,0.3 mM DTT). The cell suspension was centrifugedat 12,000 x g for 10 min. The cell pellet was resus-

pended in 125 ml ofbuffer I plus 20 ml of 25-,tm glasspowder (Heat Systems). The cell suspension was

disrupted for 10 min with a sonifier (Bronson modelS110) at 10 A. The temperature during cell breakagewas held at 8°C or below by alternating the samplefrom an ice to ethylene glycol-dry ice bath. Fortymilligrams ofPMSF freshly dissolved in 4 ml of 95%ethanol was added to the cell extract after min 1 ofbreakage. The extract was centrifuged at 12,000 x g

for 1.5 h. The supernatant fraction (143 ml) was

designated fraction I and retained.(ii) Phase partition. Fraction I was partitioned

between phases of dextran and PEG by the Shoren-stein and Losick (19) modification of the Babinet (2)procedure. Thirty-five milligrams of PMSF and 0.3

ml of 0.1 M DTT were added per 100 ml of fraction I.Fraction I (143 ml) was treated with 45.9 ml of 30%(wt/wt) PEG and 16.5 ml of 20% (wt/wt) dextran,both dissolved in water. After stirring for 30 min,the mixture was centrifuged at 14,000 x g for 10min. Two phases were obtained; the upper phase,containing the PEG, was discarded. To the dextranphase (26 ml), 62 ml of buffer I, 9 mg of PMSF, 0.075ml of 0.1 M DTT, 25.4 ml of 30% PEG, and 13.2 g ofNaCl were added. The mixture was stirred for 30min and centrifuged as before. The PEG phase wasdiscarded. To the dextran phase (22 ml), 52.6 ml ofbuffer I, 8 mg of PMSF, 0.07 ml of 0.1 M DTT, 21.6ml of 30% PEG, and 22.7 g of NaCl were added. Themixture was stirred for 45 min and centrifuged asbefore. The dextran phase was discarded. The PEGphase (80 ml) containing the PNPase activity wasdialyzed for 2 h against two changes of 4 volumes ofbuffer I plus 0.05 M NaCl and 5% (vol/vol) glycerol.

(iii) (NH4)2SO4 fractionation. After dialysis,(NH4)2SO4 (16.3 g/100 ml) was added, and 1 N NaOHwas used to maintain the pH at 7.6. The mixturewas stirred for 30 min and centrifuged 5 min at10,000 x g. Two phases were obtained; the upperphase, containing the PEG, was discarded. To thelower phase, (NH4)2SO4 (7 g/100 ml) was added; themixture was stirred for 30 min and centrifuged 45min at 23,500 x g, and the precipitate was dis-carded. The 30 to 52% (NH4)2SO4 precipitate wasmade by adding (10 g/100 ml) (NH4)2SO4 to the su-pernatant fraction. The mixture was stirred andcentrifuged as described before, and the precipitatewas dissolved in 4 ml of buffer II (0.01 M Tris-hydrochloride, pH 8, 1 mM EDTA, pH 7, 0.01 MMgCl2, 0.3 mM DTT, 10% [vol/vol] glycerol, and 0.1M NaCl) and designated AS 52.

(iv) Agarose gel filtration. AS 52 was applied to aPharmacia K26/40 column of Bio-Gel A-1.5-m aga-rose and eluted with buffer II plus 0.1 M NaCl at aflow rate of 5 ml/h. Fractions of 2.2 ml were col-lected, and 5 ,ul was assayed for enzyme activity.The effluent in tubes 29 to 34 (PNPase I) and tubes40 to 42 (PNPase II) showed activity (See Fig. 1).These two activities were pooled separately and di-alyzed overnight against storage buffer (0.01 M Tris,pH 8.4, 0.01 M MgCl2, 0.1 mM EDTA, pH 7,0.03 mMDTT, 25 mM NH4Cl, 50% [vol/vol] glycerol).

(v) DEAE-Sephadex chromatography. PNPase Iactivity from the agarose gel filtration step wasdialyzed into buffer II containing 0.05 M NaCl. Thismaterial was applied to a column of DEAE-Sepha-dex (Pharmacia K9/15) at a flow rate of 10 ml/h. Thematerial was fractionated by stepwise elution withbuffer II, which contained 0.05 M, 0.1 M, 0.3 M, and0.5 M NaCl. Fractions 50 to 56 eluted with 0.5 MNaCl contained the PNPase I activity. This materialwas pooled and dialyzed into storage buffer.

(vi) Polyacrylamide gel electrophoresis. Sodiumdodecyl sulfate (SDS)-gel electrophoresis was per-formed as described by Weber and Osborn (22) ongels (8 cm long) containing 7.5% acrylamide and0.1% SDS. Five to twenty microliters from eachfraction containing PNPase activity were diluted 1:1into a gel buffer containing 0.01 M sodium phos-phate, pH 7.2, 3% SDS, 4 M urea, and 0.002 M DTT

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B. AMYLOLIQUEFACIENS POLYNUCLEOTIDE PHOSPHORYLASE

and heated for 2 min in a bath of boiling water. Thesamples were then dialyzed overnight at room tem-perature against gel buffer. To the dialysate wasadded glycerol to a concentration of 10% (vol/vol)and 0.003% bromophenol blue. One hundred fiftymicroliters of sample was applied to each gel. Sam-ples were stacked at 1 mA per tube for 2 h andsubjected to electrophoresis at 15 mA per gel for 3.5h. Gels were stained with a 0.25% (wt/vol) solutionof Coomassie brilliant blue R in methanol-aceticacid-water (5:1:5, vol/vol/vol) for 2 h. The gels wereelectrophoretically destained in 7.5% acetic acid.The molecular weight of PNPase I was determinedby SDS-gel electrophoresis as described previously(22).

RESULTS

Separation of two PNPase activities by aga-rose gel filtration. When the material fromfinal phase partition was fractionated by differ-ential solubility in 32 to 52% (NH4)2S04 and theresulting precipitate (AS 52) was dissolved inbuffer and fractionated by agarose gel filtra-tion, two peaks of PNPase activity were re-solved (Fig. 1). The major peak (PNPase I) waseluted first and was followed by a second ortho-phosphate-sensitive small peak of activity(PNPase II). The fractions demonstratingPNPase I and PNPase II activities were pooledseparately, and the degree of purity was as-sessed by SDS-polyacrylamide gel electrophore-sis. The PNPase I fraction showed significantlyfewer bands than PNPase II (Fig. 2A) and also

j10040

so la

60

~40 so0I

~~~~~~60

j20 lt40~

20 1a

Fracffon NumberFIG. 1. Agarose gel filtration on AS 52 fraction of

BaM-2 cell extracts obtained as described in Materi-als and Methods. Symbols: Solid line, percent ofabsorbance at 260 nm, (0) micromoles ofGMP incor-porated per 50-,pl sample in 20 min.

B A C=~~~~~~~~~~~~~~~~~~~.I iFIG. 2. SDS-polyacrylamide gel electrophoresis of

samples ofPNPase activity. (A) Pooled agarose frac-tions of PNPase I activity, (B) pooled agarose frac-tions of PNPase II activity, (C) PNPase I activityeluted from DEAE-Sephadex column.

demonstrated the reactions of a true PNPase.The relationship between the two activities is,at present, not clear, and PNPase II will be thesubject of future investigations. The remaininganalyses pertain to the further purification andcharacterization of PNPase I.DEAE-Sephadex chromatography. The

pooled PNPase I material described above wasfractionated by stepwise elution on DEAE-Sephadex columns. The active material waseluted from the column in the presence of 0.5 MNaCl (Fig. 3). Gel electrophoresis in the pres-ence of SDS and urea produced a single band ofprotein (Fig. 2C) that migrated at a rate char-acteristic of a protein of molecular weight 7.4 x104 (Fig. 4). PNPase I is composed of a singlepolypeptide chain or oligomers thereof. Thespecific activity of this material was calculatedto be 170 nmol of AMP incorporated per mg ofprotein per min.Effect of pH and divalent cations on polym-

erization activity. Maximum polymerizationactivity for the incorporation of ADP into acid-insoluble material was observed in cacodylatebuffer at pH 5.0 (Fig. 5A), and pH dependenceappeared as a relatively sharp peak. The syn-thesis of (guanylic acid)n [(G),,] from GDP, onthe other hand, produced a broad pH rangewith maximal polymerization at pH 7.0 in Tris-hydrochloride buffer (Fig. 5B). The concentra-tion of the Tris-hydrochloride buffer (i.e., 1.0 to10 mM) did not alter the extent of polymeriza-tion, whereas the cacodylate buffer was foundto be inhibitory at a concentration above 2.0mM.The polymerization reaction is completely de-

pendent upon the presence of divalent cations.Both the syntheses of (A),, and (G),, exhibit thisdependence: (A),, synthesis is optimal in 10 mMMgCl2, and (G). synthesis is optimal at 5 mM

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872 ERICKSON & GROSCH

E=0

020a

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15'

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0.75 K10

a00.50 "0

0.25 2

Inv SOC00 v

0 10 20 30 40 50 60

Fraction NumberFIG. 3. DEAE-Sephadex chromatography ofPNPase L. Symbols: Solid line, percentage of absorbance at

260 nm; (0) nanomoles of AMP incorporated per 50-sl sample in 20 min; numbers above bars signifymolarity ofNaCl used for elution at designated points in fractionation.

9-

8~

7-

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4-AC

a

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b PNPeSa I

%b BSA

O' IgO (H chain)

6, ovalbumin

o " 19 (L chain)\ trypsin

I I I I

O .2 .4 .8 .8 1.0Mobility

FIG. 4. Estimation ofmolecular weight ofPNPaseI by SDS-polyacrylamide gel electrophoresis.

MgCl2. At higher concentrations, Mg2+ be-comes inhibitory in both systems (Fig. 6). Man-ganese can serve as the cation in both

I

syntheses, but the final amount of polymeriza-tion is only 0.5 of that observed in the presenceof Mg2+.

Inorganic [32P]ADP exchange reaction. Inthe presence of NDP and phosphate, PNPasecan exchange the 8-phosphate of the nucleotidewith orthophosphate (7, 21). This reaction is adistinguishing property of PNPase. Table 1shows that PNPase I of B. amyloliquefacienscarries out this reaction as efficiently as thewell-characterized PNPase of E. coli.PNPase I activity as a function of time of

incubation. The reaction stoichiometry ofPNPase indicates that as the polymerizationreaction proceeds, the accumulation of ortho-phosphate will tend to drive the reaction in theopposite direction. The polymerization reactionstudied as a function of time demonstrates thatthis is the case for both (A),, and (G), syntheses(Fig. 7). In the case of (A). synthesis, the for-ward reaction continues for 30 min at a pointwhen approximately 45% of the added ADP hasbeen rendered acid insoluble. At this point, thephosphorolysis reaction becomes dominant andpolymer degradation becomes evident. In thecase of (G), synthesis, only about 5% of theadded NDP is polymerized before the reactionis inhibited. The cause of this pronounced inhi-bition is not known. In addition, there is only aslight amount of (G),, degradation during thetime course of the experiment.

Phosphorolysis reaction of PNPase I. Thephosphorolysis reaction evident in the preceed-ing experiments can be studied by following the

0

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0.1M I I Ia ~I'I A

00CM_S LX _ X _ |IIl

~~~~~~~~~~~~~~ I

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B. AMYLOLIQUEFACIENS POLYNUCLEOTIDE PHOSPHORYLASE 873

~0

0

0

c

a.

4t

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a0.5

0

Ec

0

.~0.4

h.0600.0

-C

ILa 0.2

9.0 8.0 7.0 6.0 5.0

p HFIG. 5. Dependence ofpolymerization reaction on

pH. With the exception ofpH, the standard polymeri-zation assay was used in the presence of 1.0 pg ofenzyme. (A) Response for (A),, synthesis; (B) synthe-sis of (G),,. Symbols: (@) Tris-hydrochloride buffer,(0) sodium cacodylate buffer.

degradation of [3H](A),, in the absence of theNDPs. The stoichiometry of the reaction indi-cates that the degradative pathway would re-

quire PO42-, and this is shown to be the case inFig. 8A. The purified PNPase I shows no degra-dation of [3H](A),L after 30 min of incubation inthe absence of orthophosphate, indicating no

contaminating ribonuclease activity. The addi-tion of as little as 2.0 nmol Of P042- initiatesphosphorolysis, and the degradation is com-

plete in the presence of 20 nmol. The resistantisotope remaining in the presence ofhigh levelsof orthophosphate may be related to the resist-ant fraction described previously (11), whichwas postulated to be residual oligomeric mate-rial that cannot bind to the enzyme and is,therefore, not degraded.The dependence of phosphorolysis upon pH

is shown in Fig. 8B, and the data suggest an

Q.5

0

0

0.4

~50.3

0

02.2z

.!0.10

EIC

0 I

0 10 20 40 80

[MgCI2I mM

FIG. 6. Dependence ofpolymerization reaction on

divalent cation concentration. PNPase I was added tostandard polymerization reaction at a concentrationof 0.5 pg per 50-pl assay. Symbols: (O) (A)n synthe-sis, (0) (G),, synthesis.

TABLE 1. Exchange reaction between inorganicphosphate and the /-phosphate ofADP catalyzed by

PNPase I and E. coli PNPase

Sample descriptiona cpm retained on fil-ter

PNPase I from BaM-2 89,000PNPase from E. coli 67,000No protein 400

a Concentrations of the enzymes as PNPase I, 10,ug per assay, and E. coli, 2.8 U per assay, as ob-tained from Miles Research Products.

explanation for the pH optimum of the polym-erization reaction of ADP. The polymer is rap-idly degraded at neutral or alkaline pH, but thephosphorolysis reaction is almost completelyinhibited in cacodylate buffer at pH 5.0 to 4.5.Inhibitory studies on PNPase I. The effects

of various inhibitors of PNPase I are summa-rized in Fig. 9. As expected, inorganic PO42-markedly inhibits the reaction at levels above5.0 nmol per assay. Rifamycin SV, a potentinhibitor of B. subtilis deoxyribonucleic acid(DNA)-dependent RNA polymerase, demon-strates partial inhibition of the reaction, butonly at relatively high concentrations. Unex-pectedly, heparin proved to be a very potentinhibitor of PNPase I. It is also of interest tonote that both (A),, and (G),, inhibited the syn-thesis of (A),, by PNPase I. The reaction provedrelatively insensitive to N-ethylmaleimide andhigh concentrations of KCI.

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874 ERICKSON & GROSCH

DISCUSSIONThe presence of PNPase in extracts of B.

subtilis has been previously described (3, 21)but was not studied in detail. PNPase I of B.amyloliquefaciens, as described in the presentinvestigation, is an apparently primer-inde-pendent form ofthe enzyme that can be purifiedto a single protein band upon SDS-polyacryl-amide gel electrophoresis. The primer inde-

22.5

CL

2

0

~01.5

z

a o1.0

0

Ec

0.5

0 L0 30 60 120

Minutes IncubationFIG. 7. Polymerization as a function ofincubation

time. Enzyme was added at a level of 1.0 pg perassay. Symbols: (a) (A)n synthesis, (0) (G)n synthe-sis.

100

so3)c

-C 60

-0 40

0

o 2

n 0

0 1 2 3-2

p4J, nmoles log)

pendence of our preparation must be qualified,since we have not rigorously proved the ab-sence of nucleic acid contamination, and verysmall amounts of nucleic acid tightly bound tothe enzyme could escape detection by conven-tional means. The immediate initiation of (A),and (G)" synthesis at the maximal rate mightbe indicative of the existence of such a contami-nant (7).The enzyme possesses many of the properties

and characteristics of the well-studied PNPaseofMicrococcus luteus (5, 7, 10, 11, 21). PNPase Ican carry out the polymerization, phosphoro-lysis, and inorganic [32P]ADP exchange reac-tions that are characteristic of this class of en-zyme. The divalent cation requirement, as wellas the inhibition by high concentration of thecations, is comparable to that described for theother enzymes (7, 21). The major difference inthe reaction conditions utilized in studies withB. amyloliquefaciens PNPase I is the relativelylow pH optimum. Although the pH optimum isdependent upon reaction conditions, the valueis usually reported to be between a pH of 8 and9 for other enzymes. In addition, the molecularweight value of 7.4 x 104 for PNPase I is signifi-cantly smaller than the 2.5 x 105 value reportedfor the M. luteus enzyme (10).The existence of two separable PNPase activ-

ities is not unique to B. amyloliquefaciens. ThePNPase of M. luteus is purified as a primer-independent form which, upon limited tryptichydrolysis, yields a slightly smaller primer-dependent form of the enzyme (10). B. amyloli-quefaciens produces large quantities of extra-cellular proteases, and these enzymes may act

9 7 6 5

pH

FIG. 8. Phosphorolysis reaction as a function ofP042- concentration and pH. Reaction conditions were asdescribed in Materials and Methods in the presence of0.5 pg ofPNPase Iper assay. (A) Reaction studied as afunction ofpo42- concentration, and (B) phosphorolysis reaction as a function ofpH. The reactions carriedout in the pH range of9.5 to 7.0 utilized Tris-hydrochloride as a buffer, and sodium cacodylate was used inthe acidic pH values.

A B

01

-C<60-

, 40-

2 2

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VOL. 130, 1977 B. AMYLOLIQUEFACIENS POLYNUCLEOTIDE PHOSPHORYLASE

04. 0A 0.4 0.3

; 0.3 0.3 0.3 0.1

.') I0 1.0 3.0 0 4 66 0 LO 3.0 0 1 a 4 5

(P04) a me(MO) Rilemysim SV,yg me"ds AsP.IYm.1,,I s. ofm(Seg)

FIG. 9. Inhibitors of PNPase I polymerization activity. Concentrations shown represent the amount ofcompound per assay (50 ud). The symbols for (D) are: (-) (A)n as inhibitor, (0) (G), as inhibitor.

on the enzyme to yield two forms that can beseparated by agarose gel filtration. Alterna-tively, the two activities may be of diverse ori-gin, and this observation would be a first reportof two distinct PNPase enzymes in a given cell.The inhibition data summarized in Fig. 9 are

of interest for several reasons. Rifamycin SV isa potent inhibitor of DNA-dependent RNA po-

lymerase, and it has been found that the B.subtilis RNA polymerase is completely in-hibited by 0.1 ug/ml (1). PNPase I is inhibitedless than 50% by 160 ug of rifamycin SV per ml,and similar results have been obtained withrifampin. It has been shown that these drugsinteract with the /8 subunit of RNA polymerase(8). Heparin is also a potent inhibitor of bacte-rial RNA polymerase, and it has been postu-lated that the site of this interaction is the ,3'subunit and may convert /3' to /8 (24). Syntheticpolyribonucleotides also can inhibit RNA po-lymerase by competing with DNA molecules forthe template binding site on the /3' subunit (9).These data suggest that PNPase I and the /3'(and ,3) subunit appear to share some proper-ties relating to polynucleotide binding. Theserelationships are of interest, since a (A),,-syn-thesizing enzyme has been purified in E. coli(14), and it was suggested that this protein wasthe a subunit ofRNA polymerase. The enzymewas sensitive to phosphate, not inhibited byrifampin, and had a molecular weight in therange of 2.0 to 4.0 x 104 (13, 14).As previously mentioned, B. amyloliquefa-

ciens produces large quantities of extracellularenzymes, as much as 100 times the amount ofprotease synthesized by B. subtilis (4). Contin-

ued de novo synthesis of these enzymes is ob-served in the absence ofmRNA synthesis (4, 6),which was interpreted to indicate that a largepool of mRNA specific for the extracellular en-zymes had accumulated. If PNPase acts to in-crease RNA turnover (15), one might have ex-

pected a very low level or absence of PNPasein B. amyloliquefaciens. On the other hand, ifPNPase is involved in extending the half-life ofmRNA (11) then PNPase may play an impor-tant role in RNA metabolism in bacterialspecies that produce extracellular enzymes. In-vestigations to elucidate a possible role forPNPase in B. amyloliquefaciens are in prog-

ress.

ACKNOWLEDGMENTS

We would like to thank Marilou Heck for her advice andassistance in the work on polyacrylamide gel electrophore-sis; Michael Kotick, Blake Ingle, and Borek Janik for criti-cally reading the manuscript.

LITERATURE CITED

1. Avila, J., J. M. Hermoso, E. Vinuela, and M. Salas.1971. Purification and properties of DNA-dependentRNA polymerase from Bacillus subtilis vegetativecells. Eur. J. Biochem. 21:526-535.

2. Babinet, C. 1967. A new method for the purification ofRNA polymerase. Biochem. Biophys. Res. Commun.26:639-644.

3. Beers, R. F. 1959. Inhibition of polynucleotide phospho-rylase by degraded ribonucleic acid: its reversal byvarious agents. Nature (London) 183:1335-1337.

4. Both, G. W., J. L. McInnes, J. E. Hanlon, B. K. May,and W. G. Elliott. 1972. Evidence for an accumula-tion of messenger RNA specific for extracellular pro-tease and its relevance to the mechanism of enzymesecretion in bacteria. J. Mol. Biol. 67:199-217.

5. Chou, J. Y., M. F. Singer, and P. McPhie. 1975. Kineticstudies on the phosphorolysis of polynucleotides by

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polynucleotide phosphorylase. J. Biol. Chem.250:508-514.

6. Gould, A. R., B. K. May, and W. H. Elliott. 1973.Accumulation of messenger RNA for extracellularenzymes as a general phenomenon in Bacillus amylo-liquefaciens. J. Mol. Biol. 73:213-219.

7. Grunberg-Manago, M. 1963. Polynucleotide phospho-rylase. Prog. Nucleic Acid Res. 1:93-133.

8. Heil, A., and W. Zillig. 1970. Reconstitution ofbacterialDNA-dependent RNA polymerase from isolated sub-units as a tool for the elucidation of the role of thesubunits in transcription. FEBS Lett. 11:165-168.

9. Hunt, D., and H. G. Klemperer. 1970. Effect of polyri-boinosinic acid on RNA chain initiation. Biochim.Biophys. Acta 204:260-262.

10. Klee, C. B. 1969. The proteolytic conversion of polynu-cleotide phosphorylase to a primer-dependent form.J. Biol. Chem. 244:2558-2566.

11. Klee, C. B., and M. F. Singer. 1968. The processivedegradation of individual polyribonucleotide chains.II. Micrococcus lysodeikticus polynucleotide phospho-rylase. J. Biol. Chem. 243:923-927.

12. Nakazato, H., S. Venkatesan, and M. Edmonds. 1975.Polyadenylic acid sequences in E. coli messengerRNA. Nature (London) 256:144-146.

13. Ohama, S., and A. Tugita. 1972. Poly A synthesizingactivity in- a constitutiveusubnit ofRNA polymerase.Nature (London); 240:35-3$.

14. Ohasa, S., A. Tsugita, and S. Mii. 1972. Isolation anicharacterization ofpoly A polymerase from cell debrisof E. coli. Nature (London) 240:39-41.

15. Raue, H. A., and M. Cashel. 1974. Regulation of RNAsynthesis in Escherichia coli. II. Polynucleotide phos-phorylase activity in cold shocked cells. Biochim. Bio-phys. Acta 340:40-51.

16. Reiner, A. M. 1969. Isolation and mapping ofpolynucle-otide phosphorylase mutants of Escherichia coli. J.Bacteriol. 97:1431-1436.

17. Rogers, S., and P. Pfuderer. 1968. Use of viruses ascarriers of added genetic information. Nature (Lon-don) 219:749-751.

18. Sekiguchi, M., and S. S. Cohen. 1963. The selectivedegradation of phage-induced ribonucleic acid by poly-nucleotide phosphorylase. J. Biol. Chem. 238:349-356.

19. Shorenstein, R. G., and R. Losick. 1973. Purificationand properties of the sigma subunit of ribonucleicacid polymerase from vegatative Bacillus subtilis. J.Biol. Chem. 248:6163-6169.

20. Soreq, H., U. Nudel, R. Salomon, M. Revel, and U. Z.Littauer. 1974. In vitro translation of polyadenylicacid-free rabbit globin messenger RNA. J. Mol. Biol.88:233-245.

21. Steiner, R. F., and R. F. Beers. 1961. Polynucleotides,p. 94-149. Elsevier, New York.

22. Weber, K., and M. Osborn. 1969. The reliability ofmolecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem.244:4406-4412.

23. Williamson, R., J. Crossley, and S. Humphries. 1974.Translation of mouse globin messenger ribonucleicacid from which the poly(adenylic acid) sequence hasibe? nve. Biochem. 13:704-707.

24. Zillig, W., E. Fuchs, P. Palm, D. Rabussay, and K.Zechel. 1970. On the different subunits of DNA-de-pendent RNA polymerase from E. coli and their rolein the complex function of the enzyme, p. 151-157. InL. Silvestri (ed.), RNA polymerase and transription.North-Holland Publishing Co., Amsterdam.

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