Michael E. Salvucci* and Steven J. Crafts-Brandner · tions were assayed for activity, and peak fractions were stored individually at -80°C. HPLC Determination of Reaction Products

$Page 1: Michael E. Salvucci* and Steven J. Crafts-Brandner · tions were assayed for activity, and peak fractions were stored individually at -80°C. HPLC Determination of Reaction Products$
Plant Physiol. (1 995) 108: 1269-1 276

Purification and Properties of a Unique Nucleotide Pyrophosphatase/Phosphodiesterase I That Accumulates in

Soybean Leaves in Response to Fruit Removal'

Michael E. Salvucci* and Steven J. Crafts-Brandner

United States Department of Agriculture, Agricultural Research Service, Western Cotton Research Laboratory, 41 35 East Broadway Road, Phoenix, Arizona 85040-8830

Severa1 unique proteins accumulate in soybean (Clycine max) leaves when the developing fruits are removed. In the present study, elevated levels of nucleotide pyrophosphatase and phosphodiesterase I activities were present in leaves of defruited soybean plants. The soluble enzyme catalyzing these reactions was purified nearly 1 000-fold, producing a preparation that contained a single 72-kD polypeptide. The molecular m a s of the holoenzyme was approximately 560 kD, indicating that the native enzyme was likely oc- tameric. The purified enzyme hydrolyzed nucleotide-sugars, nucleotide di- and triphosphates, thymidine monophosphate pni- trophenol, and inorganic pyrophosphate but not nucleotide mono- phosphates, sugar mono- and bisphosphates, or NADH. The subunit and holoenzyme molecular masses and the preference for substrates distinguish the soybean leaf nucleotide pyrophosphatase/phosphodiesterase I from other plant nucleotide pyrophosphatase/phosphodiesterase I enzymes. Also, the N-terminal sequence of the soybean leaf enzyme exhibited no similarity to the mammalian nucleotide pyrophosphatase/phosphodiesterase I, soybean vegetative storage proteins, or other entries in the data bank. Thus, the soybean leaf nucleotide pyrophosphatase/phosphodiesterase I appears to be a heretofore undescribed protein that i s physically and enzymatically distinct from nucleotide pyrophosphatase/phosphodiesterase I from other sources, as well as from other phosphohydrolytic enzymes that accumulate in soybean leaves in response to fruit removal.

A myriad of changes occurs in soybean (Glycine max) plants in response to the disruption of senescence-associated events (Staswick, 1994). Early work by Wittenbach (1982,1983) showed that a number of proteins accumulate in the leaves when developing fruits are continuously removed. These proteins, now known as VSPs, are thought to serve as reservoirs for the amino acids produced from degradation of Rubisco and other leaf proteins (Witten- bach, 1982, 1983). More recently, other proteins have been identified that accumulate in soybean leaves in response to the removal of developing fruits. These proteins include the Rubisco complex protein, a cytosolic protein that forms a specific association with the Rubisco holoenzyme in vitro (Crafts-Brandner and Salvucci, 1994), and an acid phosphatase (Staswick et al., 1994).

This study was conducted while the authors were affiliated

* Corresponding author; e-mail msalvuccQasrr.arsusda.gov; fax with the Kentucky Agricultural Experiment Station.

1-602-379-4509.

Fruit removal perturbs the normal progression of events associated with the remobilization of carbon and nitrogen from the leaves to the developing seeds. In response, acid phosphatases and other hydrolytic enzymes accumulate in the leaves, presumably to adjust metabolism to the altered source-sink status. The most abundant of these enzymes are the VSPs, which were previously thought to be simply for storage but are now known to be active catalytically as polyphosphatases (DeWald et al., 1992) and lipoxygenases (Tranbarger et al., 1991).

Both the VSPs and the Rubisco complex protein accumulate, albeit transiently, during the course of normal senescence (Wittenbach, 1983; Staswick, 1989, 1990; Crafts- Brandner et al., 1991). The appearance of these proteins during normal development and their accumulation in response to sink removal suggest that they may be part of the normal metabolic machinery of protein degradation and remobilization during senescence. Here we describe a nucleotide pyrophosphatase/phosphodiesterase I that accumulates in soybean leaves in response to sink removal. The physical and enzymatic properties of this enzyme differ from nucleotide pyrophosphatase/phosphodiesterase I enzymes from mammalian (Evans et al., 1973; Byrd et al., 1985; Yano et al., 1985) and plant (Balakrishnan et al., 1977; Bartkiewicz et al., 1984) sources, as well as from other soybean leaf phosphohydrolytic enzymes (DeWald et al., 1992; Staswick et al., 1994).

MATERIALS AND METHODS

Chemicals

[8-3H]ATP and [U-'4ClGlc were purchased from Amer- sham2 and used to synthesize [8-3H]ADP-[U-'4C]Glc (Preiss and Greenberg, 1972; Drake et al., 1989). Biochemi- cals including nucleotides and nucleotide-sugars were obtained from Sigma.

Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable.

Abbreviations: FPLC, fast protein liquid chromatography; TMP- pNP, thymidine monophosphate p-nitrophenol; VSP, vegetative storage protein.

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1270 Salvucci and Crafts-Brandner Plant Physiol. Vol. 108, 1995

Plant Material

Soybean (Glycine mux L. cv McCall) plants were grown in the greenhouse and defruited as described previously (Crafts-Brandner et al., 1991). The second or third leaf below the last fully expanded leaf was used for compara- tive measurements of enzyme activity. Control (i.e. untreated) plants and plants that were continuously defruited were sampled 3 to 4 weeks after the start of fruit removal.

Enzyme Assays

For measurement of enzyme activity in desalted leaf extracts, leaf discs were extracted at 4°C in a Ten Broeck glass homogenizer containing 50 mM Hepes-KOH, pH 7.5, 5 mM MgCl,, 2 mM EDTA, 1 mM PMSF, 10 p~ leupeptin, 5 mM 2-mercaptoethanol (buffer A), and 1% (w/v) PVP-40. Extracts were centrifuged for 3 min at 13,OOOg and the supernatant fluid was desalted in 50 mM Hepes-KOH, pH 7.5, and 5 mM 2-mercaptoethanol by centrifugal gel filtra- tion (Salvucci et al., 1992). For some experiments, the 13,OOOg supernatant fraction was further centrifuged at 237,OOOg for 1 h to separate soluble components from microsomes.

Nucleotide pyrophosphatase activity in crude and purified preparations was determined at 30°C by measuring Glc-1-P formation in a two-stage assay. In the first stage, the nucleotide-sugar was incubated with the enzyme in 100-pL reactions containing 100 mM acetate-NaOH, pH 4.5, or other buffers as indicated in the text. After 5 to 10 min, the reaction was terminated by boiling for 2 min. Precipi- tated protein was removed by centrifugation for 1 min at 13,00Og, and an aliquot of the supernatant fluid was assayed to determine the amount of Glc-1-P formed. The amount of Glc-1-P was determined spectrophotometrically at 35°C by monitoring the increase in A340 caused by re- duction of NADE'+ in a 500-pL reaction mixture containing 100 mM Hepes-NaOH, pH 8.0,5 mM MgCl,, 1 mM NADP+, 5 mM DTT, 2.5 IU of phosphoglucomutase, and 2 IU of Glc-6-P dehydrogenase. Maximal activities and K , values were determined by nonlinear regression analysis of the dependence of activity on substrate concentration using the GraFit program (Leatherbarrow, 1992), after correction for boiled enzyme controls. The results presented are the means * SD of duplicate assays of representative experiments.

Nucleotide di- and triphosphatase and inorganic pyrophosphatase activities were determined at 30°C by measuring Pi formation (Chifflet et al., 1988). Reaction mixtures contained 100 mM acetate-NaOH, pH 4.5 (or other buffers as indicated), phosphorylated substrate at the concentrations indicated in the text, and enzyme in a total volume of 150 pL. Reactions were conducted for 3 min and terminated by boiling. Phosphodiesterase I activity was measured continuously at 30°C by monitoring the increase in

that occurred upon hydrolysis of TMP-pNP. The reaction mixture contained 100 mM Tricine-NaOH, pH 7.5, 5 mM MgCl,, TMP-pNP, and enzyme in a total volume of 500 pL. Maximal activities and K,,, values were determined as described above and the results presented are the means 2 SD of duplicate assays.

Agarose gels were used to detect exo- and endonuclease activity (Sambrook et al., 1989). Double-stranded (plasmid DNA with no RNase treatment) and single-stranded (M13 phage DNA) circular DNA and linear double-stranded DNA (@X174/HueIII fragments) were incubated separately with the enzyme for 4 h at 30°C in reactions containing 50 mM Hepes-KOH, pH 7.5, and 5 mM MgC1,.

Purification of Nucleotide Pyrophosphatase/Phosphodiesterase I

A11 steps were performed at 4°C. Nucleotide pyrophosphatase/phosphodiesterase I was purified from leaves of defruited soybean plants. Following removal of the midrib, 800 g of leaves were homogenized in a Waring blender in 2 L of buffer A containing 1 mM DTT and 1% (w/v) polyvinylpolypyrrolidone. The homogenate was filtered through four layers of cheesecloth and two layers of Mira- cloth (Stratagene), and the filtrate was centrifuged for 15 min at 19,OOOg. Four hundred milliliters of DEAE-Sephacel, equilibrated in 20 mM Hepes-KOH, pH 7.2, and 5 mM 2-mercaptoethanol (buffer B), were added to the supernatant fluid and incubated with occasional mixing for 20 min. The suspension was poured into a Biichner funnel and the DEAE-Sephacel was collected on Whatman No. 1 filter paper. The DEAE-Sephacel cake was rinsed sequentially with 500 mL of buffer B, 1 L of buffer B containing 0.15 M

KC1, and 1.5 L of buffer B containing 0.5 M KCl. The 0.5 M KCl eluant was supplemented with saturated ammonium sulfate, pH 7.0, to 25% (w/v) and centrifuged for 10 min at 20,OOOg to remove precipitated protein. The supernatant fraction was treated with solid ammonium sulfate to a final concentration of 45% (w/v), and precipitated protein was collected by centrifugation for 10 mili at 20,OOOg.

Protein precipitating between 25 and 45% (w/v) ammonium sulfate was resuspended in buffer B and desalted in buffer B on a 2.5- X 100-cm column of Sephadex G-25-fine. The desalted protein was loaded onto a 2.5- X 20-cm column of Cibracon-blue (Bio-Rad) and incubated overnight in the column by stopping flow. After 10 h, the column was eluted at 1.5 mL min-' sequentially with buffer B, buffer B containing 0.15 M KCl, and buffer B containing 0.5 M KCI. During each step, the eluant was monitored for A,,, to ensure complete removal of adsorbed protein before addition of the next eluant buffer. Protein in the 0.5 M KC1 eluant was precipitated by addition of ammonium sulfate to 50% (w/v) and collected by centrifugation for 10 min at 15,OOOg. The pellet was resuspended in buffer A and desalted by passage through a 40-mL column of Sephadex G-50-80, equilibrated in buffer A. The desalted protein was fractionated by anion-exchange FPLC (Pharmacia) on a 10- x 1-cm Mono Q column. The column was equilibrated in 25 mM Hepes-KOH, pH 7.8, and eluted at 2 mL min-l by application of a linear KCl gradient from O to 1 M over 160 mL. Column fractions were assayed for nucleotide (UDP- Glc) pyrophosphatase activity. Two peaks of activity were resolved on the anion-exchange column, and fractions corresponding to these peaks were pooled separately. The pooled fractions were diluted with 3 volumes of 50 mM Mes-KOH, pH 6.0, and then chromatographed at 1 mL

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Soybean Leaf Nucleotide PyrophosphataseJPhosphodiesterase I 1271

min-' on a 0.5- X 5-cm cation-exchange FPLC column (Mono S, Pharmacia). The column was eluted with KC1 using a linear gradient from O to 0.5 M over 20 mL. Frac- tions were assayed for activity, and peak fractions were stored individually at -80°C.

HPLC Determination of Reaction Products

Products of the nucleotide pyrophosphatase reaction were determined by anion-exchange HPLC (Salvucci and Crafts-Brandner, 1991) after reaction of [8-3H]ADP-[U- ''C]Glc with the enzyme. The eluant from the HPLC column was monitored for radioactivity and A,,,. Products of the nucleotide pyrophosphatase reaction were also determined by HPLC on a boronate column (Salvucci and Crafts-Brandner, 1991; Salvucci et al., 1992) after reaction of unlabeled UTP or UDP for 1 h with the enzyme. Reaction products were detected by their A,,,.

N-Terminal and Peptide Sequencing

Purified nucleotide pyrophosphatase/phosphodiesterase I was electrophoresed in a 10% SDS-PAGE minigel and electroblotted to a polyvinylidene difluoride-Q (Millipore) membrane (Salvucci et al., 1990, 1992). The electroblotted 72-kD polypeptide was detected by staining the margins of the membrane with Coomassie blue. Unstained portions of the membrane containing the electroblotted polypeptide were excised, and portions were submitted to the Univer- sity of Kentucky Macromolecular Facility for N-terminal sequence analysis. Tryptic peptides were generated from the remaining portions of the membrane (Bauw et al., 1989). Peptides were separated by reverse-phase HPLC (Salvucci et al., 1992), and those that were well resolved were submitted for sequencing. Peptides were sequenced at the University of Kentucky Macromolecular Facility using an Applied Biosystems 477A pulse liquid protein sequencer with on-line 120A phenylthiohydantoin identification.

Molecular Mass Determination

Rate zona1 centrifugation in Suc gradients was used to estimate the molecular mass of the soybean nucleotide pyrophosphatase/phosphodiesterase I. The procedure was similar to that described previously (Salvucci et al., 1990), except that the gradients were centrifuged in a Beckman SW-55 rotor instead of a SW-40 rotor, with appropriate adjustments for the smaller gradient size and higher centrifugal force. The position of the nucleotide pyrophos-

phatase/phosphodiesterase I was determined by measuring UDP-Glc pyrophosphatase activity and confirmed by SDS-PAGE.

Miscellaneous Techniques

Protein was determined by the dye-binding assay of Bradford (1976) using BSA as the standard. Chl was measured in 80% (v/v) acetone by the method of Arnon (1949). SDS-PAGE was performed in full-format, 7.5 to 150/ O 1' inear gradient gels or 10% minigels as described previously (Chua, 1980). Nondenaturing PAGE was performed at 4°C in 4 to 7% linear gradient minigels by omitting SDS from the standard gel formulation. Native gels were pre-electrophoresed for 30 min with 10 mM thioglycolate. Phospho- diesterase activity was detected by incubating the gels with 1 mM TMP-pNP in 50 mM Tricine-NaOH, pH 7.5. The yellow activity band was excised and boiled in the pres- ente of SDS, and the solution was electrophoresed in a 10% SDS-PAGE minigel.

RESULTS

Detection of Nucleotide pyrophosphatase and Phosphodiesterase I Activities in Soybean Leaves

While measuring ADP-Glc pyrophosphorylase activity in soybean leaf homogenates, we found that extracts pre- pared from the leaves of defruited soybean plants also catalyzed PPi-independent Glc-1-P formation from ADP- Glc. To verify that the reaction involved a nucleotide pyrophosphatase, product analysis was conducted after incubating [8-3H]ADP-[U-14C]G1~ with a purified protein fraction from defruited soybean leaves (see below). Sepa- ration of the labeled products by anion-exchange HPLC showed that [8-3H]AMP and [U-'*C]Glc-P were produced in the reaction (data not shown).

Nucleotide pyrophosphatase activity was much higher in leaves from plants that were defruited compared to untreated plants (Table I). Three weeks after the defruiting treatment was started, control and continuously defruited plants had similar amounts of leaf Chl and soluble protein, whereas nucleotide pyrophosphatase activity was 12-fold higher in the leaves from defruited plants (Table I). Leaves from defruited plants also contained significantly higher phosphodiesterase I activity compared to untreated control plants. Nucleotide pyrophosphatase and phosphodiesterase I activities increased with time, provided the plants were continuously defruited (data not shown). Centrifuga- tion of extracts for 1 h at 237,OOOg showed that the activity

Table I. Chl, soluble protein, nucleotide pyrophosphatase, and phosphodiesterase I activities in leaves of control and defruited soybean plants

measured at pH 7.5 with 5 mM TMP-p-NP. Nucleotide pyrophosphatase activity in desalted extracts was measured at pH 4.5 with 10 mM ADP-Glc. Phosphodiesterase I activity was

Parameter Control DeDodded DeDoddedKontrol

Chl (mg cm-l) 0.035 0.039 1.1 Soluble protein (mg cm-') 0.37 2 0.08 0.45 2 0.07 1.2 Nucleotide pyrophosphatase (lu mg-' protein) 0.046 ? 0.001 0.548 5 0.020 11.9 Phosphodiesterase I (lu mg-' protein) 0.01 5 2 0.000 0.100 ? 0.002 6.7




present in the low-speed supernatant fraction of leaf ex-tracts was soluble and not associated with a microsomalfraction (data not shown).

Purification of Nucleotide Pyrophosphatase from Leaves ofDefruited Soybean Plants

The soybean nucleotide pyrophosphatase/phosphodies-terase I was purified from leaves of defruited plants by theprocedure described in Table II. Nucleotide pyrophos-phatase activity eluted in two separate peaks when chro-matographed on an anion-exchange FPLC column. Thesepeaks were chromatographed separately on the cation-exchange column, but activity eluted in a similar positionfrom the Mono S column. After cation-exchange chroma-tography, nucleotide pyrophosphatase activity was en-riched nearly 1000-fold to a final specific activity of morethan 2100 IU mg"1 protein.

Figure 1 shows the polypeptide profile at the varioussteps in the purification procedure. The Coomassie blue-stained SDS-PAGE gel revealed that a polypeptide of 72 kDwas enriched throughout the purification regime and wasthe only polypeptide visible in the most highly purifiedfractions from the cation-exchange (peak 1) column (lane7). The gel also showed that the two peaks from the anion-exchange column (lanes 6 and 8) were similar except for theamounts of a contaminating polypeptide at 34 kD. How-ever, to avoid complications, further analysis was con-ducted on enzyme from the cation-exchange (peak 1) col-umn, the most highly purified material.

Fractions from the cation-exchange columns containingthe purified nucleotide pyrophosphatase were stored sep-arately at — 80°C without further addition. No loss of ac-tivity was detected after 2 months of storage at -80°C orafter repeated freezing and thawing of the fractions. In fact,residual enzyme activity was detected in glass cuvetteseven after rinsing with water and acetone and air dryingfor 24 h at room temperature. However, no activity wasdetected after heating the enzyme for 2 min at 100°C.Activity of the purified enzyme was unaffected by thepresence of the divalent metal cations Mg2+, Ca2+, andMn2+ or sulfhydryl reagents (data not shown).

1 2 3 4 5 6 7 8 9

116 •*•97.4 -*•

66 -»>

45 •*

31 •*

21 -»•

74.4 -»•

fl

Figure 1. SDS-PAGE of the polypeptides present at the various steps(Table II) in the purification of the soybean leaf nucleotide pyrophos-phatase/phosphodiesterase I. Samples were taken of the crude ho-mogenate (lane 2) and crude supernatant (lane 3) and after chroma-tography on DEAE-Sephacel (lane 4), Cibracon-blue (lane 5), anion-exchange FPLC (peak 1, lane 6; peak 2, lane 8), and cation-exchangeFPLC (peak 1, lane 7; peak 2, lane 9) and electrophoresed in 7.5 to15% linear gradient gels. Molecular mass standards were electropho-resed in lane 1 and their apparent molecular masses in kD areindicated in the margin. Polypeptides were visualized by stainingwith Coomassie brilliant blue R-250.

Nondenaturing Gel Electrophoresis of the NucleotidePyrophosphatase/Phosphodiesterase I

Fractions containing purified nucleotide pyrophos-phatase also exhibited phosphodiesterase I activity. Phos-phodiesterase I activity in these fractions was enrichedmore than 1000-fold to a final specific activity of about 350jamol TMP-pNP hydrolyzed min"1 mg"1 protein (see be-low). At 0.5 mM TMP-pNP, phosphodiesterase activity ofthe purified enzyme was inhibited markedly by ADP-Glc,a substrate for the nucleotide pyrophosphatase reaction

Table II. Purification of soybean leaf nucleotide pyrophosphatase/phosphodiesterase INucleotide pyrophosphatase activity was measured at pH 4.5 with 10 mM UDP-Glc.

Purification Fraction

Crude supernatantDEAE-SephacelAmmonium sulfate (25-45%)Cibracon-blueAnion-exchange FPLC

Peak 1Peak 2

Cation-exchange FPLCPeak 1Peak 2

Proteinmg

7,7512,216

58220

2.65.8

0.30.2

Activity

llf

17,26112,32010,3822,276

1,000661

656390

Specific ActivityIU mg~ ' protein

2.25.6

17.8114

385114

2,1871,950

Purification

-fold12.58.1

52

17552

994886



Soybean Leaf Nucleotide Pyrophosphatase/Phosphodiesterase I 1273

(Fig. 2). These results indicated that purified soybean nucleotide pyrophosphatase also possessed phosphodiesterase I activity.

To verify the dual activities of the enzyme, the purified protein was electrophoresed in 4 to 7% nondenaturing polyacrylamide gradient gels, and the gels were stained for phosphodiesterase I activity. The results showed that phosphodiesterase I activity co-migrated with the stained band near the top of the gel (data not shown). When the yellow activity band was excised and the contents were subjected to SDS-PAGE, protein in the band migrated as a single 72-kD polypeptide (data not shown).

p H Dependence of Nucleotide Pyrophosphatase and Phosphodiesterase I Activities

The pH optimum of the soybean leaf nucleotide pyrophosphatase was approximately 4.5 for UDP-Glc and 5.5 for UDP (Fig. 3). In contrast, phosphodiesterase I activity exhibited a broad but relatively well-defined pH optimum of about 7.5. The soybean nucleotide pyrophosphatase/ phosphodiesterase I also hydrolyzed PPi. The pH optimum for inorganic pyrophosphatase activity was about 4.5.

Substrate Specificity of the Soybean Nucleotide Pyrophosphatase/Phosphodiesterase I

The act iv i ty of p u r i f i e d soybean nuc leot ide pyrophosphatase in the presence of saturating concentrations of five Glc-based nucleotide-sugars is shown in Table 111. The highest activities occurred with two pyrimidine-nucleotides, UDP-Glc and CDP-Glc. These nucleotide-sugars were hydrolyzed at almost twice the rates of the purine- based nucleotide-sugars. However, of the nucleotide-sugars tested, the pyrimidine sugar TDP-Glc was hydrolyzed at the lowest rate. The apparent K , values for ADP-Glc and UDP-Glc were 1.5 and 4.2 mM, respectively, indicating that

I I I I I I I I

I I I I I I I I I O 2 4 6

[ADP-Glc], (mM) Figure 2. lnhibition of the phosphodiesterase I activity of purified soybean leaf nucleotide pyrophosphatase by ADP-Clc. Phosphodi- esterase I activity was measured at pH 7.5 in the presence of 0.5 mM TMP-pNP and the indicated concentrations of ADP-Clc.

750

500

250

O

60

40

20

O

3 4 5 6 7 8 9 1 0

PH Figure 3. The effect of pH on the activity of purified soybean leaf nucleotide pyrophosphatase/phosphodiesterase I. Nucleotide pyrophosphatase, inorganic pyrophosphatase, and phosphodiesterase I activities were measured at the indicated pH in the presence of 10

TMP-pNP (A). Reactions contained 1 O 0 mM of the following buffers: acetate-NaOH, pH 3.5, 4.5, and 5.5; Mes-NaOH, pH 5.5 and 6.5; Mops-NaOH, pH 6.5 and 7.5; Tricine-NaOH, pH 7.5 and 8.5; and Gly-NaOH, pH 8.5 and 9.5.

mM UDP-Clc (A), 10 mM UDP (O), 10 mM PPi (H), and 0.5 mM

the apparent affinity for ADP-Glc was 3-fold higher than for UDP-Glc.

The K , and V,,, values of purified soybean nucleotide pyrophosphatase/phosphodiesterase I for various nucleotide substrates, as well as for PPi and TMP-pNP, are shown in Table IV. Maximum rates of hydrolysis in acetate buffer at pH 4.5 were slightly higher for nucleotide diphosphates compared to nucleotide triphosphates, whereas the apparent K , values for nucleotide triphosphates were con- siderably lower than for nucleotide diphosphates. PPi was hydrolyzed at rates equivalent to approximately one-half the rate with nucleotide di- and triphosphates. However, the purified enzyme was unable to hydrolyze the nucleotide monophosphate AMP at appreciable rates and the rate of UMP hydrolysis was less than 1% of the rate with UDP. The purified enzyme exhibited no detectable activity toward the sugar phosphates Glc-1-P, Fru-6-P, Glc-6-P, or Fru-1,6-bisP or the pyridine nucleotide NADH (data not shown). Similarly, the enzyme exhibited no nuclease activity when incubated with total RNA, single- or double- stranded circular DNA, or double-stranded linear DNA (data not shown).

Table 111. Activity o f purified soybean nucleotide pyrophosphatase/phosphodiesterase I towards various nucleotide-sugars

All assays were conducted at pH 4.5 in the presence of saturating concentrations of the indicated nucleotide-sugar.

Nucleotide-Sugar Activity

/U mg- protein UDP-Clc 1652 2 90 CDP-Glc I224 5 3 ADP-Clc 721 2 26 GDP-Clc 613 2 27 TDP-Clc 311 ? 8



1274 Salvucci and Crafts-Brandner Plant Physiol. VOI. 108, 1995

Table IV. Activity and apparent affinity of purified soybean nucleotide pyrophosphatase/phosphodiesterase I for various nucleotides, PPi, and TMP-pNP

Pyrophosphatase activities and apparent K,,, values were determined at pH 4.5 from the response of activity to substrate concentration. Phosphodiesterase activity was determined at pH 7.5 using TMP-pNP as the substrate.

Substrate

UDP ADP UTP ATP IDP UMP AMP PPi TMP-pNP

Maximal Activity

/u mg- protein 255 2 12 214 t 1 2 180 2 8 144 2 7 305 t 1 1.9 5 2

O 110 t 8 356 t 7.6

Km

0.45 0.1 8 0.1 1 0.05 ND" ND"

mM

0.64 2.61

a Not determined.

The purified nucleotide pyrophosphatase/phosphodiesterase I exhibited significant phosphodiesterase I activity (Fig. 2; Table IV). At a saturating substrate concentration and an optimal pH of 7.5, the rate of TMP-pNP hydrolysis exceeded that for nucleotide diphosphates. However, the apparent affinity of the enzyme for TMP-pNP was consid- erably lower than for nucleotide di- and triphosphates, as indicated by a K, value of 2.6 mM. This value was similar to the value obtained for the nucleotide-sugars (see above). Purified soybean nucleotide pyrophosphatase/phosphodiesterase I also hydrolyzed the phosphatase substrate p - nitrophenol-phosphate, but at pH 7.5 the rate was less than 10% of the phosphodiesterase I rate (data not shown). At pH 4.5, purified nucleotide pyrophosphatase/phosphodiesterase I exhibited no detectable activity toward p-nitrophenol-phosphate (data not shown).

Product analysis showed that UDP and Pi were the initial products of UTP hydrolysis, followed later by the appearance of UMP (data not shown). Similarly, UMP and Pi were produced when UDP was supplied as the substrate. These results indicate that hydrolysis occurred at a PPi bond and, for nucleotide triphosphates, specifically the PPi bond between the p- and y-phosphates.

Relative Molecular Mass of the Soybean Nucleotide Pyrophosphatase/Phosphodiesterase I

The relative molecular mass of the soybean nucleotide pyrophosphatase/phosphodiesterase I was determined by rate zonal centrifugation in SUC gradients (Fig. 4). The purified enzyme migrated farther into the gradient than Rubisco, the 530-kD molecular mass standard. The position of the nucleotide pyrophosphatase/phosphodiesterase I extrapolated to a relative molecular mass of about 560 kD. A relatively large molecular mass for the nucleotide pyrophosphatase was also suggested by electrophoresis in nondenaturing polyacrylamide gels. When electrophoresed in a 4 to 7% polyacrylamide gel under nondenaturing condi- tions, nucleotide pyrophosphatase activity and Coomassie- stained protein were detected near the top of the gel,

migrating slower that the Rubisco standard (data not shown). Thus, the soybean leaf nucleotide pyrophosphatase/phosphodiesterase I is multimeric, probably an octamer comprising eight 72-kD subunits.

Analysis of the N Terminus of the Soybean Nucleotide Pyrophosphatase/Phosphodiesterase I

The first eight amino acids of the amino terminus of the soybean nucleotide pyrophosphatase/phosphodiesterase were determined by automated Edman degradation analysis of the purified enzyme after transfer to a polyvinylidene difluoride membrane. When submitted for comparison to the BLAST server, the amino acid sequence showed no similarity to entries currently in the data bank (data not shown). Visual inspection confirmed that the sequence did not correspond to any portion of the VSP (Staswick, 1990) or mammalian pyrophosphatase/phosphodiesterase I (van Driel and Goding, 1987; Funakoshi et al., 1992) sequence. The amino acid sequences of two interna1 tryptic peptides from the pyrophosphatase/phosphodiesterase I were also obtained, but the sequences of these peptides also failed to match entries currently in the data bank (data not shown).

DlSCUSSlON

A number of different types of nucleotide pyrophosphatases and associated activities have been described from a variety of organisms (Drummond and Yamamoto, 1971; Evans et al., 1973; Beacham and Wilson, 19821, including plants (Balakrishnan et al., 1977; Bartkiewicz et al., 1984). Of greatest similarity to the soybean leaf enzyme described here is the nucleotide pyrophosphatase from

600 L L j

200

O

O 1 2 3

Volume, (mL) Figure 4. Molecular mass of the soybean leaf nucleotide pyrophosphatase/phosphodiesterase I determined by rate zonal centrifugation on Suc gradients. The arrow and open symbol (O) indicate the position of nucleotide pyrophosphatase activity and protein. The closed symbols (A) represent the position of the M, standards Cyt c (1 2,400), carbonic anhydrase (29,000), BSA (66,000), alcohol dehydrogenase (1 50,000), P-amylase (200,000), and Rubisco (530,000). The molecular mass of the soybean leaf nucleotide pyrophosphatase/ phosphodiesterase I (560,000) was extrapolated from the regression line.



Soybean Leaf Nucleotide PyrophosphataseJPhosphodiesterase I 1275

potato tuber (Bartkiewicz et al., 1984). The potato tuber enzyme exhibits a subunit molecular mass of 74 kD, compared to 72 kD for the soybean leaf enzyme, and like the soybean enzyme hydrolyzes TMP-pNP, as well as nucleotide di- and triphosphates. However, the potato tuber enzyme is distinct from the soybean leaf nucleotide pyrophosphatase/phosphodiesterase I, exhibiting much lower specific activities, a lower holoenzyme molecular mass, and differences in the relative specificity toward various nucleotide di- and triphosphate substrates compared to the soybean leaf enzyme. Unfortunately, nucleotide-sugars, the best substrates for the soybean leaf nucleotide pyrophosphatase/phosphodiesterase I, were not examined as possible substrates for the potato tuber enzyme.

The best studied of the nucleotide pyrophosphatases is the mammalian plasma membrane nucleotide pyrophosphatase/phosphodiesterase I (Evans et al., 1973; Byrd et a]., 1985; Yano et al., 1985). This enzyme has been purified from human placenta (Yano et al., 1987), rat and mouse livers (Evans et al., 1973; Elovson, 1980), and cultured mouse plasmacytoma cells (Rebbe et al., 1991), and the genes for the mouse plasmacytoma and human fibroblast enzymes have been cloned and sequenced (van Driel and Goding, 1987; Rebbe et al., 1991; Funakoshi et al., 1992). The enzyme has been identified as the PC-1 antigen (Rebbe et al., 1991,1993), a plasma membrane protein expressed at a late stage in B-cell differentiation. Excess activity of this enzyme causes Lowes syndrome, a genetic disorder char- acterized by undersulfation of glycosaminoglycans in the fibroblasts (Yano et al., 1987, and refs. therein). In addition, Oda et al. (1991) have shown that the mammalian nucleotide pyrophosphatase/phosphodiesterase I possesses Thr-specific ectoprotein kinase activity, which is stimulated by acidic fibroblast growth factor.

On the basis of limited sequence comparison, the soybean leaf nucleotide pyrophosphatase/phosphodiesterase I differs structurally from the mammalian plasma membrane enzyme. The two enzymes also exhibit different physical and catalytic properties. For example, the soybean enzyme is soluble, whereas the mammalian plasma membrane nucleotide pyrophosphatase/phosphodiesterase I is primarily membrane bound, although soluble forms of the mammalian plasma membrane enzyme have been detected (Belli et al., 1993). Also, the soybean enzyme is likely to be an octamer comprised of 72-kD subunits, in contrast to the mammalian enzyme, which is dimeric and comprised of two 115- to 130-kD subunits (Evans et al., 1973; Elovson, 1980; Byrd et al., 1985; Yano et al., 1985, 1987). Finally, the soybean leaf and mammalian plasma membrane nucleotide pyrophosphatase/phosphodiesterase I exhibit sev- era1 important differences in substrate specificity, including differences in the ability to hydrolyze NADH, nucleotide diphosphates, and PPi, as well as differences in the pH optimum for phosphodiesterase activity (Evans et al., 1973; Elovson, 1980; Yano et al., 1985, 1987).

The 25- to 31-kD VSPs that accumulate in soybean leaves in response to sink remova1 exhibit polyphosphatase activity (DeWald et al., 1992). In addition, Staswick et al. (1994) have described a 51-kD acid phosphatase that accounts for

the majority of the phosphatase activity in leaves from defruited soybean plants. The 72-kD subunit molecular mass of the nucleotide pyrophosphatase/phosphodiesterase I distinguishes it from both the VSP and the soybean leaf acid phosphatase. Also, there are a number of important differences in substrate specificity between the nucleotide pyrophosphatase/phosphodiesterase I and the other two soybean leaf enzymes. These differences include (a) much lower rates of hydrolysis of the artificial phosphatase substrate, p-nitrophenol-phosphate, compared to the leaf acid phosphatase (Staswick et al., 1994), (b) much higher specific nucleotide di- and triphosphatase activities than the VSPs (DeWald et al., 1992), and (c) unlike the VSPs (DeWald et al., 1992), an inability to hydrolyze sugar mono- and bisphosphates.

Like other nucleotide pyrophosphatases, the precise function of the nucleotide pyrophosphatase/phosphodiesterase I in the leaves of defruited soybean plants is un- known. Remova1 of flowers and pods perturbs the normal course of leaf senescence by eliminating sinks at a time when leaf enzymes are being degraded. In response to this treatment, nitrogen remobilization is inhibited and excess amino acids from Rubisco and other degraded leaf proteins are used for de novo synthesis of proteins like the VSPs (Wittenbach, 1982). It is interesting that severa1 of the VSPs are glycoproteins, since one of the proposed roles of the mammalian nucleotide pyrophosphatase/phosphodiesterase I is in regulating glycoprotein synthesis through modulation of glycosyltransferase substrate availability (Rebbe and Hickman, 1991; Rebbe et al., 1993). It is conceivable that the nucleotide pyrophosphatase/phosphodiesterase I may play a similar role in leaves, considering the large flux of glycoprotein synthesis in defruited soybeans.

The mammalian nucleotide pyrophosphatase/phosphodiesterase I may also be involved in regulating the availability of substrates like 3’-phosphoadenosine 5’- phosphosulfate for biosynthetic processes (Yano et al., 1987) and in scavenging nucleotides for mineralization and other cellular processes (Huang et al., 1994). Although there is little information concerning the metabolic changes that occur in soybean leaves in response to removal of developing fruits, it is likely that the changes are consid- erable as leaf metabolism adjusts to a new role as a storage tissue. These changes may require scavenging of nucleotides for Pi and carbon skeletons to synthesize metabolites needed for the increased flux of amino acids into storage proteins. Further study of the metabolic events that occur in soybean leaves in response to defruiting may help to elucidate the function of the soybean leaf nucleotide pyrophosphatase/phosphodiesterase I.

ACKNOWLEDCMENTS

We would like to acknowledge the expert technical assistance of J.C. Anderson, L. Staples, and G. Sievert. We thank Dr. F. van de Loo for his help with measuring nuclease activity.

Received February 27, 1995; accepted April 13, 1995. Copyright Clearance Center: 0032-0889/95/108/1269/08.




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Michael E. Salvucci* and Steven J. Crafts-Brandner · tions were assayed for activity, and peak fractions were stored individually at -80°C. HPLC Determination of Reaction Products