Light and dark reduction of nitrite in a reconstituted enzymic system*

THE JOURNAL. OF BIOLOGICAL CHEMISTRY Vol. 239, No. 6, June 1964 P in ed in U.S.A. Ligh and Da k Reduc ion o Ni i e in a Recons i u ed Enzymic Sys em* A. PANEQUE, J. M. RAMIREZ, F. F. DEL CAMPO, AND $1. LOSADA F om he Secci6n de Bioquimica y FisiologCa Celula , Cen o de In es igaciones BioMgicas, C.S.I.C., Mad id, Spain (Recei ed o publica ion, No embe 19, 1963) Kessle (1) has s udied he educ ion o ni i e o ammonia by he g een alga Ankis odesmus bo h in he ligh in an a mosphe e o ni ogen and in he da k unde hyd ogen. No speci ic con- clusion could be d awn as o he mechanism o he p ocess, al hough i was assumed ha educed py idine nucleo ides migh se e as he immedia e hyd ogen dono s o he educ ion o ni i e and ha ene gy- ich phospha e bonds we e speci ically equi ed o he occu ence o he eac ion. The p oduc ion o “assimila o y powe ” by noncyclic pho ophospho yla ion (2) was, he e o e, conside ed a p e equisi e o ni i e educ ion. Nicholas (3), wo king wi h cell- ee enzyme p epa a ions om Neu ospo a, ound also ha phospho yla ions we e in ol ed in he educ ion o ni i e. Roussos and Nason (4) pu i ied om ex ac s o soybean lea es a soluble “py idine nucleo ide-ni i e enzyme” which, in he p esence o an uniden i ied, hea -s able, o ganic ac o ob- ained om he same ex ac s, equi ed ni i e in o de o ca alyze he oxida ion o educed di- o iphosphopy idine nucleo ide. Howe e , hey could no demons a e any disap- pea ance o ni i e concomi an wi h he enzymic oxida ion o py idine nucleo ide, and he a e o ni i e emained unknown. Mo e ecen ly, Hageman, C eswell, and Hewi (5) ha e shown ha he educ ion o ni i e o ammonia by ma ow lea enzymes occu s wi h educed benzyl iologen alone o in ca aly ic amoun s wi h DPNH, bu no wi h TPNH o DPNH in he absence o he dye. Mo enson, Valen ine, and Ca nahan (6) ob ained om Clos idium pas eu ianum a wa e -soluble, nonheme, non la in, i on-con aining p o ein, named e edoxin, which linked hy- d ogenase wi h a a ie y o elec on accep o s, among hem ni i .e. Acco ding o Valen ine e al. (7), spinach e edoxin subs i u ed o Clos idium e edoxin in he educ ion o ni i e by ex ac s o his bac e ium wi h hyd ogen gas as he elec on dono . Huzisige and Sa oh (8) isola ed om spinach lea es a soluble enzyme p epa a ion, “pho osyn he ic ni i e educ ase,” which was equi ed in addi ion o g ana o he pho ochemical educ ion o ni i e. In p e ious wo k om ou labo a o y (9, lo), i has been shown, howe e , in spinach chlo oplas s, ha he educ ion o ni i e i sel is a da k eac ion. The mechanism in ol ed in he p ocess was ound o be simila o he one implica ed in he pho osyn he ic educ ion o TPN+ (11). To ake place, he educ ion o ni i e equi ed, in addi ion o ligh - o da k- educed * Aided by G an AM 06848-01 om he Na ional Ins i u es o Heal h, Uni ed S a es Public Heal h Se ice, and by a g an om he J. Ma ch Founda ion. spinach e edoxin, a he molabile ac o (ni i e educ ase) also p esen in he chlo oplas ex ac . The p esen epo is conce ned wi h he pu i ica ion o he enzymes in ol ed in he da k and ligh educ ion o ni i e, and wi h he cha ac e iza ion o he p ocess in a econs i u ed enzymic sys em. O pa icula in e es in he p esen in es iga- ion is ha ene gy- ich phospha e is no equi ed o he educ- ion o ni i e; bu when ni i e is educed in he ligh , adenosine iphospha e is p oduced simul aneously in s oichiome ic amoun s. EXPERIMENTAL PROCEDURE P epa a ion o Chlo oplas F agmen s and Fe edoxins-B oken chlo oplas s o once-washed b oken chlo oplas s we e p epa ed om spinach acco ding o Wha ley, Allen, and A non (12). Spinach and C. pas eu ianum e edoxins we e ob ained as desc ibed by Tagawa and A non (11). Pu i ica ion o Spinach Ni i e Reduc ase-A c ude homogena e o spinach lea es was p epa ed as desc ibed by San Pie o and Lang (13) excep ha , as indica ed by Tagawa and A non (II), he pH was adjus ed wi h T is bu e o pH 7.3. P ecipi a ion wi h ace one, ex ac ion o he p ecipi a e, and dialysis we e also ca ied ou acco ding o San Pie o and Lang (13), bu a pH 7.3. The dialyzed ex ac was supplemen ed wi h su icien NaCl o gi e a inal concen . a ion o 0.2 M, and passed h ough a DEAE-cellulose bed, 3 X 6 cm, equilib a ed wi h 0.15 M T is (pH 7.3)-0.2 M [Cl-l in o de o adso d he e edoxin. The ni . i e educ ase ac ion which wen h ough he column was p ecipi a ed wi h ammonium sul a e be ween 0 and 70% sa u a- ion. A e s anding o 20 minu es, he suspension was cen i- uged o 10 minu es a 27,000 X g, and he supe na an solu ion was disca ded. The sedimen was suspended in T is 0.05 M, pH 7.3, and dialyzed o e nigh agains he same bu e in he cold. This pu i ied spinach ni i e educ ase p epa a ion e i- denced e edoxin-TPN educ ase ac i i y. (14) as assayed by he me hod o A on and Jagendo (15), bu was ee o e e- doxin. Pu i ica ion o C. pas eu ianum Hyd ogenase-C. pas eu ianum s ain W-5 was ob ained om he Ame ican Type Cul u e Col- lec ion, Washing on, D. C. The lyophilized cul u e was ans- e ed o he po a o medium o Jensen and Spence (16) and hen o he medium o Ca nahan and Cas le (17), wi h ammonia as he ni ogen sou ce. A c ude ex ac o C. pas eu ianum was p epa ed by au olysis o he d ied cells acco ding o he ech- nique o Ca nahan e al. (18), excep ha 0.05 M T is bu e , pH 7.3, was used. In o de o adso b he e edoxin, he c ude 1737 a FAC BIOLOGIA/BIBLIOTECA on May 22, 2017h p://www.jbc.o g/Downloaded om 1738 Ligh and Da k Reduc ion o Ni i e Vol. 239, No. 6 ex ac was passed h ough a DEAE-cellulose bed equilib a ed wi h he same bu e . The hyd ogenase-con aining ac ion which came h ough he column was ea ed wi h calcium phos- pha e gel (2 mg pe mg o p o ein), allowed o s and o 10 minu es, and cen i uged o 5 minu es a 2000 x g. The supe na an liquid was disca ded, and he sedimen washed by suspending i in T is, 0.7 M, pH 7.3. The suspension was allowed o s and o 10 minu es wi h occasional mixing and cen i uged as be o e, and he supe na an solu ion was disca ded. The pelle was suspended in 0.1 M sodium py ophospha e bu e , pH 7.0, in o de o elu e he hyd ogenase, and he suspension was allowed o s and o 10 minu es wi h occasional s i ing. A e cen i uga ion a 2000 x g o 5 minu es, he sedimen was disca ded; he esul ing supe na an solu ion con ained he hyd ogenase. All s eps we e pe o med a O-4” in he p esence o 3 mM cys eine. In he p esence o e edoxin he pu i ied Clos idium hyd ogenase did no ca alyze ei he TPN o ni i e educ ion wi h molecula hyd ogen (6, 7, 19). The enzyme was also unable o educe me hylene blue; howe e , i libe a ed HP in he p esence o educed me hyl iologen. Chemicals-ADP, TPN , TPNH, DEAE-cellulose, and n-cys eine we e pu chased om Sigma Chemical Company. Me hyl iologen was pu chased om Mann Resea ch Labo a- o ies. NazH32P01 was ob ained om he J. E. N., Mad id, Spain. The calcium phospha e gel was p epa ed acco ding o Keilin and Ha ee (20), bu Na2HP04 was used ins ead o Na3P04, and NaOH was added o adjus he pH o 7.4. Analy ical Me hods-Oxygen e olu ion o hyd ogen up ake was es ima ed in Wa bu g manome e lasks. When he eac- ions we e ca ied ou in he ligh , illumina ion was p o ided om below by a loo-wa luo escen lamp and om abo e by a bank o loo-wa e lec o lood lamps a dis ances o 3 and 14 cm, espec i ely, om he eac .ion essels. Ni i e was es ima ed by he me hod o No ak and Wilson (21). Ammonia was de e mined by nessle iza ion a e di usion and abso p ion o he gas in 0.01 N H&04 in Conway uni s (22). Radioac i e adenosine iphospha e was measu ed by he echnique o A non, Wha ley, and Allen (23). P o ein con- cen a ion was assayed by he me hod o Low y e al. (24). RESULTS AND DISCUSSION The expe imen s o be desc ibed on he mechanism o ni i e educ ion in a econs i u ed enzymic sys em con i m p e ious in es iga ions om his labo a o y (9, 10) and e eal ha e e- doxin is always in ol ed in he p ocess. Fe edoxin i sel is no he enzyme which ca alyzes he educ ion o ni i e, bu he elec on ca ie which unc ions in collabo a ion wi h he spinach ni i e educ ase in he ans e o elec ons o ni i e. Pho ochemical Reduc ion o Ni i e-The educ ion o ni i e in he ligh equi ed spinach e edoxin and spinach ni i e educ ase in addi ion o illumina ed g ana (Table I). No s imula ion o ni i e educ ion by Mn++ ions was obse ed (4,5). Subs a e amoun s o ni i e we e educed o ammonia, and oxygen was e ol ed, in acco dance wi h Equa ion 1 (Table II). 2 NO, + 6 Hz0 + 2 H+ ligh ~2 NH, + 3 02 + 4 H,O (1) Unde he app op ia e expe imen al condi ions and in he p esence o o hophospha e and ADP, he educ ion o 2 moles o ni i e o ammonia was accompanied by he p oduc ion o 6 TABLE I Cha ac e iza ion o ni i e pho o educ ion in econs i u ed chlo oplas sys em The comple e eac ion mix u e included, in a inal olume o 3 ml, once washed b oken chlo oplas s con aining0.2 mg o chlo o- phyll; spinach e edoxin, 1 mg; spinach ni i e educ ase, 2 mg; T is bu e , pH 8.0, 150 pmoles; and sodium ni i e, 6 moles. The gas phase was a gon. The mix u e was incuba ed a 20” o 15 minu es. O he expe imen al condi ions a e indica ed unde “Expe imen al P ocedu e.” Reac ion sys em Non- educed Oxygen e ol ed Comple e Spinach e edoxin omi ed. ............ Ni i e educ ase omi ed ............... Ni i e omi ed ......................... Comple e, da k ......................... .moles pa om 1.4 4.8 0.2 0.7 0.1 0.9 0 0.7 0.1 0 TABLE II Equi alence o oxygen e olu ion and ammonia o ma ion in pho o educ ion o ni i e The expe imen al condi ions we e he same as in Table I, excep ha 0.5 mg o spinach e edoxin was used, and ni i e was added as indica ed. The eac ion was un o comple ion. NO*- added Oxygen e ol ed Ammonia p oduced 0 0 0 0.5 1.3 0.4 1.5 4.2 1.3 c a 3- 4 O2 ATP 2 4 6 8 Minu es FIG. 1. S oichiome y o oxygen e olu ion and ATP o ma ion in noncyclic pho ophospho yla ion wi h ni i e as he e minal elec on accep o . The expe imen al condi ions we e he same as in Table I, excep ha b oken chlo oplas s con aining 0.2 mg o chlo ophyll we e used, and he ollowing we e added: MgC12, 10 moles; Na,Hz2P04, 10 moles; and ADP, 10 pmoles. moles o ATP and he e olu ion o 3 moles o oxygen, in ac- co dance wi h Equa ion 2 (Fig. 1). 2 NO,- + 6 ADP + 6 POdHa + 6 H,O + 2 H+ -.&@ (2) 2 NH3 + 6 ATP + 3 02 + 10 Hz0 The dec ease o he a e o ATP o ma ion as compa ed wi h O2 a FAC BIOLOGIA/BIBLIOTECA on May 22, 2017h p://www.jbc.o g/Downloaded om June 1964 A. Paneque, J. M. Rami ex, F. F. Del Campo, and M. Losada 1739 e olu ion is explained by he uncoupling e ec o ammonia on ATP o ma ion du ing he pho ochemical educ ion o ni i e. Ni i e i sel does no a ec he pho ophospho yla ion coupled o e icyanide educ .ion, bu i s educ ion p oduc , ammonia, e ec i ely uncouples pho osyn he ic phospho yla ion om e icyanide (25) and TPN+ educ ion (26). The educ ion o ni i e in he ligh is hus a new ype o noncyclic, e edoxin-dependen pho ophospho yla ion eac ion in chlo oplas s, simila o bu no iden ical wi h he one s udied by A non e al. (2, 27) wi h TPN+ as he e minal oxidan . A he elec on dono end, he simila i ies a e o al, since bo h TPN+ (27, 28) and NO%- (9, 10) can use ei he wa e o asco ba e- dichlo ophenol indophenol as he educ an . A he elec on accep o end he di e ence is mos p onounced since, as shown in Table III, he noncyclic elec on low was no a ec ed by 1 nn KCN in he TPN sys em (c . A on and Jagendo (15)), whe eas i was comple ely inhibi ed in he NO1 sys em (c . Roussos and Nason (4) and Hageman, C eswell, and Hewi (5)). Bo h sys ems a e, he e o e, iden ical up o he e edoxin le el. Reduced e edoxin has been ecen ly iden i ied by Wha ley, Tagawa, and A non (29) as he ea lies chemically isola ed educ an o med a he expense o adian ene gy apped du ing pho osyn hesis. Once e edoxin has been educed, he elec- ons can be ans e ed ei he o TPN+ by TPN educ ase (14) o o ni i e by ni i e educ ase. Tagawa and A non (11) ound ha he p o eins om g een plan s (13) and pho osyn he ic bac e ia (27), p e iously known as pho osyn he ic py idine nucleo ide educ ases, a e e edoxins which sha e chemical and unc ional simila i ies wi h he e e- doxins om nonpho osyn he ic mic oo ganisms (19), and a e capable o ans e ing he elec ons coming om hyd ogen gas o om ligh -ac i a ed chlo ophyll o he chlo oplas TPN+-TPN educ ase sys em. I was in e es ing, he e o e, o de e mine whe he Clos idium e edoxin is capable o eplacing spinach e edoxin in he pho ochemical educ ion o ni i e. As shown in Fig. 2, Clos idium e edoxin can subs i u e o spinach e edoxin bu is a a less e ec i e elec on ca ie . Da k Reduc ion o Ni i e-The in es iga ion o he da k educ ion o ni i e o ammonia wi h he hyd ogen gas-Clos id- ium hyd ogenase sys em as he sou ce o elec ons also showed ha he p ocess was e edoxin-dependen and equi ed he p esence o spinach ni i e educ ase (Fig. 3). In his da k sys em, by con as wi h he pho ochemical one, Clos idium e edoxin was mo e ac i e han spinach e edoxin in media ing TABLE III E ec o cyanide on noncyclic elec on $0~ wi h TPN and NOz- as e minal elec on accep o s The eac ion mix u e included, in a inal olume o 3 ml, once washed b oken chlo oplas s con aining 0.3 mg o chlo ophyll, 0.5 mg o spinach e edoxin, 4 mg o spinach ni i e educ ase, and 200 pmoles o T is bu e , pH 8.0. Whe e indica ed, 4 pmoles o NaN02, 4 pmoles o TPN+, o 3 pmoles o KCN we e added. O he expe imen al condi ions we e he same as in Table I. Accep o Addi ion NOz- NOz TPN+ TPN+ Cyanide Cyanide T Oxygen e ol ed /Aa oms 3.1 0 3.2 3.1 5 I / Fd (Clos idium) 3 6 9 12 15 Minu es CFIG. 2. Fe edoxins (Fd) as elec on ca ie s in he ligh e- duc ion o ni i e by chlo oplas s. Expe imen al condi ions we e as in Table I, excep ha 1.5 mg o Clos idium e edoxin we e used whe e indica ed. A-A Fd (Clos idium) -MV w-+ Fd (spinach) -no NO; U--E no ni i e educ ase - no ca ie J E 5 10 15 Minu es FIG. 3. Fe edoxins (Fd) and me hyl iologen (MV) as elec- on ca ie s in he da k educ ion o ni i e wi h hyd ogen gas as he elec on dono . The eac ion mix u e con ained, in a inal olume o 3 ml, Clos idium hyd ogenase, 1 mg; spinach ni i e educ ase, 10 mg; T is bu e , pH 8.0, 200 pmoles; cys eine, 10 pmoles; and sodium ni i e, 1.5 pmoles. Whe e indica ed, 1 mg o spinach e edoxin, 1.5 mg o Clos idium e edoxin, o 1 hmole o me hyl iologen was added. Gas phase, hyd ogen. Tempe a u e, 26”. he ans e o elec ons (c . Valen ine e a2. (7)). The speci ici y o Clos idium and spinach e edoxin is, he e o e, mo e igid o he elec on dono sys em han o he ni i e-spinach ni i e educ ase sys em. a FAC BIOLOGIA/BIBLIOTECA on May 22, 2017h p://www.jbc.o g/Downloaded om 1740 Ligh and Da k Reduc ion o Ni i e Vol. 239, No. 6 Wi h he hyd ogen-Clos idium hyd ogenase sys em, i was possible o demons a e ha spinach ni i e and TPN educ- ases di e ed no only in hei sensi i i ies o KCN (Table III), bu also in hei inac i a ion by hea ing. As shown in Table IV, ni i e educ ase ac i i y disappea ed a e he enzyme was hea ed o 10 minu es a 60”, whe eas TPN educ ase was no a ec ed by he same eabmen . Losada e al. (10) ound ha he da k educ ion o ni i e wi h TPNH, ca alyzed by he spinach chlo oplas ex ac , was e edoxin-dependen . They sugges ed ha TPNH ans e ed i s elec ons o e edoxin wi h he TPN educ ase also p esen in he chlo oplas ex ac in a eac ion which would be he e e se o ha s udied by Tagawa and A non (11) ; i.e. spinach e edoxin could media e he ans e o elec ons om he TPNH-TPN educ ase sys em o he ni i e-ni i e educ ase sys em. Recen epo s om A non’s labo a o y (14) ha e shown ha spinach TPN- educ ase in ac ca alyzes he e e sible educ ion o TPN+ by e edoxin. In C. pas eu ianum, howe e , he eac ion seems no o be e e sible. Acco ding o Valen ine e edoxin does no media e he e olu ion o Hz (19) o he educ ion o ni i e (30) om TPNH. Since ou spinach ni i e educ ase p epa a ion con ained also TPN educ ase, we ha e es ed i s abili y o educe ni i e om TPNH. As Table V shows, he enzymic oxida ion o TPNH by ni i e equi ed he p esence o spinach e edoxin. TABLE IV E ec o hea ing on spinach ni i e educ ase and TPN educ ase The expe imen al condi ions we e he same as in Fig. 3, excep ha all he essels included 1 mg o Clos idium e edoxin, and 4 moles o NaNOz o TPN+ we e added as indica ed. The hea ea men o he spinach ni i e educ ase p epa a ion con aining TPN educ ase was ca ied ou a 60” o 10 minu es. Accep o NOz- NO, TPN+ TPN+ TPN+ T ea men None Hea None Hea Hz up ake pm&s 2.6 0.3 3.0 3.0 0.0 TABLE V Fe edoxin as elec on ca ie in da k oxida ion o TPNH wi h ni i e as elec on accep o The eac ion mix u e included, in a inal olume o 3 ml, spin- ach e edoxin, 0.5 mg; spinach ni i e educ ase (con aining TPN educ ase) 2 mg; T is bu e , pH 8.0, 150 pmoles; sodium ni i e, 6 pmoles; and TPNH, 4 pmoles. The eac ion was un a 20”, unde a gon, o 20 minu es. The oxida ion o TPNH was measu ed by he change in op ical densi y a 340 np. The en- dogenous oxida ion in he absence o ni i e was sub aced om he obse ed a es. Reac ion sys em TPNH oxidized pmoles Comple e................................. 1.0 Spinach e edoxin omi ed. 0 Ni i e educ ase omi ed. 0.2 NO; ni i e educ ase H2 Hyd ogenase *FERREDOXIN * TPN educ ase TPNH e- (chlo oplas l 0 LIGHT FIQ. 4. Diag amma ic ep esen a ion o he ole o e edoxin in he educ ion o ni i e. The ans e o elec ons om e e- doxin o ni i e is a da k eac ion ca alyzed by he chlo oplas ni i e educ ase. Fe edoxin i sel can be educed ei he in he ligh (by g ana) o in he da k (by hyd ogen gas o educed i- phosphopy idine nucleo ide). The ole o e edoxin in he pho ochemical and da k educ ion o ni i e ca alyzed by spinach ni i e educ ase is diag amma i- cally ep esen ed in Fig. 4. SUMMARY Reduc ion o ni i e by a econs i u ed enzymic sys em bo h in he da k and in he ligh has been in es iga ed. In he ligh , and in he p esence o spinach g ana, e edoxin, and spinach ni i e educ ase, ni i e can ac as he e minal elec on accep o in a new ype o noncyclic pho ophospho yla- ion. Subs a e amoun s o ni i e a e educed o ammonia, and his eac ion is accompanied by he e olu ion o oxygen and he o ma ion o adenosine iphospha e. The ela ion be ween moles o ni i e educed, moles o ammonia p oduced, moles o o hophospha e es e i ied, and a oms o oxygen e ol ed is 1:1:3:3. In he da k, and in he p esence o e edoxin and spinach ni i e educ ase, ni i e can be educed ei he by he hyd ogen- Clos idium hyd ogenase sys em o by he educed iphospho- py idine nucleo ide-spinach iphosphopy idine nucleo ide e- duc ase sys em. Clos idium e edoxin subs i u es o spinach e edoxin in he educ ion o ni i e by spinach ni i e educ ase, he i s being a mo e e ec i e elec on ca ie in he da k wi h he hyd ogen- hyd ogenase sys em, and he second in he ligh , when he elec- ons a e supplied by he illumina ed g ana. Noncyclic pho o educ ion o TPN+ is no a ec ed by cyanide unde condi ions which comple ely inhibi he elec on low o ni i e. Spinach ni i e educ ase is des oyed by hea ing a 60” o 10 minu es, whe eas spinach TPN educ ase is no a ec ed by he same ea men . Acknowledgmen -The au ho s wish o acknowledge he able echnical assis ance o Miss Ma ia D. Alcain. REFERENCES 1. KESSLER, E., Symposia So . Exp l. Biol., 13, 87 (1959). 2. ARNON, D. I., WHATLEY, F. R., AND ALLEN, M. B., Science, 127, 1026 (1958). 3. NICHOLAS, D. J. D. Ann. Bo any (London), 21, 587 (1957). 4. ROUSSOS, G. G., AND NASON, A., J. Biol. Chem., 236, 2997 (1960). 5. HAGEMAN, R. H., CRESWELL, C. F., AND HEWITT, W. J., Na u e, 193, 247 (1962). a FAC BIOLOGIA/BIBLIOTECA on May 22, 2017h p://www.jbc.o g/Downloaded om June 1964 A. Paneque, J. M. Rami ex, F. F. Del Campo, and M. Losada 6. MORTENSON, L. E., VALENTINE, R. V., AND CARNAHAN, J. E., Biochem. and Biophys. Resea ch Communs., 7, 448 (1962). 7. VALENTINE, R. C., BRILL, W. J., WOLFE, R. S., AND SAN PIETRO, A.. Biochem. and Biophys. Resea ch Commons., 10, 73 (1963). ’ _ 8. 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Bac e ial.. 86. 1114 (1963). ’ ’ a FAC BIOLOGIA/BIBLIOTECA on May 22, 2017h p://www.jbc.o g/Downloaded om A. Paneque, J. M. Rami ez, F. F. Del Campo and M. Losada Ligh and Da k Reduc ion o Ni i e in a Recons i u ed Enzymic Sys em 1964, 239:1737-1741.J. Biol. Chem. h p://www.jbc.o g/con en /239/6/1737.ci a ion Access he mos upda ed e sion o his a icle a Ale s: When a co ec ion o his a icle is pos ed• When his a icle is ci ed• o choose om all o JBC's e-mail ale sClick he e h p://www.jbc.o g/con en /239/6/1737.ci a ion. ull.h ml# e -lis -1 This a icle ci es 0 e e ences, 0 o which can be accessed ee a a FAC BIOLOGIA/BIBLIOTECA on May 22, 2017h p://www.jbc.o g/Downloaded om