Preparation and properties of soybean ribonuclease

BIOCHIMICA ET BIOPHYSICA ACTA

431

Q

P R E P A R A T I O N AND P R O P E R T I E S OF SOYBEAN RIBONUCLEASE A. J. MEROLA* AND FRANK F. DAVIS Departments o] Bacteriology and Agricultural Biochemistry**, Rutgers, The State University ol New Jersey, New Brunswick, N.J. (U.S.A.) (Received July 3ist, 1961)

SUMMARY A ribonuclease has been partially purified from soybean sprouts. Purine and pyrimidine nucleoside 2',3'-cyclic phosphates and 3'-mononucleotides are produced by its action on RNA. Adenosine 2',3'-cyclic phosphate is cleaved to the 3'-nucleotide but cyclic pyrimidine nucleotides were not acted upon by the enzyme. Soluble RNA is less readily hydrolyzed than is more highly polymerized RNA. Polynucleotide material remaining after 85 % hydrolysis of the soluble RNA was found to contain the bulk of the base-methylated nucleotides originally present in the soluble RNA.

INTRODUCTION Crude extracts from m a n y plant sources have been shown to degrade ribonucleic acids 1-5. Ribonucleases from spinach e, pea leavesL s, ryegrassg,10 and tobacco leaves n, 12 have been purified extensively and their modes of action determined. This paper reports the partial purification and characterization of the ribonuclease from soybean originally described b y SCHLAMOWlTZ AND GARNER1. Differences in the enzyme's ability to hydrolyze various RNA fractions are reported. MATERIALS AND METHODS Ribonucleic acids

RNA was isolated from fresh baker's yeast (Saccharomyces cerevisiae) b y the method of CRESTFIELD, SMITH AND ALLEN18. This RNA, having been precipitated with I M NaC1, henceforth is referred to as the insoluble fraction. RNA in the supernatant from the I M NaC1 treatment was purified as described by DAVIS AND ALLEN14 and is referred to as the soluble fraction. Synthetic compounds

Adenosine 2'-benzyl phosphate and adenosine 3'-benzyl phosphate were prepared according to BROWN, HEPPEL AND HILMOE16. Adenosine 5'-benzyl phosphate was Nomenclature: The system of nomenclature for the methylated purine bases is the one used previously by ADLERet al. 25to show the relationship of the compounds to guanine and adenine. * Present address: Institute for Enzyme Research, University of Wisconsin, Madison (U.S.A.). ** Paper of the Journal Series, New Jersey Agricultural Experiment Station. Biochim. Biophys. Acta, 55 (1962) 431-439

432

A.J.

MEROLA, F. F. DAVIS

synthesized b y the method of DAvis AND ALLEN17. Adenosine 2',3'-cyclic phosphate, uridine 2',3'-cyclic phosphate and cytidine 2',3'-cyclic phosphate were obtained from Schwarz Biochemical Corporation. Small amounts of contaminating mononucleotides were removed b y paper chromatography before use. NZ-dimethylguanine (2-dimethylamino-6-hydroxypurine) was kindly supplied b y Dr. G. H. HITCHINGS of the Wellcome Research Laboratories, Tuckahoe, N.Y. (U.S.A.). An acidic polymer formed b y polymerizing 2,5-dihydroxybenzoic acid with formaldehyde was a gift from CIBA Pharmaceutical Products, Inc., Summit, N.J. (U.S.A.).

Enzyme assay The method of enzyme assay and the unit of enzyme activity employed were those described b y FRISCH-NIGGEMEYER AND REDD111. Protein was determined b y the spectrophotometric method of WARBURG AND CHRISTIAN18.

Enzyme puri/ication 900 g of common soybeans were immersed in distilled water for 4 h, drained, rinsed, and allowed to germinate on moist filter paper at room temperature. T h e beans were washed daily to remove microbial growth. After four days the radicles had reached a length of approx. 4 cm and were removed from the cotyledons. Since the radicles were found to have more than twice the initial specific activity of the cotyledons only the radicles were used in the preparation of the enzyme. The radicles were washed and suspended in I 1 of cold o.I M acetate buffer (Na+), p H 5.0, and homogenized briefly in a Waring blendor. The homogenate was filtered through several layers of gauze. The residue was stirred with I 1 of buffer and filtered. The combined filtrates were adjusted to p H 4-5 with acetic acid and stored under toluene at 4 ° for 30 days. During this period a large amount of protein precipitated, resulting in a four-fold increase in specific activity. As reported b y SCHLAMOWITZAND GARNER 1, most of the phosphatase activity is lost under the acid conditions of storage. At intervals the preparation was examined microscopically for microbial growth with the aid of the Gram stain. Only a negligible number of organisms contaminated the preparation. Following storage at 4 ° the straw-colored supernatant was filtered b y gravity, and fractionated with ammonium sulfate. Fractions collected were 0.5-0.6, 0.6-0.7, and 0.7-0.8 saturation. The highest total activity and specific activity was found in the fraction precipitating between 0.6 and 0.7 saturation. This fraction was dissolved in IOO ml of the p H 5 acetate buffer and precipitation with ammonium sulfate was repeated. The 0.6-0. 7 fraction was dialyzed overnight against IO 1 of cold distilled water and centrifuged. A s u m m a r y of this purification procedure appears in Table I. The supernatant was concentrated b y pervaporation to 50 ml. The concentrated solution can be stored in the liquid state under toluene at 4 ° with little loss of activity after several weeks. For prolonged periods of storage the enzyme preparation can either be lyophilized or stored in solution at -- 15 °. In an alternate procedure, the moist beans were sprouted in sand without nutrient until the appearance of two sets of true leaves. The plants were harvested, washed and treated as above. In this procedure the separation of the radicles from the cotyledons is not necessary. Biochim. Biophys. Acta, 55 (1962) 431-439

433

SOYBEAN RIBONUCLEASE AND MINOR NUCLEOTIDES TABLE I PURIFICATION OF SOYBEAN RIBONUCLEASE F o r t h e d e f i n i t i o n of e n z y m e u n i t s see FRISCH-•IGGEMEYER

Fraction

Total units

Specificactivity

AND REDD111.

Yield (%)

Cru de e x t r a c t

19o ooo

26

IOO

p H 4.5 s t o r a g e 0.6--o. 7 a m m o n i u m sulfate fractionation No. I No. 2 Dialysis and concentration

143 ooo

112

75

69 8oo 6o ooo 50 ooo

1245 145o 13oo

36 3o 25

The enzyme exhibits no deaminase activity and has no effect on highly polymerized DNA from calf thymus. There is a small amount of residual phosphatase activity in the preparation as determined chromatographically b y the appearance of nucleosides when the enzyme is incubated with the various nucleotides. This monophosphatase activity is substantially inhibited by the addition of o.oi M fluoride to the incubation mixtures.

Paper chromatography and dectrophoresis One-dimensional paper chromatography of ribonuclease digests was carried out b y the descending technique in isopropyl alcohol-ammonia-water 19. Two-dimensional chromatographic studies were carried out as described by DAVIS, CARLUCCIAND R O U B E I N 15. Bands or spots were visualized with an ultraviolet lamp, photographed, and eluted from the chromatograms with water. Major mononucleotides were identified by spectral examination, and electrophoresis as described by CRESTFIELD AND ALLEN20. Nucleoside 2',3'-cyclic phosphates were determined b y electrophoresis of eluted substances in o.I M phosphate buffer, p H 7.0, before and after treatment of the substance with o.I N HC1 at room temperature for 4 h (see ref. 12). Minor nucleotide components were separated b y electrophoresis or twodimensional chromatography 15 and their identifications confirmed b y spectral examinations. The unidentified minor ribonucleotide ("spot 2") described previously a6, has now been identified as NZ-dimethylguanosine 2' (3')-phosphate b y chromatographic and spectral comparison of the base with an authentic sample of N 2dimethylguanine (2-dimethylamino-6-hydroxypurine). A Beckman Model DU spectrophotometer was used in all spectral work. Quantitative studies were carried out b y spectrophotometry using published extinction coefficients. RESULTS

Properties o/ soybean ribonuclease Optimum pH: The activity of the enzyme reaches a maximum at pH 5.2 when tested with the insoluble fraction of yeast RNA as the substrate (Fig. i). Effect o/ionic strength: The ionic strength of reaction mixtures was varied by the addition of NaC1. Optimum enzyme activity was found in the region of o.I (Fig. 2). Effect o/activators and inhibitors: Various substances were tested for their action •on the enzyme (Table II). The acidic polymer of 2,5-dihydroxybenzoic acid and Biochim. Biophys. Acta, 55 (1962) 431-439

434

A.J.

M E R O L A , F. F. D A V I S

lO0

75 V-

~- 5 0 o

25

/ I,

/

/\ o

I

40

I

I

5.0

I

i

I

6.0

pH

!

7.0

F i g . I. A c t i v i t y o f t h e e n z y m e a s a f u n c t i o n o f p H . A s s a y s w e r e r u n i n o . i M a c e t a t e b u f f e r s . A b o v e p H 6 f i n a l a d j u s t m e n t s o f p H p r i o r to a d d i t i o n o f e n z y m e w e r e m a d e w i t h d i l u t e N a O H .

/\

I00

95

V> r- 9 0 o

85

o~

80

o

I

I

I

I

I

0.05

0.1

0.15

0,2

0.25

IONIC STRENGTH

F i g . 2. E f f e c t

of ionic strength TABLE

EFFECT

OF THE

ADDITION

OF VARIOUS

Addition

None C a 2+ M n 3+ M g 3+

on enzymic

activity.

II

ACTIVATORS AND INHIBITORS

TO T H E

REACTION

Concentration

Percen~ activity ot control

-IO - s M lO -3 M

IOO 95 96 91

Z n 3+

IO -3 M lO -3 M

C u 2+ Reduced glutathione Cysteine p-Chloromercuribenzoate Iodoacetate Acid polymer

lO -8 M lO -3 M 2 . lO -8 M 10 -4 M lO -3 M o . o i ~o

MIXTURE

85

43 lO2 ioi I oo 96 I8

Biochim. Biophys. Acta, 55 (1962) 4 3 1 - 4 3 ( )

SOYBEAN RIBONUCLEASE AND MINOR NUCLEOTIDES

435

formaldehyde, previougly shown to be a potent inhibitor of bovine pancreatic ribonuclease" is strongly inhibitory, as is Cu 2+, and to a lesser extent Zn 2+ and Mg 2+. Other potential activators or inhibitors appear to have little effect. Heat stability: The enzyme, in p H 5 . o acetate buffer, was heated in a boilingwater bath. At varying time intervals samples were cooled in an ice-water bath and assayed b y the standard method. After 20 rain at IOO° appreciable activity remains (Fig. 3). The initial rapid loss in activity followed b y the slow decrease suggests that there is substantial coprecipitation or that the enzyme preparation contains two enzymes, one more thermostable than the other. Chromatographic patterns of RNA digests using the heated enzyme appeared identical to those prepared with unheated enzyme.

,o0 7~

)i->

o

50

0~0~

o __ --0

35

t

!

!

I

I 5 MINUTES

I I0 at

I 15

/ 20

I00 ° C

Fig. 3- Effect of heating for v a r y i n g periods of time on t h e a c t i v i t y of the enzyme. Details are given in the text.

Action o/ soybean ribonuclease on the insoluble R N A /,action: The reaction mixture was made up in o.I M acetate buffer (Na+), pH 5.2, and contained, per millilitre, 5 mg of insoluble fraction, 16o units of enzyme, and lO -2 mmoles sodium fluoride. Incubation was carried out under toluene at 35 ° for 24 h, the hydrolysate then being submitted to one-dimensional chromatography. Analysis of the chromatogram showed the major products to be purine and pyrimidine nucleoside 2',3'-cyclic phosphates, and purine mononucleotides. Minor amounts of pyrimidine mononucleotides were found. Where necessary, paper electrophoresis of material eluted from chromatograms was performed to effect complete separations 2°. Time studies showed adenosine 2',3'-cyclic phosphate appearing first, followed by the 2',3'-cyclic phosphates of guanosine, uridine and cytidine. Electrophoresis of nucleotides at p H 9.2 in both o.I M borate and o.I M carbonate buffer established the absence of 5'nucleotides. Electrophoresis of the cytidylic acid in p H 6.2 phosphate buffer and the adenylic acid in p H 8.o bicarbonate buffer s° showed each mononucleotide to be the 3' isomer. A quantitative determination of the reaction products of each nucleotide in the hydrolysate gave the following percentages in the cyclic form: guanylic acid, 48, adenylic acid, 49; uridylic acid, 75; cytidylic acid, 85. No polynucleotide material was observed on the chromatograms. Biochim. Biophys. Acta, 55 (1962) 431-439

436

A.J.

MEROLA,

F. F . D A V I S

Action of the enzyme on simple substrates: The 2',3'-cyclic phosphates of adenosine, uridine and cytidine (0.5 %) were incubated singly with the enzyme under conditions used in the hydrolysis of RNA. Chromatographic examination of the reaction mixtures in the isopropanol-ammonia-water solvent revealed no detectable hydrolysis of the cyclic pyrimidine nucleotides. Approx. 18 % of the adenosine 2',3'-cyclic phosphate was cleaved by the enzyme. When adenosine 2'-, 3'- and 5'-benzyl phosphates were tested as substrates in the reaction mixture, only adenosine 3'-benzyl phosphate was acted upon. This ester was transformed in the amount of 24 %, both adenosine 2',3'-cyclic phosphate and adenosine 3'-phosphate being produced. Action o/the enzyme on the soluble R N A fraction: Yeast RNA soluble in I M NaC1 is of low molecular weight and contains a relatively large amount of minor nucleotide components 15. This fraction also incorporates amino acids using an amino acidincorporating enzyme system from yeast (unpublished observation). When the soluble fraction was incubated with the soybean nuclease under standard conditions, 85 % hydrolysis of this fraction was obtained, in contrast to practically complete ( > 97 %) hydrolysis of the insoluble fraction. Diffuse bands of polynucleotide material were observed on chromatograms of enzymic digests of the soluble fraction (Fig. 4). Material from band 8 was submitted to alkaline hydrolysis by the procedure of CROSBIE, SMELLIE AND DAVIDSON21 and chromatographed in two directions using the technique described previously 15. The chromatogram, shown in Fig. 5, revealed the presence of relatively large amounts of base-methylated nucleotides in this band. Table III presents the relative nucleotide compositions of the soluble fraction and band 8. Bands 7, 6 and 5 when t r e a ~ d in a manner similar to band 8 also were found to contain substantial, though decreasing amounts of base-methylated nucleotides, apparently present originally in the bands in small polynucleotides. Difficulties were experienced in obtaining precise data concerning the amounts of the total bas e-methylated nucleotides present in polynucleotide areas of the chromatograms, but bands 5, 6, 7 and 8 were estimated to contain about three-fourths of the total base-methylated nucleotides of the soluble fraction, and these substances were obtained as mononucleotides only after alkaline hydrolysis of the bands. 5-Ribosyluracil phosphate in contrast, appeared to be concentrated to a lesser degree in the polynucleotide fractions. The bulk of this compound was liberated as the cyclic TABLE

III

NUCLEOTIDE COMPOSITIONS OF THE SOLUBLE R~z6k FRACTION AND OF BAND 8 FROM THE NUCLEASE DIGEST Moles per zoo moles Adenylic acid Guanylic acid Cytidylic acid Uridylic acid 5-Ribosyluracil phosphate Thymine ribonucleotide Ne-Methyladenylic acid i-Methylguanylic acid N2-dimethylguanylic acid

Soluble ]~action

Band 8

2o.3 27.1 27. 7 19.3 3.9 o. 5 0. 4 o. 4 0. 4

II.9 13. 9 4o.3 2o.7 6.8 1.8 1. 7 i. 5 1.4

Biochim.

Biophys.

A c / a , 55 (1962) 4 3 1 - 4 3 9

SOYBEAN RIBONUCLEASE AND MINOR NUCLEOTIDES

437

Fig. 4- Descending chromatogram of a soybean nuclease digest of the soluble fraction. The chromatogram was submitted twice to the solvent, with drying between submissions. Bands: I, adenosine 2', 3'-cyclic phosphate; 2, cytidine 2', 3'cyclic phosphate; 3,uridine 2', 3'-cyclic phosphate; 4, guanosine 2', 3'-cyclic phosphate; 5, adenylic acid; 6, guanylic acid; 7 and 8, polynucleotides. The origin (not shown) is 9 cm above band 8. Note the presence of polynucleotide material between bands 4 and 5, 5 and 6. Traces of cytidylic acid and uridylic acid are also found between band 5 and 6.

nucleotide and was isolated from the leading edge of the guanosine 2',3'-cyclic phosphate band (band 4, Fig. I) b y electrophoresis at p H 3.4. If hydrolysis of the soluble fraction was allowed to proceed for 48 h instead of 24 h, little additional hydrolysis of the resistant material was observed. However, when material eluted from the polynucleotide-containing bands 7 and 8 was submitted to action of the soybean RNAase under the standard conditions, hydrolysis occurred, though at a lowered rate, with the production of cyclic and mononucleotides. DISCUSSION The soybean RNAase appears similar with respect to m a n y of its properties to RNAases of several other plants. It is comparable to ryegrass 9 and spinach e RNAases in its stability to heat, to tobacco leaf RNAase in its pH optimum n, and to tobacco- and pea-leaf TM,24 RNAases in its ability to hydrolyze purine, but not pyrimidine, nucleoside 2',3'-cyclic phosphates to nucleoside 3'-phosphates. Although the soybean enz y m e does not act upon free pyrimidine mononucleoside 2',3'-cyclic phosphates, pyrimidine mononucleoside 3'-phosphates appear in RNA digests. Various possibil-

Biochim. Biophys..4cta, 55 (1962) 431-439

438

A. J. MEROLA, F. F. DAVIS

Fig. 5. Two-dimensional chromatograms of alkaline hydrolysates of the soluble fraction (left) and of band 8, Fig. 4 (right). U, uridylic acid; G, guanylic acid; C, cytidylic acid; A, adenylic acid; i, 5-ribosyluracil 2'(3')-phosphate; 2, i-methylguanylic acid; 3, N2-dimethylguanylic acid; 4, N"-methyladenylic acid (6-methylaminopurine ribotide); 5, 5-methyluridy lic acid (ribothymidylic acid). The dashed lines indicate boundaries between fluorescing and non-fluorescing areas. ities exist to explain these findings. The enzyme may be capable of hydrolyzing cyclic pyrimidine nucleotides when they are attached through their 5' positions to the ends of polynucleotide chains. A portion of the 5'-phosphodiester bonds m a y be cleaved without the concomitant formation of cyclic nucleotides. Other possibilities are that the enzyme preparation contains a small amount of a second RNAase that cleaves at the 5' position without tile production of cyclic nucleotides, or that the pyrimidine nucleotides were present originally at the ends of polynucleotide chains. The finding that, under similar conditions of hydrolysis, the enzyme liberates over half of the adenine of the insoluble fraction as tile 3'-nucleotide while hydrolyzing only 18 % of adenosine 2',3'-cyclic phosphate can also be explained by the possibilities outlined above. The likelihood of contamination with a second RNAase is considered remote in view of the similarities of results using unheated and heated enzyme. The resistance of base-methylated nucleotides to enzymic liberation may be a steric effect, in which the methyl groups hinder approach of the enzyme to internucleotide linkages. Contrariwise, base-methylated nucleotides m a y occupy specific sites in RNA, possibly near the ends of chains. If initial hydrolysis occurs in such a manner as to liberate these compounds as small polynucleotides, increasing inhibition of the enzyme by reaction products or the decreased ability of the enzyme to hydroBiochim. Biophys. Acta, 55 (1962) 431-439

439

SOYBEAN RIBONUCLEASE AND MINOR NUCLEOTIDES

lyze internucleotide links of small polynucleotides (as evidenced by the slow rate of transesterification of adenosine 3'-benzyl phosphate), would cause their accumulation during transesterification and hydrolysis. Inhibition of plant ribonucleases by reaction products has not been reported, though bovine pancreatic ribonuclease has been shown to be inhibited strongly by various ribonucleotides2z. NOTE ADDED IN PROOF STOCKX AND VANDENDRIESSCHE 26-28 have recently published comprehensive studies on a ribonuclease isolated from soybean. With regard to its general properties, and mode of action relative to tile major nucleotides, their enzyme appears similar to the enzyme described here. ( R e c e i v e d F e b r u a r y 2oth, 1962 ) ACKNOWLEDGEMENTS This work

was supported

of Health,

U.S. Public

(G-6428) and

in part

Health

by research

Service

by an institutional

grant

grants

from

the National

(C-4223), The National from The American

Institutes

Science Foundation

Cancer

Society.

REFERENCES M. SCHLAMOWlTZ AND R. L. GARNER, J. Biol. Chem., 163 (1946) 487 . N. W . PIRIE, Biochem. J., 47 (195 O) 614. B. AXELROD, J. Biol. Chem., 167 (1947) 57. L. SCHUSTER AND N. O. KAPLAN, J. Biol. Chem., 2Ol (1953) 535. B. BHEEMESWAR AND M. SREENIVASAYA,J. Sci. Ind, Research (India), 12 B 529. T. W . TUVE AND C. B. ANI~INSEN, J. Biol. Chem., 235 (196o) 3437. W . S. PIERPOINT, Biochim. Biophys. Acta, 2I (1956) 136. 8 M. HOLDEN AND N. W . PIRIE, Biochem. J., 60 (1955) 39. 9 L. SHUSTER, J. Biol. Chem., 229 (1957) 289. 10 L. SHUSTER,H. G. KHORANA AND L. A. HEPPEL, Biochim. Biophys. Acta, 33 (1959) 452. 11 W. FRISCH-NIGGEMEYER AND K. K. REDDI, Biochim. Biophys. Acta, 26 (1957) 4 °. 13 K. K. REDDI, Biochim. Biophys. Acta, 28 (1958) 386. la A. M. CRESTFIELD, K. C. SMITH AND F. W. ALLEN, J. Biol. Chem., 216 (1955) 185. 14 F. F. DAVIS AND F. W. ALLEN, J. Biol. Chem., 227 (I957) 907. 15 F. F. DAVIS, A. F. CARLUCCI AND I. F. ROUBEIN, J . Biol. Chem., 234 (1959) 1525. 16 D. M. BROWN, L. A. HEI'PEL AND R. J. HILMOE, J. Chem. Soc., (1954) 4 o. 17 F. F. DAVIS AND V. W. ALLEN, Biochim. Biophys. Acta, 21 (I956) 14. 18 O. WARBURG AND W . CHRISTIAN, Biochem. Z., 31o (1941) 384 . l0 R. MARKHAM AND J. D. SMITH, Biochem. J., 52 (1952) 5523 o A. M. CRESTFIELD AND F. W. ALLEN, Anal. Chem., 27 (1955) 424 • 31 G. W . CROSBIE, R. M. S. SMELLIE AND J. N. DAVlDSON, Biochem. J., 54 (1953) 287. ~3 F. F. DAVIS AND F. W. ALLEN, J. Biol. Chem., 217 (1955) 13. z3 I-L H1~YMANN, Z. R. GULICK, C. J. DEBOER, G. DE STEVENS AND R. L. MAYER,Arch. Biochem. Biophys., 73 (1958) 366. ~4 R. MARKHAM AND J. D. STROMINGER, Biochem. J., 64 (1956) 46 P. 35 M. ADLER, B. WEISSMAN AND A. B. GUTMAN, J. Biol. Chem., 23o (1958) 717 . ~6 j . STOCKX AND L. VANDENDRIESSCHE,Arch. internat. Physiol. Bioch., 69 .(1961) 493. 37 j. STOCKX AND L. VANDENDRIESSCHE,Arch. internat. Physiol. Bioch., 69 (1961) 521. • 8 j . STOCKX AND L. VANDENDRIESSCHE, Arch. internat. Physiol. Bioch., 69 (1961) 545. I 3 a 4 5 6

Biochim. Biophys. Aaa, 55 (1962) 4 3 1 - 4 3 9