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Purification and Characterization of Polynucleotide Phosphorylase From Proc. Natl. Acad. Sci. USA Vol. 82, pp. 1311-1315, March 1985 Biochemistry Purification and characterization of polynucleotide phosphorylase from cucumber (polynucleotide synthesis/RNA-dependent RNA polymerase/cucumber mosaic virus) ZULFAQAR ALAM KHAN AND H. FRAENKEL-CONRAT* Department of Molecular Biology, University of California, Berkeley, CA 94720; and Naval Biosciences Laboratory, Oakland, CA 94625 Contributed by H. Fraenkel-Conrat, October 15, 1984 ABSTRACT Polynucleotide phosphorylase (polyribonu- CL-6B, QAE-Sephadex A-25, and Blue-Sepharose CL-6B cleotide:orthophosphate nucleotidyltransferase, EC 2.7.7.8) were from Pharmacia. activity has been found in many prokaryotes and studied in Plant and Virus Strain. Cucumber (Cucumis sativus L.) detail since 1955. Such enzymes have been detected also in plants were grown in a greenhouse. The yellow strain of cu- plants. We now describe the purification of polynucleotide cumber mosaic virus (CMV-Y) was inoculated onto cucum- phosphorylase from cucumber cotyledons and leaves. This en- ber cotyledons by rubbing with the aid of Carborundum (600 zyme is a complex of three subunits, possibly not identical, of mesh), and the infected as well as healthy plants were kept in about Mr 50,000. Its enzymatic properties are similar to those the greenhouse (20-30'C). About 7-day-old cotyledons or 15- of the tobacco enzyme. Unlike the prokaryotic enzymes, the to 20-day-old leaves were harvested, virus infected or not, plant enzyme shows activity in the absence of primer but is to and the harvests were stored frozen at -70'C. various extents stimulated by various ribopolynucleotides or Assay of Polynucleotide Phosphorylase Activity. Enzyme RNAs. RNA-dependent RNA polymerase, not previously activity was measured in terms of incorporation of [3H]NDP shown to exist in non-virus-infected cucumber, has been found into high molecular weight polynucleotides bound by to be present at a low level and was separated from the much DEAE-cellulose paper discs (Whatman DE-81). The reac- greater amount of polynucleotide phosphorylase, although tion mixture contained (final volume, 25 /Ld) besides the en- some of the physical properties of the two enzymes are rather zyme; 78 mM Tris HCl (pH 8.0), 1.62 tkCi (1 Ci = 37 GBq) of similar. [8-3H]GDP (specific activity, 10.8 Ci/mmol), 906 /iM UDP, 6.5 mM MgCl2, 32.5 mM NH4Cl, 13 mM KCl, 26 mM In the course of a continuing study of the RNA-dependent (NH4)2SO4, and 10 ,g of poly(U) as primer. A unit of poly- RNA polymerase of cucumber cotyledons and leaves, it be- nucleotide phosphorylase is defined as the amount that in- came evident that these plants, be they virus-infected or not, corporates 1 nmol of GDP during 30 min at 30°C under these contained much of another enzyme, polynucleotide phos- conditions. Specific activity is defined as units of enzyme phorylase (polyribonucleotide:orthophosphate nucleotidyl- per milligram of protein. Protein concentration was deter- transferase, EC 2.7.7.8). Polynucleotide phosphorylase was mined by the method of Bradford (10) with bovine serum first discovered and purified from Lactobacillus and has albumin as the standard. since been isolated from many other aerobic and anaerobic Purification of the Cucumber Polynucleotide Phosphoryl- bacteria (1-4). It has proved to be a most useful tool in many ase. All preparations were performed at 0-40C. Healthy or molecular biological studies requiring polynucleotides of CMV-infected cucumber leaves or cotyledons (100-200 g) known composition, with one of the first important applica- were homogenized in a Waring Blendor for 3 min with 30-sec tions serving in the establishment of the genetic code. How- intervals after the addition, for each gram of plant material, ever, the role of these enzymes in prokaryotes has not yet of 1 ml of extraction buffer (100 mM Tris HCl, pH 8.0/200 been identified. It has been suggested that they could serve mM NH4Cl/180 mM 2-mercaptoethanol/4 mM EDTA/10% as alternate means to nucleases for the degradation of un- glycerol) mixed with an equal volume of saturated ammoni- wanted RNA, but only in systems in which the inorganic um sulfate. The homogenized material was squeezed phosphate concentration is sufficiently high to reverse the through three layers of cheesecloth and centrifuged at 10,000 reaction. x g for 10 min. The pellet was resuspended in the original Polynucleotide phosphorylase was detected, character- volume of extraction buffer that was 50% saturated with am- ized, and partially purified from tobacco by Brishammar and monium sulfate and again was centrifuged. Then the pellet Juntti (5). Its presence in animal cells is scanty and dubious was extracted with 0.2 ml (per gram of plant material) of 1:1 (5-7). Del'vig and co-workers (8, 9) have studied the particu- diluted extraction buffer lacking ammonium sulfate, and the lar association of such enzymatic activity with polysomes. suspension was centrifuged at 10,000 x g for 10 min; and this We now have isolated an almost pure enzyme of proper- low-salt extraction was repeated with 0.15 ml. The two ex- ties similar to those of tobacco (5) from cucumber and report tracts were combined and used for the following steps. here its properties. These procedures are the same as those used for the solubili- zation of RNA-dependent RNA polymerase (ref. 11 and cur- rent work). MATERIALS AND METHODS Step 1. PEG precipitation. For RNA-dependent RNA Materials. [8-3H]GDP, [5-3H]UDP, [8-3H]GTP and [5- polymerase precipitation, 11% PEG 6000 was used by Ku- 3H]UTP were purchased from Amersham; unlabeled nucleo- marasamy and Symons (12), while Gill et al. (13) and Takan- side di- and triphosphates were from Sigma; homopolynu- ami and Fraenkel-Conrat (11) used 22% PEG for complete cleotides were from P-L Biochemicals; DEAE-Sepharose precipitation of RNA polymerase. We adopted the higher concentration of PEG (22%) also for polynucleotide phos- The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviation: CMV, cucumber mosaic virus. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 1311 Downloaded by guest on September 27, 2021 1312 Biochemistry: Khan and Fraenkel-Conrat Proc. NatL Acad Sd USA 82 (1985) phorylase precipitation. Therefore, an equal volume of 44% Table 1. Summary of purification scheme of polynucleotide (wt/wt) PEG 6000 in 30 mM 2-mercaptoethanol/1 M NH4Cl phosphorylase from cucumber leaves and/or cotyledons was added to the initial extract and stirred for 40 min at 00C. Total Total The precipitate was collected by centrifugation at 12,000 x g protein,* activity,t Specific for 15 min and extracted with about 0.1 ml (per gram of leaf Scheme mg units activity* material) of 20 mM Tris HCl, pH 8.0/5% PEG 6000/2 M NH4Cl/1 mM EDTA/30 mM 2-mercaptoethanol. This ex- Initial extract, ammonium tract was subjected to centrifugation at 10,000 x g for 10 min sulfate precipitation 109 9047 83 and stored frozen at -70'C until used. The preferred purifi- Step 1, PEG 6000 cation procedure is as follows. precipitation 60.5 6534 108 Step 2. The extract was thawed and dialyzed against two Step 2, DEAE-Sepharose changes of 800 ml of A-buffer (40 mM Tris HCl, pH 8.0/30 chromatography 10.16 8819 868 mM NH4Cl/25% glycerol/0.5 mM dithiothreitol) for at least Step 3, QAE-Sephadex 4 hr. The dialysate contained insoluble material, which was chromatography 3.25 8187 2,519 centrifuged at 5000 x g for 10 min. The pellet was resus- Step 4, Blue-Sepharose pended in about 5 ml of A-buffer and centrifuged at 10,000 x chromatography§ 0.5 5541 11,082 g for 10 min, and the supernatants were combined and ap- Step 5, Glycerol gradient plied onto a column (3 x 14 cm) ofDEAE-Sepharose CL-6B, centrifugation 0.048 1636 34,083 which had been equilibrated with the same A-buffer. After *Batch size was 150 g of leaves. The amount of protein was the column was washed with 150 ml of A-buffer, the enzyme determined by the method of Bradford (10). was eluted with a linear 0.03-0.8 M NH4Cl gradient in the tThe total activity is given in nmol of GDP incorporated during 30 same buffer (total 200 ml); 2.2-ml fractions were collected min at 30'C with poly(U) as primer. under gravity. tThe specific activity is expressed in nmol of GDP incorporated per Step 3. Active fractions from step 2 were dialyzed against mg of protein. §The specific activity of the peak tube is about 30,000. This peak A-buffer as described above, and the dialysate was loaded fraction was mostly used for further characterization. onto a strongly basic anion exchanger, QAE-Sephadex A-25 column (1.4 x 14 cm). After the column was washed with 40 ml of A-buffer, the enzyme was eluted with a linear 0.3-0.7 fectual or harmful to the enzyme, such as carboxymethylcel- M NH4Cl gradient in the same buffer (total 80 ml); 2.2-ml lulose, phosphocellulose, and poly(U)-Sepharose, are not fractions were collected under gravity. A significant amount described. of unwanted protein was separated from the sharp activity The use of high ammonium sulfate in the initial extraction peak by this step. followed by PEG precipitation was found advantageous for Step 4. The active fractions from step 3 were dialyzed the isolation of the RNA-dependent RNA polymerase (ref.
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