Plant Physiol. (1973) 52, 580-584

Adenosine 5'-Triphosphate-Sulfurylase in Corn Rootsand Its Partial Purification1

Received for publication January 11, 1972

FUNMILAYO D. ONAJOBI,2 C. V. COLE, AND CLEON RossDepartment of Agronomy and Agricultural Research Service, United States Department of Agriculture andDepartment of Botany and Plant Pathology, Colorado State University, Fort Collins, Colorado 80521

ABSTRACT

ATP-sulfurylase (ATP-sulfate adenyltransferase, EC2.7.7.4) was found in nonparticulate fractions of both rootsand leaves of Zea mays L. seedlings using two detection meth-ods. Addition of exogenous pyrophosphatase was essential formaximum rates of conversion of 3SO2- to labeled adenosinephosphosulfate in unpurified root extracts, but not in unpuri-fied leaf extracts. In the presence of exogenous pyrophospha-tase, the enzyme from roots exhibited specific activities as highas those obtained with the leaf enzyme. The root enzyme waspurified 33-fold by centrifugation and column chromatographyprocedures. Its molecular weight obtained by Sephadex gelfiltration was about 42,000. Its Kit for pyrophosphate was 7AM, while for adenosine phosphosulfate, the Km was 1.35 uM.None of the enzyme fractions studied converted adenosinephosphosulfate into detectable amounts of 3'-phosphoadeno-sine-5'-phosphosulfate. ATP-sulfurylase was also found in rootsof corn seedlings grown aseptically. The data suggest that atleast the first reaction in sulfate reduction might proceed aseffectively in roots as in shoots.

The mechanisms of sulfate reduction in microorganismshave been studied extensively (10, 14, 15, 17, 23-25). Thesestudies showed that the first step in the metabolism of sulfateis the activation of sulfate by ATP-sulfurylase (EC 2.7.7.4)according to the following:

ATP + SO42= APS + PPi

In some microorganisms, an enzyme APS3-kinase (EC 2.7.1.25)further activates APS to PAPS (8, 15). In studies with the greenalga (Chlorella pyrenoidosa), Schiff and his collaborators (9)identified PAPS as a probable intermediate in the reduction ofsulfate.

Studies of the mechanisms involved in the reduction of sul-

1 Contribution from Colorado State University Experiment Sta-tion and Agricultural Research Service, United States Departmentof Agriculture. This work was supported in part by the Inter-national Atomic Energy Agency. Published with the approval ofthe Director of the Colorado State University Experiment Stationas Scientific Series No. 1786.

2 Present address: Department of Biological Sciences, Universityof Ife, Ile-Ife, Nigeria.

'Abbreviations: APS: adenosine-5'-phosphosulfate; PAPS: aden-osine-3'-phosphate-5'-phosphosulfate; DEAE: diethylaminoethyl.

fate in higher plants have been few (19). The presence of ATP-sulfurylase in plant tissues has been demonstrated by Asahi(3), Adams and Johnson (1), Ellis (7), Balharry and Nicholas(4), and Shaw and Anderson (18). Most of these investigatorsfailed to detect any APS-kinase, but Mercer and Thomas (13)reported some incorporation of 3SO- into PAPS by maize andbean chloroplast fragments. Tsang et al. (20), on the basis ofwork with Chlorella, suggested that APS-kinase, where it ex-ists, participates in a side reaction of sulfate ester formationand not in the direct pathway of sulfate reduction.Most studies of sulfate reduction in higher plants have been

limited to leaves. However, Ellis (7) and Balharry and Nicholas(4) demonstrated substantial ATP-sulfurylase activities in to-mato roots and root tips of oat seedlings, respectively. We havediscovered that excised corn roots show as much ATP-sul-furylase activity as corn leaves. This paper presents a methodfor partial purification and some properties of the ATP-sul-furylase of corn roots.

MATERIALS AND METHODS

Radioactive materials (H2I'SO, and H332P04) were obtainedfrom New England Nuclear Corporation. APS and ATP wereobtained from Sigma Chemical Company. Firefly lantern ex-tract was obtained in a dried form from Sigma Chemical Com-pany. Inorganic pyrophosphatase was obtained from Worthing-ton Biochemical Corp.Corn plants were grown in modified Hoagland's solution in

a growth chamber with a 16-hr photoperiod as described byBledsoe et al. (5). The corn plants were used for enzyme prep-aration when they were 2 to 3 weeks old. In certain instances,4- to 5-day-old seedlings were used.

Aseptic corn seedlings were grown as follows. Seeds weresoaked briefly in 95% ethanol to remove fungicide and thensoaked in sterile-distilled water overnight at about 5 C. Aftertwo sides of each seed were scraped off to expose the endo-sperm tissue, the seeds were sterilized with 1% NaOCl undervacuum for 5 min with two changes of similarly diluted NaOCland rinsed many times with sterile-distilled water. The seeds(5 per Petri dish) were then aseptically plated on Hoagland-peptone-glucose agar (22) and incubated at 35 C for 4 days.The roots of these seedlings were freed of agar and used forthe preparation of sulfurylase enzyme under sterile conditions.

Preparation of Corn Leaf or Root Extract for SulfurylaseAssay. Homogenates of corn leaves and roots were preparedaccording to the method of Ellis (7) using a solution containing200 mm tris-HCl buffer, 10 mm cysteine, and 10 mm Na2EDTAat pH 8.0 (medium I). The homogenate was strained throughfour layers of nylon to obtain the crude fraction, which was

then centrifuged at 30,000g for 30 min. The precipitate was

discarded.580

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ATP-SULFURYLASE IN CORN

The supernatant was further centrifuged at 105,000g for 1

hr to obtain a microsomal pellet and supernatant-soluble frac-

tion. The pellet was suspended in half-strength medium I and

homogenized with a glass rod. All enzyme preparations were

dialyzed against a solution containing 20 mm tris-HCl, 1 mM

cysteine, and 1 mm Na,EDTA at pH 8.0 (mediumII) prior to

enzyme assay. All operations were carried out between 0 and

4 C.Enzyme Assays. Two methods were used to estimate ATP-

sulfurylase activity. The first (method A) involved the incor-

poration of35SO42- into APS; in the second (method B) the

amount of ATP formed in the presence of APS and pyrophos-

phate was determined by a firefly luminescence procedure.Method A. The enzyme preparation (0.4 ml) was incubated

with 10,umoles of ATP, 101umoles of MgCl2, 1 tumole of

Na2'SO4 (25-50,tc), 65,umoles of tris-Cl (pH 8.0), and 1 unit

of pyrophosphatase in a final volume of 0.65 ml at 35 C for

60 min. The reaction was stopped by placing the incubationtubes in a boiling water bath for 1 min, and the protein pre-

cipitate was removed by centrifugation. The APS product was

separated either by two-dimensional TLC (6) or by paper elec-

trophoresis and detected by radioautography.Paper electrophoresis of aliquots (10-25,l) of the reaction

mixture was carried out in 30 mm citrate buffer, pH 5.9, at 400v for 4 hr at 2 C. Radioactive compounds were detected either

by exposure to x-ray films or by cutting the dried paper into1-cm segments and counting the 3S radioactivity in each seg-

ment with a liquid scintillation counter after adding toluenecocktail (containing 4 g PPO and 0.05 g POPOP/l toluene).Method B. The firefly assay for ATP-sulfurylase was adapted

from that used by Balharry and Nicholas (4). Light emissionwas measured using a Nuclear Chicago Mark I liquid scintil-lation spectrometer with circuits switched out of coincidenceso that light events detected by either of the photomultiplierswere registered. A preparation containing soluble extract from250 mg of dried lanterns (Sigma FLE-250) was extracted for 4hr with 10 ml of ice-cold water and centrifuged. Aliquots ofthe extract were stored at -15 C. Just before use aliquots were

diluted 10-fold with 10 mm phosphate buffer at pH 7.3.The assay was conducted in liquid scintillation counting vials

containing: 0.05 ml of 1 M MgCI2; 1 ml of 10 mm phosphatebuffer, pH 7.3; 1 ml of 50 mm sodium arsenate buffer, pH 7.3;1 ml (or less) of water; 10 ,ul of 3.0 mm sodium pyrophosphateand 10 Al of 0.15 mm APS. A 0.1-ml aliquot of the firefly en-

zyme preparation was rapidly mixed with the assay mixture ina vial and immediately positioned in the counting chambermaintained at 10 C. Exactly 30 sec after the firefly enzyme was

added, recording of consecutive 0.1-min counts was started.The activity of the firefly preparation was routinely checked

by additions of 0 to 300 pmoles ATP per assay in the absenceof APS or pyrophosphate in the mixture. The 0.1-min countsrecorded 0.5 min after the addition of the firefly enzyme were

directly related to the amount of ATP added over this con-

centration range. At this dilution of the enzyme preparation thesensitivity ranged from 500 to 850 counts per pmole ATP de-pending on the age of the enzyme preparation. Higher concen-

trations of the firefly enzyme were avoided because of ATP-sulfurylase activity in the firefly preparation. To measure

ATP-sulfurylase activities, 10 to 200 ,LI of the enzyme extractwere added to the assay mixture and mixed at time zero. After30 sec, 0.1 ml of the firefly enzyme was added. Consecutive 0.1-min counts were recorded 1 min after addition of tissue en-

zyme extract.The time of incubation of the assay mixture with the corn

root enzyme prior to the addition of firefly enzyme was variedbetween 30 sec and 5 min. A linear relationship between timeand counts, and hence of ATP formation, was observed. More-

over, when the amount of ATP formed per min was calculated,the same results were obtained whether the calculation wasbased on the 30-sec incubation period or the slope of the timeversus ATP plot. Therefore, the 30-sec incubation period wasused in all subsequent assays.

Partial Purification of ATP-Sulfurylase of Corn Roots. Thecorn root enzyme was partially purified by gel filtration onSephadex G-150 followed by ion-exchange chromatography on

DEAE-Sephadex A-50. A Sephadex G-1 50 (Pharmacia) col-umn (2.5 X 32 cm) was equilibrated with 0.05 M tris-HClbuffer, pH 7.4, containing 0.2% sodium azide and 0.2 mM di-thiothreitol. Sucrose (0.5 g) was dissolved in 5 ml of the

105,000g supernatant of corn root extract, and this solutionwas layered on top of the column. Protein contents in each5-ml fraction were estimated by the method of Lowry et al.(12). ATP-sulfurylase activity of each fraction was assayed bythe firefly method. Fractions showing sulfurylase activity were

pooled and dialyzed against 0.1M tris-HCl buffer, pH 7.4, con-taining 0.2% sodium azide and 0.2 mm dithiothreitol and thenfurther fractionated on a DEAE-Sephadex A-50 column.DEAE-Sephadex A-50 was swollen in 0.1M tris-HCI buffer,

pH 7.4, containing 0.2% sodium azide for 4 days at 4 C. Acolumn (1 cm X 33 cm) was prepared and equilibrated withthe same tris buffer in the presence of 0.2 mm dithiothreitol.The enzyme was eluted with a 0.1 M to 0.5M buffer gradient.The flow rate of the column was about 10 ml/hr, and 5-mlfractions were collected. Sulfurylase activity was estimated bythe firefly method.

RESULTSFormation of APS. When the 30,000g supernatant of corn

roots or leaves was incubated with Na2'SO, in the presence ofATP and MgCl,, paper electrophoresis of the incubation mix-tures revealed two radioactive bands in control incubations andthree radioactive bands in the presence of enzyme (Fig. 1). Themost rapidly migrating band was radioactive sulfate. A secondband not visible in the figure but indicated by a dotted lineran just ahead of ATP. Radioactivity in this band was shownto be 32P by liquid scintillation spectrometry and by its disap-pearance from autoradiograms made after allowing time for'P decay.A third band ran between ADP and ATP and had the same

mobility as authentic APS. This radioactive band was cut out,eluted, and mixed with authentic APS. A portion of this mix-ture was analyzed by two-dimensional TLC (6), and anotherportion was electrophoresed on paper. Radioactivity movedprecisely with authentic APS in both systems. Careful exami-nation of radioautographs showed no evidence for formationof other labeled compounds (i.e. PAPS) during incubation withroot or leaf extracts.The leaf preparation contained about 10 to 15 times more

ATP sulfurylase activity/mg protein than did the root prep-aration when no pyrophosphatase was added to either incuba-tion mixture. However, when 1 unit of pyrophosphatase wasadded during incubation, the activity of the root extract in-creased at least 10-fold, but the activity of the leaf preparationwas not greatly changed (Table I). In all subsequent assays in-volving APS formation, 1 unit of pyrophosphatase was added.

Formation of APS by Sterilized Corn Roots. It is possiblethat the AP3S formation from ATP and Na2,SO4 in the pres-ence of corn root extracts was due to associated microorgan-isms. To eliminate this possibility corn seedlings were grownunder aseptic conditions, and the root homogenate was pre-pared under sterile conditions. The 30,000g supernatant fromthis homogenate yielded no microbial counts. In two separateexperiments using assay method A, we found formation of 6.3

581Plant Physiol. Vol. 52, 1973

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ONAJOBI, COLE, AND ROSS

........

35SfA

32

ri . L. ixauoauuwgrapns oi 3- oaDe±ea proaucts separatec oy

paper electrophoresis following incubation with leaf and root en-zyme extracts.

and 6.4 nmoles APu5S/mg protein -hr, respectively, in this su-pernatant fraction. Similar enzyme preparations from unsteri-lized roots gave 3.1 and 3.5 nmoles AP3"S/mg protein in thetwo experiments.

Sulfurylase Activity in Various Subcellular Fractions ofCorn Roots. As a preliminary to the purification of the sul-furylase in corn roots, the 30,000g supernatant was furthercentrifuged at 105,000g for 1 hr. The supernatant was sep-arated, and the microsomal pellets were homogenized withbuffer. After dialysis the various fractions were assayed forATP sulfurylase activity by method A. Most of the ATP sul-furylase activity was in the 1 05,000g supernatant and verylittle activity was in the microsomal pellets (Table II).

Purification of ATP-Sulfurylase of Corn Roots. Purification

j^ of the corn root enzyme was attempted, first by gel filtration'Xj of the 105,000g supernatant on G-150 Sephadex. Three major

RQcitb - protein-containing peaks were eluted (Fig. 2a). In some of theexperiments, fractions under each peak were combined, di-alyzed, and the three pooled fractions were assayed for ATP-sulfurylase activity by method A. Only fraction II possessedsulfurylase activity (data not shown). Furthermore, 0.1-ml ali-quots of each of the 5-ml fractions collected from the G-150Sephadex column were assayed for sulfurylase activity by

4 S method B. A peak of activity coincided with part of the middleprotein peak, in agreement with results obtained when thefractions under each peak were pooled (Fig. 2a).The fractions from gel-filtration possessing sulfurylase ac-

tivity (fraction S, Fig. 2a) were pooled, dialyzed, and furtherpurified by ion-exchange chromatography on DEAE-SephadexA-50. Two distinct peaks of protein were obtained, but thefirst peak possessed a shoulder (Fig. 2b). When the fractionswere assayed for sulfurylase activity by method B, a peak of

c', -activity coinciding with the shoulder of the first protein peakwas obtained. The ionic strength at which the sulfurylase peakwas eluted was between 0.15 M and 0.2 M tris-Cl. Table IIshows that a 33-fold purification of the sulfurylase enzyme wasachieved from centrifugation through ion-exchange chromatog-raphy.

Fractions from the ion-exchange column possessing sulfur-ylase activity were pooled and stored at -15 C. This fractiondesignated "purified" ATP-sulfurylase was later used for study-ing the following properties of the corn root enzyme:

Molecular Weight. The G-200 Sephadex column was cali-brated using several proteins of known molecular weight andplotting Ve/Vo against the logarithm of molecular weight ofthe proteins (2). From the volume at which the ATP-sulfurylaseactivity was eluted from the G-200 Sephadex column, the mo-

Table I. Effect of Pyrophosphatase oii the Formnationof APS by Cornl Leaf anid Root Extract

Experiment Corn Tissue Pyrophosphatase APS35 Formed2Extract' Added

itnit mn,ole/nzg lur

I Leaf 1 2.44I Leaf None 3.04I Root 1 2.20I Root None 0.18

II Leaf 1 3.2II Root 1 3.9

1 Extracts used are the 30,000g supernatant.2 Calculations based on specific activity of 35SO42- in the assay

mixture.

Table II. Purification of Corni Root ATP-Sulfurylase

APS Formed ATP Formed

Enzyme Fraction Total Protein Recovery PurificationSpecific Total Specific Totalactivity activity activity activity

nig nmolesln/g-/r nmole/hr nmole/mg-stin nmPzoles/Inin per cent

Whole homogenate 14.0 1.5 21.0 100 130,000g supernatant 5.0 4.1 20.5 1.23 6.8 98 2.74105,000g supernatant 4.0 4.9 19.5 1.52 6.1 88 3.3-3.4105,OOOg pellet 0.80 0.4 0.3SephadexG-150activefraction' 0.46 6.07 2.8 40 13.5DEAE-Sephadex active fraction' 0.08 [ 14.80 1.2 17 33.0

1 These are fractions from the respective columns which possessed ATP-sulfurylase activities.

582 Plant Physiol. Vol. 52, 1973

r%f 354Z 1,-,Ik-l-A

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ATP-SULFURYLASE IN CORN

lecular weight of corn root ATP-sulfurylase was estimated as42,000 + 3,000.

Kinetics of Corn Root ATP-Sulfurylase. Kinetic studies wereconducted with crude and purified enzyme extracts usingmethod B. The formation of ATP was linear over the time in-terval of 30 sec to 5 min and linear with regard to enzyme con-centration from 7.5 to 150 /tg protein/assay. The concentra-tion of APS was varied in assay mixtures containing 0.5 ,ig ofprotein of partially purified corn root ATP-sulfurylase, phos-phate buffer, MgCl2, sodium arsenate, pyrophosphate, and thefirefly enzyme in the usual concentrations. The amount of ATPformed with each APS concentration was measured, and thereciprocal of ATP formed was plotted against the reciprocalof APS concentration. Calculation of the Km for APS gavea value of 1.35 ,uM (Fig. 3a). The concentration of pyrophos-phate was also varied between 2 and 50 /uM using 0.5 ,ig ofprotein of the "purified" ATP sulfurylase and other standardconstituents of the assay mixture. The calculated Km for pyro-phosphate was 7 ,iM (Fig. 3b).

Stability of the ATP-Sulfurylase. The sulfurylase activity ofthe 105,000g supernatant of corn roots is stable for at least 2months at -15 C. The partially purified fractions were, how-ever, less stable; the active fraction from the DEAE-Sephadexcolumn lost about 80% of its sulfurylase activity within 1

Lna)0E

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0

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cx

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FIG. 3. Lineweaver-Burke plots for APS and pyrophosphate.0

Assay mixture contained the standard amount of reagents forsulfurylase assay by the FFL method except for (a) APS whoseconcentration was varied, (b) pyrophosphate whose concentration

25 was varied.

month at -15 C. The enzyme is unstable to heat. When fresh20 enzyme was prepared and heated in a boiling water bath for 1

min, no sulfurylase activity could be detected by method B.

E

a)

0-

Tube Number

FIG. 2. a: Gel-filtration of 105,000g supernatant of corn rootson a G-150 Sephadex column, eluting with 0.05 M tris-HCl buffer,pH 7.4. b: Ion exchange chromatography of fraction S from a on

DEAE-Sephadex A-50, eluting with a gradient of 0.1 to 0.5 M

tris-HCl buffer, pH 7.4. Protein was estimated by 280/260 nm

ratios.

DISCUSSION

The results show that ATP-sulfurylase activity exists in corn

leaves and roots, and that in the presence of exogenous pyro-phosphatase this activity is similar in both organs. No PAPSwas detected by any of the methods used for the analysis ofthe incubation mixtures from corn leaves or roots. The absenceof PAPS supports the findings of Asahi (3), Ellis (7), and Bal-harry and Nicholas (4), who could not detect any APS-kinaseactivity in higher plant tissues. However, the report by Mercerand Thomas (13) that maize and bean chloroplast fragments in-

corporate 5SOQ2- into both APS and PAPS contradicts our re-

sults. This discrepancy needs to be resolved. It is possible that

PAPS may be present in quantities too small to be detected byeither the chromatographic or electrophoretic method em-

ployed, or that either PAPS or APS-kinase is labile under the

assay conditions.Whether or not plant tissues possess APS-kinase is important

regarding further details of mechanisms of sulfate reduction.

40 50I10cbeNm20 30Tube Number

b

- ATP-- Protein

Plant Physiol. Vol. 52, 1973 583

*A'

p1p(Pmole)-l

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ONAJOBI, COLE, AND ROSS

It has been concluded from work with microorganisms thatPAPS is the substrate for further reduction by assimilatorysulfate reducers, whereas APS is the substrate used by dissimi-latory sulfate reducers (19). If higher plants, which are assimi-latory sulfate reducers, possess no APS-kinase, APS must bethe substrate for further reduction, and the mechanisms bywhich reduction occurs need to be investigated. In fact, theconclusion that PAPS is the substrate which is reduced to sul-fite by yeast and other assimilatory microorganisms (19) needsto be re-examined in light of recent findings by Tsang et al.(20) that APS was an intermediate in the reduction of sulfurin PAPS to acid-volatile substances in Chlorella pyrenoidosa.

Gel-filtration studies of the ATP-sulfurylase of corn rootsindicate a molecular weight of around 42,000, or a lower valuethan those reported for this enzyme in bakers' yeast (16), Peni-cillium chrysogenum (21), and rat liver (11) of 100,000,430,000, and 850,000, respectively. The differences in mo-

lecular weight may reflect differences in the nature of the ATP-sulfurylase in the different organisms and tissues, but it is alsopossible that the enzyme exists as aggregates of a basic sub-unit and that different aggregates are found, depending on theconditions of investigation (21).

LITERATURE CITED

1. ADAMS, C. A. AND R. E. JOH-NSON. 1968. ATP-sulfurylase activity in tlhe soy-bean (Glycinie max (L.) MIerr.). Plant Physiol. 43: 2041-2044.

2. AN.DREWS, P. 1965. The gel-filtration behaviour of proteins related to their

molecular weights over a wide range. Biochem. J. 96: 595-606.

3. ASAHI, T. 1964. Sulfur metabolism in higher plants. IV. Mechanism of sul-

fate reduction in chloroplasts. Biochim. Biophys. Acta 82: 58-66.

4. BALHARRY, G. J. E. AND D. J. D. NICHOLAS. 1970. ATP-sulphurylase in

spinach leaves. Biochim. Biophys. Acta 220: 513-524.

5. BLEDSOE, C., C. V. COLE, AND C. Ross. 1969. Oligomycin inhibition of phos-phate uptake and ATP labeling in excised maize roots. Plant Physiol. 44:

1040-1044.6. COLE, C. V. AND C. Ross. 1966. Extraction, separation, and quantitative esti-

mation of soluble nucleotides and sugar phosphates in plant tissues. Anal.

Biochem. 17: 526-539.7. ELLIS, R. J. 1969. Sulphate activation in higher plants. Planta 88: 34-42.

8. GREGORY, J. D. AN-D P. W. ROBBINS. 1960. Metabolism of sulfur compounds(sulfate metabolism). Annu. Rev. Biochem. 29: 347-364.

Plant Physiol. Vol. 52, 1973

9. HODSON, R. C., J. A. SCHIFF, A. J. SCARSELLA, AND M. LEVINTHALL. 1968.Studies of sulfate utilization by algae. 6. Adenosine-3'-phosphate-5'-phos-phosulfate (PAPS) as an intermediate in thiosulfate formation from stul-fate by cell-free extracts of Chlorella. Plant Physiol. 43: 563-569.

10. KLINE, B. C. AND D. E. SCHOENHARD. 1970. Biochemical characterization ofsulfur assimilation by Salrnonella pullorum. J. Bact. 102: 142-148.

11. LEVI, A. S. AND G. WOLF. 1969. Purification and properties of the enzyme

ATP-sulfurylase and its relation to vitamin A. Biochim. Biophys. Acta 178:262-282.

12. LOWRY, 0. H., N. J. RoSEBROUaGH, A. L. FARR, AND R. J. RANDALL. 19.51.Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275.

13. MERCER, E. I. AND G. THOUAS. 1969. The occurrence of ATP-adenylsul-phate-3' -phosphotransferase in the chloroplasts of higher plants. PhNyto-chemistry 8: 2281-2285.

14. PASTERNAK, C. A., R. J. ELLIS, M. C. JONES-MORTIMER, AND C. E. CRIGHTON.1965. The control of sulphate reduction in bacteria. Biochem. J. 96: 270-275.

15. PECK, J. D., JR. 1962. Comparative metabolism of inorganic sulfur compoundsin microorganisms. Bact. Rev. 26: 67-94.

16. ROBBINS, P. W. AND F. LIPMIANNN. 1958. Enzymatic synthesis of adenosine-.a

phosphosulfate. J. Biol. Chem. 233: 686-690.17. Roy, A. B. AND P. A. TRoDINGER. 1970. The Biochemistry of Inorganic Com-

pounds of Sulfur. Cambridge University Press, New York.18. SHAW, W. H. AND J. W. ANDERSON. 1972. Purification, properties, and substrate

specificity of adenosine triphosphate sulphurylase from spinach leaf tissue.Biochem. J. 127: 237-247.

19. THOMPSON, J. F. 1967. Sulfur metabolism in plants. Annu. Rev. Plant Plhysiol.18: 59-84.

20. TSANG, MI. L., E. E. GOLDSCHNIIDT, AND J. A. SCHIFF. 1971. Adenosine-5phosphosulfate (APS35) as an intermediate in the conversion of adenosine-3'-phosphate-5'-phosphosulfate (PAPS35) to acid-volatile radioactivity.Plant Physiol. 47: S20.

21. TWEEDIE, J. W. AND I. H. SEGAL. 1971. Adenosine triphosphate sulfurylasefrom Penicillium chrysogetnum. II. Physical, kinetic, and regulatory proper-

ties. J. Biol. Chem. 246: 2438-2446.22. WELLS, T. R., W. A. KREUTZER, AND D. L. LINDSEY. 1972. Colonization of

gnotobiotically grown peanuts by Aspergilluts flavris andt selected interacting

fungi. Phytopathology 62: 1238-1242.23. WHELDRAKE, J. F. 1967. Intracellular concentration of cysteine in E. colt and

its relation to repression of the sulphate-activating enzymes. Biochem. J.105: 697-699.

24. WHELDRAKE, J. F. AN-D C. A. PASTERN-AK. 1965. The control of sulphate acti-v-ation in bacteria. Biochem. J. 96: 276-280.

25. WXILSON, L. G., G. T. ASAHI, AND R. S. BANDDURSKI. 1961. Yeast sulfate-re-ducing system. 1. Reduction of sulfate to sulfite. J. Biol. Chem. 236: 1822-

1829.

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