comparative enzymology ofthe adenosine triphosphate ...mm tris-hcl buffer, ph 8.0, containing 10 mm...

Plant Physiol. (1974) 53, 180-186

Comparative Enzymology of the Adenosine Triphosphate

Sulfurylases from Leaf and Swollen HypocotylTissue of Beta vulgaris

MULTIPLE ENZYME FORMS IN HYPOCOTYL TISSUE'

Received for publication June 21, 1973 and in revised form September 13, 1973

D. I. PAYNTER2 3 AND J. W. ANDERSONDepartment of Botany, La Trobe University, Bundoora, Victoria, 3083, Australia

ABSTRACT

ATP sulfurylase activity was partially purified from theswollen hypocotyl of beetroot (Beta vulgaris); activity wasmeasured by sulfate-dependent PPi-ATP exchange. The ATPsulfurylase activity was separated from pyrophosphatase andATPase activities which interfere with the assay of ATP sul-furylase activity. The ATP sulfurylase activity from hypocotyltissue was invariably resolved into two approximately equal ac-tivities (hypocotyls I and II) by ion exchange chromatographyand polyacrylamide gradient gel electrophoresis. Both enzymescatalyzed selenate- and sulfate-dependent PPi-ATP exchange;the affinity of hypocotyl II for these substrates was greaterthan for hypocotyl I. It is unlikely that the two activities ariseby allelic variation or as an artifact of purification; they aremost probably isoenzymes. Studies of the subcellular localiza-tion of the two hypocotyl enzymes were inconclusive.ATP sulfurylase was also purified from leaf tissue. Ion ex-

change chromatography resolved the ATP sulfurylase fromleaf tissue into a major activity (which accounted for 98% ofthe total leaf activity) and a minor activity. The major leafand hypocotyl II ATP sulfurylases were indistinguishable asjudged by the properties investigated.

In plants, fungi, and the assimilatory sulfate-reducingbacteria, sulfate is the principal form of sulfur required forgrowth (24). In yeast, it is well established that sulfate is ac-tivated by ATP in two separate reactions catalyzed by the en-zymes ATP sulfurylase (ATP-sulfate adenylyl transferase, EC2.7.7.4) and APS4 kinase (ATP-adenylyl sulfate 3'-phospho-transferase, EC 2.7. 1 .25), respectively:

'This work was supported by a grant from the Australian Re-search Grants Committee.

2 Present address: Attwood Veterinary Research Laboratory,Mickleham Rd., Westmeadows, Vic. 3047, Australia.

3D.I.P. was the holder of a Victorian State Department ofAgriculture Cadetship.

I Abbreviations: APS: adenosine 5'-sulfatophosphate; PAPS:adenosine 3'-phosphate 5'-sulfatophosphate; DEAE-cellulose: di-ethylaminoethyl cellulose.

Mg2+ATP + S04,< APS + PPi

APS + ATP IMg2+ PAPS + ADP

In yeast, PAPS is the activated form of sulfate; the sulfatemoiety of PAPS is reduced and used to form the various com-pounds containing sulfur in the -2 valency state which areessential for the growth and function of all living cells. Thecapacity of an organism to synthesize APS and PAPS frominorganic sulfate, however, does not necessarily imply thatthe organism is able to reduce sulfate since it is well establishedthat animals synthesize APS and PAPS yet are unable to re-duce sulfate in significant quantities (24). In animals, PAPSis used to form a wide range of sulfate esters in reactionscatalyzed by the sulfotransferases (24).ATP sulfurylase activity has been demonstrated in a wide

range of plants (1, 2, 14). The activated form of sulfate re-duced by plants, however, is uncertain. In several studies APSkinase activity could not be demonstrated (5, 6, 14), and thishas led to the suggestion that APS might be the activatedform of sulfate (6, 14), as has been described for the dis-similatory sulfate-reducing bacteria (22). APS kinase activityhas been described in Chlorella (16), but Tsang et al. (29) andSchmidt (25) have determined that APS is the activated form ofsulfate for reduction in this organism. APS kinase activityhas been reported in higher plants (7, 20); the enzyme fromspinach only catalyzed the synthesis of PAPS in the presenceof 3'-AMP (7). Since the incorporation of sulfate into cysteine/cystine by isolated chloroplasts was almost entirely dependenton the addition of 3'-AMP, Burnell and Anderson (7) suggestedthat PAPS is an intermediate in sulfate reduction in spinach.The ATP sulfurylase from spinach leaf tissue has been

highly purified (6, 27); the enzyme catalyzes APS-dependentsynthesis of ATP (6), sulfate- and selenate-dependent PPi-ATPexchange and the synthesis of APS when coupled with Mg2+-dependent alkaline pyrophosphatase (27). In leaf tissue, ATPsulfurylase and other enzymes associated with sulfate activa-tion have been described in chloroplasts (5-8).

It is well established that nonphotosynthetic tissues containATP sulfurylase activity (2, 14) and incorporate sulfate intocompounds containing sulfur in the -2 valency state (13, 18,21). Slices of the nonphotosynthetic hypocotyl tissue of beet-root incorporate sulfate into cysteine (13); the incorporationwas enhanced by serine, implying that sulfate is reduced tosulfide and incorporated into either serine or O-acetylserine ina reaction catalyzed by serine sulfhydrase or O-acetylserine

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Plant Physiol. Vol. 53, 1974 MULTIPLE FORMS OF ATP SULFURYLASE IN BEETROOT

sulfhydrase, respectively. This suggests that the ATP sulfuryl-ase activity of nonphotosynthetic tissue is associated with thereduction of sulfate. Since the ATP sulfurylase activity of leaftissue is associated with chloroplasts, this raises the questionas to whether two forms of the enzyme exist in plants. Thispaper describes a comparative study of the ATP sulfurylasesfrom the leaf and swollen hypocotyl tissues of the beetrootplant (Beta vulgaris), a plant which has previously been demon-strated to carry out nonphotosynthetic reduction of sulfatein hypocotyl tissue (13). Leaf tissue contains one major ATPsulfurylase, but hypocotyl tissue contains two enzymes of ap-proximately equal activity. One of the hypocotyl enzymes wasindistinguishable from the leaf enzyme.

MATERIALS AND METHODS

Chemicals and Enzymes. Chemicals were obtained from thesources described previously (26, 27). In addition, bovineserum albumin was obtained from the Commonwealth SerumLaboratories, Parkville, Vic., Australia and polyacrylamideslab gels (7.2 X 7.2 x 0.3 cm) containing 5 to 20% poly-acrylamide gradient were obtained from Gradient Pty. Ltd.,Lane Cove, N.S.W., Australia. 9PPi was prepared from 'Piby pyrolysis (17), and the specific radioactivity was adjusted to0.25 c/mole with unlabeled Na4P2O7.

Mg2'-dependent alkaline pyrophosphatase was purified fromspinach as described by Shaw and Anderson (27); the activityof purified enzyme was 60 units/ml, the definition of an en-zyme unit described by Shaw and Anderson (27) being used.ADP sulfurylase, ATP sulfurylase and ATPase activities wereabsent from purified pyrophosphatase although the purifiedenzyme is known to contain some 3'-nucleotidase activity (7).

Plant Material. Beetroot plants (Beta vulgaris) with welldeveloped swollen hypocotyls were obtained direct frommarket gardens; only plants of good quality were used. De-ribbed upper leaves were used as the source of leaf tissue.Hypocotyls were washed in distilled water, and the outer 0.5cm was removed by peeling. The peeled tissue was diced intocubes (approx. 8 cm3) and used as the source of hypocotyltissue.

Extraction and Purification of the Hypocotyl ATP Sul-furylases. Diced hypocotyl tissue was extracted with 100mM tris-HCl buffer, pH 8.0, containing 10 mM potassiumthioglycolate (medium 1) in a Waring Blendor using 0.5 mlof medium 1 per g fresh weight. The homogenized materialwas strained through muslin to remove cell debris. Particulatematerial was removed from the filtered solution by centrifuga-tion (20,000g for 20 min); the supernatant solution containingthe ATP sulfurylase activity is referred to as crude extract.Solid (NH4)2S04 (0.561 g/ml, equivalent to 80% saturation)was added to the crude extract. Precipitated protein was re-covered by centrifugation and redissolved in medium 1 (0.2ml/g of original weight of tissue); this procedure effected theseparation of protein from the bulk of the phenolics in thecrude extract and also served to concentrate the extract. Thisfraction is referred to as concentrated crude extract.

Concentrated crude extract was fractionated by the additionof 0.176 g of solid (NH4)2SO,/ml of extract (equivalent to 30%saturation). The supernatant solution was recovered, and afurther 0.127 g/ml of solid (NH4,)S04 was added (50% satura-tion); precipitated protein was recovered by centrifugation anddissolved in 0.04 ml of 20 mm tris-HCl buffer, pH 8, con-taining 5 mM potassium thioglycolate (medium 2) per goriginal weight of tissue. The (NH4,)S04 fraction was ex-tensively dialyzed against medium 2 to remove residual(NH4)2S0. The dialyzed fraction was subjected to gel filtrationon a Sephadex G-200 column (60 x 2.5 cm) equilibrated with

20 mM tris-HCl, pH 8 (medium 3). Fractions containing ATPsulfurylase activity were applied to a DEAE-cellulose column(10 X 2 cm) equilibrated with medium 3, and the column wasdeveloped with a linear gradient of KC1 (0-240 mM). Twopeaks of ATP sulfurylase activity were eluted from the column(Fig. 1). The ATP sulfurylase activity eluted by 110 mM KCIis designated hypocotyl I enzyme, and the activity eluted by150 mm KC1 is referred to as hypocotyl II enzyme. In some in-stances isolated hypocotyl I and II enzymes were treated on asecond DEAE-cellulose column as described by Shaw andAnderson (27); this treatment effected a 4-fold concentrationof activity. All operations were conducted at 2 to 4 C.

Extraction and Purification of the Leaf ATP Sulfurylase.Crude extracts were prepared as described for hypocotyltissue except that the volume of medium 1 used to extract theleaf enzyme was increased to 2 ml/g fresh weight. The crudeextract was not concentrated by precipitation with (NHj4)20,but was directly fractionated by four separate additions (0.176,0.062, 0.063, and 0.066 g) of solid (NHI)2SO/ml of each re-spective supernatant solution. The protein precipitates from thethird and fourth additions of (NHI)2SO, containing most of theATP sulfurylase activity, were dissolved in 0.1 ml of medium 2

035[

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-Ea,

0o00

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0.

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FIG. 1. Ion exchange chromatography of the hypocotyl and leafATP sulfurylases on a DEAE-cellulose column. The enzymes weresubjected to chromatography following (NH4)2SO4 fractionationand gel filtration on Sephadex G-200 as described in the text. TheKCI gradient was applied in a volume of 1 liter. A: Sulfate-de-pendent PPi-ATP exchange; 0: protein. The bold line depicts theconcentration of KCI.

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PAYNTER AND ANDERSON P

per g fresh weight of leaf tissue, and the solutions were com-

bined. The combined fractions were dialyzed to remove

(NH4)2SO4 and subjected to gel filtration and ion exchange chro-matography as described for the purification of the hypocotylenzymes, except that the linear gradient of KCl used to developthe DEAE-cellulose column was 0 to 280 mm. Most of theATP sulfurylase activity (98%) was eluted from the DEAE-cellulose column by approximately 150 mm KCl; a smallamount of activity was eluted by approximately 80 to 110 mMKCI (Fig. 1). All operations were conducted at 2 to 4 C.

Assay of Enzyme Activities. ATP sulfurylase activity was

measured by sulfate-dependent PPi-ATP exchange (26, 27).Assays were conducted at 35 C for 15 min and contained 2,umoles of Na2K2ATP; 2 /.moles of Na432P2OP (0.5 14c); 10

pumoles of MgCI2; 10 ,umoles of NaF; 40 ,imoles of K2SO4; 100,unmoles of tris-HCl buffer, pH 8.0; and enzyme in a final volumeof 1 ml. Fluoride was added to inhibit pyrophosphatase activity(26) but was omitted from assays of enzyme preparationswhich were known to be uncontaminated with pyrophosphataseactivity. Control incubation mixtures contained 120 ,rmoles ofKCI in lieu of K2S04 to maintain the ionic strength. Reactionswere terminated with 2 ml of trichloroacetic acid (7.5%,w/v). 32P-ATP was separated from 32PPi as described byAnderson (4) and counted in a gas flow planchet counter atinfinite thickness. Enzyme activity was calculated by themethod of Davie et al. (12) and is expressed as sulfate-de-pendent PPi-ATP exchange in nmoles/min (ATP sulfurylaseunits); specific activity is expressed as units/mg of protein.

Polyacrylamide Gradient Gel Electrophoresis of the ATPSulfurylases. The method was essentially that of Skyring et al.(28); ATP sulfurylase activity on the gels was detected by themolybdate-dependent formation of Pi (31). Electrophoresiswas performed in tris-glycine buffer (30 mm with respect to

glycine), pH 8.2, at 4 C and 150 v; the gels were prerun for25 min prior to applying the sample. ATP sulfurylase samples,adjusted to 5% (w/v) sucrose and containing not less than 0.15unit of activity, were applied to the gels in a volume not ex-ceeding 35,ul. The samples were subjected to electrophoresisfor either 3 or 15 hr; the amperage generally decreased from15 to 8 mA during electrophoresis.

Following electrophoresis, the gels were sliced in two,parallel to their face. One half was incubated at 35 C for30 min in a complete mixture (with molybdate) containing 5

mM Na2K2ATP; 20 mm MgCl2; 10 mm Na2MoO4; 100 mM tris-HCl buffer, pH 8.0; and purified spinach pyrophosphatase (finalconcentration 6 units/ml) in a total volume of 20 ml. Thegels were washed with distilled water, and Pi produced by theaction of ATP sulfurylase (and ATPase if present) was de-tected by the method of Allen (3); gels were placed in 20 mlof HCl04 (6%, w/v), and 4 ml of amidol reagent and 2 ml of6.6% (w/v) ammonium molybdate were added in that order.After10 min the treated gels were transferred to distilled waterand photographed. The other half of the gel was treatedidentically except that molybdate was omitted from the in-cubation mixture. The formation of any Pi in gels incubatedwithout molybdate was assumed to be due to ATPase ac-tivity.

Determination of Protein. Protein in crude extracts and(NH4)2SO4 fractions was determined by the method of Ellman(15) using bovine serum albumin as standard. Purer proteinfrom Sephadex G-200 and DEAE-cellulose columns wasmeasured by the method of Warburg and Christian (30).

RESULTS

Optimal Conditions for the Extraction and Assay of ATPSulfurylase from Hypocotyl tissue. The ATP sulfurylase ac-

tivity of concentrated crude extracts prepared without thio-glycolate was extremely low. The activity recovered in con-centrated crude extracts was increased when thioglycolatewas included in the extracting medium (Fig. 2). Higher con-centrations of thioglycolate were inhibitory.

In preliminary studies with concentrated crude extract,KCI was omitted from the control incubations. Under theseconditions, PPi-ATP exchange was frequently greater in thecontrols than in incubations containing sulfate. The endogenousexchange was not decreased by dialysis. Addition of KCl(equivalent in ionic strength to the substrate concentrationof K2S04) to control incubations decreased endogenous PPi-ATP exchange (Fig. 3). Accordingly, 120 mm KCI was in-cluded in all subsequent assays of crude extracts and (NH4)2SO4fractions.

Purification of the ATP Sulfurylases from Beetroot Hypo-cotyl and Leaf Tissue. The estimation of ATP sulfurylaseby sulfate-dependent PPi-ATP exchange is subject to inter-ference by pyrophosphatase and ATPase activities (26, 27).Concentrated crude extracts of beetroot hypocotyl tissue con-tain pyrophosphatase and ATPase activities (Table I). Frac-tionation with (NH4)2SO4 removed much inactive protein butfailed to effect a satisfactory separation of ATP sulfurylasefrom the two interfering enzymes. Fractionation with(NH4)2SO4 also failed to decrease the very high endogenousexchange activity in control incubations. Gel filtration of the30 to 50% (NH4)2S04 fraction on Sephadex G-200 effectedseparation of the pyrophosphatase and ATP sulfurylase ac-tivities and decreased the endogenous exchange to a negligiblelevel. A single peak of ATP sulfurylase activity was elutedfrom Sephadex G-200 columns. ATPase activity was largelyremoved by ion exchange chromatography on DEAE-cellu-lose. The small endogenous PPi-ATP exchange of ATP sul-furylase fractions purified by gel filtration was not decreasedbyKCl.

Ion exchange chromatography of the fractions subjectedto gel filtration resolved the ATP sulfurylase activity into

r-\

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a-a-

25

20

1i5

10

0*5

o Lo 20 40 60 80 100

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FIG. 2. Effect of concentration of potassium thioglycolate inthe extracting medium upon the ATP sulfurylase activity ex-tracted from hypocotyl tissue. Cubes of randomized hypocotyl tis-sue (20 g) were extracted with 20 ml of 100 mM tris-HCl buffer,pH 7.5, containing 20mM MgCl2 and the concentrations of thio-glycolate specified. The crude supematant solutions were recoveredand concentrated crude extracts were prepared as described in thetext except that the (NH4)2S04 precipitates were resuspended anddialyzed against the appropriate medium used to extract thetissue. After dialysis, activity was measured by sulfate-dependentPPi-ATP exchange. Concentrated crude extracts did not catalyzethioglycolate-dependent PPi-ATP exchange.

182 Plant Physiol. Vol. 53, 1974

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two approximately equal activities (Fig. 1). Crude extractsof hypocotyl tissue, depigmented by passage through a Sepha-dex G-50 column equilibrated with medium 1, were also re-solved into two approximately equal ATP sulfurylase activitieswhen directly subjected to ion exchange chromatography ona DEAE-cellulose column. ATP sulfurylase purified by frac-tionation with acetic acid was also resolved into two approxi-mately equal activities when subjected to chromatography on aDEAE-cellulose column. In all cases one peak of activity(hypocotyl I) was eluted by 110 mm KCI, and the other peak(hypocotyl II) was eluted by 150 mM KCI.

Leaf tissue contained much higher ATP sulfurylase ac-tivity than hypocotyl tissue; typical activities were 60 units/gfresh weight and 1.3 units/g fresh weight for leaf and hypo-cotyl tissue, respectively. Crude extracts of leaf tissue con-

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30

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10 _0 0-2 0-4 0*6

Ionic strength

FIG. 3. Effect of ionic strength on the PPi-ATP exchangecatalyzed by dialyzed concentrated crude extracts of hypocotyl tis-sue in the presence (0) and absence (A) of 40 mM K2S04. The ionicstrength refers to the components KC1 and KS04 only. Incubationswith and without K2504 were adjusted to the ionic strength specifiedwith KCI.

Table I. Typical Purificationi of the ATP Suilfuirylases fromHypocotyl Tissue of Beetroot and Their Separationi from

Pyrophosphatase anid ATPase ActivitiesThe purification procedure and the conditions of the enzyme

assays were as described in the text except that the ATP sulfuryl-are activities of fractions eluted from Sephadex G-200 and DEAE-cellulose columns were measured in the absence of fluoride andKCl was omitted from control incubations. All activities are ex-

pressed with respect to 500 g of hypocotyl tissue. Pyrophosphataseactivity in all fractions was completely inhibited by 10 mm NaF.

Enzyme Activity

Treatment Protein ATP sul- Pyrophos- ATPase

furylase phatase

mg units/mg protein

Dialyzed crude extract 770 0.75 4.29 0.043(NH 4) 2SO4 fraction (30-50%c 204 1.19 5.03 0.041

saturation)Sephadex G-200 79 2.31 0.01 0.07DEAE-cellulosePeak I (hypocotyl I) 0.61 11.6 0 0Peak II (hypocotyl II) 0.41 16.3 0 0

Table II. Typical Purification of the ATP Sulfurylase from LeafTissue of Beetroot antd Its Separation from Pyrophosphatase

anid ATPase ActivitiesThe purification procedure was as described in the text. All ac-

tivities are expressed with respect to 200 g of leaf tissue. All otherconditions were as described in Table I.

Enzyme activity

Treatment ProteinAATP Pyro- ATP-

sulfury- phos- APlase phatase ase

mg units/mg of protein

Dialyzed crude extract 1180 10.1 8.2 0.02(NH4)2SO4 fraction (combined 496 8.5 8.2 0.023rd and 4th fractions)

Sephadex G-200 253 22.0 1.4 0.03DEAE-cellulose 3.09 61.2 0.01 0

tained an active pyrophosphatase and some ATPase activity.The purification procedure for the beetroot leaf ATP sulfuryl-ase, which was essentially the same as the procedure used to ef-fect a 1000-fold purification of the spinach leaf enzyme (27),removed the pyrophosphatase activity but effected only a 6-fold increase in specific activity of the beetroot ATP sulfurylase(Table II). In all experiments essentially one peak of activitywas eluted from the DEAE-cellulose column (Fig. 1); the con-ditions for the elution of the leaf enzyme were identical tothe conditions required to elute the hypocotyl II enzyme. Asmall amount of ATP sulfurylase activity was eluted prior tothe major leaf enzyme during ion exchange chromatographybut this activity did not exceed 2% of the major activity. Asimilar elution pattern was obtained when the (NHU),S04 frac-tionation procedure for purifying the leaf enzyme was re-placed with a single step precipitation of crude extract by(NH,)SO,. In both cases, the major leaf enzyme was elutedby 150 mM KCl.Some Properties of the Hypocotyl and Leaf ATP Sul-

furylases. The rates of sulfate-dependent PPi-ATP exchangecatalyzed by purified hypocotyl I, hypocotyl II and leaf ATPsulfurylases were constant at 35 C for at least 15 min. The pHoptimum of the leaf and hypocotyl II enzymes was 8 althoughthe optimum for the leaf enzyme was relatively broad (Fig. 4).The pH optimum of the hypocotyl I enzyme was 9. All threeenzymes were virtually inactive below pH 5.5; the low activityof the two hypocotyl enzymes below this pH was presumablydue to enzyme denaturation since these enzymes were inactiveat pH 8.0 if pretreated at pH 5.5.

Spinach leaf enzyme catalyzes both selenate- and sulfate-dependent PPi-ATP exchange (27). The effect of concentrationof selenate and sulfate on exchange catalyzed by the threebeetroot ATP sulfurylases was studied in standard assays inwhich 40 mm sulfate was replaced with specified concentra-tions of K,SO, and K2SeO,. All three beetroot enzymes cata-lyzed selenate-dependent exchange; the effects of selenate andsulfate concentration on exchange were consistent withMichaelis-Menten kinetics. The Km values for selenate andsulfate were similar for the leaf and hypocotyl II enzymes; theKm values of the hypocotyl I enzyme for selenate and sulfatewere higher than for the other two enzymes, but the ratios forV (selenate)/ V(sulfate) and Km (selenate)/Km (sulfate) wereapproximately the same for all three enzymes (Table III). Allthree enzymes were approximately saturated by 40 mm K2SO,and 20 mm K,SeO,; higher concentrations were not inhibitory.

All three purified enzymes were virtually inactive in the ab-sence of MgCl2. Exchange was approximately saturated by

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PAYNTER AND ANDERSON

5

4

31

-E

Etc

\atow

E

c

u

t)

CL

-C

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2

0

FIG. 4. Effectsulfurylases froiII enzyme; A:scribed in the tepH 8, was replavalues specified.

Table III. SumiHypocotyl I,

BeetrKm values w(

(27) and are exj

Property

E concentrations of polyacrylamide than the hypocotyl I en-zyme (Fig. 6); the resolution of hypocotyl I and II activities at15 hr was less than at 3 hr, suggesting that although the two

E enzymes differed significantly in charge at pH 8.2, the dif--30 > ferences in molecular weight were small as judged by poly-

acrylamide gradient gel electrophoresis.Margolis and Kenrick (19) have related the concentration of

polyacrylamide at which a protein ceases to migrate in apolyacrylamide gradient gel to the molecular weight of the

20 - protein. This relation, together with the results of 15-hr elec-trophoresis experiments, indicated that the molecular weightsa\F of the leaf and hypocotyl II enzymes were slightly less thanthe molecular weight of hypocotyl I; the molecular weights

a- of the three enzymes were approximately 200,000 to 230,000.10 ._ Concentrated crude extracts and 30 to 50% (NH4),S04 frac-

tions of hypocotyl tissue were subjected to electrophoresis for3 hr. Two molybdate-dependent bands were resolved. The fastmigrating band stained slightly more heavily in the presence

O ,,II-. of molybdate than the slower moving band, but the fast mov-07~5 85 95 ' ing band stained lightly in control incubations without molyb-

date, suggesting that the fast moving band was not separatedpH from ATPase activity. The two ATP sulfurylases in unpurified

.ofpHnteativtyfteprifed TPfractions migrated at approximately the same rate as hypocotyltof pH on the activity of the purified ATP IadI T ufrlssm beetroot. 0: Hypocotyl I enzyme; *: hypocotyl I and II ATP sulfurylases.leaf enzyme. Conditions of the assay were as de- Studies of the ATPSuffurlase Activity ofHitochondriaxt for purified enzymes except that tris-HCl buffer, from Hypocotyl Tissue. Experiments designed to determine theiced with 120 ,tmoles of tris-HCl buffer at the pH subcellular localization of the two hypocotyl ATP sulfurylases

were inconclusive. A mitochondria-enriched fraction wasprepared by the method of Raison and Lyons (23); 8% of the

mary of the Kinetic Properties of the Purified total ATP sulfurylase activity was associated with the particu-Hypocotyl II, and Leaf ATP Sulfurylases from late fraction. This fraction was homogenized with medium 3root as Measured by PPi-ATP Exchange in a Potter-Elvejham homogenizer; only 48% of the ATPere computed as described by Shaw and Anderson sulfurylase activity associated with the particulate fraction waspressed :+ SEM. recovered in the soluble fraction. The soluble fraction con-

tained both hypocotyl I and II activities when examined by ionATP Sulfurylase exchange chromatography on a DEAE-cellulose column, but

the relative activities of the two enzymes varied between ex-Hypocotyl I i Hypocotyl II I Leaf periments.

mm

Km (sulfate) 5.3 i 0.3 2.21 i 0.03 2.13 i 0.02Km (selenate) 1.39 ± 0.04 0.51 i 0.01 0.50 i 0.01V (selenate)/V (sul- 0.28 0.27 0.27

fate)Km (ATP) 0.21 i 0.02 0.21 + 0.05

5 mM MgCl2; higher concentrations (up to 20 mM) were notinhibitory. The effect of MgCl, concentration on sulfate-de-pendent PPi-ATP exchange was similar for all three enzymes.The effect of ATP concentration on the sulfate-dependentexchange catalyzed by the leaf and hypocotyl I enyzmes wasconsistent with Michaelis-Menten kinetics. The Km valtues forthe two enzymes were similar (Table III).

Polyacrylamide Gradient Gel Electrophoresis of the Beet-root ATP Sulfurylases. Electrophoresis was performed foreither 3 or 15 hr. The rate of migration of each enzyme wasconstant for 4 hr, indicating that the polyacrylamide gradienthad no effect on electrophoretic mobility during this time.After 4 hr the rate of migration decreased; by 15 hr the en-

zymes ceased to migrate any further into the gel.In 3-hr experiments, purified leaf and hypocotyl II enzymes

migrated at the same rate; purified hypocotyl I enzyme mi-grated at a slower rate (Fig. 5). The separation of a mixture ofpurified hypocotyl I and hypocotyl II enzymes was greatestafter 3 to 4 hr of electrophoresis. In 15-hr experiments theleaf and hypocotyl II enzymes compacted into the gel at higher

DISCUSSION

Studies of the hypocotyl ATP sulfurylases in crude extractswere complicated by the very low activity of the tissue, inac-tivation of the enzymes during extraction, pyrophosphataseand ATPase activities, and very high endogenous PPi-ATPexchange. Inactivation of the enzyme was minimized by in-cluding 10 mm thioglycolate in the extracting medium (Fig. 2).Pyrophosphatase was inhibited by 10 mM NaF. Since endog-enous PPi-ATP exchange was inhibited by ionic strength (Fig.3), thereby causing an apparent K,S04-dependent inhibition ofendogenous exchange in some experiments, KCI of equiva-lent ionic strength to the substrate level of KSSO, was alwaysadded to control incubations when monitoring for ATP sul-furylase activity. The effect of ATPase on sulfate-dependentPPi-ATP exchange was minimized by using dilute extracts ofenzyme; under these conditions, hydrolysis of ATP by ATPasewas insufficient to cause a significant decrease in sulfate-de-pendent PPi-ATP exchange.

Purification only effected a 15- to 25-fold increase in specificactivity of the hypocotyl ATP sulfurylases. The more im-portant aspect of purification, however, was the separationof ATP sulfurylase from enzymes which interfered with theassay of ATP sulfurylase activity. Purified ATP sulfurylasewas uncontaminated with pyrophosphatase and ATPase ac-tivities, and the endogenous PPi-ATP exchange of purifiedenzyme was negligible (Table I). Similar remarks also applyto the leaf enzyme (Table II).

184 Plant Physiol. Vol. 53, 1974

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- we

L Hi Hu+ve

FIG 5 (3h)

- ve

4w-. I--~~~~

L LHI HI Hi Hu+ve

FIG 6 Q5h)

FIG.5. Polyacrylamide gradient gel electrophoresis

purified ATP sulfurylases from beetroot. Electrophoresis

ducted for 3 hr. The gel was incubated with molybdate

scribed in the text to detect molybdate-dependent

Pi. Pi was not detected on control gels of the purified

cubated without molybdate. L: leaf enzyme;

enzyme; HII: hypocotyl II enzyme.

FIG. 6. Polyacrylamide gradient gel electrophoresis

Generally speaking, the properties of the three beetrootATP sulfurylases were similar to each other and to the enzymesdescribed in spinach leaf (27) and the leaf tissue of fourspecies of Astragalus (W. H. Shaw and J. W. Anderson, un-published data). All enzymes were inactive in the absence ofMg2+, have pH optima from 7.5 to 9.0, and catalyze bothsulfate- and selenate-dependent exchange. The affinity of allthe enzymes for selenate was greater than for sulfate and theV (selenate)/ V (sulfate) ratios fall within the range 0.23 to0.29 regardless of the origin of the enzyme.

There are, however, several quantitative properties of thebeetroot hypocotyl I enzyme which distinguish it from theother two beetroot enzymes. The most obvious of these dif-ferences was a difference in charge which permitted separationof the hypocotyl enzymes by ion exchange chromatography onDEAE-cellulose columns at pH 8.0 (Fig. 1) and by electro-phoresis at pH 8.2 (Figs. 5 and 6). The hypocotyl I enzyme alsohad a lower affinity for sulfate and selenate (Table III) and aslightly higher pH optimum than the leaf and hypocotyl IIenzymes (Fig. 4). The differences in molecular weight of thethree enzymes was small; the enzymes were retarded but notseparated by gel fitration on Sephadex G-200 columns butwere resolved by electrophoresis for 15 hr on a polyacrylamidegel gradient. The leaf and hypocotylII enzymes, on the otherhand, were indistinguishable by electrophoresis at 3 or 15 hr,ion exchange chromatography, or their kinetic properties.

Hypocotyl tissue invariably contained hypocotyl I andhypocotylII enzymes in approximately equal proportions; thisrules out the possibility of allelic variation. Several lines of evi-dence suggest that it is unlikely that the two hypocotyl en-zymes arise as an artifact. Firstly, the two activities were re-

solved in approximately equal amounts irrespective of whetherthe extracts were purified and irrespective of the method ofpurification. Secondly, the differences in molecular weight ofthe two hypocotyl enzymes is barely significant, but themolecular weight of both enzymes is in excess of 200,000.Thirdly, hypocotylII enzyme was indistinguishable from theleaf enzyme. While we cannot exclude the possibility that leaftissue contains an additional ATP sulfurylase (a small amountof ATP sulfurylase was eluted prior to the major leaf ATPsulfurylase on DEAE-cellulose columns), this activity was

never greater than 2% of the major leaf enzyme. We concludethat the multiple forms of ATP sulfurylase in hypocotyl tis-sue are most probably isoenzymes.

In leaf tissue, sulfate reduction and the enzymes of thesulfate reduction pathway (including ATP sulfurylase) areassociated with chloroplasts (6, 7, 24). Presumably the ATPsulfurylase of beetroot leaf tissue is a chloroplastic enzyme.

We are unable to comment on the subcellular localization ofthe two hypocotyl enzymes, but the similarity of the leaf andhypocotyl II enzymes warrents a thorough study of the plastidsof the nonphotosynthetic hypocotyl tissue for ATP sulfurylaseactivity. The studies on the association of the hypocotyl en-

zymes with mitochondria were inconclusive, especially sincethe differential centrifugation technique used to prepare themitochondria does not adequately resolve the large numberof subcellular particles of size and density similar to mito-chondria which occur in nonphotosynthetic tissue. It is im-possible to attribute a physiological role to the two enzymes

until their subcellular localization has been clearly established;in view of the problems in extracting subcellular organelles

purified ATP sulfurylases from beetroot. Electrophoresis was con-ducted for 15 hr and ATP sulfurylase activity was detected as de-scribed in Fig. 5. At 15 hr the enzymes ceased to migrate anyfurther into the gradient. L: leaf enzymes; HI: hypocotyl I en-zyme: HII: hypocotyl H enzyme.

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186 PAYNTER AN

from beetroot hypocotyl, it would seem prudent to chooseanother nonphotosynthetic tissue to pursue this problem.

Nitrite, like sulfate, is reduced by both photosynthetic andnonphotosynthetic tissues. Dalling et al. (10) reported thatphotosynthetic tissue contains a single nitrite reductase whichis associated with chloroplasts. Nonphotosynthetic tissue, onthe other hand, contains two nitrite reductase activities; one ofthese enzymes was indistinguishable from the leaf enzyme (9).Both of the nonphotosynthetic activities were associated withproplastids (11). While the subcellular distribution of the twoATP sulfurylases of beetroot hypocotyl in uncertain, there is astriking similarity in the distribution of isoenzymes of ATPsulfurylase and nitrite reductase in photosynthetic and non-photosynthetic tissue.

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