Plant Physiol. (1968) 43, 547-554

Studies of Sulfate Utilization by Algae. 4. Properties ofa Cell-Free Sulfate-Reducing System from Chlorellal

Jerome A. Schiff and Mark Levinthal2Department of Biology, Brandeis University, Waltham, Massachusetts 02154

Received December 22, 1967.

Abstract. A cell-free system from Chlorella pyrenoidosa Chick (Emerson strain 3) whichproduces acid-volatile radioactivity from 35SO42- is described. A high speed supernatant fromcells broken in the French Press at pH 7.0 shows maximal activity when fortified with ATP,an ATP-generating system (creatine phosphate and creatine phosphokinase), TPN, a TPN-reducing system (glucose-6-phosphate and glucose-6-phosphate dehydrogenase) and MgCl,.This system is quite labile and is not stable to dialysis. Addition of low concentrations of2,3,-dimercaptopropan-1-ol (BAL) to the buffers used for enzyme preparation stabilize theextracts and permit them to be dialyzed for 4 hours without loss of activity. If additionalBAL is also added to the incub-ation mixtures it can replace TPNH as a reductant. DPNHalso shows appreciable activity.

The system prepared with BAL-containing buffers shows maximal activity at pH 9.0.At this pH, the system requires only ATP, Mg2- and additional BAL and has high activitvand stability compared with the other conditions tried. The optimum concentrations of thesereactants has been determined and the kinetics of production of acid-volatile radioactivity aredescribed. Nucleoside triphosphates other than ATP are not appreciably active in thissystem. In all cases, anaerobic conditions are required for maximal activity, the enzymeextracts are labile to heat, and no unequivocal requirement for thioctic acid can be demonstrated.

There is consideraibile evidence from nuttritionaland genetic studies that plants and most micro-organismis can reduce stulfate to satis,fy their entirestulfur requirements (5, 15). Cell-free stulfate re-duicing systems have been o,btained from yeast(1,6, 16, 18), Desulfovibrio (8), and Salmitonella(4), and several intermediates and enzymes involvedin the process have been identified. Eviden,ce isalso available concerning the prodtucts formed fromstullfate in these and other organissms in zivo (5, 15).

Chlorell(l pyrenoidosaz was selected for thepresent invest:gation becatu-se it combines the ad-vantages o,f sttudying stilfate reducttion in a greenplant with the ease of maniptulation of a micro-organism. We have previously shown that theprodutcts formed from sulfate by intact Chlor ellacells are: cysteine, glutathione, homocysteine,.S-adeno'syl methionine, and adenosine-3'->phosphate-5'-phosphosutlfate (PAPS) (11, 13, 14). Some ofthese compotunds were also identified sutbsequientlyby others (3,17).

In prelliminary work, we adopted an assav basedon acid volatility oif the productts formed, since

1 Supported by grants from the National ScienceFoundation.

2 Supported during part of the period in which thiswork was conducted by a Predoctoral Research Felilow-ship from the National Institutes of Health. Presentaddress: National Institutes of Health, Bethesda, 'Mary-land.

several of the inorganic species which might beproduiced reduictively from )sulfate yield gases onacidification, while sulfate does not. Thtus sulfitte(SO32-) and thioistulfate (S.,O520 ) wouild be expectedto vield suilftur dioxide (SO<) on acidification, whilesulIfide (S2-) would yield hydrogen sulfide (H,S).Using this assay, it was fotund that an active cruidebroken cell preparation fTrom Chlorella cotuld befuirther fract;onated by centrifugation without lossof activity (12). (The natuire of the acid volatileproduct is described in an accompanying paper.)This active high-speed stupernatant fraction couldbe prepared in imidazole or phosphate butffers, btttris proved to yield more active extracts, althouighrelatively high concelntrations of ithis btuffer hadto be tused when working in the range of pH 7.0.This system appears to be rather insensitive to ionicstrength, howvever, since buffers in the range of0.05 to 0.5 Mr yield active extracts if the buffercapacity is adequlate. Havinig found the conditionsfor obtaining active extracts from Chlorella, weturned to defining the optimum condditions foractivity and the minimum requiremenits for co-factors. This is the sulbject of the present pa,per.

Materials and Methods

Growth of Organismit. Chlorella pyrenoidosaClhick (Emerson strain 3) was grown on themedium shown in talble I (Dr. R. C. FuXller, personalcommunication) under aseptic conditions. The cul-

547

PLANT PHYSIOLOGY

Table I. MUedium1t For the Growth of Chlorella

Substance Conc Volume added

M1\A 1111/1/KH9PO4 0.5MgSO4 2.0KNO3 5.0K,HPO4 0.5Ca(NO)., 0.25Fe-versenoll 1.0Trace elements2 1.0

To make Fe-versenol, dissolve 59 grams Versenol-120in 500 ml water, and add 24.9 g FeSO4*7H O0. Diluteto 1 liter and aerate overnight. Filter and use fil-trate.

2 One liter of trace elements solution contains: H BO3,1.43 g; MnSO4.H90, 1.05 g; ZnCl, 50 mg; CuSO4*5H10, 40 mg; H2MoO+4H20, 10 mg.

tulres were shaken in a waiter bath at 260 over"daylight" fluorescent tuibes providing 800 ft-c atthe lower surface, and were continuiously bubbledwith 5 % CO, in air during growth.

Preparation of Extracts. Foulr types of extractswere employed and were prepared and maintainedat 40 uinless sitored, as described below, for sllbse-qtient use.

A) "7" extracts. A 1 liter culture late in the logphase of growth was harvested by centrifugationat 1060 X g for 10 minuites yielding abouit 4 ml ofpacked cells. After resu,spension of the pellet in0.5 as itris-HCI ('pH 7.0), the cells w-ere resedimentedat 1860 X g for 10 minutes and were resuispended in20 ml of the same buffer. This suspension wasthen passed through a chilled French Pressure Cell(Aminco) at 9 tons of pressure and was thencentrifuged for 10 minuites at 1860 X g. The suiper-natant fluiid was then centrifuiged at 86,000 X g for1 hiouir in a Spinco Model L2 preparative ultra-centrifuge and the supernatant fluid constituited theundialyzed enzyme preparation. For dialysis, thispreparation was uised to fill ttubing previouslycleaned by boiling in distilled water 3 times, followedeach time by a distiilled water rinse, boiling once ina diilute soilution of disoditum ethyl,enediamine tetra-acetic acid and rinsing in distililed water, and fina,llyboiling and rinsing exhaustively in distilled water.Dialysis was carried otut againist 2000 ml of 0.5 Mitris-H,Cl (pH 7.0) for 4 hours with 1 change ofbuffer. The contents of the dialysis ttubing con-stituted the di,alyzed extract.

B) The same procedures were eimployed as inA, bhut 2,3-dimercaptopropan-1-ol (BAL) ait a con-centration of 1.5 mm was included in alil buffers.

C) "7-9" extracts. The same procedure wasempltoyed as in A, but the initial lbuffer used was50 mM tri!s-H,Cl (pH 7.0) containing 1.5 mm BAL.The high speed supernatanit fluiid so prepared wasthen dialyzed against 50 mm tris-HCI (pH 9.0)containing 1.5 mm BAL, for 4 hours (wilth 1 changeof buiffer) -ais described in A above. The conteinTts

o,f the dialysis tubing constituted the enzyme prep-aration.

D) "9-9" extracts. The same procedtire, wasemployed as in C, btut the initial buffer was 0.1 M%tri,s-HCl (ipH 9.0) containing 1.5 ml's BAL, whichwas also used, subseqtuently, for dialysis.

If not tised immediately, extracts prepared ac-cording to methods B, C, and D were frozen andstored at -200 in a series o)f small tubes to avoidrepeated freezing and thawing of the same extractwhich progressively reduced the activliity. Extractsprepared according to procedure A were allways usedimmediately since they deteriorated rapidly evenwhen frozen. Whole cells could be kept frozenfor some time and still yield active extracts if theywere first waished and resuspended in the initialbuffer to be uised for breakage in lprocedtlres B, C,and D. Again, these !cell stuspensions were dispensedinto small tulbes to avoid more thiani 1 freeze-thawcycle for any stusp,ension.

Intcutbationl of Ex-tracts. All experimeints werecarried otut in Thinfberg ttubes exvactuated twice,filled with n,itrogen to remove and exclude atmo,s-pheric oxygen and finally left tinder a vacuutm.Failtu,re to -do this resulited in a loss of half or moreolf the activity. The tubes were incuibated at 300in a constant temperature room for 2 houirs uinlessotherwise indicated. At the end of the incuibationperiod, 1.0 ml oif the reaction mixtuire together with100 jumo,lels of sodium thiosulfate as carrier (omis-sion of carrier gave low recoveries of acid-volatileradioactivity from radioiaictive thio,stilfate or sulffitein control experiments) was placed in the middleconcenitric well of a Conway diffulsion dish w\Nith aseparatedi drop of acid (1.0 ml of 2 \- HOSO, conl-taininig 0.1 % v/v octanol). 'rhe ceniter welIl ofthe di,sh received 1.0 ml of 2 N NaOH containing0.1 % (v/v) octtanol and the otutermost concentricring sea,l was the H,SO4-octano,l soluttion. Afterthe dish was covered, it was carefully rotated to mixthe acid anid the sample, and wa2s then allowed tostand overn'ight. An aliqtiot (uisuially 0.1 mln) ofthe NaOH wa,s then dried on a glass planchot withthe aid o,f an infra-red la,mp and was couinted in aNuiclear Chicago Model D-47 automatic planichetcounter operating in the proportional range.

For kinetic experiments, a nulmber of identicalincuibation mixtures were constitui'tedl from a sing-leenzyme extract in individuial Thulniberg tulbes, eachof which wais opened and assayeid at the indicatedtime after the beginning of the experiment.

For the determination of pH o,ptima, dloutblestrength extracts were prepared by resuspendingthe cells in one-half the usuial volulme of buffertised foir breakage. The buiffer nsed in all caseswas 0.05 M tris-H,Cl (pH 7.0). When BAI, wasomitted each aliquoit of the high speed supernatantfltlid was di'luted with an eqtual voluime of 0.5 Mtris-HCl or MES-HCI of the appropriate pH.When BAL was incduded in tPhe buiffer u-sed forbreakage (at a conc of 1.5 m-r') the high speed

548i

SCHIFF AND LEVINTHAL-SULFATE REDUCING EXTRACT FROM CHLORELLA

supernatant fluid was dialyzed against the samebuffer in the manner descrilbed above before ali- Equots of it were diluted wilth an equal volume of °130 "pH 7" EXTRACT _ _0.5 M tri,s-HCl or MES-HCl oif the appropriate pH. -,The final pH was checked, after mixing, in all acases. By diluting many aliquots of the same x 110odouble strength extract, an entire pH cutrve could ,be measured with a single preparation. o

Reagents. ATP (diso,dium salt), TPN, TPNH, g90DPN, DPNH, thioctic acid, creatine-P, gluco'se-6-P, screatine phosphokinase, and glucose-6-P dehydroge- >nase were purchased from the Sigma Chemical 70Company. The glu,cose-6-P dehydrogenase solution /

0

was dialyzed against 0.1 M tris-HCl (pH 7.0) over- a

night at 40 before being frozen for further use. < 50When high ATP concentrations were to be uised w

-j

(as in the ATP optimum experiments) 10 pmolesof ATP were dissolved per 0.1 ml of 0.1 M trisbefore being added to the reaction mixtures. Nudle- o /oside 'triph,osphates other than ATP were purchased >10from the Schwartz Chemical Company (UTP andCTP), Cal Biochemicals (dATP and TTP), and <Pabst (GTP). Two-(N-morphol'ino) ethanesuil- TIME (HRS.)

fonic acid (MES) an(d 2,3-dimercaptopropan-1-ol FIG. 1. Kinetics of formation of acid-volatile radio-

(BAL) were obtained from Cal Biochemicals. activity from 35S042 by an undialyzed Chlorella ex-

Sodium tri, n-hydroxyethyl-et'hylene-diamilne triace- tract at pH 7 lacking BAL. Conditions are the same as

tate (Versenol-120) was purchased from the Fisher in table II, except incubation times other than 2 hoursScientific Company. were employed by using several identical incubation

mixtures in separate T,hunberg tubes.Results

and Discussion stimttlation observed in the absence of ATP, but

in the presence of an ATP generating systemExtracts Prepared at pH 7.o. Table II shows (creatine phosphate and creatine phosph-okinase)

the results obtained with a typical extract prepared indicates that there is probably some endogenousby method A l(pH 7.0). The undialyzed extract ADP. The addi,tion of TPNH or TPN and a re-

shows requirements for ATP and Mg2+. The small dtucing system (glucose-6-P and glucose-6-P dehy-

Table II. Reduction of Sulfate at pH 7: Cofactor Requirements and Lability to Dialysis in the Absence of BALThe complete system contained: carrier-free 35SO42- (5 X 108 cpm), TPN (1.56 ,m,oles), gluoose-6-P (10

nmioles), glucose-6-P dehydrogenase (0.05 units), ATP (10 gmoles), creatine-P (10 umodes), creatine phospho-kinase (10 ug), MTgCl2 (50 ,tmoles), thioctic acid (0.49 Atmoles), Chlorella extract (method A) (1.0 ml) and 0.5 M

tris-HCl, pH 7.0 (0.1 ml) in a total volume of 2.0 ml.

Acid-volatile radioactivity ofUndialyzed Dialyzed

extract extract

CPM/2 mnl CPM/2 mnlComplete system 66,680 460O") "y -TPN 23,780 0IV "y -TPN, glucose-6-P

and glucose-6-P dehydrogenase 15,060 0"y "p -TPN, gluoose-6-P

aild glucose-6-P dehydrogenase;+TPNH (1.56 pmoles) 35,800 0

"yly -ATP 3500 0Pt "1 -ATP, creatine-P

and creatine phosphokinase 0 0-MgGl2 26 0-thioctic acid 67,040 120Chlorella extract heated

at 700 for 3 min 0 0

549

PLANT PHYSIOLOGY

drogenase) is quiite stimtulatory andcl implicatesTPNH as a reductant. The restlts also indicatethat a smalll a,mount of endogeno,us TPN is probablypresent since the added reducing system, in theabsence o,f added TPN results in some increase inactivi)ty. Thioctic acid appears to have little effectin this system, aithough the effect of this compo-undis quiite variable, occasionally it is stimulatory orinhibitory, to some extent, butt it has not beenpos,sible to show an absolute requ1irement. The heatlability of the extract is consistent with a requiire-ment for enzymes contained in the Chlirella extract.

The requirement for ATP and Mg2+ is nottunexpected since most suilfate-uitilizing sys)tems relyon 1 or 2 activation steps to form successively,adleno,sine-5'-phosphosul fate (APS) aind adenoisilne-3'-pho!sphate-5'-wphosphosulfate (PAPS) (2, 9, 10).The formation of PAPS from suilfate by thliis speciesof Chilorella has already been mentionied (11, 17).TPNH has also been implicated in sulfate-redutciing,systemis from yeast (6, 18).

Extracts prepared in this way are quiite labileand rapidly lose activity. Activity is lost almostco,mpletely on dialysis for 4 houirs as shown-i initable II, Where dialyzed and uindialyzed samples oftfhe same extract are compared.

The uindialyzed extracts show a brief lag fol-lowed by essentially liinear kinetics from aboutt 1hour to 12 houirs but beyond this time the activityprogressively dimini,shes (fig 1). \Vhile MES-1HC1is a solmewhat inferior buiffer to tris-HCI as jutdgedfrom the overlap r-gion in figure 2, 9 pH optimaare (listinguishable, 1 in the regionl of pH 7.0, theother at pH 8.5 indicating that s,everal enzymes areprobably involved in the prolduction of acid-volatileradioactivity whose interactions provide the (loubleop)tima.

It was sulbsequently !found that the inclusioi oflow concentrations (1.5 mM) of BAL in the buifferused to prepare the extracts (method B) increasedtheir activity and protected' thtnnmfrom deterioration.Table III shows that the undia,lyzed extracts are

4 5 6 7 8 9 10p H

FIG. 2. pH optima for formlation of acid-volatileradioactix itv fronm 35SO42- by ani undialyzed Cli/oreliaextract lackin- BAL. Conditions are the same as intable II, except that the pH of the enzynme extracts wasadjusted witlh either MES or tris buffer as describedin M\Jaterials and AMethods an(l additional buiffer wasnot added to the incubatioln mixtuires.

Table I ll. Ncdction ofSo latit, ait pit 7.0: Cofactor Requiremlenlts and Stabilitv to Dialysis in t12PIresenicc of BJIA

Tlu compllete system conit line(l: carrier-free 35SO42- (5 X 106 cpiin), TP\ (1.56 1amoles). g-lutcose-6-P (10,umoles), glucose-6 P (ellvdro-eiiase (0.05 unit), ATP (10 kmiole-), creatine-P ('10 Aiiioles), creatiine phosphokinase(10 u), AMg-Cl, (50 uLmoles), thioctilc acid (0.49 lkmole), C/i/ore//al extract (method B) (1.0 ml) and 0.5 -m tris-HCi.pH 7.0 (0.1 ml) in a total volume of 2.0 ml.

Complete system,. . -TPN

-TPN, glucose-6-P andlg,lucose-6-P dehydrogen,ase

-TPN, glucose-6-P and-lucose-6-P dehydrogenase:+TPNH (1.56 Amoles)

-ATP-ATP, creatine-P and

creatine phosphokinase-MgCl,-thioctic acidEnzyme heated at 700 for 3 min

Acid-volatileEnzymle

undialyzecl

CPM/2 mil102,30058.060

2440

129,9604480

10601800

67,4.800

radioactivity ofEnzymedialyze,d

CPM4'/2 inl175,90067,720

28,660

71,060880

1602000

99.5800

550

SCHIFF AND LEVINTHAL-SULFATE REDUCING EXTRACT FROM CHLORELLA 551

Table IV. Reducing .4genits Active i7t Systeml (it pH 7.0The complete system conltained: ATP (10 jkmoles), MgCl, (50 ,moles), thioctic acid (0.49 ,nmole), Chlorella

extract (method B) (1.0 ml), carrier-free 35SO42- (4 X 106 cpm), creatine-P (10 umoles), creatinle phosphokinase(10lg) and 0.5 M tris-HCl, pH 7.0 (0.1 ml) in a total volume of 2.0 ml.

Acid-volatile radioactivity

Complete system,. . +TPN (1.56 jkmoles), glucose-6-P

(10 Am'oles) and glucose-6-Pdehydrogenase (0.05 unit)+TPNH (1.56 Amoles)+DPNH (1.56 pAmoles)+glucose-6-P (10 gtmoles) glucose-6-P dehydrogenase (0.05 unit)and BAL (3 gmoles)+BAL (3 pumoles)+BAL (3 umoles), -ATP+BAL (3 pmoles), -MgCl,+BAL (3 umoles), -thioctic acid+BAL (3,moles), -ATP, creatine-Pand creatine phosphokinase+BAL (3 tmoles), enzyme heatedat 700 for 3 min

CPM/2 ml12,000

39,02031,02021,980

49,18071,600

1801780

78,760

0

0

1000r

0800-N

01-x

0

,r 400-:

Lu

-J

'I-J

0

,200-

<

/l I

I'

I II

0

MIEI

II

I1

Io

/ I/~~~~~~~~~

b'

0 o

5 6 7 8 9 10opH

FIG. 3. pH optima for formation of acid-volatileradioactivity from 35S042- by a dialyzed Chlorella ex-tract containing 1.5 mM BAL. Each incubation mixturecontained: ATP (10 *moles), MgCl, (50 jumoles), thi-octic acid (0.49 /mole), carrier-free 35SO42- (4 X 106cpm), creatine-P (10 ,umoles), creatine phosphokinase(10 sg), BAL (3 ,*moles), and Chlorella extract broughtto proper pH as described in Materials and Methods(1.0 ml), all in a total volume of 2.0 ml.

quite active and clearly requ-ire TPNH as a re-ducitant. ATP is still reqtuired as is Mg2+. In thisparticular experiment, thioctic acid was somewhatstimulatory. The system retained its lability toheat. Under BAL protection, the extracts couldl bedialyzed for 4 hotirs wiithout lo,ss of activity, indeedan increase in activity was frequently dbservedas thotigh some inhibitory material was being re-moved. The general pattern of requirements re-mained essentially the same as dicd the heat labilityof the extracts. The proteotive effect of BALmight be aittribufted to its maintenance of sulffhydrylgrouips of the enzyme extract in the reduiced formor to its vell-known properties as a chelator ofheavy metals. Perhaps it acts in both capacities.

To ouir suirprise, when the concentration of BAIwas increased by adlding it to the incubation mix-tuires already constituted with enzyme extracts con-taining BAL, thi,s compound could replace TPNHas a redluctant. Table IV demonstrates that BALis even more effective when the TPN-redutcingsystem is omitted. DPNH is somewhat less activethan TPNH as a reductant but when BAL replacesthese compounds, the system still requires ATPand Mg2+ and retains its lability to heat. Thiocticaci(l is somewhat inhibitory in this experimenit.The participation of BAL might reflect a thio-redoxin-4ike mechanism of electron transfer fromTPNH as in desoxy-riboside biosynthesis (7).

Figure 3 shows the pH cuirve 'for activity ofdialyzed extracts prepared in BAL-containing btuf-fers where additional BAL is uise,d as the redtuctant,rather than pyridine inucleotides. Unider these con-ditions, MES-HCl and tris-HGl appear to be eqllallygoodl butffers. There is the sulggestion of a smallshoul-der in the region of pH 7 to 8, but the majoractivity is found at pH 9.0. These extracts em-

PLANT PHYSIOLOGN-

T-iblI V. _1[iiii'mal Rcquiremic elits For S. it/fate Reduction (It pI-I 9.0The complete system Ccontinue(l in a total -oltumiie of 2.35 nil ATP (10 /Amol s), I-Cl, (50 gtmoles) BAL

(3.0 umclleis), 1.0 mI tris-HCl (pH 9.0) (0.1 ml), carrier-free 35SO2- (3.8 X 106 cpm), and Cliloiell(a extract, eithermietlhod C ("7-9") or method D ("9-9") as indicated (1.0 m!).

\cid-volatile radioactivity of"9-9" cxtract "7-9" extract

CPif11/2.35 mlliComplete s stclml

. ATlI. A- i -CI.,

I I,-BAL (3.0 ymlnlei)". ' lEliime hecated 3 mini at 70

465.088423634

49350

1.053.9750

376510995

235

ploying BAL at pH 9.0 were so muichl more aictivNeand stable than any obtailled previously, that t1hewere a,do,pte!d for fturther study.

Extracts Prep(ared (t pH g.o. Two types ofextracts were employed in these stutdiei and thedetails of their preparationi are given in metllodsC anid D of Materials and MIethods. Both containBAL at a concentration o,f 1.5 m-i btt "7-9" ex-

tracts are prepar,ed by h)reaking the cell's at pH 7.0and (lialyzing them against pH 9.0 butffer, whille"9-9" extracts are prepared by us1ing pH 9.0 buifferfor 'both steps. The 7-9 extracts are tisuialily more

:-I

E

to>

0

I)

'a

D

0

0r

r

0

C)

a'

C)-v

N)

LO

3

0

i

20 40 60 80 100ATP (Mu MOLES /2 35 ml)

FIG. 4. Optimnim concentration of ATP for theformation of acid-volatile radioactivity from 35SO42-by 2 types of Chlor ella extract at pH 9. Conditionsthe same as in table V, except that ATP concentrationwas varied.

0.

ix

T

0

'I

-J

I1-

0

0

J

>

9

30 50 70 90 100Mg C12 (~L MOLES/2 35 ml)

C)

0a-

-I

m

ao

0G'

C-)

x

3'D

9W

FIG. 5. Optimum concentrations of \IgCl9 for theformationi of acid-volatile radi-oactivity from 35SO42-by 2 types of Clilorclla extract at pH 9. Conditions thesame as in tahle except that MTgCI., collcentrationwas varied.

active, perhaps hecatise pH 9 buffeer extracts someinhibitory materials from the cells; these latterextracts generally contain mtuch more pigment than7-9 extracts. As is evi.dent, however, the 2 typesof extract seem to h)e equivalent in their general

properties, and are used interchangeably by tus.Taible V shows the minimal requtirements for

formation of acid-volatile radioactivity by theseextracts. ATP, MIg2+ and adidtiona-l BAL are allessential an!d the system remains labile to heat.Figure 4 shows tha't 'both types of extracts yieldoptimal acid-volatile ra%dioactivity at abo-ut 10 ytmolesof ATP per 2.35 ml of reaction mixttire, high

552

SCHIFF AND LEVINTHAL-SULFATE REDIUCING EXTRACT FROMI CHLORELLA

concentrations of ATI'P are markedly inhibitory.Mg'2 (fig 5) displays a broad optimum in botlhtypes of extract at 50 jumoles per 2.35 ml. Add(i-tional BAL, as a reducitan(t, is active and relativelynon-inhibitory over a wide range of concentrations(fig 6), with the b)est activity between approxi-

* '9-9' EXTRACTSE x "7-9" EXTRACTS

300- 0O0

Q. mlx ~~~~~~~~~~900">

T ~~~~~~~~~~~~0~~~~~xI 0

,/ x-X'

> 150- /X, 600~

< I0

-300

I~x--xl

0 10 10 100 200

R~~~~~AL(M~MOLES/2 35ml)

FIG. 6. Optimum concentrationis of BAL for theformation of acid-volatile radioactivity from 35SO42- by2 types of Chlorclla extract at pH 9. Conditions thesame as in table V, except that BAL concentrationwas varied.

* 99-9" EXTRACTSx "7-9" EXTRACTS

E 1,500 X

IC)

x0

Q- ~~~0/~0/X */

_ 1,000

0C)

500w

0

0 12 24 48TIME (HRS.)

FIG. 7. Kinetics of formation of acid-volatile radio-activity from 35SO42- for 2 types of Chlorella extractat pH 9. Conditions the same as in table V, but identicalsamples in separate tubes were allowed to incubate fordifferent times before being assayed.

mately 3 and 10 to 12 jLmoles per 2.35 ml. Theregion of rise in the BAL concentration cuirve issomewhat different from exper.ment to experimenitand proba,bly reflects the basal amount of BALremaInIng in the initial enzyme extract, which isvariable, since BAL is continuiaflly removed throughair oxidation (luring the prel)aration of the enzymeextracits. Figlure 7 shows that the kinetics offormation of aci,d-volatile radioactivity is essentiallylinear from time zero uintil abou,t 8 houirs in bothtypes of extract. For this reason, 2 hotiri o,f inlcuI-bation was adopted as a convenilent time of assayin most exl)eriments. The acti:.ty c,f the systemw%-as also proportionial to the amoulnit of enzymeadded, over a considerable range, wiheni concentratedenzyme extracts were employed.

Table VI shows that ATP is the preferrednucleoside triphosphate in this systemn, as in othersvstems involving sulfate activat on (2).

Table VI. Nu cleotide Specificity for- the Pr-odu ctiont ofAcid-Volatile Radioactivity

The complete system contained: BAL (3.0 pnmoles),MgCl, (50 /Lm-oles), carrier-free 35SO42- (1 X 106cpm), 10 umoles of the indicated nucleotide, 1 ml ofClilor-ello extract (metho D) and ciuou-oh 0.1 Mi tris-HCl (pH 9) to bring total volullme of incubation milix-ture to 2.0 ml.

Nucleotide added tocomplete system

No additionATPDesoxyATPGTPTTPUTPCTP

Acid-volatile radioactivity

CP/1112.0 nl580

383.0203440

13.02010,92028.3003560

Acknowledgment

The expert technical assistaiice ofLemieux is gratefully acknowledged.

M\iss Jeaininette

Literature Cited

1. ASAHI, T., R. BANDURSKI, AND L. WILSON. 1961.Yeast sulfate reducing system. II. Enzymaticreduction of protein disulfide. J. Biol. Chem.236: 1g30-35.

2. BANDURSKI, R., L. \NXILSON, AND C. SQUIRES. 1956.The mechanism of "active sulfate" formation.J. Am. Chem. Soc. 78: 6408-09.

3. CANTONI, G. 1960. Sulfoniium compounds. In:Comparative Biochemistry. Florkin and Mason,eds. Academic Press, New York. 1: 206.

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