Biochimica et Biophysica Acta, 998 (1989) 145-150 Elsevier

145

BBAPRO 33454

Ribonuclease in tomato vacuoles: high-performance liquid chromatographic analysis of ribonucleolytic activities

and base specificity

Stef fen Abe l ~, G e r d - J o a e h i m Kraus s z a n d K o n r a d G l u n d 1

t Martin-Luther-Universitiit Halle-Wittenberg, Sektion Biowissenschaften, WB Biochemie, Pflanzenbiochemische Abtei!un~ and z Biotechnikum, Abteilung Bioanalytik, Halle (G. D. R.)

(Received 7 March 1989)

Key words: HPLC; Ribonucleolytic activity; Base specific:'ty; Vacuolar ribonuclease; ( L. esculentum)

We have developed isocratic high-performance liquid chromatographic techniques for assaying monomeric products of action of ribonudeolytic enzymes using RNA and diribonucleoside monophosphates as substrates. The methods allow detection of phosphodiesterase, phosphotransferase, cyclic nucleotide phosphodiesterase and nucleofidase activities. In addition to a dassification of ribonudeases under study, quantitative measurements can be used to assess enzyme specificity with respect to bases adjacent to the cleavage site. The methods have been used to characterize a tomato vacuolar nimm_~ease. The enzyme sldits phosphoester bonds by a phosphotransferase reaction yielding 2',3'-cyclic nucleoside monophosphates. Only the respective purine nudeotides were further decycfizod to the 3'-nuclcoside monophosphates with 2' ,3 '-c~MP as the best substrate. Analysis of products of yeast RNA hydrolysis revealed that the enzyme spfits preferentially adjacent to purine residues, in particular to guanine (G >> A - U > C). The same order of base preference was essentially found using diribonudeoside monophosphates as substrates (G >> A > U > C and G > U - C > A for bases in 5 ' and 3' position, respective|y).

Introduction

Although plant tissues have often a profusion of ribonucleolytic enzymes [1,2], many aspects of RNA-hy- drolyzing enzymes (e.g., compartmentation, specificity and regulation of enzyme activity, structural data on protein and gene organization) are unknown. This is in contrast to the well-studied E. col| [3] and pancreatic- type ribonucleases (RNases) [4,5]. These plant activities can be classified conventionally by their substrate specificity (nucleases, RNases), mode of action (endo-, exoribonucleolytic), and reaction products (phospho- diesterase, phosphotransferase) [2,6].

Vacuoles of higher plant cells contain most of the intracellular activity of many acid hydrolases [7], thus displaying homology to the animal lysosomes [8] which are involved in the cellular turnover of macromolecules [9]. In an attempt to elucidate the role of plant vacuoles in cellular RNA degradation, we have previously dem- om',trated the occurrence of RNase activity in these

Correspondence: S. Abel, Martin-Luther-Unive~itiit, Sektion Bio- wissenschaften, WB Biochemie, Pflanzenbiochenfische Abteilung, Neuwerk 1, DDR-4020 Halle, G.D.R.

organelles [10], using methods of protoplast and vacuole isolation [11] from cultured tomato cells [12]. The vacuolar enzyme was purified and characterized as a phosphotransferase acting specifically on single- stranded RNA by an endonucleolytic mode of action [13]. According to Wilson [2] the enzyme was classified as RNase I (EC 3.1.27.1).

An intriguing feature of RNA-degrading enzymes is their relative base specificity for nucleotides at the cleavage site [1,2,5,6]. In this study we have investigated the base specificity of the tomato vacuolar RNase by (i) following the rate of hydrolysis of diribonucleoside monophosphates (NpN) and (ii) monitoring the forma- tion of mononucleotides (NMP) during hydrolysis of yeast RNA. For that purpose we have developed new isocratic ~,gh-performance liquid chromatographic (HPLC) techifiques which are suitable for a rapid analy- sis of monomeric products in ribonucleolytic reaction mixtures.

Materials and Methods

Plant material Cells of tomato (Lycopersicon escu~!entum cv. Lukul-

lus) were grown heterotrophically in batch culture [12].

0167-4838/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

146

Chemicals Diribonucleoside monophosphates were purchased

from Sigma (St. Louis, MO, U.S.A.). High molecular weight RNA from yeast, the 2',3'-cyclic, 2'-, 3'- and 5'-isomers of common ribonucleotides and ribonucleo- sides were obtained from Serva Feinbiochemica (Heidelberg, F.R.G.). All other chemicals were of ana- lyrical grade.

Purificatio n and assay of tomato vacuolar RNase The tomato vacuolar RNase was purified from sus-

pension-cultured cells as described [13]. Enzyme from the last purification step was used (affinity chromatog- raphy on agarose-5-(4-aminophenyl-phosphoryl) 2',3'- cyclic uridine monophosphate). RNase activity was as- sayed spectrophotometrically at 260 nm and 1 unit was defined as the amount of enzyme causing an increase in absorbance of ethanol soluble products of 1.0 (rain. ml)-I [10].

HPLC systems The HPLC equipment consisted of a precision pump

(Liquochrom 307, Budapest, Hungary) maintaining a flow rate of 1.5 ml- min -1, a variable UV detector (type OH-308, Labormim, Budapest, Hungary) adjusted to a sensitivity of 0.02 AUFS (absorption unit at full scale) and a recorder (type OH-814/1, Radelkis, Budapest, Hungary). Prepacked columns (4.6 × 250 mm) were purchased from Serva Feinbiochemica (Heidelberg, F.R.G.). Sample volumes of 5-30 /tl were injected, eluates monitored at 254 nm and products identified by comparing their retention times with those of authentic standards. For calibration curves, linearity was obtained in the range of 0.1-1.5 nmol. The following isocratic HPLC systems were used at room temperature: system A: reversed-phase HPLC on Octyl-Si 100 Polyol (5/tm) using 0.02 M (NH4)HePO 4 (pH 6.2) as mobile phase; system B: reversed-phase HPLC on Octadecyl-Si 100 Polyol (10 Itm), mobile phase as in system A; system C: boronate affinity HPLC on Dihydroxyboryl-Si 100 Polyol (5/~m) with 0.01 M KH 2 PO4 (pH 6.0), as mobile phase [14]; system D: ion-exchange chromatogrephy on DEAE-Si 100 Polyol (5/tm) with HeO, adjusted with NaOH to pH 7.0, as mobile phase.

Hydrolysis of 3'-and 5"-nucleoside monophosphates 3'- and 5'-nueleotidase activities were measured at

pH 5.6 (50 mM acetic acid-NaOH) and pH 8.8 (50 mM Tris-HCl) using 3'-UMP, 3'-GMP, 5'-UMP and 5'-GMP as substrates. In a final volume of 200/zl, the reaction mixture contained 5 mM substrate and 4 units of puri- fied enzyme (for controls heat-inactivated enzyme was used). Incubations were run at 37 o C for 24 h, terminated by diluting into ice-cold distilled water (1:20) and analyzed by reversed-phase HPLC (system A; substance

(retention time in min): 3'-UMP, 5'-UMP (1.3), 3"-GMP (2.5), 5'-GMP (2.0), uridine (2.8), guanosine (10.0).

Hydrolysis of 2",3'-cyclic nucleoside monophosphates and diribonucleoside monophosphates

2',3'-Cyclic nucleotide phosphodiesterase reactions and cleavage of diribonucleoside monophosphates were performed in a total volume of 50/~1 containing 50 mM acetic acid-NaOH (pH 5.6), 1 mM substrate and ap- propriate amounts of enzyme activity (0.005-0.5 units) (control with heat-inactivated enzyme for each sub- strate). Incubations were carried out at 37 °C and reac- tions were terminated by injecting a 10 lgl aliquot directly onto the HPLC column (system A was used for cyclic nucleotide hydrolysates and system C for digests of diribonucleoside monophosphates).

Analysis of RNA hydrolysates For enzymatic RNA hydrolysis, reaction mixtures

contained in a total volume of 2 ml 50 mM acetic acid-NaOH (pH 5.6), 10 mg yeast RNA as substrate and 0.4 units of purified RNase. After appropriate times, 100/tl were withdrawn and pipetted into 900 pl cold ethanol. After chilling ( - 2 0 ° C, 12 h) and centrif- ugation (11000×g, 15 rain), the supernatants were evaporated. Samples were dissolved using the HPLC eluent and analyzed with system B.

The base composition of the RNA was determined after hydrolysis according to [15]. Ten mg of yeast RNA were suspended in 200 /~1 12 M HCIO 4 and kept at 100 °C for 1 h. After cooling, resuspension to I ml with water and centrifugation (11000 × g, 10 rain), the su- pernatant was neutralized with KOH. KCIO 4 precipi- tated at 4 °C was removed by centrifugation. The super- natant was finally diluted with water and used for quantitative analysis of base composition (HPLC sys- tem D, nucleobases (retention time in rain): cytosine (2.7), uracil (3.2), guanine (4.6), adenine (5.2)). The relative base composition of the yeast RNA used was determined to adenine (22%), guanine (28~), uracil (20%) and cytosine (22~) with only trace amounts of minor bases.

Results

Products of ribonucleolytic action To study the base specificity of a ribonucleolytic

enzyme activity at the level of mononuc!eotides released from natural or synthetic substrates, it is necessary to know precisely the sequence of products formed. For the tomato vacuolar RNase, the hydrolysis of GpU was followed to monitor the appearance of hydrolytic prod- ucts. As can be seen in Fig. lc, the immediate products of GpU hydrolysis were 2',3'-cGMP and uridine. After prolonged incubation (24 h, Fig. ld), detectable amounts of 3'(2')-GMP appeared. Other product combinations,

147

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I

I I I i

13 0 2

Time (rain)

4

A I I ; t

2

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d

4

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4 6 1 3

Fig. 1. Hydrolysis of GpU by the tomato vacuolar RNase. (a) Run with authentic substances, 1 nmol each of substrate and possible split products. (b) Control, incubation with heat-inactivated enzyme for 24 h. (c and d) Reaction time 30 min and 24 h, respectively. Peak identities: 1,

3"(2")-GMP and 5'-UMP; 2, 2',3'-cGMP; 3, GpU; 4, uridine; 5, guanosine. HPLC system C was used.

namely Y(2')-GMP and uridine only, or guanosine and 5'-UMP, as expected for phosphodiesterase activities, were not detectable. In addition, no dephosphorylation of Y(2')-GMP to guanosine could be observed (Fig. ld), indicating absence of nucleotidase activity. This

was directly proved with 3'- and 5'-NMP as substrates (not shown).

To study the decyclization reaction (Fig. ld) in more detail, 2',Y-cyclic NMP were used as substrates. The HPLC conditions described (system A) allowed the

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Fig. 2. Enzymatic hydrolysis of 2',3'-cyclic nucleosid~, monophosphates. (a-c) Run with authentic substances, l nmol each of substrates and possible products of decyclizafion. (d-f) Incubations with vacuolar RNase (substrate used, reaction time): (d) 2',3'-cGMP, 10 min; (e) 2',3'-cAMP, 30 min; (0 2',3'-cUMP and 2",3'-cCMP, 24 h. Peak identifies: 1, 3'-GMP; 2, 2"GMP; 3, 2",3'-cGMP; 4, 3'-AMP; 5, 2'-AMP; 6, 2",3'-cAMP; 7,

3"(2")-CMP; 8, 2',3"-cCMP; 9, 3"(2')-UMP; 10, 2',3'-cUMP, HPLC system A was used.

148

TABLE I

Retention times (min) on Dihydroxyboryi-Sl 100 Polyol (HPLC system C) of diribonucleoside monophosphates and authentic substances of possible products after nucleolytic hydrolysis

5" Substrates (NpN) Products

3': C U G A N 2',3'-cNMP 3'(2')-NMP 5'-NMP Nucleoside

U 2.7 2.7 3.~ 3.8 U 2A 2.2 2.3 5.6 C 2.7 3.0 3.8 4.3 C 2A 2.1 2.3 5.8 G 3.1 3.2 4~ ~1 G 2.6 2.3 2.5 13~ A 3.6 3.6 ~2 4.2 A 2.9 2.7 2.8 ~

discrimination between the 2'- and 3'-isomers of purine nucleoside monophosphates. The elution profiles in Fig. 2 reveal that the 2',3'-cyclic purine nucleoside

TABLE I1

Enzymatic hydrolysis of diribonucleoside monophosphates

Incubations were performed and reaction products determined as described in Materials and Methods. Reaction velocities were calcu- lated on the basis of generation of 2',3'-cyclic NMP and are given as per cent of GpG hydrolysis (100~ correspond to 132 nmol 2',3'-cGMP rain- i. unit-enzyme- i). For each experiment, six time points were taken; data are from one out of two independent experiments.

5"-Residue 3"-Residue

Activity (~) G A U C

G 1 ~ 8.5 ~ . 2 ~ .9 A 14.1 10.2 3~ 3.2 U ~7 1.2 2.4 0.3 C 1.2 1.0 0.g ~1

monophosphates were hydrolyzed by the vacuolar en- zyme to 3'-GMP and 3'-AMP. On the contrary, 2",3'- cUMP and 2',3'-cCMP were not degraded by the en- zyme. Decyclization reaction kinetics revealed that product formation varied linearly with time and enzyme concentration (not shown). Enzyme activity on purine substrates was greater on 2',3'-cGMP (0.15 nmol- rain -1. unit. enzyme -1) than 2',3'-cAMP (0.06 nmol. rain- ]. unit. enzyme- 1).

Enzyme specificity for diribonucleoside monophosphates All possible 16 combinat ions of the four c o m m o n

R N A bases present in diribonucleoside monophos - phates were used to s tudy the base specificity of the tomato vacuolar RNase . Enzymat ic digests were analyzed directly by H P L C . The condit ions used (sys- tem C) were sufficient to separate all substrates f rom their corresponding products after enzymatic hydrolys is (see Table I), thus allowing quantif icat ion of the clea-

6

2 3

1

5

a I I i l , , l I I ~ i J

0 5 10 20 30 40 Time (min)

Fig. 3. Elation profile of ribonuclcoside monophosphates after hydrolysis of yeast RNA with tomato vacuolar RNase. For assay conditions see Materials and Methods, incubation time was 30 nfin. Peak identifies: 1, 3'(2')-CMP; 2, 2',3'-cCMP; 3, 3'(2')-UMP; 4, 2'3'-cUMP; 5, 3'-GMP; 6,

2',3'-cGMP; 7, 3'-AMP; 8, 2',3'-cAMP. HPLC system B was used.

vage reaction. This was performed for the vacuolar RNase by following the formation of 2',3'-cyclic NMP. During the short reaction times considered, decycliza- tion of 2',3'-cyclic purine nucleoside monophosphates was not detectable. Table II shows the rates of reaction for the hydrolysis of 16 diribonucleoside n-ionophos- phates. From the data presented, the following conclu- sions can be drawn: Summing up all reaction velocities referring to a given nucleoside X, irrespective of its position in the substrate (i.e., XpN + NpX, N stands for any of the four nucleosides), it becomes evident that the enzyme hydrolyzes preferentially bonds adjacent to purine bases, in particular guanine (G >> A = U = C). A more detailed inspection reveals that the enzyme ex- hibits a relative guanine specificity for both nucleobases flanking the phosphodiester bond to be cleaved, sug- gesting two different guanine-preferring nucleotide- binding sites on the enzyme molecule surface. Purine bases (guanine much more than adenine) in the 5'-posi- tion considerably promote the susceptibility of di- ribonucleoside monophosphates for enzymatic hydroly- sis. This base preference applies if guanine or a pyrimi- dine is in the 3'-position. However, the relative base specificity with adenine as the 3'-residue is different; diribonucleoside monophosphates of that structure are

2.0

'f.5

E o,,

"f.O

0.5

10 20 30 40

Time ( rain )

Fig. 4. Enzymatic release from yeast RNA of nucleoside monophos- phates by the tomato vacuolar RNase. Incubations were analyzed as described. Each time point represents the sum of cycfic and noncyclic nucleoside mouophospkates released at a given time (e.g., G = 2',3'-

cGMP + 3'-GMP).

149

considerably less susceptible to enzymatic cleavage (the preference of guanine as 5"-residue is lower than that for adenine as 5'-residue). The relative order of base preferences found for the 5"- and Y-position are G >> A > U > C and G > U = C > A, respectively.

Monomeric products of enzymatic RNA hydrolysis To investigate further the relative base specificity of

the tomato vacuolar RNase, a natural substrate (yeast RNA) was subjected to hydrolysis. Using HPLC system B, the four 2',3'-cNMP and the respective 3'-NMP were identified as monomeric products in enzymatic RNA digests. Moreover, as illustrated in Fig. 3, separation of the eight mononucleotides formed was sufficient to allow their quantification. Data presented in Fig. 4 reveal that guanosine monophosphates (sum of 2',Y- cGMP and Y-GMP) were preferentially released fol- lowed by adenosine-, uridine- and cytidine monophos- phates. About 80~ of the mononucleotides formed were purine nucleotides with guanosine nucleotides the most abundant (60%).

Discussion

Several approaches have been in use to analyze the base specificity of RNA-degrading enzymes. Synthetic homopolyribonucleotides and diribonucleoside mono- phosphates are preferred substrates in such studies [16,17-20]. However, results obtained with nucleotide homopolymers may not necessarily reflect the base specificity towards natural RNA [1,21,22], e.g., investi- gation of the tomato vacuolar RNase revealed that it can hydrolyze poly(U), poly(I) and poly(A), but not poly(G) and poly(C) [13]. The more adequate approach is the analysis of hydrolytic products in enzymatic digests of natural RNA substrates. Owing to the low base specificity found for the tomato vacuolar RNase (all bonds are eventually hydrolyzed, see Table II), it was not possible to draw precise conclusions of base preferences by analyzing hydrolytic products of end- labeled 5 S RNA (E. coli) on sequencing gels (not shown). Therefore, we have developed new isocratic HPLC methods to quantitatively analyze monomeric products of RNase action on synthetic (diribonucleo- side monophosphates) and natural (yeast RNA) sub- strates which are generally applicable for the charac- terization of RNA-degrading enzymes and have been used in comparative studies of tomato secretory RNases (unpublished data).

These HPLC techniques take account of the follow- ing activities of RNA-degrading enzymes: phospho- diesterase and phosphotransferase activities (using di- ribonucleoside monophosphates as substrates) analyzed by employing boronate affinity HPLC (system C, Fig. 1), and cyclic nucleotide phosphodiesterase (side activ- ity of phosphotransferases [6,22]) and nucleotidase ac-

150

tivities (side activity of plant nucleases [6]) analyzed by reversed-phase HPLC (system A, Fig. 2). The limited combinations of the expected products obtained after enzymatic hydrolysis of diribonucleoside monophos- phates allow for the rapid determination of the type of the cleavage reaction (phosphodiesterase or phos- photransferase activities; see Fig. 1). Moreover, all the 16 diribonucleoside monophosphates can be used to determine the base specificity, regardless if the enzyme produces 5'-, 3'- or 2',3'-cyclic NMP (Table I). In addition, we succeeded in a single-run separation of mononucleofides liberated from yeast RNA by the tomato vacuolar RNase (2',3'-cyclic NMP and 3'-NMP, HPLC system B, Fig. 3). A sufficient ~eparation of a mixture of 5'-NMP was achieved under ~he same condi- tions (not shown; retention times in min: 5'-CMP, 2.4; 5"-UMP, 2.8; 5"GMP, 3.8; 5'-AMP, 8.2~), thus proving as an additional approach for studying the base specific- ity of RNA-degrading enzymes.

The tomato vacuolar RNase has been characterized as a phosphotransferase, producing 2',3'-cyclic NMP as obligate monomeric products which are further hydro- lyzed in a second reaction [13] (Fig. 1). Enzymatic hydrolysis of 2',3'-cyclic NMP occurs only for the purine-containing substrates and this leads to the re- spective 3'-NMP (Fig. 2). The decyclization step pro- ceeds considerably slower than the generation of the respective 2',3'-cyclic NMP from diribonucleoside monophosphates (see Table II). This further char- acterizes the vacuolar RNase as a 2',3'-cyclic nucleo- tide-2'-phosphodiesterase (EC 3.1.4.16) which is con- sistent with the RNase I-type according to Wilson [6]. A systematic use of diribonucleoside monophosphates for studying base specificity of the tomato vacuolar RNase revealed that purine-purine and guanine-pyrimidine in- ternucleotide bonds are especially susceptible to en- zymatic hydrolysis. The guanine preference found for both residues of the cleaved phosphodiester bond is more striking for nucleobases in the 5'- than the 3'-posi- tion (Table If). The results of the cleavage of di- ribonucleoside monophosphates are in general agree- ment with those obtained by using yeast RNA as the substrate. Comparing both groups of substrates reveals similar ratios of enzymatic release of mononucleotides (see Table II and Fig. 4), being GMP >> AMP = UMP > CMP. A purine (guanine) base preference was found for other soluble acid plant RNases using diribonucleo- side monophosphates [16,23,24] or RNA [16,24-26] as substrates. This provides further evidence that the solu- ble major plant endoribonucleases identified as RNase I are most probably of vacuolar origin [1,6,13].

Acknowledgements

Authors are indepted to I. Duessel for technical assistance. We are grateful to Dr. L. O'Garro of the University of the West Indies (Biology Department), Barbados, for critical reading of the manuscript.

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