Bivehimica et BiophysicuActa, 1077(1991)47-55 © 1991 ElsevierSciencePublishersB.V.0167-4838/91/$03.50 ADONIS 016748389100126B
47
BBAPRO 33849
Purification and characterization of nucleoside diphosphate kinase from spinach leaves Toshiko Nomura, Tetsuya Fukui and Atsushi Ichikawa Departnwntof PhysiologicalChemistry, Facultyof PharmaceuticalSciences, Kyoto University, Kyoto (Japan)
(Received22 August1990) Key words: Nucleosidediphosphatekinas¢;GYP bindingprotein;Thiophosphoryla~ion;(Spinachleaf) Two ~T.es of nncleoslde &plmsphate kinase (NDP kinase ! and NDP kinase !!) have been purified from splnaeh leaves to electropberetlc honmgeneity. The enzymes were copurifled with apparent 13SSlGTP¥S-binding activities. NDP kinase !, which was not adsorbed to a bydmxyapafite column, and NDP kinase IlL,which was adsorbed, had molecular weights of 16000 and 18000, respectively, as judged by polyacrylamide gel electrophoresls in the presence of sodium dodecyl sulfate. The molecular weights determined by gel Cdtration were 92000 and 110000, respectively, suggesting that both enzymes are composed of six identical subunits. Minor ,~ifferences in some amino acids between NDP kinase ! and NDP kinase H were observed when both enzymes were analyzed for amino acid composition. The apparent [3SSlGTP*/S-binding activity of purified NDP kinase ! and NDP kinase II was found to be due to the formation of a 13sSlthioplmsphorylated enzyme, which is the intermediate of the NDP kinase reaction. Introduction Nucleoside diphosphate klnase (NDP kinase, EC 2.7.4.6.) plays a role in control of the metabolic flow of phosphate groups among nucleoside (or deoxynucleoside) di- and uiphosphates, and in maintaining their functional levels. The NDP kinases were purified to homogeneity from various sources [1-6]. NDP kinase cDNA from Dictyostelium discoideurn [7] and Myxococcus xanthus [8] have recently been cloned and sequenced. Until now this enzyme from higher plants has not been isolated to electrophoretic homogeneity, but only fractionated in extracts of pea seed [9] and Lily pollen [10]. NDP Kinase, along with enzyme activity, has also been proposed as having a role in regulating GTP-dependent processes, since NDP kinases were shown to associate with proteins having an affmity specific for guanine nucleotides; such as Gs protein from rat liver [4,11], elongation factor elF2 [12], and 21 kDa GTPbinding proteins from Ehrlich ascites tumor cells [13] and human plateiets [14]. However, recent pubfished results are inconsistent. Lacombe et al. [7] dem-
Correspondence: A. lchikawa, Departmentof PhysiolosicalChemistry, Faculty of Pharmaceuucal Sciences,Kyoto University,Kyoto 606, Japan.
onstrated that thiophosphorylation of NDP kinase from extracts of bacteria transformed with a plasmid allowed the detection of GTP?S binding activity, :rod MufiozDorado et al. [8] reported that the 16 kDa GTP-binding protein from Myxococcus xanthus was NDP kinase, these suggesting that GTP-binding activity in NDP kinase may be generated from thiophosphate bound onto the enzyme molecule as an intermediate of the phosphate group-transfer reaction. The present report describes the molecular properties of purified NDP kinase coporified with GTP3,S-binding activity from spinach leaves and demonstrates that OTPyS-binding activity is generated from thiophnsphorylated enzyme formation. Materials and Methods Materials
The following materials were purchased from the sources indicated: DEAE-Sepharose CL-6B, Ultrogel AcA 44, Mono S HRS/5 and an eiectrophoresis calibration kit from Pharmacia LKB Biotechnology (Uppsala, Sweden); hydroxyapatite from Bio-Rad Laboratories (Richmond, CA, U.S.A.); Reactive blue-2 agarose, eytochrome c, ovalbumin, bovine serum albumin, sodium thiophosphate and all nudeotides from Sigma (St. Louis, MO, U.S.A.); lactate dehydrogenase from rabbit muscle, malate dehydroganase from pig heart and nucleoside
48
diphosphate kinase from beef heart from Boehringer (Mannheira, F.R.G.); silver staining kit from Wako Pure Chemical Industries (Osaka, Japan); [35S]GTPTS (1350 Ci/mmol), [¥-32p]GTP (22 Ci/mmol), [332P]ATP (30 Ci/mmol) and [8-3H]GDP (8.1 Ci/mmol) from New England ..XYuclear(Boston, MA, U.S.A.).
Enzyme assays NDP kinase activity was assayed by two different methods, i.e., by monitoring the formation of radioactive GTP from ATP and radioactive GDP (a, whole enzyme reaction), or of the phosphorylated enzyme, the intermediate of the enzyme reaction (b, partial enzyme reaction). (a) Radioactive GTP formation. The activity of the whole enzyme reaction was assayed by the method of Kimura and Shimada [15] with a slight modification. The reaction mixture (50 lai) comprised 5 nmol of [8-3HIGDP (50-100 Ci/mol), 50 nmol of ATP, 0.25 Fmol of MgCI 2 and 2.5 pmol of Tris-HC1 (pH 7.4). The reaction was initiated by adding the enzyme, conducted at 37°C for 5 min and terminated by adding 5 pl of 10 M formic acid followed by 5 pl of 100 mM GTP. Products formed in the reaction mixture were separated by thin-layer chromatography on polyethyleneiraine-cellulose sheets with 2 M formic acid/1.2 M LiCI 0:1, v/v), detected with an ultraviolet lamp, scraped off, and extracted with 20 mM Tfis-HC1 (pH 7.4) containing 0.7 M MgCI 2 for I h at room temperature. Radioactivity in the extracts was counted in 0.5~ 2,5-diphenyloxazole (PPO) in toluene/Triton X-100 (2:1, v/v) with a liquid scintillation counter. (b) Phosphoenzyme formation. Formation of the enzyme-boand phosphate intermediate was measured by a minor modification of the method of Koyama et al. [16]. The reaction mixture (100/~1) comprised 0.2 nmol of [,/-32p]ATP (750-1000 Ci/mol), 0.1 ~tmol of MgCI 2, 60 /tmol of KCI, 0.1 /tmol of EDTA, 0.4 pmol of dithiothreitol and 2 /tmol of Tris-HCl (pH 7.5). The reaction was initiated by adding 0.1/tg of the purified enzyme, or an aliquot from each fraction which shows the activity corresponding to that of 0.1 ttg of purified enzyme for Table I, carried out at 0 ° C for 10 rain and terminated by the addition of 1.0 ml of 0.1 M sodium pyrophosphate containing 10 mM EDTA. The sample was filtered through a nitrocellulose membrane (Schleicher and Schull; BA 85, 0.45/tin), and then the filter was washed with 3 × 2 ml of buffer, dried and subjected to radioactivity counting using 0.5~ 2,5-diphenyloxazole in toluene.
pmol of NaCL 0.06/tmol of EDTA, 0.3 pmol of 2-mercaptoethanoL 1.2/zmol of Tris-HCi (pH 7.5) and 0.1 pg of the purified enzyme or an aliquot of each fraction which shows the activity corresponding to that of 0.1 Fg of the purified enzyme for Table 1. Incubation was carried out at 30°C for 60 min and the reaction was terminated by dilution of the reaction mixture with ice-cold reaction mixture without [35S]GTP¥S, followed by rapid fdtration of the mixture through a nitrocellulose fdter. The fdter was washed, dried and then subjected to radioactivity counting as described above. Nouspecific binding was taken as the binding which was not competed for by non-labelled 100 p M GTP¥S. The values in the figures represent specific binding, which was defined as total binding minus nouspecific binding, unless otherwise stated.
Other assays Protein was determined using Folin-Ciocalteu reagent with bovine serum alb,min as a standard or from the absorbance at 280 nm, as described by Layne [18]. When interference by sulfhydryl compounds of the protein assay was not negligible, protein was also determined according to Ross and Schatz [19] using iodoacetate for carboxymethylation of the sullhydryl compounds.
Preparation of [8-JH]GTP¥S [8-3H]GTP¥S was prepared by the enzymatic transfer of thiuphosphate from GTPvS to [8-3H]GDP using beef fiver NDP kinase as described by Goody et al. [20].
Purification of the enzyme
[35S]GTPyS binding assay
All purification steps were carried out at 0 - 4 ° C . Step 1. Crude extract. 3 kg of freshly harvested spinach (Spinacia oleracea L.) leaves were cut into approx. 2-cm wide pieces, which were homogenized with a blender for 1 vain in 6000 ml of 20 mM Tris-HCl (pH 7.5) containing EPM (1 mM EDTA, 0.2 mM PMSF and 5 mM 2-mercaptoethanol), fdtered through cheese cloth, and then centrifuged at 2500 × g for 10 rain. The resulting supematant was centrifuged at 10000 × g for 10 rain and the precipitate (chloroplast-rich fraction) obtained was resuspended in 900 ml of the same buffer. Aliquots of a 10~ solution of Lubrol PX and a 2 M solution of NaCi were then added to the pellet suspension to obtain final concentrations of 1~ and 0.1 M, respectively, and then protein solubilization was carried out by incubating the suspension at 0 ° C for 60 rain. After centrifugation at 100000×g for 60 min, the resulting supematant was used as the crude extract.
The GTP,/S binding assay was carded out by a modification of the method of Northup et ai. [17]. The standard reaction mixture (60/tl) comprised 60 pmol of [3sS]GTP¥S (1000-1500 Ci/mol), 1.5 pmol of MgC! 2, 6
crude extract was diluted with 4 volumes of 20 mM Tris-HCi (pH 7.5) containing EPM, and then applied to a column (5.5 x 21 cm) of DEAE-Sepharose CL-6B
Step 2. DE ° ~.-Sepharose CL-6B chromatography. The
49 precquifibrated with 20 m M Tris-HCl containing EPM, 20 mM NaCI and 0.1~ Lubrol PX. After washing the column with 750 ml of the same buffer, elution was carried out with a 3400-ml linear gradient of NaCi (20 to 500 raM) in the same buffer. The active fractions eluted at approx. 0.2 M NaCI were combined. Step 3. Hydroxyapatite chromatography. The combined active fractions were directly appfied to a column (2.6 x 20 cm) of hydroxyapatite preequilibrated with 10 m M potassium phosphate (pH 7.0) containing EPM and 1~ Na-cbolate. The column was washed with 300 ml of the same buffer and eluted with a 600-ml linear gradient of 10 to 300 m M potassium phosphate (pH 7.0) containing EPM and 1~ Ha-cholate. At this step, the enzyme activity was separated into two peaks; the major peak of enzyme activity (NDP kinase-l) appeared in the pass-through fraction, the other second peak (NDP khmse-ll) being eluted with about 200 m M potassium phosphate. The two fractions were separately pooled. Step 4. Uitrogel Ac,4 44 chromatography. The two active fractions were separately concentrated to 25 ml in an Anficon filtration apparatus, separately applied to a column (3 × 98 cm) of Ultrogel AcA 44 pr~.-equilibrated with 10 m M potassium phosphate (pH 7.0) containing EPM, 1~ Na-cholate and 0.1 M NaCI, and eluted with the same buffer. The peak fractions of N D P kinases I and II were separately pooled. Step 5. Reactive bive-2 agarose chromatography. The N D P kinase I and II fractions from step 4 were desahed by passage through a column (6 × 35 cm) of Sephadex G-25 preequllibrated with 20 m M potassium phosphate (pH 6.3) containing EPM and 0.1~; Lubrol PX. The desalted fractious were separately applied to a column (0.86 × 17 cm) of Reactive blue-2 agarose preequilibrated with the same buffer. The column was washed with 50 ml of the same buffer and eluted with a 200-ml linear gradient of 0 to 3 M potassium chloride in the same buffer. The active fractious were combined and pooled. Step 6. Mono S HPLC. The two active fractions from step 5 were desalted by passage through a column (4 × 18 cm) of Sephadex G-25 preequllibrated with 50 m M 2-(N-Morpholino)ethanesulfonic acid (Mes, p H 5.5) containing EPM and 0.1~ Lubrol PX. The desalted fractions were separately applied to a column (0.5 × 5 cm) of Mono S H R S / 5 precquilibrated with the same buffer. The column was washed and eluted with an 18-ml linear gradient of 0 to 0.3 M NaCI in the same buffer. The tictive fractions were combined and stored at -20°C.
Electrophoresis and molecular weight determination Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was performed on slab gels (12.5~ polyacrylamide) as described by Laemmfi [21]
and protein was stained with a silver staining kit. An elect~ophoresis cafibration kit, composed of phosphorylas¢ b (94000), bovine serum albumin (6"]000), ovalbumin (43000), carbonic anhydrase (30000), soybean trypsin inhibitor (20100) and a-lactalbumin (14400), was used as molecular weight standards. Calibrated gel filtration on Ultrogel AcA 44 was performed according to the method of Andrews [22], with lactate dehydrogenase from rabbit muscle (149000), malate dehydrogenase from pig heart (67000), ovalbumin (45 000) and cytochrome c from horse heart (13000) as standards.
Amino acid analysis 300 pmol (estimated as the subunit contenO each of N D P kinase I or N D P kinase II was hydrolyzed in 6 M HCI at l l 0 ° C for 24 h and then analyzed with a Hitachi 835 amino acid analyzer. Results
Purification of the enzyme For N D P kinase activity, the amount of the stable phosphorylated enzyme intermediate of the reaction or [32p]GTP produced from G D P and [32p]ATP through the whole enzyme reaction was measured. Throughout the whole purification procedure, apparent [3sS]GTPTSbinding activity was also assayed in order to compare its column elution prof'de with that of N D P kinase activity. The N D P kinase, which was assayed as pbosphate-incosporating protein, was einied as a single peak on the DEAE-Sepharose CL-6B column, this material also exhibiting [3SS]GTP~,S-binding activity (Fig. 1). However, the material having N D P kinase activity together with [3sS]GTP-i,S-binding activity, was resolved into two peaks on the hydroxyapatite column (Fig. 2). The material which appeared in the pass-through frac8j
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Fraction Number
Fig. 1. Elation of NDP kinase from a DEAE-SepharoscCL-6B column. The crude extract was diluted with 4 vols. of 20 mM Tds-HCi (pH 7.5) containing EPM, then applied to a DEAE-Sepharose CL-6Bcolumn(5.5 × 21 cm), Elution of NDP kinasewas earned out with a linear gradient of NaCI, from 20 to 500 raM, in 20 mM Ttis-HCI containing EPM, 20 mM NaCI and 0.l~ Lubrol PX. (O) [3ZP]phosphate-incorporatingactivity;(e) [3SS]GTPyS-binding activity; ( . . . ) concentration of protein; and (-) concentration of NaCI.
50
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47
BBAPRO 33849
Purification and characterization of nucleoside diphosphate kinase from spinach leaves Toshiko Nomura, Tetsuya Fukui and Atsushi Ichikawa Departnwntof PhysiologicalChemistry, Facultyof PharmaceuticalSciences, Kyoto University, Kyoto (Japan)
(Received22 August1990) Key words: Nucleosidediphosphatekinas¢;GYP bindingprotein;Thiophosphoryla~ion;(Spinachleaf) Two ~T.es of nncleoslde &plmsphate kinase (NDP kinase ! and NDP kinase !!) have been purified from splnaeh leaves to electropberetlc honmgeneity. The enzymes were copurifled with apparent 13SSlGTP¥S-binding activities. NDP kinase !, which was not adsorbed to a bydmxyapafite column, and NDP kinase IlL,which was adsorbed, had molecular weights of 16000 and 18000, respectively, as judged by polyacrylamide gel electrophoresls in the presence of sodium dodecyl sulfate. The molecular weights determined by gel Cdtration were 92000 and 110000, respectively, suggesting that both enzymes are composed of six identical subunits. Minor ,~ifferences in some amino acids between NDP kinase ! and NDP kinase H were observed when both enzymes were analyzed for amino acid composition. The apparent [3SSlGTP*/S-binding activity of purified NDP kinase ! and NDP kinase II was found to be due to the formation of a 13sSlthioplmsphorylated enzyme, which is the intermediate of the NDP kinase reaction. Introduction Nucleoside diphosphate klnase (NDP kinase, EC 2.7.4.6.) plays a role in control of the metabolic flow of phosphate groups among nucleoside (or deoxynucleoside) di- and uiphosphates, and in maintaining their functional levels. The NDP kinases were purified to homogeneity from various sources [1-6]. NDP kinase cDNA from Dictyostelium discoideurn [7] and Myxococcus xanthus [8] have recently been cloned and sequenced. Until now this enzyme from higher plants has not been isolated to electrophoretic homogeneity, but only fractionated in extracts of pea seed [9] and Lily pollen [10]. NDP Kinase, along with enzyme activity, has also been proposed as having a role in regulating GTP-dependent processes, since NDP kinases were shown to associate with proteins having an affmity specific for guanine nucleotides; such as Gs protein from rat liver [4,11], elongation factor elF2 [12], and 21 kDa GTPbinding proteins from Ehrlich ascites tumor cells [13] and human plateiets [14]. However, recent pubfished results are inconsistent. Lacombe et al. [7] dem-
Correspondence: A. lchikawa, Departmentof PhysiolosicalChemistry, Faculty of Pharmaceuucal Sciences,Kyoto University,Kyoto 606, Japan.
onstrated that thiophosphorylation of NDP kinase from extracts of bacteria transformed with a plasmid allowed the detection of GTP?S binding activity, :rod MufiozDorado et al. [8] reported that the 16 kDa GTP-binding protein from Myxococcus xanthus was NDP kinase, these suggesting that GTP-binding activity in NDP kinase may be generated from thiophosphate bound onto the enzyme molecule as an intermediate of the phosphate group-transfer reaction. The present report describes the molecular properties of purified NDP kinase coporified with GTP3,S-binding activity from spinach leaves and demonstrates that OTPyS-binding activity is generated from thiophnsphorylated enzyme formation. Materials and Methods Materials
The following materials were purchased from the sources indicated: DEAE-Sepharose CL-6B, Ultrogel AcA 44, Mono S HRS/5 and an eiectrophoresis calibration kit from Pharmacia LKB Biotechnology (Uppsala, Sweden); hydroxyapatite from Bio-Rad Laboratories (Richmond, CA, U.S.A.); Reactive blue-2 agarose, eytochrome c, ovalbumin, bovine serum albumin, sodium thiophosphate and all nudeotides from Sigma (St. Louis, MO, U.S.A.); lactate dehydrogenase from rabbit muscle, malate dehydroganase from pig heart and nucleoside
48
diphosphate kinase from beef heart from Boehringer (Mannheira, F.R.G.); silver staining kit from Wako Pure Chemical Industries (Osaka, Japan); [35S]GTPTS (1350 Ci/mmol), [¥-32p]GTP (22 Ci/mmol), [332P]ATP (30 Ci/mmol) and [8-3H]GDP (8.1 Ci/mmol) from New England ..XYuclear(Boston, MA, U.S.A.).
Enzyme assays NDP kinase activity was assayed by two different methods, i.e., by monitoring the formation of radioactive GTP from ATP and radioactive GDP (a, whole enzyme reaction), or of the phosphorylated enzyme, the intermediate of the enzyme reaction (b, partial enzyme reaction). (a) Radioactive GTP formation. The activity of the whole enzyme reaction was assayed by the method of Kimura and Shimada [15] with a slight modification. The reaction mixture (50 lai) comprised 5 nmol of [8-3HIGDP (50-100 Ci/mol), 50 nmol of ATP, 0.25 Fmol of MgCI 2 and 2.5 pmol of Tris-HC1 (pH 7.4). The reaction was initiated by adding the enzyme, conducted at 37°C for 5 min and terminated by adding 5 pl of 10 M formic acid followed by 5 pl of 100 mM GTP. Products formed in the reaction mixture were separated by thin-layer chromatography on polyethyleneiraine-cellulose sheets with 2 M formic acid/1.2 M LiCI 0:1, v/v), detected with an ultraviolet lamp, scraped off, and extracted with 20 mM Tfis-HC1 (pH 7.4) containing 0.7 M MgCI 2 for I h at room temperature. Radioactivity in the extracts was counted in 0.5~ 2,5-diphenyloxazole (PPO) in toluene/Triton X-100 (2:1, v/v) with a liquid scintillation counter. (b) Phosphoenzyme formation. Formation of the enzyme-boand phosphate intermediate was measured by a minor modification of the method of Koyama et al. [16]. The reaction mixture (100/~1) comprised 0.2 nmol of [,/-32p]ATP (750-1000 Ci/mol), 0.1 ~tmol of MgCI 2, 60 /tmol of KCI, 0.1 /tmol of EDTA, 0.4 pmol of dithiothreitol and 2 /tmol of Tris-HCl (pH 7.5). The reaction was initiated by adding 0.1/tg of the purified enzyme, or an aliquot from each fraction which shows the activity corresponding to that of 0.1 ttg of purified enzyme for Table I, carried out at 0 ° C for 10 rain and terminated by the addition of 1.0 ml of 0.1 M sodium pyrophosphate containing 10 mM EDTA. The sample was filtered through a nitrocellulose membrane (Schleicher and Schull; BA 85, 0.45/tin), and then the filter was washed with 3 × 2 ml of buffer, dried and subjected to radioactivity counting using 0.5~ 2,5-diphenyloxazole in toluene.
pmol of NaCL 0.06/tmol of EDTA, 0.3 pmol of 2-mercaptoethanoL 1.2/zmol of Tris-HCi (pH 7.5) and 0.1 pg of the purified enzyme or an aliquot of each fraction which shows the activity corresponding to that of 0.1 Fg of the purified enzyme for Table 1. Incubation was carried out at 30°C for 60 min and the reaction was terminated by dilution of the reaction mixture with ice-cold reaction mixture without [35S]GTP¥S, followed by rapid fdtration of the mixture through a nitrocellulose fdter. The fdter was washed, dried and then subjected to radioactivity counting as described above. Nouspecific binding was taken as the binding which was not competed for by non-labelled 100 p M GTP¥S. The values in the figures represent specific binding, which was defined as total binding minus nouspecific binding, unless otherwise stated.
Other assays Protein was determined using Folin-Ciocalteu reagent with bovine serum alb,min as a standard or from the absorbance at 280 nm, as described by Layne [18]. When interference by sulfhydryl compounds of the protein assay was not negligible, protein was also determined according to Ross and Schatz [19] using iodoacetate for carboxymethylation of the sullhydryl compounds.
Preparation of [8-JH]GTP¥S [8-3H]GTP¥S was prepared by the enzymatic transfer of thiuphosphate from GTPvS to [8-3H]GDP using beef fiver NDP kinase as described by Goody et al. [20].
Purification of the enzyme
[35S]GTPyS binding assay
All purification steps were carried out at 0 - 4 ° C . Step 1. Crude extract. 3 kg of freshly harvested spinach (Spinacia oleracea L.) leaves were cut into approx. 2-cm wide pieces, which were homogenized with a blender for 1 vain in 6000 ml of 20 mM Tris-HCl (pH 7.5) containing EPM (1 mM EDTA, 0.2 mM PMSF and 5 mM 2-mercaptoethanol), fdtered through cheese cloth, and then centrifuged at 2500 × g for 10 rain. The resulting supematant was centrifuged at 10000 × g for 10 rain and the precipitate (chloroplast-rich fraction) obtained was resuspended in 900 ml of the same buffer. Aliquots of a 10~ solution of Lubrol PX and a 2 M solution of NaCi were then added to the pellet suspension to obtain final concentrations of 1~ and 0.1 M, respectively, and then protein solubilization was carried out by incubating the suspension at 0 ° C for 60 rain. After centrifugation at 100000×g for 60 min, the resulting supematant was used as the crude extract.
The GTP,/S binding assay was carded out by a modification of the method of Northup et ai. [17]. The standard reaction mixture (60/tl) comprised 60 pmol of [3sS]GTP¥S (1000-1500 Ci/mol), 1.5 pmol of MgC! 2, 6
crude extract was diluted with 4 volumes of 20 mM Tris-HCi (pH 7.5) containing EPM, and then applied to a column (5.5 x 21 cm) of DEAE-Sepharose CL-6B
Step 2. DE ° ~.-Sepharose CL-6B chromatography. The
49 precquifibrated with 20 m M Tris-HCl containing EPM, 20 mM NaCI and 0.1~ Lubrol PX. After washing the column with 750 ml of the same buffer, elution was carried out with a 3400-ml linear gradient of NaCi (20 to 500 raM) in the same buffer. The active fractions eluted at approx. 0.2 M NaCI were combined. Step 3. Hydroxyapatite chromatography. The combined active fractions were directly appfied to a column (2.6 x 20 cm) of hydroxyapatite preequilibrated with 10 m M potassium phosphate (pH 7.0) containing EPM and 1~ Na-cbolate. The column was washed with 300 ml of the same buffer and eluted with a 600-ml linear gradient of 10 to 300 m M potassium phosphate (pH 7.0) containing EPM and 1~ Ha-cholate. At this step, the enzyme activity was separated into two peaks; the major peak of enzyme activity (NDP kinase-l) appeared in the pass-through fraction, the other second peak (NDP khmse-ll) being eluted with about 200 m M potassium phosphate. The two fractions were separately pooled. Step 4. Uitrogel Ac,4 44 chromatography. The two active fractions were separately concentrated to 25 ml in an Anficon filtration apparatus, separately applied to a column (3 × 98 cm) of Ultrogel AcA 44 pr~.-equilibrated with 10 m M potassium phosphate (pH 7.0) containing EPM, 1~ Na-cholate and 0.1 M NaCI, and eluted with the same buffer. The peak fractions of N D P kinases I and II were separately pooled. Step 5. Reactive bive-2 agarose chromatography. The N D P kinase I and II fractions from step 4 were desahed by passage through a column (6 × 35 cm) of Sephadex G-25 preequllibrated with 20 m M potassium phosphate (pH 6.3) containing EPM and 0.1~; Lubrol PX. The desalted fractious were separately applied to a column (0.86 × 17 cm) of Reactive blue-2 agarose preequilibrated with the same buffer. The column was washed with 50 ml of the same buffer and eluted with a 200-ml linear gradient of 0 to 3 M potassium chloride in the same buffer. The active fractious were combined and pooled. Step 6. Mono S HPLC. The two active fractions from step 5 were desalted by passage through a column (4 × 18 cm) of Sephadex G-25 preequllibrated with 50 m M 2-(N-Morpholino)ethanesulfonic acid (Mes, p H 5.5) containing EPM and 0.1~ Lubrol PX. The desalted fractions were separately applied to a column (0.5 × 5 cm) of Mono S H R S / 5 precquilibrated with the same buffer. The column was washed and eluted with an 18-ml linear gradient of 0 to 0.3 M NaCI in the same buffer. The tictive fractions were combined and stored at -20°C.
Electrophoresis and molecular weight determination Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was performed on slab gels (12.5~ polyacrylamide) as described by Laemmfi [21]
and protein was stained with a silver staining kit. An elect~ophoresis cafibration kit, composed of phosphorylas¢ b (94000), bovine serum albumin (6"]000), ovalbumin (43000), carbonic anhydrase (30000), soybean trypsin inhibitor (20100) and a-lactalbumin (14400), was used as molecular weight standards. Calibrated gel filtration on Ultrogel AcA 44 was performed according to the method of Andrews [22], with lactate dehydrogenase from rabbit muscle (149000), malate dehydrogenase from pig heart (67000), ovalbumin (45 000) and cytochrome c from horse heart (13000) as standards.
Amino acid analysis 300 pmol (estimated as the subunit contenO each of N D P kinase I or N D P kinase II was hydrolyzed in 6 M HCI at l l 0 ° C for 24 h and then analyzed with a Hitachi 835 amino acid analyzer. Results
Purification of the enzyme For N D P kinase activity, the amount of the stable phosphorylated enzyme intermediate of the reaction or [32p]GTP produced from G D P and [32p]ATP through the whole enzyme reaction was measured. Throughout the whole purification procedure, apparent [3sS]GTPTSbinding activity was also assayed in order to compare its column elution prof'de with that of N D P kinase activity. The N D P kinase, which was assayed as pbosphate-incosporating protein, was einied as a single peak on the DEAE-Sepharose CL-6B column, this material also exhibiting [3SS]GTP~,S-binding activity (Fig. 1). However, the material having N D P kinase activity together with [3sS]GTP-i,S-binding activity, was resolved into two peaks on the hydroxyapatite column (Fig. 2). The material which appeared in the pass-through frac8j
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Fraction Number
Fig. 1. Elation of NDP kinase from a DEAE-SepharoscCL-6B column. The crude extract was diluted with 4 vols. of 20 mM Tds-HCi (pH 7.5) containing EPM, then applied to a DEAE-Sepharose CL-6Bcolumn(5.5 × 21 cm), Elution of NDP kinasewas earned out with a linear gradient of NaCI, from 20 to 500 raM, in 20 mM Ttis-HCI containing EPM, 20 mM NaCI and 0.l~ Lubrol PX. (O) [3ZP]phosphate-incorporatingactivity;(e) [3SS]GTPyS-binding activity; ( . . . ) concentration of protein; and (-) concentration of NaCI.
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