UDP-Glucose Pyrophosphorylase from Potato Tuber: Purification and Characterization1

Transcription

1 J. Biochem. 106, (1989) UDP-Glucose Pyrophosphorylase from Potato Tuber: Purification and Characterization1 Kenichi Nakano,2 Yasuko Omura,3 Mitsao Tagaya, and Toshio Fukui4 The Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567 Received for publication, May 10, 1989 UDP-glucose pyrophosphorylase from potato tuber was purified 243-fold to a nearly homogeneous state with a recovery of 30%. The purified enzyme utilized UDP-glucose, but not ADP-glucose, as the substrate, and was not activated by 3-phosphoglyceric acid. Product inhibition studies revealed the sequential binding of UDP-glucose and MgPP1 and the sequential release of glucose-1-phosphate and MgUTP, in this order. Analyses of the effects of Mg2+ on the enzyme activity suggest that the MgPP, and MgUTP complexes are the actual substrates for the enzyme reaction, and that free UTP acts as an inhibitor. The enzyme exists probably as the monomer of an approximately 50-kDa polypeptide with a blocked amino terminus. For structural comparison, 29 peptides isolated from a tryptic digest of the S-carboxymethylated enzyme were sequenced. The results show that the potato tuber enzyme is homologous to UDP-glucose pyrophosphorylase from slime mold, but not to ADP-glucose pyrophosphorylase from Escherichia coli, and provide structural evidence that UDP-glucose and ADP-glucose pyrophosphorylase are two different protein entities. UDP-glucose plays a central role as a glucosyl donor in numerous cellular metabolic pathways, including the synthesis of sucrose in higher plant tissues. ADP-glucose is also important in the synthesis of starch. These nucleotide sugars are synthesized from a-d-glucose 1-phosphate (glucose-1-p) and nucleoside triphosphates by the action of pyrophosphorylase. Potato tuber contains pyrophosphorylase activities for UDP-glucose and ADP-glucose. Sowokinos (1) suggested that the two activities are separate protein entities, based on the following observations. During tuberization the ADP-glucose pyrophosphorylase activity markedly increases concomitantly with starch synthesis, while the UDP-glucose pyrophosphorylase activity rises only 2- to 3-fold. The two activities can be separated by ammonium sulfate fractionation of the crude extract. ap Rees et al. (2) have examined the relative contributions of ADP-glucose and UDP-glucose to starch synthesis in non-photosynthetic cells and reached the conclusion that starch is synthesized via ADP-glucose rather than UDPglucose. Thus the starch synthesis depends upon a supply of glucose-1-p for ADP-glucose pyrophosphorylase in amyloplasts. In cytosol the incoming sucrose is first degraded to UDP-glucose and fructose by sucrose synthase, and the nucleotide sugar formed is then converted to glucose-1-p by 1This study was supported in part by a Grant -in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. Present addresses: 2National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries, Tsukuba, Ibaraki 305. Central Research Institute, Mitsubishi Kasei Co., Ltd., Yokohama, Kanagawa To whom correspondence should be addressed. Abbreviations: glucose-1-p, glucose 1-phosphate; glucose-6-p, D-glucose 6-phosphate. UDP-glucose pyrophosphorylase. Recently, ap Rees (3) has shown that carbon can cross the amyloplast envelope as glucose-1-p rather than triose-phosphate. This view is supported by two additional pieces of evidence (4, 5), and emphasizes the importance of UDP-glucose pyrophosphorylase acting both in the synthesis and degradation of UDP-glucose. The present paper describes the purification and characterization of UDP-glucose pyrophosphorylase from potato tuber as the first step of our studies on this enzyme. The purified enzyme utilizes UDP-glucose, but not ADPglucose, as the substrate, and is not activated by 3-phosphoglycerate, confirming that UDP-glucose and ADP-glucose pyrophosphorylases are different protein entities. The partial amino acid sequence of the potato tuber enzyme is homologous to UDP-glucose pyrophosphorylase from slime mold (6) but not to ADP-glucose pyrophosphorylase from Escherichia coli (7), providing the structural evidence for the above view. EXPERIMENTAL PROCEDURES Reagents-Phosphoglucomutase (rabbit muscle) and UDP-glucose dehydrogenase were purchased from Sigma Chemical, and D-glucose 6-phosphate (glucose-6-p) dehydrogenase (yeast) from Oriental Yeast. Ultrafiltration membrane (UK02, mol. wt. cut off, 20,000) was from Advantec Toyo. DEAE-cellulose was the product of Brown, Sephadex G-100 of Pharmacia, and Butyl-Toyopearl of Tosoh. Tosylphenylalanyl chloromethyl ketone-treated trypsin was obtained from Millipore Corporation. All the other reagents were of the highest grade available. Enzyme Assays-Four different assay methods were used. Assay A: This method was carried out routinely during 528 J. Biochem.

2 UDP-Glucose Pyrophosphorylase from Potato Tuber 529 enzyme purification. The UDP-glucose degradation was monitored by the initial rate of glucose-1-p formation determined by spectrophotometric measurement of the NADPH formation accompanying the enzyme-coupled conversion to 6-phosphogluconate through glucose-6-p, essentially according to Sowokinos (1). The reaction mixture contained, in 0.74ml, 50 mm Tris-HCl (ph 8.0), 7 mm MgCl2, 0.5 mm mercaptoethanol, 10 mm NaF, 2 mm UDP-glucose, 0.02 mm glucose-1, 6-bisphosphate, 0.3 mm NADP, 5 mm PP1, 1 unit each of phosphoglucomutase and glucose-6-p dehydrogenase, and the UDP- glucose pyrophosphorylase to be assayed. The reaction was started by the addition of pyrophosphorylase, and the NADPH formation at 30 Ž was continuously monitored at 340 nm. One unit was defined as the amount of enzyme that catalyzes the formation of 1ƒÊmol of glucose-1-p per minute under the conditions employed. Assay B: Assay A was separated into two steps: first, UDP-glucose degradation was carried out for a fixed time; and, second, the amount of glucose-1-p formed was measured according to the same principle as in Assay A. This method was used to analyze the effect of the Mg2+ concentration on the activity in the UDP-glucose degradation direction. The first reaction was carried out, in 1ml, in 50 mm Tris-HCl (ph 8.0), 2 mm UDP-glucose, 2 mm PP1, and MgCl2, if added. The reaction was started by addition of the enzyme. After incubation at 30 Ž for 2min, the reaction was stopped by immersing in boiling water for 2 min. An aliquot of 200ƒÊl was used for the second reaction. The second reaction was started by the addition of phosphoglucomutase and glucose-6-p dehydrogenase, and the absorbance difference at 340 nm before and after the second reaction was used to calculate the amount of glucose-1-p formed in the first reaction. Assay C: This method was applied to the determination of Km for UTP and glucose-1-p, and the effect of the Mg2+ concentration on the UDP-glucose synthesis direction. The first reaction was performed at 30 Ž in 0.5ml of 20 mm Tris-HCl (ph 8.0) containing 5 mm glucose-1-p, 2 mm UTP, and 10 mm MgCl2. The reaction was started by addition of the enzyme, and, after 5 min, stopped by immersing the tube in boiling water for 2min. The amount of UDP-glucose formed was quantified according to the method of Franke and Sussman (8) using UDP-glucose dehydrogenase. Assay D: This method was employed to study the product inhibition of the enzyme in the UDP-glucose degradation direction, because the glucose-1-p originally added hindered the exact quantification of the sugar phosphate formed when Assay A was applied. The enzyme reaction was performed as the first reaction in assay B. An aliquot of 50ƒÊl was applied on a HPLC reverse-phase column (C18, 4.6 ~150mm). The mobile phase was 50 mm KH2PO4 (ph 6.4) containing 0.6 mm tetrabutylammonium bromide and 7% methanol. The amount of UTP formed was quantified by calculating the peak area. Determination of Protein Concentration-During purification, the concentration of protein was determined by the method of Bradford (9) using ovalbumin as the standard. The absorption coefficient of the purified enzyme at 280 nm and at 1 mg/ml was determined to be 0.49 on the basis of amino acid analysis of the protein. The molar absorption coefficient was calculated by assuming a molecular weight of 50,000. Amino Acid Analysis-Amino acid composition was determined, after hydrolysis with 6 N HCl in an evacuated tube, on a Hitachi Amino Acid Analyzer 835. Sequence Analysis-The amino acid sequence was determined with an Applied Biosystems model 470A Protein Sequencer, linked to a model 120A PTH-Analyzer. RESULTS AND DISCUSSION urification-potato tuber P(2.6 kg) was cut into slices and soaked in cold 0.7% sodium dithionite and 0.7% trisodium citrate for 1 h. The slices were processed with an electric juicer to obtain the extract. The cloudy liquid was centrifuged at 8,000 ~g for 10min to remove the cell debris and starch. The ph was adjusted to 7.5 with 1 N NaOH. The crude extract was fractionated from 40 to 65% saturation of ammonium sulfate. The protein precipitate after saturation to 65% was dissolved in a minimal volume of 30 mm Tris-HCl (ph 7.5) containing 1.2 mm EDTA and 5 mm 2-mercaptoethanol, and was dialyzed against 10 liters of the same buffer overnight at 4 Ž. The dialyzed solution was centrifuged to obtain a clear solution. The solution obtained was applied at 4 Ž on a DEAEcellulose column (4.0 ~25cm) which had been equilibrated with 30 mm Tris-HCl buffer (ph 7.5) containing 1.2 mm EDTA and 5 mm 2-mercaptoethanol, and the enzyme was eluted with the same buffer. The pass-through fractions containing the enzyme activity were pooled and dialyzed overnight at 4 Ž against 5 mm Tris-HCl buffer (ph 7.5) containing 0.2 mm EDTA and 2 mm 2-mercaptoethanol. The dialyzed solution was clarified by centrifugation, if necessary. The dialyzed solution was then applied at 4 Ž on a DEAE-cellulose column (3.0 ~45cm) which had been equilibrated with 5 mm Tris-HCl buffer (ph 7.5) containing 0.2 mm EDTA and 2 mm 2-mercaptoethanol. The column was washed with 500 ml of the same buffer, and the protein was eluted with a linear gradient from 0 to 0.1 M NaCl in the same buffer (800ml/800ml each). The fractions containing the enzyme activity were pooled and TABLE I. Purification of UDP-glucose pyrophosphorylase from potato tuber.a adata are from 2.6 kg of potato tuber, Vol.106, No.3, 1989

3 530 K. Nakano et al. Fig.1. SDS-PAGE of UDP-glucose pyrophosphorylase purified from potato tuber. Lane 1, the purified potato tuber enzyme; lane 2, the purified enzyme plus marker proteins; lane 3, marker proteins. The marker proteins were potato tuber phosphorylase (molecular weight, 110K), bovine serum albumin (67K), ovalbumin (45K), and carbonic anhydrase (30K). TABLE II. Types of product inhibition in the potato tuber UDP-glucose pyrophosphorylase reaction under different reaction conditions. concentrated by ultrafiltration with a UKO2 membrane. The concentrated solution was applied on a Sephadex G-100 column (3.0 ~85cm) which had been equilibrated with 25 mm Tris-HCl (ph 7.5) containing 1 mm EDTA and 5 mm 2-mercaptoethanol. The fractions containing the enzyme activity were pooled. The pooled enzyme solution was brought to 40% saturation of ammonium sulfate by addition of the solid salt. The sample was applied on a Butyl-Toyopearl 650S column (2.0 ~15cm) which had been equilibrated with 25 mm Tris-HCl (ph 7.5) containing 1 mm EDTA and 40% saturation of ammonium sulfate. The enzyme was eluted by a linear gradient from 40 to 0% saturation of ammonium sulfate in the same buffer. The final preparation was stored at 4 Ž. Table I shows the summary of the purification, and Fig. 1 the results of SDS-PAGE of the final preparation. The enzyme was purified 243-fold to a nearly homogeneous state by the combination of ammonium sulfate fractionation, and column chromatographies on DEAE-cellulose, Sephadex G-100, and Butyl-Toyopearl. Although the enzyme requires no sulfhydryl-reducing reagent for the enzyme activity, we added 2-mercaptoethanol to the buffer to prevent oxidation of polyphenol pigments, especially in the early stages of the purification. The purified enzyme was stable for months at 4 Ž in Tris-HCl buffer (ph 7.5). The enzyme did not show any activity loss after dilution to the order of ƒêg/ml. The specific activity of the final preparation ( `900 units/mg protein) is comparable to those of the enzymes purified from Sorghum seedling (11) and Typha latifolia pollen (12). Kinetic Studies The purified enzyme utilized UDPglucose but not ADP-glucose as substrate, and was not activated by 3-phosphoglyceric acid (data not shown). These properties are characteristic of the UDP-glucose Fig.2. Effect of magnesium ion concentration on the enzyme activity of potato tuber UDP-glucose pyrophosphorylase. (A) Initial velocities of glucose-1-p formation were measured with different fixed concentrations of substrates. ( ) 1 mm UDP-glucose and 1 mm PP1; ( ) 1 mm UDP- glucose and 0.1 mm PP1; ( ) 0.1 mm UDP-glucose and 1 mm PP1. (B) Initial velocities of UDP-glucose formation were measured with different fixed concentrations of substrates. ( œ) 2 mm UTP and 2 mm glucose-1-p; (A) 2 mm UTP and 0.2 mm glucose-1-p; ( ) 0.2 mm UTP and 2 mm glucose-1-p. Fig.3. Elution profile of the tryptic digest of the carboxymethylated enzyme on reversed phase HPLC. The digest was chromatographed on a Gilson HPLC system using a column (M & S Pak C18, 4.6 ~150mm) at a flow rate of 0.5 ml/min at the room temperature. The peptides were eluted with a linear gradient of 0 to 50% acetonitrile in 0.1% trifluoroacetic acid. The eluent was monitored by the absorption at 215 nm over a period of 120 mm. Only the peak numbers of the peptides used for the sequence comparisons (see Fig. 4) are shown in this figure. J. Biochem.

4 UDP-Glucose Pyrophosphorylase from Potato Tuber 531 Fig.4. Sequence comparison between UDP-glucose pyrophosphorylases from slime mold and potato tuber. The tryptic peptides contained in the peaks on HPLC (see Fig.3) were sequenced either directly or after further purification by HPLC. The peptides are aligned below the sequence of the slime mold enzyme (6), on the basis of more than 35% identity, except for peptide 48 (with 30% identity). Other peptides are not shown. Pairs of identical residues between the two enzymes are shown in boldface. The numbers in parentheses show the original peak numbers (see Fig.3) from which the peptides were isolated, and the subsequent letters the order of elution in the further purification on HPLC (data not shown). pyrophosphorylase activity detected by Sowokinos (1), suggesting that UDP-glucose and ADP-glucose pyrophosphorylases may be distinct proteins. The Km values for substrates were determined from the results of initial velocity studies. The double reciprocal plots of the enzyme activity against the substrate concentrations were all linear (data not shown). The calculated values for the different substrates were 0.12 mm for UDP-glucose, 0.11 mm for PP,, 0.17 mm for UTP, and 0.18 mm for glucose-1-p. The double reciprocal plots of the enzyme activity against the concentration of UDP-glucose or PP1 gave a set of straight lines intercepting at a single point (data not shown), suggesting that the enzyme follows a sequential mechanism. Product inhibition studies were carried out further to obtain information regarding the sequence of the binding of substrates and the release of products from an enzyme during catalysis. Table II summarizes the results of those studies. The simplest mechanism which qualitatively satisfies these results would be the sequential binding of UDP-glucose and MgPP, and the sequential release of glucose-1-p and MgUTP, in this order, as proposed for the enzymes from other sources (11-13). The effects of the Mg2+ concentration on the enzyme activity were examined with different fixed concentrations of substrates (Fig.2). The enzyme showed an absolute requirement for the divalent ion for catalysis of both synthesis and degradation of UDP-glucose. In the degradation of UDP-glucose, maximum rates were obtained when Mg' was added at approximately the same concentration as PP1, independent of the UDP-glucose concentration, suggesting that the MgPPI complex is the actual substrate. In the synthesis of UDP-glucose, a similar relationship was observed between the concentrations of Mg2+ and UTP, suggesting that the MgUTP complex is the actual substrate. Furthermore, the velocities with 2.0 mm UTP were less than with 0.2 mm UTP at Mg2+ concentrations of less than 1.0 mm. These results indicate the inhibition by noncomplexed, free UTP. Similar observations were made with the erythrocyte enzyme (13). Gillett et al. (14) reported that the calf liver enzyme forms stable complexes with UDP-glucose or UTP. These complexes could be isolated by gel filtration on a Sephadex G-25 column. When the potato enzyme preincubated with radioactive UDP-glucose at 20 Ž for 12 min was subjected to centrifugal filtration on a Sephadex G-50 column, no radioactivity was recovered in the protein fraction. This implies that the potato enzyme does not form a stable complex with substrate. Structural Studies The molecular weight of the purified enzyme was determined under reducing and denatured conditions by SDS-PAGE to be 53,000. When the analysis was performed under nonreducing and native conditions by chromatography on Superose 12, the enzyme was eluted as a single peak with the apparent molecular weight of 47,000. These results suggest that the enzyme exists as the monomer of an approximately 50 kda polypeptide. A similar conclusion has been obtained for the pollen enzyme (12), although the mammalian liver enzyme exists as the octamer of identical subunits with a molecular weight of 50,000 (15). The amino acid sequences of UDP-glucose pyrophosphorylase 1 from slime mold ( 6) and ADP-glucose pyrophosphorylase from E. coli (7) have been deduced from the Vol.106, No.3, 1989

5 532 K. Nakano et al. nucleotide sequences of the DNAs encoding these enzymes. To reveal the structural relationship between these two enzymes and potato enzyme, we determined the partial amino acid sequence of the latter enzyme. Gas-phase sequencer analysis of the intact carboxymethylated protein gave no positive results, suggesting that the N-terminus of the enzyme is blocked. The purified enzyme (1 mg) was S-carboxymethylated according to the method of Crestfield et al. (10), extensively dialyzed against water, and then lyophilized. The lyophilized material was dissolved in 25 mm Tris-HCl (ph 8.0) containing 2 M urea, and digested with `8ƒÊg of trypsin. After incubation overnight at 37 Ž, an aliquot of 200 pl was subjected to HPLC using a C18 column (Fig.3). Some fractions contained single peptides. Other fractions needed further purification on HPLC using the same column but in an ammonium acetate system. Thus, 29 peptides were isolated, and their amino acid compositions and sequences were analyzed. The numbers of amino acid residues in those peptides were about 300 in total, although the sequence determinations of some peptides were not completed. The amino acid sequences of the peptides containing more than 9 amino acid residues were compared with the complete amino acid sequences of slime mold UDP-glucose pyrophosphorylase (6) and E. coli ADP-glucose pyrophosphorylase (7) in the SDC-GENETYX (Software Development). No significant homology was detected between the potato and E. coli enzymes, whereas there was marked homology between the potato and slime mold enzymes. Figure 4 compares the amino acid sequence of the potato enzyme with that of the slime mold enzyme. These results are consistent with the view that the potato enzyme purified is UDP-glucose pyrophosphorylase, distinct from ADPglucose pyrophosphorylase. Among the 300 amino acids determined only 4 tryptophan residues were identified. Since tryptophan has a high absorption coefficient at 215 nm, it is unlikely that we missed isolating tryptophan-containing peptides in our procedure. This low tryptophan content might explain the low absorption coefficient at 280 nm of this protein. REFERENCES 1. Sowokinos, J. R. (1976) Plant Physiol. 57, ap Rees, T., Leja, M., Macdonald, F. D., & Green, J. H. (1984) Phytochemistry 23, ap Rees, T. (1989) Proc. Ann. Meeting & 29th Symp. of Jpn. Soc. Plant Physiol. Nagoya, pp Keeling, P. L., Wood, J. R., Tyson, R. H., & Bridges, I. G. (1988) Plant Physiol. 87, Tyson, R. H. (1987) Ph. D. thesis, University of Cambridge 6. Ragheb, J. A. & Dottin, R. P. (1987) Nucleic Acids Res. 15, Baecker, P. A., Furlong, C. E., & Preiss, J. (1983) J. Biol. Chem. 258, Franke, J. & Sussman, M. (1971) J. Biol. Chem. 246, Bradford, M. M. (1976) Anal. Biochem. 72, Crestfield, A. M., Moore, S., & Stein, W. H. (1963) J. Biol. Chem. 238, Gustafson, G. L. & Gander, J. E. (1972) J. Biol. Chem. 247, Hondo, T., Hara, A., & Funaguma, T. (1983) Plant Cell Physiol. 24, Tsuboi, K. K., Fukunaga, K., & Petriccinai, J. C. (1969) J. Biol. Chem. 244, Gillett, T. A., Levine, S., & Hansen, R. G. (1971) J. Biol. Chem. 246, Turnquist, R. L., Gillett, T. A., & Hansen, R. G. (1974) J. Biol. Chem. 249, J. Biochem.