Plant Physiol. (1991) 96, 693-6980032-0889/91 /96/0693/06/$01 .00/0

Received for publication November 30, 1990Accepted February 27, 1991

Purification and Characterization of GalactinolSynthase from Mature Zucchini Squash Leaves1

Patrick T. Smith*, Tsung Min Kuo, and C. Gerald CrawfordSeed Biosynthesis Research Unit, United States Department of Agriculture, Agricultural Research Service, National

Center for Agricultural Utilization Research, 1815 North University Street, Peoria, Illinois, 61604

ABSTRACTGalactinol synthase catalyzes the first committed step in the

biosynthesis of raffinose sugars. Previous attempts to purify theenzyme have proven difficult and have resulted in low quantitiesof unpurified enzyme. Galactinol synthase was purified 752-foldfrom mature zucchini (Cucurbita pepo L. cv Burpee Hybrid) leavesusing sequential liquid chromatography on DE 52, Octyl-Sepha-rose CL-4B, and Sephacryl S-200. This isolation scheme resultedin an 18.6% recovery of the initial activity. The purified enzymehad a specific activity of 23.3 micromoles per minute per milligramprotein, a pH optimum of 7.5, and the activity was enhanced bydithiothreitol and MnCI2. The enzyme was only half as active withMgCI2 as with MnCI2. Na+, K+, and Ca2+ cations had little effecton the enzyme activity, while Co2+, Zn2+, Cu2 , and Fe3+ cationswere strongly inhibitory at 10 millimolar concentrations. Purifiedgalactinol synthase bound specifically to the substrates myo-inositol and UDP-galactose (Km = 6.5 and 1.8 millimolar, respec-tively), while exhibiting little affinity for an alternative glycosyldonor (UDP-glucose) or inositol epimers (epi- and scyllo-). Tenmillimolar concentrations of UMP, UDP, UTP, AMP, ADP, ATP,NAD+, NADH, NADP+, UDP-xylose, and UDP-mannose, or 20 mil-limolar sucrose, talose, galactose, glucose, xylose, and melibioseexhibited various degrees of inhibitory effects. Twenty millimolarstachyose, raffinose, fructose, and mannose, and 10 millimolarUDP-glucuronic acid and UDP-galacturonic acid had little or noeffect on the enzyme activity. The purified galactinal synthase isa monomer of Mr 42,000 with an isoelectric point of 4.1.

The raffinose family ofoligosaccharides is the second largestgroup of sugars present in plant tissues (5). In many species,particularly the Cucurbita, these oligosaccharides (raffinose,stachyose, and verbascose) serve as transport sugars (19).Raffinose saccharide biosynthesis is initiated by the transferof a galactosyl unit from galactinol (O-a-D-galactopyranosylmyo-inositol) to Suc to produce raffinose; this reaction isfurther iterated to yield stachyose and verbascose (15). Gal-actinol is synthesized from UDP-Gal and myo-inositol byUDP-D-a-galactose:inositol galactosyltransferase or GS2 (15)as shown below:

UDP-Gal + myo-inositol *-* galactinol + UDP

'Supported in part by a grant from the American Soybean Asso-ciation to T. M. K.

2 Abbreviations: GS, galactinol synthase; EG, ethylene glycol;FPLC, fast protein liquid chromatography; Mel, melibiose; OS, Octyl-Sepharose CL-4B; RB, running buffer; Tal, talose; UDP-GalUA,UDP-galacturonic acid.

The synthesis of galactinol is the first committed step inthe synthesis of the raffinose saccharides; however, little isknown of the enzyme which catalyzes this step.Frydman and Neufeld (6) first detected GS in crude extracts

from maturing pea seeds. Pharr et al. (20) and Webb (23)partially purified GS from mature cucumber and squashleaves, respectively. The subcellular localization ofGS has yetto be established in either leaf tissue or developing seeds.Galactinol synthase activity has a pH optimum between 7.0and 8.0 and is enhanced by both f-mercaptoethanol (or DTT)and MnCl2 (8, 20, 23). Handley and Pharr (8) also showedthat concentrations of MnCl2 greater than 1.0 mm couldinhibit enzyme activity. Galactinol synthase is highly specificfor UDP-Gal and myo-inositol and demonstrates little activitywith alternate substrates, such as UDP-, ADP-, GDP-, CDP-,and TDP-sugars or epimers of inositol (6, 23). Galactinolsyntheses have been partially purified from cucumber andstraight-necked squash leaves with specific activities of 0.81and 1.56 ,umol min-' mg protein-', respectively (8, 23). Basedon the elution profile from gel permeation chromatography,Webb (23) suggested that only one isozyme ofGS was present.However, these enzyme preparations contained five majorand five minor bands on SDS-PAGE.

This paper details a new strategy for the purification of GS,resulting in the highest specific activity yet reported. Addi-tionally, an estimated mol wt and isoelectric point of zucchinileaf GS are presented for the first time, as well as a moreextensive characterization of the purified enzyme.

MATERIALS AND METHODS

Chemicals

Redi Earth was obtained from Grace3 (Cambridge, MA).Osmocote and Micro Max were acquired from Sierra Chem-ical (Milpitas, CA). UDP-[U-14C]Gal and UDP-[U-'4C]Glcwere purchased from New England Nuclear (Boston, MA).EG was purchased from Fisher Scientific (Fair Lawns, NJ).Hepes was obtained from Research Organics (Cleveland, OH)and Ecoscint from National Diagnostics (Manville, NJ). Pres-wollen microgranular DE 52 was obtained from WhatmanBioSystems (Maidstone, Kent, UK) and Sephacryl S-200 fromPharmacia (Piscataway, NJ). Water used in this study wasdistilled and further purified by a Nanopure II ion-exchange

3 The mention of firm names or trade products does not implythat they are endorsed or recommended by the U.S. Department ofAgriculture over other similar products not mentioned.

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Plant Physiol. Vol. 96, 1991

system (Sybron/Barnstead; Boston, MA). All remainingchemicals were purchased from Sigma Chemical Co. (St.Louis, MO).

Plant Growth

Seeds of zucchini squash (Cucurbita pepo L. cv BurpeeHybrid) were planted in 2 gallon pots containing Redi EarthPeat-Lite Mix, Osmocote (2 oz/ft3) and Micro Max (20 g/ft3).The plants were grown in a greenhouse for 4 weeks andtransferred to an environmental chamber (Percival Manufac-turing, Boone, IA) 1 week prior to harvesting. Plants weresubjected to a 14 h photoperiod (291 ItE m-2 s-') at 27/20'C(day/night) and were watered once daily. Growth chamberswere used to decrease the possible environmental variables(light and temperature) that can influence GS activity in leafmaterial (2, 21).

Enzyme Assay

GS activity was determined by a modification of the iso-topic assay of Handley and Pharr (8). Reactions were per-formed in 1.5 mL microcentrifuge tubes in a final volume of55 1AL containing: 50 mM Hepes-Na(salt) (pH 7.5), 5.0 mMMnCl2, 3.0 mm DTT, 20 mm myo-inositol, 10 mM UDP-Gal,1.5 ,mol UDP-[U-'4C]Gal (0.041 ,1Ci ,smol-'), and 10 1uL ofenzyme. The reaction was initiated by the addition of sub-strates and incubated at 320C in a shaking water bath for 20min. The reaction was stopped by the addition of 0.2 mL ofcold ethanol and 1.0 mL of cold H20. Unreacted UDP-[U-'4C]Gal was removed by the addition of 0.3 g of DE 52, andthe tubes were shaken at room temperature (25°C) for 20min. After centrifuging at 6000g for 5 min (25°C), 0.5 mL ofthe supernatant was transferred to a 20 mL scintillation vial.Ten milliliters of Ecoscint scintillation liquid was added toeach vial. The samples were counted on a Beckman LS-9800scintillation counter, using the unbound isotope as a measureof galactinol formed by the reaction. Boiled enzyme extractswere used as controls for all enzyme assays. The enzymepreparations were regularly diluted during the purification toensure that the activity determinations were linear with re-spect to time.

Enzyme Purification

The following steps were performed on ice or at 4°C.

Enzyme Extraction

Mature leaves were deveined and cut into small pieces, andapproximately 180 g of leaf tissue was ground in a chilledmortar and pestle with 4 volumes (w/v) 50 mM Hepes-Na(salt)(pH 7.5) containing 10% EG and 1.0 mM DTT. The homog-enate was centrifuged at 25,000g for 15 min. The resultingsupernatant was gently decanted and used for enzymepurification.

Anion Exchange Chromatography

The crude extract (800 mL) was applied to a DE 52 column(4.5 i.d. x 4.5 cm) that was preequilibrated with RB (50 mM

Hepes-Na(salt) [pH 7.5], containing 10% EG, 1.0 mm DTT).The column was washed at a flow rate of 5.0 mL min-' with3 bed volumes of RB, followed by 12 bed volumes of RBcontaining 100 mm NaCl. The enzyme was eluted from thecolumn with RB containing 175 mm NaCl. Fractions contain-ing the highest activity were pooled and solid NaCl added toincrease the NaCl concentration to 500 mM.

Hydrophobic Interaction Chromatography

The pooled enzyme was applied to an Octyl-Sepharose CL-4B column (2.5 i.d. x 4.0 cm) that had been prepared before-hand by sequentially washing with: 10 bed volumes of RBcontaining 500 mM NaCl, 4 bed volumes of RB containing500 mM NaCl and 0.05% Triton X-100, and 8 bed volumesof RB containing 500 mm NaCl. The enzyme, which is notretained under these conditions, was eluted with RB contain-ing 500 mm NaCl at a flow rate of 6.0 mL min-'. The activeenzyme fractions were pooled and concentrated to 0.5 mLusing a Centriprep-10 Concentrator (Amicon, Danvers, MA).

Size Exclusion Chromatography

Concentrated enzyme was applied to a Sephacryl S-200column (1.5 i.d. x 100.0 cm) preequilibrated with RB. Theenzyme was eluted at a flow rate of 0.2 mL min-' with RB,and the fractions containing the enzyme were pooled andused for enzyme characterization. Purified GS was preparedfor SDS-PAGE using a modified procedure of Laemmli (13).

Native Molecular Mass Estimation

The native Mr was determined on a Superose 12 (HR 10/30) column (Pharmacia) using the Pharmacia FPLC system.One hundred microliters of the pooled S-200 sample wereapplied to the column operating at a flow rate of 0.2 mLmin-'. The native molecular mass estimation was determinedfrom multiple runs with the pooled enzyme, pooled enzymewith dextran blue, and pooled enzyme with molecular massstandards. The native molecular mass estimation was derivedfrom a plot of the V,/Vo versus log Mr. The molecular massstandards were as follows: dextran blue (2000 kD), alcoholdehydrogenase (150 kD), BSA (66 kD), carbonic anhydrase(29 kD), and Cyt c (12.4 kD). Column fractions were assayedfor GS activity as described above, while the protein standardswere detected with the Bradford method (1).

Isoelectric Point Estimation

Galactinol synthase from the S-200 column (4-6 mL) wasdialyzed against 4 L of 25 mM Bis-Tris (pH 6.5) containing2.0 mM MnCl2 and 1.0 mM DTT at 4°C overnight. Thissample was applied to an FPLC Mono P, (HR 5/20) column(Pharmacia) and eluted at a flow rate of 0.75 mL min-' witha pH gradient (6.5-3.5) formed by Polybuffer 74 (1:10 dilu-tion; Pharmacia) containing 2.0 mm MnCl2 and 1.0 mM DTT.The fractions were analyzed for GS activity and pH. Fractionswith the highest enzyme activity were pooled, concentratedwith a Centricon- 10 (Amicon), and analyzed by SDS-PAGE.

694 SMITH ET AL.

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GALACTINOL SYNTHASE IN ZUCCHINI LEAVES

Characterization of the Purified Enzyme

The influence of mono- (Na' and K+), di- (Mg2+, Ca2+,Co2+, Cu2+, and Zn2+), and tri- (Fe3") valent ions (10 mM) inthe chloride form, nucleotides (UMP, UDP, UTP, AMP,ADP, ATP, NAD+, NADH, and NADP+; 10 mM) and mono-(Glc, Fru, Xyl, Gal, Man, and Tal), di- (Suc and Mel), andoligo- (raffinose and stachyose) saccharides (5-20 mM) was

determined for the stable S-200 enzyme preparation. Severalalternative substrates for myo-inositol (scyllo- and epi-inositol,0-10 and 0-20 mm, respectively) and UDP-Gal (UDP-Xyl,UDP-Man, UDP-Glc, UDP-GlcUA, and UDP-GalUA; 10mM) were analyzed. Additionally, UDP-Glc, both cold and14C labeled, was substituted for UDP-Gal and tested as an

alternative substrate.

Protein Determination, Km and K. Estimation

Protein concentrations were determined on an MR-700plate reader (Dynatech, Chantilly, VA) using the method ofBradford (1) with BSA as the protein standard. Estimates ofKm and Ka values for all substrates and cofactors were obtainedfrom a minimum of three trials, from separate purifications,and were determined with a computer program for a nonlin-ear regression analysis based on the Michaelis-Menten equa-tion (4).

RESULTS

Purification of Galactinol Synthase

Galactinol synthase was purified 752-fold from maturezucchini squash leaves with an 18.6% recovery and a specificactivity of 23.3 ,umol min-' mg protein-' (Table I). Theenzyme was recovered from the DE 52 column in one sharppeak with a bulk elution using RB containing 175 mM NaCl(Fig. 1). The ion exchange column eliminated >95% of thetotal protein with a 158% recovery of GS activity (Table I).Attempts to elute GS from the DE 52 column with NaClgradients (100-300 mM) proved ineffective, and resulted in a

slow release of enzyme in a broad peak (data not shown).Chromatography on the OS column eliminated 60 to 70%

ofthe total protein, with a twofold purification. In preliminarystudies, GS bound to the OS column preequilibrated with 500mM NaCl, and could be eluted from the column with a 35%EG solution, although all activity was lost within 24 h. Pre-treatment of the column with Triton X-100 allowed theenzyme to pass directly through the column while many otherproteins were retained.

o 32

280

I- 8E

*. 6a%EI. 4

0

0._

5 10 15 20 185 190 195 200

Fraction Number

0

-6C

O._0

0

_O

1a

E

IEC

E

Is

Figure 1. Elution profile of zucchini leaf GS (A) and protein (0) fromDE 52. The enzyme activity was eluted from the column with 175mM NaCI. Pooled fractions are indicated by the filled triangles (A).

The GS preparation was successfully concentrated with theAmicon concentrator without a loss of activity. Pharr et al.(20) reported that attempts to concentrate GS resulted in a

significant loss of activity. In contrast, most (>95%) GSactivity could be recovered from the Amicon concentrator byrinsing the concentrator with RB.

Fractionation on the S-200 column resulted in a 7.5-foldpurification of GS, and increased the stability of the enzyme.

The resulting enzyme preparation contained three major (22,37, and 42 kD) and several minor protein bands on SDS-PAGE (Fig. 2). The molecular masses were estimated bycomparisons to the molecular mass markers and based on therelative mobility versus the log Mr.

Characterization of Purified Enzyme

Relative Molecular Mass and Isoelectric Point Estimations

The purified enzyme had an Mr of 42,000 based on gelpermeation analysis (Fig. 3) and an isoelectric point of 4.1(Fig. 4). Chromatofocusing the purified GS resulted in theelution of a single activity peak, which coincided with a singleprotein peak. When the pooled GS fractions were analyzedby SDS-PAGE, one major band with a molecular mass of 42kD and several minor bands were observed (Fig. 2).

DTT Requirement

The enzyme preparation was transferred to dialysis tubing(12-14 kD cut-off; Allied Fisher Scientific, Pittsburgh, PA)and dialyzed at 4°C against 4.0 L of RB (without DTT)

Table I. Isolation Scheme of Galactinol Synthase from Mature Zucchini LeavesValues represent the average of five separate experiments.

Total Total Specific Purifi-Steps Activity Protein Activity cation Recovery

Wnmollmin mg jimol/min/ -fold %mg protein

Crude 57.75 1892.48 0.03 100.0DE 52 91.16 47.76 1.91 61.6 157.9Octyl-Sepharose 47.08 15.11 3.12 100.5 81.5Sephacryl S-200 10.72 0.46 23.30 751.7 18.6

695

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Plant Physiol. Vol. 96, 1991

.,

Ib -.

o .05I 4

0 .040-1

I .03Ea, .02E. .01

10a. 0

4

110.4 a, 4

v

E -"0.3 ° LI

0.2 <0 .ca .E

0.1 0 E'a

O X45

Fraction Number

overnight. GS activity was enhanced with increasing concen-

trations (0-3.0 mM) of DTT.

Effects of Mn2+ and Other Cations

Galactinol synthase activity was greatly enhanced by MnCl2(Fig. 5). The MnCl2 response saturated at 3.0 mm, whileMnCl2 concentrations up to 10 mm did not adversely affectGS activity. The enzyme was only 50% as active when MgC12was substituted for MnCl2 (Fig. 5). GS had a higher affinityfor MnCl2 (Ka = 0.19 mM) than MgCl2 (Ka = 0.86 mM).Interestingly, supplementing the grinding buffer and/or theRB with MnCl2 resulted in a total loss of enzyme activitywithin 24 h. Na', K+, and Ca2+ ions had little effect on GSactivity, yet Co2+, Zn2+, Cu2+, and Fe3` were found to beinhibitory (Table II).

5.25a

o 5.002

o 4.75

6 4.502

0 4.25

4.00

1.0 1.2 1.4 1.6 1.8 2.0

Ve/ VO2.2

Figure 3. Native molecular mass estimation of zucchini leaf GS basedon the elution profile from a Pharmacia FPLC Superose 12 (HR 10/30) column.

Figure 4. Elution profile of zucchini leaf GS (A) and protein (0) fromPharmacia FPLC Mono P (HR 5/20) chromatofocusing column. ThepH gradient (6.5-3.5) was formed with Polybuffer 74, containing 2.0mM MnCI2 and 1.0 mm DTT at a flow rate of 0.75 mL min-.

pH Optimum

Galactinol synthase had a broad pH optimum from 7.0 to8.0, with activity decreasing dramatically below 6.5 and above8.0 (Fig. 6). The decrease in GS activity at high pHs may haveresulted, in part, from the precipitation of MnCl2.

Substrate Specificity

Galactinol synthase exhibited a high specificity for bothmyo-inositol and UDP-Gal with Kms of 6.5 and 1.8 mm,respectively. Only 10 and 8% ofthe activity could be achievedwhen substituting the respective epimers, epi- and scyllo-inositol, for myo-inositol. When substituting UDP-Glc (un-labeled and [U-'4C]) for UDP-Gal in the standard assay

mixture, UDP-Glc did not act as an effective alternativesubstrate.

Effects of Various Metabolites

All nucleotides (UMP, UDP, UTP, AMP, ADP, ATP,NAD+, NADH, and NADP+) decreased GS activity to variousdegrees as compared to the control (Table II). UDP-GlcUAand UDP-GalUA had no effect on GS activity (Table II).UDP-Man decreased GS activity by 22%, whereas UDP-Glc

2.4W-

'

IJ 2.0i E2 ' 1.6

.. E 1.2o0 _

6 , 0.8

0.4

0 2 4 6 8 10

Cation (mM)

Figure 5. The activity of zucchini leaf GS in increasing concentrationsof MnCI2 (0) and MgCI2 (A). Most SE bars are smaller than the symbolsize.

MnCI2 0' MgCI2 A

0

/ s0I0-A

696 SMITH ET AL.

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GALACTINOL SYNTHASE IN ZUCCHINI LEAVES

Table II. Influence of Various Substances on Zucchini LeafGalactinol Synthase Activity

Various substances were added to the standard assay mixture.Galactinol synthase activity in the standard assay was used as thecontrol (100%). The reaction concentrations of cations, nucleotides,and UDP-sugars were 10 mm. Carbohydrates were analyzed from5.0 to 20 mm, although only the 20 mm data were presented. Eachvalue is the average of one to two trials with four replicates per trial.

Metabolite Activity Metabolite ActivityRemaining Remaining

Na+ 100 UDP-Xyl 49K+ 100 UDP-Glc 56Ca2+ 100 UDP-Man 78co2+ 24 UDP-GlcUA 100Cu2+ 0 UDP-GalUA 100Zn2+ 1 Xyl 90Fe3+ 3 Fru 100AMP 75 Glc 89ADP 39 Gal 43ATP 33 Tal 64UMP 66 Man 95UDP 16 Suc 83UTP 38 Mel 26NAD+ 81 Raffinose 100NADH 87 Stachyose 100NADP+ 56

and UDP-Xyl decreased the total activity by 44 and 51%,respectively (Table II). Xyl, Glc, and Man slightly decreased(less than 10%) GS activity, whereas Suc reduced GS activityby 17% as compared to the control. Tal, Gal, and Meldecreased GS activity over the entire range tested (5.0-20.0mM), resulting in a total reduction in activity of 36, 57, and74%, respectively, at the 20 mm level (Table II). Fru, raffinose,and stachyose did not affect GS activity (Table II).

DISCUSSION

This isolation scheme represents the highest purification(752-fold) of GS published to date, based on specific activity(23.3,umol min-' mg protein-'). Previously, Pharr et al. (20),Handley and Pharr (8), and Webb (23) had purified GS 41-,105-, and 86-fold, respectively, with the corresponding specificactivities of 0.41, 0.81, and 1.56 Imol min-' mg protein-'.Our purification scheme, which reverses the order of previousGS isolation schemes, places an ion exchange column beforethe size exclusion and adds a hydrophobic (OS) column (TableI). There was a 158% recovery ofGS activity with the DE 52step (Table I). This increase in GS activity is believed to be aresult of the removal of inhibitory substances, such as cations,nucleotides, and carbohydrates (Table II) from the crudeenzyme preparation.

Zucchini GS demonstrated many properties common toUDP-glycosyl transferases and the less purified preparationsof GS, such as a broad pH optimum, low isoelectric point,and high specificity for both UDP-Gal and myo-inositol (3,6-8, 10, 12, 22, 23). Changes of the hydroxyl configurationat the second (scyllo-) or sixth (epi-) carbon (17) of inositolcan result in a >90% reduction in total activity. Altering the

hydroxyl configuration at the fourth carbon (UDP-Glc) orsubstituting an acid group at the sixth position (UDP-GalUA)ofthe UDP-Gal drastically reduced the activity ofthe purifiedzucchini GS (Table II). The Kms for UDP-Gal (1.8 mM) andmyo-inositol (6.5 mM) determined from this study are similarto the Kms (0.2 and 4.0 mm, respectively) previously reportedby Handley and Pharr (8).The activity of purified zucchini GS was reduced in the

presence of all nucleotides examined in this study, with UDPexhibiting the most inhibitory effect (Table II). Handley andPharr (8) found that UDP inhibition was competitive, withrespect to UDP-Gal, whereas the inhibition toward myo-inositol was noncompetitive. This UDP competitive/non-competitive inhibition is a common trait among UDP-glyco-syl transferases (11, 12, 22). Handley and Pharr (8) suggestedthat this nucleotide inhibition may serve to regulate GSactivity in vivo.Of all sugars examined from 0 to 20 mm, Gal and Mel had

the greatest reducing effect on GS activity, whereas raffinoseand stachyose had no effect on GS activity (Table II). All foursugars contained at least one galactosyl residue; it is possiblethat raffinose and stachyose have steric hindrance whichprevents their interaction with the enzyme. The lack of inhi-bition by raffinose and stachyose suggests that there is nofeedback inhibition from the biosynthetic intermediates inthe raffinose pathway.

Zucchini GS activity was greatly enhanced in the presenceof Mn2+ (Fig. 5). Unlike cucumber GS (8), higher levels ofMnCl2 (3-10 mM) did not inhibit the purified zucchini GSactivity. The reason for this discrepancy is not clear at thispoint. The MnCl2 requirement of zucchini GS was clearlyshown in the chromatofocusing experiments. The Polybuffersgenerally used to elute proteins from the chromatofocusingcolumns are known to act as chelators of divalent ions (16).Supplementing the Polybuffer with a 2.0 mm MnCl2 wasnecessary for the recovery of some GS activity from thechromatofocusing column. Similar observations on the diva-lent cation requirement in chromatofocusing were made withCa2+ for a-amylase (9, 14, 18) and Mn2+ for the Fe-protein ofnitrogenase (16, 24). Omitting the respective ions from the

2.0

L-

o -EE1.6

%. T 1.2-

._-Z - 0.8

o

0 Bis-TrisA^ \, ^A Hepes-NaOH

0 Trizma0

0~O tI -~~~~~~~~~~~~~~~~~~

6.0 7.0 8.0 9.0pH

Figure 6. Determination of pH optimum of zucchini leaf GS in 100mm of Bis-Tris (0), Hepes-Na(salt) (A), and Trizma (E) buffers. Thebuffer concentration of the enzyme preparation was diluted to 3.0mm (1 6.7-fold) prior to the assay for activity. Most SE bars are smallerthan the symbol size.

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Plant Physiol. Vol. 96, 1991

Polybuffer resulted in a significant to complete loss of activitywhen eluting the enzyme from the column.

Zucchini leaf GS appears to be a single monomeric unitwith native molecular mass of 42 kD (Fig. 2). Numerousstudies have reported monomeric UDP-glycosyl transferaseswith Mrs ranging from 30 to 68,000 (3, 7, 22), althoughmultiple subunits and isozymes have also been reported (10,12). Our data indicated the presence of only one form ofzucchini GS (Figs. 3 and 4), but we cannot rule out thepossibilities that multiple isoforms do exist without furtherstudies.

ACKNOWLEDGMENTS

We would like to thank Thomas Cheesbrough, Michael Muhitch,and Donn Warner for their helpful suggestions with the chromatog-raphy and in the preparation of this manuscript; we also thankJoAnne Toohill for growing and maintaining the plant material.

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3. Dixon SC, Martin RC, Mok MC, Shaw G, Mok DWS (1989)Zeatin glycosylation enzymes in Phaseolus. Plant Physiol 90:1316-1321

4. Duggleby RG (1981) A nonlinear regression program for smallcomputers. Anal Biochem 110: 9-18

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6. Frydman RB, Neufeld EF (1963) Synthesis of galactosylinositolby extracts from peas. Biochem Biophys Res Commun 12:121-125

7. Gross GG (1983) Partial purification and properties of UDP-glucose:vanillate -0-glucosyl transferase from oak leaves. Phy-tochemistry 10: 2179-2182

8. Handley LW, Pharr DM (1982) Ion stimulation, UDP inhibitionand effects of sulfhydryl reagents on the activity of galactinolsynthase from leaves of cucumber, Cucumis sativus L. ZPflanzenphysiol 108: 447-455

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10. Jourdan PS, Mansell RL (1982) Isolation and partial character-ization of three glucosyl transferases involved in the biosyn-thesis of flavonol triglucosides in Pisum sativum L. ArchBiochem Biophys 213: 434-443

11. Jurgens D, Shalaby FYYI, Fehrenbach FJ (1985) Purificationand characterization of camp-factor from Streptococcus aga-lactiae by hydrophobic interaction chromatography and chro-matofocusing. J Chromatogr 348: 363-370

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13. Laemmli UK (1970) Cleavage ofstructural proteins during assem-bly of the head bacteriophage T4. Nature 227: 680-685

14. Lecommandeur D, MacGregor AW, Daussant J (1988) Purifi-cation of germinated barley a-amylase isozymes and limit-dextrinase by chromatofocusing and affinity chromatography.J Chromatogr 441: 436-442

15. Lehle L, Tanner W (1972) Synthesis of raffinose-type sugars. InV Ginsberg, ed, Methods in Enzymology, Complex Carbohy-drates, Part B, Vol 28. Academic Press, New York, pp 522-529

16. Ljungstrom E, Yates MG, Nordlund S (1989) Purification of theactivating enzyme for the Fe-protein of nitrogenase from Azos-pirillum brasilense. Biochim Biophys Acta 994: 210-214

17. Loewus FA, Dickinson DB (1982) Cyclitols. In FA Loewus, WTanner, eds, The Encyclopedia of Plant Physiology, Vol 13A.Springer-Verlag, Berlin, pp 193-216

18. MacGregor AW, Marchylo BA, Kruger JE (1988) Multiple a-amylase components in germinated cereal grains determinedby isoelectric focusing and chromatofocusing. Cereal Chem65: 326-333

19. Pharr DM, SoxHN (1984) Changes in carbohydrate and enzymelevels during the sink to source transition of leaves of Cucumissativus L., a stachyose translocator. Plant Sci Lett 35: 187-193

20. Pharr DM, Sox HN, Locy RD, Huber SC (1981) Partial char-acterization of the galactinol forming enzyme from leaves ofCucumis sativus L. Plant Sci Lett 23: 25-33

21. Robbins NS, Pharr DM (1987) Regulation of photosyntheticcarbon metabolism in cucumber by light intensity and photo-synthetic photoperiod. Plant Physiol 85: 592-597

22. Schulz M, Weissenbock G (1988) Three specific UDP-glucuron-ate:flavone-glucuronosyl-transferases from primary leaves ofSecale cereal. Phytochemistry 27: 126 1-1267

23. Webb JA (1982) Partial purification of galactinol synthase fromleaves of Cucurbita pepo. Can J Bot 60: 1054-1059

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