Tm JOURNAL of Bmmorcn~ CH~I~.TRY Vol. 237, No. 11, Novembar lQ62 Prinfd in U.S.A. Hydrolysis of Ribonucleoside 2’) 3’-Cyclic Phosphates by a Diesterase from Brain* GEORGE I. DRUMMOND, N. T. IYER, ASD JACQUELI~UE KEITH From the Department of Pharmacology, Faculty of Medicine, University of British Columbia, Vancouver, Canada (Received for publication, July 19, 1962) Enzymes that are capable of hydrolyzing ribonucleoside 2’, 3’- cyclic phosphates but that do not attack intemucleotide bonds are available from various sources (1). Several of these enzymes convert ribonucleoside 2’,3’-cyclic phosphates to the corre- sponding ribonucleoside 2’-phosphates. The first die&erase of this type was studied in calf spleen by Whitfeld, Heppel, and Markham (2). Davis and Allen (3) later described a highly specific enzyme from beef pancreas that converted purine and pyrimidine 2’,3’-cyclic phosphates to the 2’-phosphates. In a previous report from this laboratory (4), it was briefiynoted that rabbit brain extracts catalyzed a rapid hydrolysis of these compound8 and that the product of hydrolysis of adenosine 2’,3’-cyclic phosphate was adenosine 2’-phosphate. This paper describe8 further studies concerning the properties and the specificity of this enzyme in the central nervous system. EXPERIMENTAL PROCEDURE M&i&--Ribonucleoside 2’, 3’-cyclic phosphates were pre- pared by the method of Smith, Moffatt, and Khorana (5). The barium salts were converted to potassium salts before use by treatment with Amberlitc IR-120 (potassium form). Ribo- nucleoside 3’,5’-cyclic phosphates were synthesized by the pro- cedure of Smith, Drummond, and Khorana (6). p-Nitrophenyl thymidine 3’-phosphate, p-nitrophenyl thymidine 5’-phosphate, TpT, TpTp, pTpTp, adenosinc 2’- and 3’-propyl phosphate, and guanosine 2’- and 3’-propyl phosphate were provided by Dr. G. M. Tener. Adenosine 2’- and 3’-benzyl phosphate were synthesized by the method of Tener and Khorana (7). ApU- cyclic-p, ApC-cyclic-p, GpU-cyclic-p, and GpC-cyclic-p were isolated from a short term ribonuclease digest of RNA, as described by Markham and Smith (8). The final electrophoretic eluates were freeze-dried. CpC-cyclic-p, CpCp, and CpC were formed enzymically and isolated by the method of Heppel, Whitfeld, and Markham (9). Cytidine 2’,3’-cyclic phosphate, 90 pmoles, and cytidine, 30 pmoles, were incubated with 3 pg of crystalline ribonuclease for 8 hours at 2”. The reaction products were separated by paper chromatography, and the final eluates were freeze-dried. Each compound was dissolved in 0.4 ml of water. Chromatographic Solvent Systems-The chromatographic sol- vent systems used were: (a) isopropanol-ammonium hydroxide- water (7:1:2 by volume), (b) saturated ammonium sulfate- isopropanol-ar sodium acetate (80 :2 : 18 by volume), (c) 5% * This work was supported by a grant from the Life Insurance Medical Research Fund, Philadelphia, Pennsylvania. disodium hydrogen phosphate overlaid with isoamyl alcohol as described by Carter (lo), and (d) isopropanol-water (70:30 by volume) with 0.35 ml of ammonium hydroxide for each liter of gas space (11). Enzyme Assay and Unit-The assay of the die&erase depends on the difference in electrophoretic mobility between adenosine 2’,3’-cyclic phosphate and the reaction product, adenosine 2’-phosphate, at pH 7.5. In the standard assay, adenosine 2’,3’-cyclic phosphate (1.5 pmoles) was added to 0.1 ml of 0.1 M Tris-HCl (pH 7.5) containing 0.25% egg albumin, and water added to a final volume of 0.2 ml. Brain extract (1 to 20 pg of protein) was added,-and the solution was incubated at 30” for 20 minutes. A sample containing substrate but no enzyme served as a control. Glacial acetic acid (0.02 ml) was added to stop the reaction, and the tubes were chilled in ice. An aliquot (0.025 ml) was subjected to paper electrophoresis with a field strength of 25 volts per cm for 90 minutes with 0.05 M potassium phosphate, pH 7.5. Unreacted substrate spots were cut out and eluted. Each eluate was brought to 1.5 ml, and the ab- sorbancy was determined at 260 rnp in a Beckman model DU spectrophotometer, with cuvettes of 0.5-cm light path. The rate of the reaction was proportional to enzyme concentration over a satisfactory range, provided hydrolysis did not exceed 60%. One unit of enzyme is defined as the amount that pro- duces 50% hydrolysis under these conditions. The specific activity is expressed as unite per mg of protein. Protein was determined by the method of Lowry et al. (12). Enzyme Preparation-Beef brain is an excellent source of the enzyme, and in all the studies reported here, except those regard- ing tissue distribution, use made of this material. The enzyme in brain is firmly bound to insoluble particulate material and has resisted purification. The isolation procedure described below serves to remove lipid and water-soluble protein. All operations were conducted in a cold room at 2”, unless otherwise indicated. Fresh frozen beef brain, 100 g, was homogenized for 4 minutes with 500 ml of acetone in a Servall Omni-Mixer at -15”. The homogenate was rapidly filtered, and the extraction was re- peated two more times with 500~ml portions of acetone. The final residue was thoroughly dried under vacuum. The resulting powder was further extracted by homogenization for 4 minutes with 200 ml of n-butanol at 0”. An additional 156 ml of n-buta- no1 was added, and the suspension was stirred in a closed flask for 5 hours. After centrifugation at 10,000 X g for 10 minutes, the supernatant solvent was discarded. The precipitate was then homogenized with 200 ml of petroleum ether (b.p. 30-60”) by guest on December 8, 2020 http://www.jbc.org/ Downloaded from
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Tm JOURNAL of Bmmorcn~ CH~I~.TRY
Vol. 237, No. 11, Novembar lQ62
Prinfd in U.S.A.
Hydrolysis of Ribonucleoside 2’) 3’-Cyclic Phosphates
by a Diesterase from Brain*
GEORGE I. DRUMMOND, N. T. IYER, ASD JACQUELI~UE KEITH
From the Department of Pharmacology, Faculty of Medicine, University of British Columbia, Vancouver, Canada
(Received for publication, July 19, 1962)
Enzymes that are capable of hydrolyzing ribonucleoside 2’, 3’- cyclic phosphates but that do not attack intemucleotide bonds are available from various sources (1). Several of these enzymes convert ribonucleoside 2’,3’-cyclic phosphates to the corre- sponding ribonucleoside 2’-phosphates. The first die&erase of this type was studied in calf spleen by Whitfeld, Heppel, and Markham (2). Davis and Allen (3) later described a highly specific enzyme from beef pancreas that converted purine and pyrimidine 2’,3’-cyclic phosphates to the 2’-phosphates. In a previous report from this laboratory (4), it was briefiynoted that rabbit brain extracts catalyzed a rapid hydrolysis of these compound8 and that the product of hydrolysis of adenosine 2’,3’-cyclic phosphate was adenosine 2’-phosphate. This paper describe8 further studies concerning the properties and the specificity of this enzyme in the central nervous system.
EXPERIMENTAL PROCEDURE
M&i&--Ribonucleoside 2’, 3’-cyclic phosphates were pre- pared by the method of Smith, Moffatt, and Khorana (5). The barium salts were converted to potassium salts before use by treatment with Amberlitc IR-120 (potassium form). Ribo- nucleoside 3’,5’-cyclic phosphates were synthesized by the pro- cedure of Smith, Drummond, and Khorana (6). p-Nitrophenyl thymidine 3’-phosphate, p-nitrophenyl thymidine 5’-phosphate, TpT, TpTp, pTpTp, adenosinc 2’- and 3’-propyl phosphate, and guanosine 2’- and 3’-propyl phosphate were provided by Dr. G. M. Tener. Adenosine 2’- and 3’-benzyl phosphate were synthesized by the method of Tener and Khorana (7). ApU- cyclic-p, ApC-cyclic-p, GpU-cyclic-p, and GpC-cyclic-p were isolated from a short term ribonuclease digest of RNA, as described by Markham and Smith (8). The final electrophoretic eluates were freeze-dried. CpC-cyclic-p, CpCp, and CpC were formed enzymically and isolated by the method of Heppel, Whitfeld, and Markham (9). Cytidine 2’,3’-cyclic phosphate, 90 pmoles, and cytidine, 30 pmoles, were incubated with 3 pg of crystalline ribonuclease for 8 hours at 2”. The reaction products were separated by paper chromatography, and the final eluates were freeze-dried. Each compound was dissolved in 0.4 ml of water.
Chromatographic Solvent Systems-The chromatographic sol- vent systems used were: (a) isopropanol-ammonium hydroxide- water (7:1:2 by volume), (b) saturated ammonium sulfate- isopropanol-ar sodium acetate (80 :2 : 18 by volume), (c) 5%
* This work was supported by a grant from the Life Insurance Medical Research Fund, Philadelphia, Pennsylvania.
disodium hydrogen phosphate overlaid with isoamyl alcohol as described by Carter (lo), and (d) isopropanol-water (70:30 by volume) with 0.35 ml of ammonium hydroxide for each liter of gas space (11).
Enzyme Assay and Unit-The assay of the die&erase depends on the difference in electrophoretic mobility between adenosine 2’,3’-cyclic phosphate and the reaction product, adenosine 2’-phosphate, at pH 7.5. In the standard assay, adenosine 2’,3’-cyclic phosphate (1.5 pmoles) was added to 0.1 ml of 0.1 M Tris-HCl (pH 7.5) containing 0.25% egg albumin, and water added to a final volume of 0.2 ml. Brain extract (1 to 20 pg of protein) was added,-and the solution was incubated at 30” for 20 minutes. A sample containing substrate but no enzyme served as a control. Glacial acetic acid (0.02 ml) was added to stop the reaction, and the tubes were chilled in ice. An aliquot (0.025 ml) was subjected to paper electrophoresis with a field strength of 25 volts per cm for 90 minutes with 0.05 M potassium phosphate, pH 7.5. Unreacted substrate spots were cut out and eluted. Each eluate was brought to 1.5 ml, and the ab- sorbancy was determined at 260 rnp in a Beckman model DU spectrophotometer, with cuvettes of 0.5-cm light path. The rate of the reaction was proportional to enzyme concentration over a satisfactory range, provided hydrolysis did not exceed 60%. One unit of enzyme is defined as the amount that pro- duces 50% hydrolysis under these conditions. The specific activity is expressed as unite per mg of protein. Protein was determined by the method of Lowry et al. (12).
Enzyme Preparation-Beef brain is an excellent source of the enzyme, and in all the studies reported here, except those regard- ing tissue distribution, use made of this material. The enzyme in brain is firmly bound to insoluble particulate material and has resisted purification. The isolation procedure described below serves to remove lipid and water-soluble protein. All operations were conducted in a cold room at 2”, unless otherwise indicated.
Fresh frozen beef brain, 100 g, was homogenized for 4 minutes with 500 ml of acetone in a Servall Omni-Mixer at -15”. The homogenate was rapidly filtered, and the extraction was re- peated two more times with 500~ml portions of acetone. The final residue was thoroughly dried under vacuum. The resulting powder was further extracted by homogenization for 4 minutes with 200 ml of n-butanol at 0”. An additional 156 ml of n-buta- no1 was added, and the suspension was stirred in a closed flask for 5 hours. After centrifugation at 10,000 X g for 10 minutes, the supernatant solvent was discarded. The precipitate was then homogenized with 200 ml of petroleum ether (b.p. 30-60”)
Hydrolysis of Ribonucleoside b’,S’-Cyclic Phosphates Vol. 237, No. 11
at O”, and after being stirred for 30 minutes, the suspension was rapidly filtered. The filter cake was washed by being stirred with 150 ml of petroleum ether, and after filtration the residue was dried under vacuum. The dry powder weighed 13 g.
This solvent powder was homogenized in a Waring Blendor (rheostat set at 50) with 350 ml of 0.02 M potassium phosphate, pH 7.5, for 3 minutes. The frothy suspension was slowly stirred for 2 hours, after which it was centrifuged (the froth included) for 90 minutes at 30,000 X g. The clear supernatant solution, which contained less than 5% of the activity and 50% of the protein, was discarded.
The precipitate was then homogenized for 3 minutes with 300 ml of 3 M NaCl in 0.02 M potassium phosphate, pH 7.5, to which had been added 20 ml of Tween 20 and 0.5 ml of 5 N KOH. The frothy suspension was stirred mechanically for 2 hours and centrifuged for 2 hours at 30,000 X g. The opalescent super- natant solution was removed, and the precipitate was re-ex- tracted with an additional 150-ml volume of the salt-Tween solution. After being centrifuged as above, the two supernatant solutions were combined (volume, 480 ml). This solution was placed in three dialysis bags (diameter, 1 & inches) and subjected to continuous flow dialysis in a 6-l&r chamber against deionized water until the dialysatc was free of chloride ion. Usually, 60 liters of water were required. The dialyzed solution was then
TABLE I
Distribution of die&erase in dog tissua
Tissues were removed from a dog under pentobarbital anes- thesia, cleared of lipid and connective tissue where necessary, and frozen until use. They were homogenized with 5 volumes of 0.02 M potaeeium phosphate, pH 7.5, for 3 minutes in a Servall Omni-Mixer. Sedimented red cells were washed twice with 0.9% NaCl solution and laked with 3 volumes of water. The re- sulting homogenates (15-ml aliquots of all those greater than this volume) were dialyzed for 6 hours against 6 liters of water at 2’; fresh water was then added, and dialysis was continued a further 6 hours. Where necessary, homogenates were redis- pereed in a Potter-Elvehjem homogenizer immediately before pipetting for assay. Dilutions were made in 0.62 M Tris-HCl, pH 7.5. The standard assay wae used.
lyophilized. To remove the Tween 20, the lyophilized residue was dispersed in 300 ml of acetone at -15” and was centrifuged at this temperature. The precipitate was extracted two more times in thii manner, then thoroughly dried under vacuum. The yield of light brown powder was 4 g. This lyophilized powder contains 90% of the original enzyme activity and has a specific activity of 350. For USC, it was suspended in 0.01 Y Tris-HCl, pH 7.5. This suspension can be repeatedly frozen and thawed without loss of activity. The enzyme is also stable in the dry powder for months at -18”.
Although this preparation has no measurable ribonuclease activity (see below), when compounds other than adenosine 2’,3’-cyclic phosphate were being used, it was routinely treated with Dowex 1 (3) to avoid any possibility of contamination with this enzyme. Thus, 200 mg of the dry powder were sus- pended in 10 ml of water and subjected to sonic oscillation for 10 minutes at 4” in a Bronwill Biosonik 20-kc oscillator. The opalescent solution was stirred for 30 minutes with 1 g of Dowex 1 (acetate form). The resin was removed by slow centrifugation and washed three times with small volumes of water. The volume of the supernatant solution and washings was 20 ml.
Suspensions of the lyophilized powder may be dispersed by treatment with deoxycholate, digitonin, or 0.5 M sodium thio- cyanate. These procedures have not assisted in further purifi- cation, however; the material reaggregates and the enzyme becomes insoluble when the dispersing agent is removed. The die&erase is rapidly inactivated on incubation with trypsin.
Distn’bution of LX&eracc-Extracts prepared from various tissues are capable of hydrolyzing adenosine 2’,3’-cyclic phos- phate. Table I shows that of those dog tissues examined, spinal cord is the most active. Various regions of the brain contain high activity, and the enzyme is present in extracts of vagus and sciat.ic nerve. Spleen, intestinal mucosa, and pan- creas, tissues in which similar activity has been reported pre- viously (2, 3), contain relatively low activity. Although the product of the reaction has been identified only in nerve tissue extracts, adrenal, liver, kidney, heart, and skeletal muscle all seem capable of hydrolyzing this substance at a slow rate. The enzyme is also present in extracts prepared from pigeon, frog, and fish brain.
Eflect of Metal Ions and Znhdbitcrs-The enzymatic hydrolysis of adenosine 2’,3’-cyclic phosphate shows no metal requirement (4). Cupric sulfate (2 mM), zinc acetate (2 mu), and mercuric chloride (1 mM> caused 26,75, and 100% inhibition respectively. EDTA, when added to the assay system, caused a 10% increase in activity above normal.
The die&erase is not inhibited by any of the following com- pounds when they are added to the assay system in the final concentrations shown: diisopropyl fluorophosphate and physo- stigmine, 0.1 mM; iodoacetamide, arsenite, and arsenate, 1 m&r; fluoride, caffeine, 3’-AMP, and PI-AMP, 10 mM; and heparin, 2 mg.
EJ’ect of pH-The enzyme is active over a broad range of pH (Fig. l), with an optimum of activity between pH 6 and 7. It is optimally active also in the presence of acetate, phosphate, and Tris-HCl and isactivatedslightly (approximately 10%) above normal when citrate and succinate are used as buffers.
Enzyme Specificihd-The die&erase attacks all the ribonucleo- side 2’,3’-cyclic phosphates tested. Table II shows that cyclic phosphates bearing purine bases are hydrolyzed somewhat more
November 1962 G. I. Drummond, N. T. Iyer, and J. Keith 3537
I I I I I I
4.0 5.0 6.0 7.0 8.0 9.C
PH FIG. 1. Effect of pH on the diestcrase. Standard resay con-
ditions were used except that the buffer was varied. There was no measurable nonenzymstic hydrolysis at any of these pH values during the incubation. The enzyme preparation used was a suspension of the lyophilized powder (specific activity, 300). 0, acetate; & phosphate; 0, Tris-HCl.
rapidly than those hearing pyrimidine bases.’ The lyophilized extract contains no ribonuclease activity as determined in the usual ribonuclease assay system (13), nor does it hydrolyze RNA “core.” In the standard die&erase assay, it does not hydrolyze simple nucleoside phosphate esters such as adenosine 2’- or 3’-propyl phosphate, adenosine 2’- or 3’-benzyl phosphate, or guanosine 2’- or 3’-propyl phosphate. It does not attack p-nitrophenylthymidine 3’-phosphate or p-nitrophenyl thymicline 5’-phosphate and is thus distinct from die&erases I and II, which were recently studied by Razzell (14, 15). The enzyme does not cleave the internucleotide bond of the dinucleotides TpT, TpTp, pTpTp, and CpC. The enzyme at this level of purity does not attack the ribonucleoside 3’,5’-cyclic phosphates of adenosine, guanosine, or uridine. It is therefore different from another die&erase in brain that converts these compounds to the nucleoside 5’-phosphates (4). In testing each of the above compounds, an amount of enzyme was used lOO-fold in excess of that required to hydrolyze completely 1.5 kmoles of adenosine 2’,3’-cyclic phosphate under standard conditions. Aliquots of each reaction mixture were not only subjected to electrophoresis at pH 7.5 as usual, but were also chromato- graphed in Solvent A, together with appropriate controls without enzyme. In every case, a single ultraviolet-absorbing spot, indistinguishable from the starting material, was present.
Ia’entifieation of Reaction Products-Each of the ribonucleoside 2’) 3’-cyclic phosphates is converted exclusively to the nucleoside
1 Inosine 2’,3’-cyclic phosphate is Sk30 hydrolyzed. We wish to thank Mr. Ken C. Wong for synthesis of this compound.
2’-phosphate. The enzymatic product formed under the stand- ard assay conditions was first separated from unreacted substrate and salt by chromatography in Solvent A. Product spots, together with authentic samples of the corresponding 2’- and 3’-nucleotides, were transferred to Whatman No. 3MM paper and subjected to ascending chromatography in Solvent B. In each case, a single ultraviolet-absorbing spot, indistinguishable from the nucleoside 2’-phosphate, was present. The product of hydrolysis of adenosine 2’,&cyclic phosphate after chroma- tography in Solvent A was also chromatographed in Solvent C and shown to be adenosine P’-phosphate. The product of hydrolysis of cytidine 2’) 3’-cyclic phosphate was also examined by ion exchange chromatography. As can be seen in Fig. 2, the product of enzymatic hydrolysis of this compound, when eluted from Dowex 1 with 0.01 M formic acid, was present as a single entity (Peak C). Furthermore, it emerged from the column at a position identical with that of P’-CMP when the commercial mixed isomers of this nucleotide were resolved by chroma- tography on the same column under identical conditions. No trace of the 3’ isomer was detected in the enzymatically produced cytidylic acid.
Action of Enzyme on Cyclic-ended L?inuckotida--It was of interest to determine whether the enzyme was capable of opening the cyclic diester linkage of cyclic-ended dinucleotides. In a typical experiment, samples of GpC-cyclic-p were incubated with excess die&erase, and the product was compared with that formed by treating the compound with ribonuclease. A third sample, incubated without enzyme, served as a control. When the three incubation mixtures were chromatographed in Solvent D (Fig. 3A), it was evident that a single compound had been formed by each enzyme. Both spots had migrated an identical distance from the origin to a position well separated from the GpC-cyclic-p control and where GpCp would be expected (8). When these spots were eluted and subjected to electrophoresis (Fig. 3B), again only one spot was present from each enzymatic incubation and both products had the same mobility. Further- more, their mobility was 1.5 times greater than that of GpC- cyclic-p, as would be expected of GpCp, which would bear a net negative charge of 3 at pH 7.5, compared with a charge of 2 for the cyclic form. This chromatographic and electrophoretic behavior indicated that the die&erase, like ribonuclease, had opened the cyclic diester linkage without attacking the inter- nucleotide bond to form the dinucleotide. When the spots ob- tained by electrophoresis were cluted, treated with alkali, and chromatographed in Solvent B, the control GpC-cyclic-p now
TABLE II
Hydrolysis of various nucleoside 2’,S’-cyclic phosphates A Dowex-treated suspension of a lyophilized powder was
used. Standard assay conditions were employed, except that the substrate wzz varied.
Fro. 2. Ion exchange chromatography of the hydrolysis product of cytidine 2’,3’-cyclic phosphate. The procedure of Cohn and Khym (10) was used. The column of Dowex l-X10, 200 to 400 mesh (1 X 16 cm) was first calibrated by chromatographing the commercial mixed isomers of cytidylic acid. A solution of 4 mg of this material in 4 ml of water at pH 9 was added to the column and, after being washed with water, wae eluted with 0.01 III formic acid. Fractions of 10 ml were collected. The elution pattern is shown by 0-C. Peak A, 2’-CMP; Peak B, 3’CMP. The column was then regenerated by being washed with 140 ml of 1 M NaOH, 90 ml of water, 200 ml of 1 M ammonium formate, 250 ml of 1 M formic acid, and 200 ml of water, in that order.
Cytidine 2’,3’-cyclic phosphate, 10 mg, was incubated with enzyme in a large scale experiment. When the reaction was complete, the mixture was streaked on Whatman No. 3MM paper and chromatographed for 12 hours in Solvent A. The band con- taining the product was eluted and freeze-dried. This material, 1.1 mg, was dissolved in 4 ml of water, brought to pH 9, applied to the column, and eluted with 0.01 M formic acid, as for cytidylic acid. The elution pattern is represented by A-h.
gave four spots, the 2’ and 3’ isomers of each nucleotide, as
expected (Fig. 3C). The product of ribonuclease action, after alkali treatment, consisted of the 2’ and 3’ isomers of guanylic
acid and 3’-CMP. The product of die&erase action had been degraded by alkali to 2’-GMP and 3’-GMP, but the cytidylic
acid in this sample, unlike that in the ribonuclease-treated material, migrated as the 2’ isomer. Thus, the action of the die&erase on this compound seems identical with its action on cyclic mononucleotides. Since the intemucleotide bond is not attacked, the product is a dinucleotide, GpCp, in which the terminal phosphate is in the 2’ position. When ApC-cyclic-p was tested by identical procedures, it was clear that the di- esterase converted this compound to the dinucleotide, ApCp. After treatment with alkali, followed by chromatography in
Solvent B, the cytidylic acid present again migrated as the 2’ isomer. By means of these methods, it was furthermore es- tablished that the cyclic die&r linkage of GpU-cyclic-p and ApU-cyclic-p were also opened to form the corresponding di- nucleotides. No attempt was made to identify the uridylic acid after alkali treatment, however.
To examine the specificity of the enzyme more thoroughly, it was necessary to test its action on a cyclic-ended dinucleotide containing only pyrimidine bases. CpC-cyclic-p was selected. This compound is degraded by ribonuclease to CMP. Samples of CpC-cyclic-p were incubated with ribonuclease and diesterase, and the products were chromatographed in Solvent D, together with samples of authentic CMP and CpCp. The results (Pig. 4A) show that while ribonuclease degraded CpC-cyclic-p to CMP, the die&erase converted it to a compound that migrated identically with CpCp. Further evidence that the product was CpCp was obtained by electrophoresis (Pig. 4.B). It is concluded that the cyclic diester linkage of CpC-cyclic-p is opened and that the die&erase, unlike ribonuclease, does not attack the inter- nucleotide bond.
A
0 00
.-.-- 1 2 3
B t
00
0
..-.-.- I23
60 Q 00 0
FIG. 3. Action of the diesterase on GpC-cyclic-p. This cyclic- ended dinucleotide isolated from 40 mg of ribonuclease digest of RNA (8) was dissolved in 0.3 ml of water, and O.l-ml samples were transferred to each of three test tubes containing 10 pmoles of Tris-HCl, pH 7.6. To one sample were added 100 pg of ribo- nuclease, and to another, 100 pg of Dowex-treated die&erase (specific activity, 240). A third sample, without enzyme, served as a control. The solutions were incubated in a final volume of 0.2 ml at 30” for 20 minutes, and 0.02 ml of glacial acetic acid was added to stop the reaction. A, The entire volume of each incu- bation mixture was spotted on Whatman No. 3MM paper and chromatographed (descending) in Solvent D. Spot I is the con- trol without enzyme; Spots 8 and 3 are the ribonucleaae- and diesterase-treated samples, respectively. B, The spots from A were transferred to No. 3MM paper, and electrophoresis was car- ried out at 2.5 volts per cm for 90 minutes in 0.05 M potassium phos- phate at pH 7.6. Spots are numbered as in A. C, Each spot from B was eluted and incubated in 0.3 M NaOH (final volume, 0.3 ml) at 37” for 22 hours. The solutions were carefully brought to pH 7 by being stirred with Amberlite IR-120 (H+ form). The entire solution from each tube was chromatographed on No. 3MM paper (ascending) in Solvent B, together with authentic samples of the mixed isomers of GMP and CMP. Spots 1, d, and .Y are numbered as in A and B.
November 1962 G. I. Drummond, N. T. Iyer, and J. Keith 3539
A
0
0 0
0 0
: % 0
0 0 -Iv.-.-.-..
I2345
B t
OoOo 0
-.-.-.-.-.m 12345
FIG. 4. Action of the enzyme on CpC-cyclic-p. The solution of CpC-cyclic-p (described under “Materials”) was added, in 0.05-ml aliquots, to each of three test tubes that contained 20 pmoles of Tris-HCl, pH 7.5, in a final volume of 0.2 ml. The first tube, without enzyme, served as a control. To the second tube were added 100 pg of ribonuclease, and to the third, 5 rg of Dowex- treated diesterase (see Fig. 3). Incubation was carried out aa in Fig. 3. A, Each incubation mixture, 0.08 ml, waa chromato- eranhed on Whatman No. 3 MM naner (descending) in Solvent D. &it 1 is the control; Spots d ad i are‘ the ribon%lease- and di- esterase-treated samples, respectively. B, The spots from A were eluted and subjected to electrophoresis as in Fig. 3. Spots 1, 2, and 3 are numbered as in A.
DISCUSSION
It is clear that the die&raze possesses considerable specificity for the 2’,3’-cyclic die&r linkage. Since it does not hydrolyze simple nucleoside phosphate esters or cause rupture of inter- nucleotide bonds, it seems similar to the pancreatic enzyme studied by Davis and Allen (3). The nature of the specificity raises interesting questions regarding the physiological substrate and the physiological function of the enzyme. Although ribo- nucleoside 2’,3’-cyclic phosphates and cyclic-ended di- and tri- nucleotides are early products of ribonucleaae action on RNA, these compounds are not known to exist in tissues in amounts detectable by present methods of nucleotide determination. Guanosine 2’, 3’-cyclic phosphate, however, haa recently been isolated from the microorganism, Chnnrwbacterium tilaceum (17). The enzyme opens the cyclic die&r linkage of cyclic- ended dinucleotidea in a manner apparently gdehtical with that in which it acts on cyclic mononucleotides. Although it is not known whether it can attack cyclic-ended polynucleotides, it may be well to consider such structures as possible physiological substrates for the enzyme. The presence in tissues of an enzyme capable of forming 2’-nucleotides should a$o focus attention on the possible physiological role of these compounds. The high activity of the enzyme in brain has no evident sign%- cance at the moment. Whether it has a bearing on any of the highly specialized functions of the central nervous system must await further studies.
The method of preparing the nucleoside 2’,3’-cyclic phosphates used here (5) gives chromatographically pure material in high yield. The availability of this highly active and specific enzyme makes possible a rapid and easy method for preparing on a large scale ribonucleoside 2’-phosphates completely free of the 3’ isomers.
SUMMARY
A highly active die&erase that converts ribonucleoside 2’,3’- cyclic phosphates to the corresponding ribonucleoside 2’-phos- phates has been prepared from beef brain.
The enzyme is most active in spinal cord and brain. It is also present in extracts of vagus and sciatic nerve. In tissues other than nerve tissue, the enzyme is much less active.
The die&erase in brain is firmly bound to insoluble particulate material. It has been extracted into solution by means of 3 Y NaCl containing Tween 20. The enzyme preparation contains no ribonuclease activity. It does not hydrolyze simple nucleo- side phosphate esters, nor does it attack the internucleotide bonds in a variety of dinucleotides. It does not hydrolyze ribonucleoside 3’,5’-cyclic phosphates.
The die&erase opens the cyclic diester linkage of several cyclic-ended dinucleotides, without rupture of the internucleotide bond, to form the corresponding dinucleotide. The evidence indicates that GpCp and ApCp, the dinucleotides formed from GpC-cyclic-p and ApC-cyclic-p, respectively, bear the terminal phosphate in the 2’ position.
Acknowledgment-We are indebted to Canada Packers, Ltd., Vancouver, for generous supplies of fresh beef brain.
REFERENCES
1. KHORANA, H.G.,inP.D. BOYER, H. LARDY,AND K. MyRB%CB (Editors), The enzymes, Vol. V, Academic Press, Inc., New York, 1961, p. 79.
3, DAVIS. F. F.. AND ALLEN, F. W., Biochim. et Biophys. Acta, 21, i4 (1956).
4. DRUMMOND, G. I., AND PERROTT-YEE, S., J. Biol. Chem., a36. 1126 (1961).
5. SMITI~, M., ‘MoF~A~, J. G., AND KHORANA, H. G., J. Am. Chem. Sot., 60, 6204 (1958).
6. SMITH, M., DRUMMOND, G. I., ASD KHORANA, H.G., J.Am. Chem. Sot., 62, 698 (1961).
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