THE JOURNAL OF BIOLOGICAL CHEMISTRY

Printed in U.S.A. Vol. 256, No. 16, Issue of August 25, pp. 8306-8309. 1981

Isolation and Characterization of Thioredoxin from the Cyanobacterium, Anabaena sp. *

(Received for publication, February 20, 1981, and in revised form, April 14, 1981)

Florence K. Gleason$ and Arne Holmgren From the Department of Chemistry, Karolinska Znstitutet, S-104 01 Stockholm 60, Sweden

Thioredoxin from Anabaena sp. has been purified 800-fold with an assay based on the reduction of insulin disulfides by NADPH and the heterologous calf thymus thioredoxin reductase. The final material was homo- geneous on polyacrylamide gel electrophoresis and had a molecular weight of 12,000; the NH2-terminal residue was serine and the COOH-terminal was leucine. Ana- baena thioredoxin-(SH)z is a hydrogen donor for the adenosylcobalamin-dependent Anabaena ribonucleo- tide reductase and is equally active with the iron-con- taining ribonucleotide reductase from Escherichia coli. Anabaena thioredoxin-Sz is a good substrate for E. coli thioredoxin reductase. We have compared the struc- ture of Anabaena and E. coli thioredoxins. Clear struc- tural differences between the proteins, compatible with the large evolutionary distance between the organisms, were seen with respect to total amino acid composition, isoelectric point, tryptic peptide maps, and a low im- munochemical cross-reactivity. However, both thiore- doxins contain a single oxidation-reduction active di- sulfide bridge with the amino acid sequence: Cy;-Gly- Pro-Cys-Lys. The tryptophan fluorescence emission of Anabaena thioredoxin-Sz increases more than %fold on reduction to thioredoxin-(SH)z. This behavior is iden- tical with that of E. coli thioredoxin, suggesting a very similar overall folding of homologous molecules.

Thioredoxin is a small (Mr s 12,000) protein, containing an active center cystine disulfide/dithiol in its oxidized and re- duced form, respectively. Initially, thioredoxin and the specific NADPH-dependent enzyme thioredoxin reductase were pu- rified from E. coli as a hydrogen donor system for ribonucleo- tide reductase and thus believed to be required chiefly for DNA synthesis (1). Thioredoxin has since been studied in a variety of organisms and shown to function in other thiol- dependent oxidation-reduction reactions (for review, see Ref. 2).

Although thioredoxin presumably occurs in all living orga- nisms, it has been purified to homogeneity and characterized in only a few. Existing data on thioredoxins from E. coli, yeast, and mammalian sources indicate considerable homol- ogy (2). A comparative study of thioredoxin from various species may help in understanding its molecular mechanism

* This work was supported by Swedish Medical Research Council Grant 13X-3529 and 13P-4292, Swedish Cancer Society Grant 961, and United States Public Health Service Grant 2R01-AM-18101 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact. * Present address, Gray Freshwater Biological Institute, P.O. Box 100, Navarre, MN 55392.

and also offer additional data on proteins and evolution com- parable to that provided by cytochrome c (3).

Other than the protein from E. coli, no bacterial thioredoxin has been isolated and described as to structure and reactivity. In this paper we report the purification and some of the properties of thioredoxin from the filamentous cyanobacte- rium Anabaena 7119. The ribonucleotide reductase from this organism has recently been purified and shown to be an adenosylcobalamin-dependent reductase (4) comparable to that found in Lactobacillus leichmannii and other prokar- yotes (5). This is in marked contrast to the iron-containing reductase found in E. coli and mammals (6). The data here reported show that the cyanobacterial thioredoxin is homol- ogous to the protein from E. coli despite the large evolutionary distance between these organisms.

EXPERIMENTAL PROCEDURES AND RESULTS’

DISCUSSION

This paper describes the isolation of a homogeneous thio- redoxin from Anabaena sp. and some of its characteristics. Thioredoxins have also been studied from E. coli (l), Lacto- bacillus leichmannii (26), the green alga Scenedesmus obli- quus (27), yeast (24), and mammalian liver (25, 28). All these thioredoxins have certain properties in common. They are heat-stable proteins with molecular weights of approximately 12,000. In the reduced form they serve as hydrogen donors for E, coli as well as their homologous ribonucleotide reductases (6). This indicates identical thiol oxidation-reduction mecha- nisms and thus the same active center structure. On the other hand, the thioredoxins differ in their reactivity with thiore- doxin reductases or antibodies, consistent with marked pri- mary structural differences. A summary of these points is given in Table 111.

Only thioredoxin from E. coli has been extensively charac- terized. Results from an x-ray crystallographic investigation to 2.8-A resolution demonstrate that this molecule represents a novel type of enzyme structure (29); the active center disulfide is located at the COOH-terminal end of a P-pleated sheet protruding out into the solution. Furthermore, the mol- ecule consists of two prominent folding domains of secondary

’ Portions of this paper (including “Experimental Procedures” and “Results,” Figs. 1 to 7, and Tables I to 111) are presented in miniprint at the end of this paper. The abbreviations used are: DTNB, 5,5” dithiobis-(2-nitrobenzoic acid); dansyl, 5-dimethylaminonaphthalene- 1-sulfonyl; Cys(Cm), carboxymethylcysteine. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 RockviUe Pike, Bethesda, MD 20014. Request Document No. 81M-387, cite author(s), and include a check for $5.60 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

8306

Thioredoxin from Anabaena 8307

structure connected by a short hinge region. The folding of Anabaena thioredoxin-S2 must be very similar to that of E. coli thioredoxin-S2, since both share reactivity properties and are substrates for the otherwise highly specific thioredoxin reductase of E. coli (2). The structure of Anabaena thiore- doxin may be represented as shown in Fig. 7. The amino acid sequence of the active center pentapeptide cleaved by trypsin and chymotrypsin digestion is identical in E. coli, Anabaena, and also in yeast thioredoxins (30). The identical tryptophan fluorescence spectra of Anabaena and E. coli thioredoxin-SZ and thioredoxin-(SH)z are a strong argument for placing the tryptophans of Anabaena in the same relative position as in E. coli (Trp-28 and Trp-31) (20). The low quantum yield of tryptophan fluorescence in thioredoxin-S2 is attributed to quenching by the disulfide (21). The large increase in fluores- cence on reduction is caused by Trp-28, which moves as the result of a localized conformational change (31).

Despite the identities in overall folding and active center residues, including conservation of thioredoxin reductase binding site(s), the peptide maps and immunological data show that Anabaena and E. coli thioredoxins have quite different primary structures. This is consistent with evolution of homologous proteins. From an evolutionary point of view, the cyanobacteria are considered to be direct descendants of an ancient group of microorganisms (32). The occurrence of an adenosylcobalamin-dependent ribonucleotide reductase in the majority of common cyanobacteria further conf i i s their ancient lineage (33). The fact that there exists a high degree of homology between the thioredoxins in a primitive photo- synthetic bacterium such as Anabaena and the geneologically distant E. coli (34) indicates that natural selection has con- served the thioredoxins throughout evolution, although the ribonucleotide reductases have diverged widely.

The role of thioredoxin in deoxyribonucleotide synthesis is presently unclear since the discovery of the glutaredoxin system in E. coli (35) and the characterization of thioredoxin- negative mutants (36). However, thioredoxin is known to have other functions. Thioredoxin has been shown to act as a cofactor for the 3”phosphoadenosine 5’-phosphosulfate sul- fotransferase system of yeast (37) and cyanobacteria (38). As a regulatory factor for photosynthetic enzymes, three thiore- doxin fractions have been purified from spinach (39). Two of these, from chloroplasts, function in specific regulation of enzymes of carbon dioxide fiation, fructose bisphosphatase and NADP-malate dehydrogenase. Both enzymes show in- creased activity after reduction by the chloroplast thioredoxin (39). Their ability to serve as hydrogen donor for E. coli ribonucleotide reductase, however, is minimal (40). Since the cyanobacteria are believed to be the evolutionary prototypes for chloroplasts (41, 42), the function of the Anabaena thio- redoxin system described here in regulation of other aspects of cyanobacterial metabolism merits further investigation.

Acknowledgments-F. K. G. would like to thank John M. Wood for his support and encouragement of her research. We are grateful to Carina Palmberg for running the amino acid analysis.

1.

2.

3.

4.

5.

6.

7.

8.

9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

23. 24.

25.

26.

27.

28.

29.

30.

31. 32. 33. 34.

35.

36.

37.

38.

39.

40.

41. 42.

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8308 Thioredoxin from Anabaena SUPPLWENTAL MATERIAL TO

ISOWITION AND CHARACTERIZATION OF THIOREMXIN FROM THE CYANOBACTERIUW, ANNIBIENA SP..

Florence X. G1eai.0~ and Arne Holmqrsn

EXPERIIIWTAL PRCCEDURES

Wterials. Dithlothceitol, 5 , 5 ~ - d 1 t h 1 o b 1 ~ l l - ~ 1 t ~ ~ b e ~ ~ ~ 1 ~ acld), NADPH, M r . a enosy cobalamln and unlabeled nuclsotldes were purchased f om the S l p a Chem- !:a1 Co!, USA. Bovlne Insulln was from VitrYm, Sweden. ,5-5Hl-Labeled CDP and

6US-Scphaross -re obtslned from Pha-cia, Sweden. Carboxymethyl cellulose 14C:-iodoaCetiC acid were obtained from -=sham, England. Sephadex G-50 and

In Ref. 7. CarboxypeptldaBe A, trypsln and chymotrypeln were from worthlngton. ICW 52) was from Whatman, Ltd., England. Amlno acld sequence reagents were as

E. coli thloredoxin was prepared as desorlbed (71. E. coli clbonucleotide re- a i c G and Anabaena rlbonucleotlde reductase were p6pared according to pub- Ilshed p r o c e d u r e s , 8 ) . The puriflcatlon of E. coli thioredoxln reductase 191 and calf thymus thloredoxin reductam (10) Fii'vXso been previously de- scribed.

Growth of the Cyanobacterlum. Anabaena .pi 7119 ( A W C 29151) was a glft of A. Nellson llnatltut far Vatten- och Luftv rdsforsknlng, StOCkholm). Culture. were routlnely maintalned on llquld medlw, CglO (11) at ro(w temperature and constant Illumlnatlon. Large quantities of cella were growl on the s- med- lum as previously described ( 4 ) .

Enzyme assays. Thiozedoxin aictlvlty was routinely determined by monitoring of oxldstlon Of NADPH In the presence of Insulin and 24 ug/ml, (28 A412 unlts/ml 110)). of thioredoxln reductase from calf thymus (10)ais describad In equatlonl:

1. Thloredoxln-S2 f NADPH thlorsdoxin- ISH) + NADP'

2. Thioredoxln-lSH)2 f lnsulln-s2 d thioradoxin-S2 + Insulin-lSH)2

Purlfled Anabaena thioredoxln serves as hydrogen donor for E. coli rlbonucleo-

m. Actlvlty Was determined by measuring the COnVBrSlOn of k-labeled tlde red"-d the h-logous adenollyl-Eobalamln-d~pe"d~t~ ctase from

CDP or CTP to the corresponding deoxyrlbonucleotlds. The assay procedure for

Anabsena enzyme, In 4. Separatlon of nucleotides warn by thin-layer chromato- the E- rlbonucleotlde reductase Is descrlbed In reference 12 and for the

graphy on polyethyleneimine plates 113).

The ablllty of thloradoxln to serve as BubStCatC for thloredoxin reductase .)as determined in the presence of NADPH and 5,5'-dithiobie 2-nItr0benzol~ acld l m ) ' . The reduction of m waa detsrnlnad by monltor- ing the change In absorbance at 412 nm 1 1 0 .

Pmteln detenlnatlon. Proteln concentration In crude extracta and partially purlfled fractions was estimated from the ratio of absorbance at 280 and 260

determined wlth a molar absorbtlvlty Of 13,700 n - 1 cm-0 mlnii7jib-nm. nm 115). The concentration of pure thloredoxln from Anabaena and E. coli was

The value for thloredoxln was also determined by amlno acld analysl..

thloredoxln

Gel eleCtr0DhoCI)sIs. NOn-denaturing slab gals I10 X 15 Cm) I161 containing 158 acrylamlde were prepared and run at 259' In 0.1 W Trls-glycine, pH 8.8. The proteln bands were stalned wlth c m . 1 ~ brilliant blue In methanol-acetic acid - H9O 145-10-45 by volume) and destained by washing wlth the s- solvent. E.

Amino acld malysls. Salt-free allquota of lyophlllred Anabaena thioredoxin (1-3 -11 were hydrolyzed with 0 . 5 ml of 6 II HCl contai-8 phenol at 1lOOC for 24 h In vacuo. The amlno acld composition was detemlned wlth a

was determlned after performic acld oxidation 118). Beckman W e 1 IT-ii-iXamlno acld analyzer. The cysteine and methionlne content

NH2-tsnlnsl anal SI.. The NH -terminal a d n o acid of Anabema thioradorin was Stermlned by theYdansyll t e c h q u e 1191 DD prevlouslyds.crlbsd 117).

COOH-terminal analy.1.. The MOH-terminal amlno acid of carboxymethylated Anabaena thloredoxln was detemlned by carboxypeptidase A digestion 120) 11: -eight of enzyme for 50 mln at 37OC) followed by amino acld analy.1..

Anabaena or E. coll 1100 -1) were dlsnolved In 200 u1 of 6 W guanidlne-HCl/ Reduction and carboXYMthylatlon. Lyophilized samples of thloredoxln frcx

thiothreltol. After InCUbdtIDn W d e l N2 for 1 h at 37O, 40 Ul Of 50 M i i % i m I s - C C m . O / l mn EDTA and reduced by addltion of 8 ul of 100 mn I

methylated thloredoxin was Isolated by chr-tography on a c o l m of Ssphadex lodoacetlc acid 12500 cw/nmell yere added. After 30 mi" at 37% the sarboxy-

G-25 I 1 x 10 m) In 0 . 5 1 NH4HC03 and lyophlllred.

ProteolYtIc dlqestlon and Peptide ~ D S . Sample. Of carboxymethylated thio='.- doxln I2 or 4 "moll were digested wlth trypsln or trypsln plus chptrypsln as descrlbed 120). After lyophlllrat10n the material Wan taken to tro-dlmen.lonal peptlde maps on thin-layer plates 117). Peptldes were vlsuallred wlth nln- hydrln stalnlng or by autoradiography for 5 days 117).

Fluorsmcsncs swctrosCOW. Fluors.ssnse ulsmion spectra were determ1n.d with

described (211. The emlssion was recorded from 290-440 M. Thloredoxln-S a modlfled zelsa 2 m 4 spectrofluorometer by excitation at 280 nm as previously

was dissolved In 250 yl of 100 W pOtassIYm phosphate, pH 7.0/1 M EDTA at2 50 ugfml. Reductlon was achleved y additlon of 1 mM dlthlothreltol.

RESULTS

Purlflcatlon of Anabaena thlorsdoxin. Approxlmstely 60 g luet weight) of frozen

Emi\vere allowed to thaw at room tmpersture. The frozen cells lysed on thaw- Anaaaena =e118 prevlOUsly suspenaea In One volume of 50 Trl.. pH 7.5/1 Iw

centrlfugad at 10.000 x g for 10 .In In a SOorvsll RC-28 centrlfugs. The pellet, Inq. The very VISCOUS lysate was sonicated for 6 mi" In a Branson Sonlflcr and

Contalning unbroken cells, heterocysts and debrls, was discarded. The super- natant lfractlon 1) was heated to 7OoC In a bolllng water bath and cooled and centrifused at 10.000 x a for 15 .In. The blue sucernstant (fraction 21 was saved and the green pellit dlscacded. Solld INH );SO4 was added to fraitlon 2

at 10.000 x g for 15 mln. The blue pellet (frastlon 31, was resuspended in to 908 (iaturatlon. The mixture was stirred at 2toC for 30 mIn and eentrlfugsd

buffer A: 10 M Trls-C1, pH 7.5/2 a EDTAfO.1 W4 dlthiothreltol and dlalyred aga ins t buffer A a t + 4 W .

After dlalysis, frsctlon 3 was adsorbed to a column of D m - Y p h a r O s e 13.5 x 16 cm) equilibrated Wlth buffer A. The column was washed wlth approximately 250 ml of buffer A and eluted wlth a gradlent Of NaC1, 0 to 0.2 (I In buffer A.

ed In an Micon ultcaflltratlon cell using a UW-2 membrane. The concentrated Fractions with thloredoxln actlvlty (at 0.1 W NaC1) were pooled and concentrat-

msterlal (fraction 41 was dlalyzsd against 50 a Trls-C1 buffer, pH 7.5. Fraction 4 was appllsd to a Ssphadex c-50 column 12.2 x 140 cml equllibratsd with 50 TrIs-C1, pH 7.5. Thioredoxin fTDCtiOnS at K A ~ I 0 . 5 I141 were pool- ed and concentrated 68 described above. The concentrated protsln Ifractlon 5) was dlslyred sgalnst 20 mn sodium acetate. pH 5 . 5 .

Fraction 5 was applied to a column of CII-cell~l~se 12.2 x 4 m) equilibrated wlth 20 mn sodlum acetate, pH 5 . 5 . Protaln was eluted wlth a gradient of sod-

pooled and concentrated IfmFtiOn 6). Fractlon 6 was dialyzed against 60 mn Ium acetate, from 20 to 50 mn. The thloredoxln fractions at -30 mn salt uere

NH4HC03 and stored at -2OoC.

TABLE I

PURIFICATION OF w1\BIEHI\ THIOREWXIN FROM 60 C OF CELLS

Fraction Total Total activity; Speclflc Droteln ~mq) AA... x mln actlvltYb

2. Heat treated 1. Crude extract 16,500 380 0.023

316 0.078 143 76 3.2 42 6.2 30 17.9

4.050 3 . AlmaDlum SYlfste precipitate 1,190 4. DEAF. pool 5. G-50 pool 6 . 8 6. CW-cellulose -1 1.7 a The Teactlon mixture contained 100 W WtaLTSIum phosphate buffer, pH 7.0.

1 mU EDTA, 480 ull NADPH and 1.6 mU InSulin. Reactlon mlxture I500 ul) and thloredoxln fracrlon 11-25 "1) were added to two Cuvettes a t 25OC. The reactlon was lnltlatsd by addition of 12 vg calf thymus thloredoxin reduc- tase to one Cuvette and the Oxldotlon of NADPH was monitored at 340 nm In a Zelss PWQ I11 spectrophotometer wlth an automatlc ampliflcarlon unit and a Servogor recorder.

AA340/mln/mq protein.

24 0.12

PurltY of thioredoxln. llaterial from frsctlon 6 was homogenous on native poly- aery am e ge e ectrophocenls (Pig. 1) at pH 8.8. Anabaena thlorsdorin-s has 1% db1:Ity than E coli thloredoxln-S or glutaredoxln (17) demo$- strating a higher l a o e l e c t ~ c ~ n t lover pH 4.31 (721.

NH and COOH-terminal residues. Danayla- -6. s Owe On y ser ne as NHtermIn.1 for An:bae:a tilored&in. Whln 1.8 nmol

was digested wlth carbxypeptldsse A, carb-mWEKYlated thioredoxln

only 1.7 n m l Of leucine was released demonstrating that this residue vas COOH- temlnal. These results confirm that the fractlon 6 material Is homogenous.

En*- activlt . The assay Of Anabaena thloredoxln du:lng pUrlflcatlOnutll(red

NADPH-thioredoxin reductase. Anabaena Its Cross-reactlvlty wlth calf thymus

thIorsdoxln-S2 gave about 208 b t t h e a c -

tivity of calf thymus thloredoxin-s as a substrate for thls marmallan enry& In the coupled reduction of Innulln. Its

E. coll thloredoxln-S 110). relatlve actlvlty was s1mlla.r to that of

thloredoxln catalyredlthe reduction of Insulin dlsulfldes wlth dlthlothreltol

"

Anabaena thloredoxln will serve as an electron donor In a reactlor, wlth

parent Of 2.0 YW was calculated from a Llneweaver-Burke plot. Anabaena

rase. thloredbin also functions as electron donor for E. soli ribonuclsorideduc-

thloredoxln 117).

rlbonuclsotlde reductase and dlthlothreltol as lletln In Flg. 2. An ap-

IS 1.2 A ese values are the s- as those prevlously publlshsd for E- for the heterologous reaftlon In t h h p G n c e Of dlthlothreltol

1 I I I I I I 2 4 6 8 10 12 14

pg THlOREDOXlNlRX

Plg. 2. Rlbonucleoside triphosphate reductase reaction In the presence of varylng m u n t s of Anabaena thloredoxln A K of 2 un was calculated from

presence of 0.1 m o t h r e l t o l . the reactlon of A n s b a e m e d o x l n wlth ;he hoblogous reductase In the

Anabaena thloredoxin-S Is also a eubatrate for E. coli NADPH-thioredoxln reduct..e as dstenlned'by monitoring the reduct& G?-EL?IwB. From the data shorn In Flv. 3, 0 358 cross-reactlvlty between the protelns was calculated.

A s-ry of the purlficatlon procedure for Anabaena thloredoxln Is s h o m In Table 1. SIXty grams wet uelght Of algal c e m d e d about 1.5 mq of pure thloredoxin wlth an apparent recovery of 88.

Thioredorin from Anabaena 8309

~ n g DTN8 as electron acceptor. TWO cuvettei-wsprepared containing 100 mM Flg. 3 . Reactmn of thloredoxin with E . coli thiocedoxin reductase UP-

TILS-HC1, pH 8.0; 4 rW E m A : 50 Yg bovine Berm albumin: 0.4 W DTNB ldis- solved in 95% ethanol); and 0.5 mM NADPH in a final volume Of 0.5 ml. Vary-

blank. The reaction was started by adding purified E- coli thioredoxin re- ing amounts Of thloredoxin were added to one cuvette; the other sewed as a

ductase (10 u9/mll to both Cuvettes and the increase i n z o r b a n c e at 412

doxin. nm was monitored. 0 - 0 - E- thioredoxin, A -A thiore-

Amino acld COm osition and s ctrm. The amino acid compasltion Of Anabaena th~oredoxin iSPshOVn in TablP2. The compDsition of E- e I201 ye= or yeast rhioredaxm. the Anabaena protein contains only one cystine S-s- and calf l ~ v e r (251 thioredoxms are included for campparinon. Like E-

bridge. The W-visible s p e m f Anabaena thioredoxln at pH 7.0 showed an

determined by A280 M absorbance.

TABLE I1

Mll t lO ACID COmSITION OF THIOREWXIN

E- & I201 yeast I1 I241 calf liver 125)

10 10 10-11 12 1 1 0 1 1 1 0 1-2

15 10 9 6 6 4 3 6 3 9 6 13 8 13 17 7 5 4 3

10

6 8 2 13

3 5 6 2 3

9 12 2 5 1 9

13 2 4

6 16 2

1 1 2 5 6 4 5

5 8 4

1 1 2 4 6 2 9

106 108 109-111 103

Tryptic peptlde maps. Tryptic peptide raps Of ["~]carbaxy.ethylated

tern compatible with the marked differences in total cOmpasitiOn. However, the autaradioqraphy showed that the active Center twotic neptide from both

and E- & thioredoxin IFlq. 41 showed large differences in pat-

t 0 t - 0

0

"c

0

b ," 5 -

Anabaena and E. coli thloredoxln. The hatched peptlde :is radioactive. 4 Flg. 4 . a1 Peptzde maps Of tryptLC dlgest Of '14 carborymethylared

n o r m a t e i i i l u a s used. bl Identlfrcatlon of cys ICm) -Gly-Pro-cys (Cm) - l y s I" the actlve Center Of both Anabaena and E. coll thloredoxin. Trypsin and Chymotrypsln dlgestlon was us- d e t a x s r t h e thinlayer separations

peptlde (hatched1 on each map. see Experxnental PrOcedUreb. Autoradiography Showed only one radioactive

A C ~ Z V ~ center sequence of Anabaena thzmedoxm. The sequence of the active center YLE aetecmlned indrrectly by m e identity of a yeptxde obtamed by combined digestion with trypsin plus chymotlypsln Of [ 4Cjcarboxymethylated rhloredoxlns (Fig. 11. This gives the peptLde C y S l C m l - G l y - P ~ O - C y s l C ~ ~ - L y s

pondmg peptide LE cleaved from Anabaena thloredoxln a6 shown by autoradro- from E- thioredoxin. The corres- 32 3s

gtaphy Of peptide maps. The pre- a Oethlonine residue in

CNBr cleavage 171. Separation Of the CNBr peptides ln 30% acetlc acid gave throredoxin, equivalent to Met37 in the E- & sequence, was tested by

the sane pattern as for E- a thioredoxin I71 (data not shown). The NH - terminal peptlde fragment of Anabaena ehloredoxin lequlvalent to rhmredozln -c-11-3711 1141 contamed b o t m p h a n s and the half-cystlnel.

Fluorescence spectra. The fluorescence spectra of Oxldlzed and reduced Anabaena thioredoxin are shown In Fig. 5. The Spectrum of oxldlted thlore- -clearly shows two m a x i m . one at 110 m and the other at 310 nm, cor- responding to tyrosine and tryptophan ernirsion, respectively. The trypto- phan fluorescence increases approxmately I-fold on rednctlon of thmre- doxin-S2 with dithiothreitol. The spectra Of the Anabaena protein are similar t o those previously reported far E- e thioredorin 1211.

Wavdapth (nm)

Fig. 5. Fluores~ence emission spectrm of Anabaena thlocedoxln. Thio- redoxin 150 uglmll was suspended in 100 MI p o t a s s i u n r p h a t e buffer, pH 7.0, in a total volume Of 250 ill. Thioredoxin was reduced by adding 1 MI dithiothreitol to the cuvette. 0 - 0 oxidized thloredoxin, A - A re- duced thioredoxin.

1munol ical cross-reactivit . Thioredoxin-S from Anabaena exhibits C ~ O B P IeaCtiVZy with antibody to E! coli thioredoxi?a-S I= as determined from the inhibition of enrymzacfivity with NADPH?;hioredoxin reductase.

homology eXiOt8 in the antigenic deteminanf Sitefsl of the two proteins. However, the results show only 101 cr096-reaCtiYity suggesting that a snall

ANTIBODY 1pl1

Fig. 6. Reaction of thioredoxm vIth antibody to E. colr thloredoxin. Antibody from goat serum was prepared as described in t h T W 5 d s . E. coli thioredoxin 120 nmol/mll and Anabaena thioredoxln 121 nmol/mll were cubated with varying amounts V y . Resldual thloredoxin activity was then determined with E. coli thloredoxm reductase ubmg DTNB as electron accePtOZ as deacribed-inthe legend to FIgUce 3 . 0 - E- thlo- redoxin, A - A thioredoxin.

TABLE Ill

2 2 E. coll Anabaena 2 2 Yeast 2 1 Calf 1l"er 4 1 - not determmed

" 1 0 0 % 10% 25%

Wlth trypsin IT), chymotrypsin IC1 a n d l l a rndlcated. The tryptophan Flg. 7. General structure Of Anabaena thloredoxln-S2. The cleavage

residues are placed in positions equvalent to E- & thmredoxin, Trp-28 and Trp-11. Residues ldentlcal ~n E- &, yeast and Rndbaena thioredoxins are underlmed.