BIOCHIMIE, 1975, 57, 647-655.
Studies on protoporphyrin biosynthetic pathway in Saccharomyces cerevisiae ; characterization of the tetrapyrrole intermediates. N. ]~ROUILLET (*), G. ARSEL1N-DE CHATEAUBODEAU (" *) a n d C. VOLLAND ( ' ) . Laboratoire d'Enzymologie, 96, Bd Raspail, 75006 Paris.
(23-10-1974). Summary. - - An acellular extract of the yeast, Saccharomgces cerevisiae, incubated with ALA (*), is able to synthesize protoporphyrin from this precursor. Several tetrapyrrole intermediates were extracted from the medium and purified by silica gel chromatography. The chromatographic behaviour and the spectral properties of the isolated seven free carboxylie porphyrins (and of the corresponding esters), show that each product has a different carboxyle number, varying from eight (uroporphyrin) to two (protoporphyrin). The identification of five of them (octa- to tetracarboxymethyl-porphyrinester) is confirmed by mass spectrometry. The effect of physical factors (temperature, pH, time) on the protoporphyrin biosynthesis system indicates that the enzymes catalysing the first steps of the pathway (ALA-)-Coproporphyrin) are more stable than those eatalysing the last steps (Coproporphyrin--> Protoporphyrin). Results obtained with some enzymatic inhibitors (EDTA, OP, pCMB) show the sensitivity of the ALA dehydratase to OP and to pCMB (confirming therefore its nature as a metallo- and sulfhydryl enzyme) and also of the overall porphyrin synthesis system to these three agents. INTRODUCTION. It has b e e n p r e v i o u s l y e s t a b l i s h e d [1] t h a t t h e aerohdcally g r o w n y e a s t Saccharomyces cerevisiae is c a p a b l e of c a t a l y s i n g p r o t o h a e m f o r m a t i o n f r o m s u c c i n y l CoA a n d g l y c i n e . T h i s s t u d y h a d d e m o n s tr.ated t h a t s e v e r a l p o r p h y r i n o g e n s w e r e s y n t h e s i z e d b y a y e a s t h o m o g e n a t e i n c u b a t e d w i t h ALA. Yet, in vivo, o n l y p r o t o p o r p h y r i n w a s e x t r a c t e d f r o m t h e cells. On t h e o t h e r h a n d , it m u s t be r e c a l l e d t h a t yeast, a n a e r o b i c a l l y g r o w n o r d u r i n g a d a p t a t i o n , e x c r e t e d c o p r o p o r p h y r i n o g e n in t h e m e d i u m [2, 3].
s e e m e d n e c e s s a r y to k n o w the n a t u r e of t h e t e t r a p y r r o l e m o l e c u l e s successiwely f o r m e d . H e n c e , t h i s p a p e r w i l l r e p o r t i s o l a t i o n a n d c h , a r a c t e r i z a t i o n of the i n t e r m e d i a t e s , w h i c h a r e a c c u m u l a t e d d u r i n g t h e i n c u b a t i o n of an a c e l h f l a r e x t r a c t of y e a s t w i t h ALA. I n f l u e n c e of s o m e p h y s i c a l f a c t o r s (pH, t e m p e r a t u r e , t i m e ) a n d of s o m e e n z y m a t i c i n h i b i t o r s w a s also s t u d i e d in o r d e r to o b t a i n m o r e i n f o r m a t i o n a b o u t the s e n s i t i v i t y of t h e d i f f e r e n t e n z y m a t i c steps to t h e s e e f f e c t o r s a n d e v e n t u a l l y to o b t a i n a l a r g e r a m o u n t of s o m e i n t e r m e d i a t e s due to t h e a c t i o n on t h e c o r r e s p o n d i n g e n z y m e s .
As a b e g i n n i n g t o w a r d the s t u d y of t h e e n z y m e s i n v o l v e d i n the p r o t o p o r p h y r i n s y n t h e s i s , it
M ATEI~IA'LS AND M E T H O D S .
(') Abbreviations. ALA : 5-aminolevulinic acid ; CO0 ME : carboxymethylester ; Copro-ME : coproporphyrin tetramethyle s t e r ; E D T A : ethylenediamine tetracetic a c i d ; O P : o rthophenanthroline ; pCMB : parachloromercuribenzoie a c i d ; Proto-ME : protoporphvrin dimethylester ; PBG : porphobilinogen ; Uro-ME :" uroporphyrin octamethylester. ALA dehydratase : EC 4.2.1.24.
ALA was purchased from Fluka ; Uro- and C o p r o p o r p h y r i n - e s t e r s ( i s o m e r s I a n d III) w e r e o b t a i n e d f r o m Sigma. E D T A d i s o d i u m salt w a s o b t a i n e d f r o m T o u z a r t and M a t i g n o n . F o l l o w i n g reagents were Merck products : benzene, ethyla c e t a t e , o r t h o p h e n a n t h r o l i n e , talc, s i l i c a gel p l a t e s F,25~ a n d s i l i c a gel 60 p o w d e r . O t h e r c h e m i c a l s were obtained fnom Prolabo.
(**) (*) Present adresses : (*) Universitd Paris VII, Tour 43, 2, place Jussieu ; 75221 Paris Cedex 05. ( ~ ) Laboratoire de Biochimie Biologie cellulaire ; Universitd Bordeaux II, 351, cours de la lib6ration, 33405 Talence.
REAGENTS.
Protoporphyrin dimethylester was from pig hemoglobin by the method by F a l k [4].
prepared described
648
N. Brouillet, G. Arselin De Chateaubodeau and C. Volland. METHODS.
The diploid w i l d s t r a i n Yeast F o a m of Saccharomyces cerevisiae was c u l t u r e d a e r o b i c a l l y using a complete m e d i u m (yeast extract (1 p. cent) a n d glucose (5 p. cent), a n d w a s harvested a c c o r d i n g to a previously described m e t h o d [1]. 1) Preparation of acellular extract S. The ceils (about 500 g w e t weight) s u s p e n d e d i n phosphate buffer 0,1 M, pH 7,2 were d i s r u p t e d w i t h a B r o n m i l l cell disintegrator (Braun), by aliquots of 20 g [1]. F o r large-scale p r e p a r a t i o n s of cell-free extract, the cells (2,5 Kg of cells made up to four liters w i t h phospha[e buffer), were disr u p t e d w i t h a refrigerated c o n t i n u o u s cell disintegrator (Dynonrill type KDL, W i l l y A. Bachofen, Basel, Switzerland) i n p r e s e n c e of one kilogram of glass heads (~ = ,0;5 ram) at 3000 r p m (flow rate : 60 m l / m i n ) . T h e acellular soluble .extract S was obtained from the b r o k e n cells s u s p e n s i o n by s p i n n i n g the s u p e r n a t a n t of a 1 5'0.0 g (15 rain) c e n t r i f u g a t i o n at 78 000 g for 1 hour. D e p e n d i n g u p o n the i n i t i a l a m o u n t of yeast ceils treated, from 1 to 6 h t e r s of the soluble .extract S (10 nag pr,oteins/ml) w e r e r e c o v e r e d ; aliquots of 10 or 2'00 ml were frozen a n d kept at - - 2,0o for several months, w i t h o u t loss of e n z y m a t i c activities. Before use a n d after thawing, they are, or not, dialysed overnight against 20 volumes of phosphate buffer ,0;1 M, pH 7,2, at 4 ° w i t h a gentle stirring. 2) Porphyrins synthesis. The acellular ,extract S was inctrbated aerobically at 35 ° i n the dark for 3 to 24 hours, in presence of 2,5 inM A,LA ; c h l o r a m p h e n i c o l (10 ~g/ml) w.as added w h e n i n c u b a t i o n lasted m o r e that 15 hours. After i n c u b a t i o n , the p o r p h y r i n s were extracted by 3N HCI a n d s p e c t r o p h o t o m e t r i e a l l y determ i n e d [13. The total a m o u n t of p o r p h y r i n s was expressed i n n a n o m o l e s of p r o t o p o r p h y r i n per ml of extract S, u s i n g EmM = 26.2 for the Soret b a n d at 4.07 n m [5]. 3) Separc~tion of porphyrins. The free p o r p h y r i n s w e r e e x t r a c t e d from the i n c u h a t i o n m e d i u m w i t h two volumes of ethylacetate-acetic acid (3:1, v/v), The p r e c i p i t a t e d p r o t e i n s w e r e centrifuged d o w n a n d the organic phase, c o n t a i n i n g the p o r p h y r i n s , was evaporated. The extracted p o r p h y r i n s a n d s t a n d a r d porp h y r i n s c o m m e r c i a l (proto-, cop'to-, a n d uroporp h y r i n ) , dissolved in c o n c e n t r a t e d a m m o n i a were
BIOCHIMIE, 1975, 57, n ° 5.
applied to a silica gel plate w h i c h w a s developed in the dark, using a 2,6-1utidin-water m i x t u r e (5:3', v/v) w i t h a r m n o n i a atmosphere [6]. M t e r development, the plate was d r i e d a n d the porp h y r i n s w e r e detected b y their red fluorescence in UV light (k ~ 366 rim). E l u t i o n of p o r p h y r i n s was realised as follows : the fluorescent spots w e r e s c r a p e d from the plates a n d the silica was s i m u l t a n e o u s l y v a c u u m - s u c k e d into the w i d e .end of a P a s t e u r pipet, the tip of w h i c h was closed w i t h glasswool ; the p o r p h y r i n s w e r e eluted from silica by 3N HC1 a n d spectrophotometTically identified a n d titrated. The following e x t i n c t i o n coeffi,cients w e r e used to calculate the c o n c e n t r a t i o n of each p o r p h y r i n : em~ = 262 ( p r o t o p o r p h y r i n ) for di- a n d tricarb o x y l i c p o r p h y r i n s ; em~ ~ 489 ( c o p r o p o r p h y r i n ) f.or tetra- a n d pentacarboxy~ic p o r p h y r i n s , a n d em~t = 541 ( u r o p o r p h y r i n ) for hexa-, hepta- a n d octacarboxylic p o r p h y r i n s (see table I for their respective km~). 4) Porphyrins esteri[ication and separation o[ esters. After i n c u b a t i o n of the e x t r a c t S d u r i n g 24 h o u r s as described above, the m e d i u m was adjusted to pH 4 w i t h glacial acetic acid. Talc was added (125 g for one liter of acellular ,extract S) whilst s t i r r i n g ; after centrifugation, the talc was w a s h e d twice with distilled w a t e r .and then d r i e d at 50 °. The p o r p h y r i n s were eluted fr,om the talc [7] w i t h the esterification m i x t u r e methanol-sulfuric acid (95:5, v / v ) ( 5 0 0 , ml for 125 g talc, 15 °, 15 h i n the dark) ; the esters were extracted with chloroform, i n presence of saturated s o d i u m acetate. The c h l o r o f o r m extract was w a s h e d several times w i t h distillated w a t e r a n d finally d r i e d w i t h a n h y d r o u s s o d i u m sulfate. About 65 per cent of the free in.trial p o r p h y r i n s were recovered as esters. F o r a n a l y t i c a l c h r o m a t o g r a p h y , - t h e esters dissolved i n c h l o r o f o r m were applied to a silica gel p l a t e ; they w e r e developed, i n the dark, w i t h a modified Doss [8] solvent as follows : benzeneethyl-acetate-methanol (42,5:13)5:0,75, v / v ) . The esters w e r e detected by the red fluorescence i n UV light a n d eluted w i t h the solvent m i x t u r e u s e d i n development, as described for the c a r b o x y l i e porphyrins. F o r p r e p a r a t i v e purpose, the esters dissoIved i n c h l o r o f o r m were separated on silica c o l u m n w i t h the same solvent as tbe one used for chromatoplates. A first c h r o m a t o g r a p h y on a small column (h = 18 cm, ¢ = 1,5 cm) allowed to e l i m i n a t e a
Characterization of porphyrins synthesized by yeast. b r o w n fraction w h i c h was a d s o r b e d on the top of the c o l u m n ; after c o n c e n t r a t i o n b y evaporation, the eluted m i x t u r e of esters w a s r e c h r o m a t o g r a p h e d on a higher c o l u m n (h = 50 c m ; ¢ = 3 cm) i n the same conditions. The elution was followed b y r e a d i n g the a b s o r p t i o n of each eluate at 405 n m a n d the p u r i t y of .each f r a c t i o n was tested b y t h i n - l a y e r c h r o m a t o g r a p h y . Only the eluates c o n t a i n i n g a single pure ester vcere collected a n d pooled ; after .evaporation, the ester was dissolved, i n a m i n i m a l a m o u n t of c h l o r o f o r m a n d kept at - - 2 0 ° i n the dark.
649
RESULTS. 1) Separation and identification of ]tee carboxylic porphyrins. T h i n - l a y e r c h r o m a t o g r a p h y (0,5-1 ~ g ) o f the porp h y r i n s synthesized b y acellular .extract S revealed the presence of seven p r o d u c t s (a to g) w i t h differ e n t Rf ; three of t h e m (a, e, g) migrate as s t a n d a r d u r o p o r p h y r i n III, c o p r o p o r p h y r i n III a n d protop o r p h y r i n IX. The 15near r e l a t i o n s h i p of Rf/Rf p,oto (this ratio was more const,ant t h a n the Rf itself) w i t h the c a r b o x y l i c groups n u m b e r o b t a i n e d for the porp h y r i n s a, e, a n d g [12] gave an estimation of the
The total a m o u n t of esters c o n t a i n e d i n chloroform solution were s p e c t r o p h o t o m e t r i c a l l y estima-
TABLE I.
Chromatographic and spectrophotometric data [or porphyrins synthesized by an acellular extract of Saccharomyces cerevisiae. Porphyrins synthesized by acellular extract I
Spots oi the chromatoplates
t
a I
[
• cOOHnumbo
).... (nm) Soret band
b I
1
i o,o4 i o,18 Ir_ . . 8. . . . . ~ _ _7,1 I 405
c
~4~
i
- -
d i - -
~e --i
f
Ig
' Standard porphyrins IUlrI° / Copro Proto III IX
i
1 o,34 !o,48 i0, l 0,ss 1 0,04 0,69 _6,1 _ 41 2,8 [. 2. . . 8 I 5,2 i 4 '!~ 2 4~-
403 - - 1402[ [ - [ -405 - - 1407 ~ - - 405 [ 402 i 407
The extract was incubated with 2.5 mM ALA, at 35 °, pH 7.2 for 3 h. Porphyrins were extracted and separated on silica gel chromatoplates (see methods for further details). The COOH number was calculated from the straight line obtained by plotting Rf,t:,,~oto against COOH number of the porphyrins a, e and g [11].
ted in micro- or m i l l i g r a m s of p r o t o p o r p h y r i n ester (en~.~~ = 171) ; after separation, each ester was estimated by using e,n~i ~ 171 for di- a n d t r i m e t h y l p.orphyrin ester, em~~ = 180 for t e t r a - a n d pentamethylester a n d em~t = 215 for hexa-, hepta- and octamethylester [5]. F l u o r e s c e n c e spectra of each ester was realized with a Safas r e c o r d i n g s p e c t r o p h o t o m e t e r equipped for fluorescence analysis. Mass spectra of esters wer~e realized w i t h a spectrometer AEI type MS9. 5) Other methods. P o r p h y r i n esters w e r e h y d r o l y s e d by 3N HC1 d u r i n g 24 hours, at 4 ~' in the dark. P r o t e i n s w e r e estimated by the m e t h o d L o w r y et al [9] w i t h lysozyme as a s t a n d a r d .
of
ALA dehydratase activity was m e a s u r e d by the method of Mauzerall [10] i n c o n d i t i o n s described by B u r n h a m [11] w i t h en,.~I of PBG given b y B u r n h a m : ~m.~ = 56 at ~ = 553 nm.
BIOCH1MIE, 1975, 57, n ° 5.
n u m b e r of c a r b o x y l i c groups for the other porp h y r i n s (table I). The spectral data of eluted porp h y r i n s confirmed their nature. 2) Separation and identification of porphyrin esters. T h i n - l a y e r c h r o m a t o g r a p h y of p o r p h y r i n esters showed also seven fluorescent spots. The relationship of Rf/Rf ~roto~m w i t h the n u m b e r of carboxymethyl groups was l i n e a r for file seven e s t e r s : this suggested that the c a r b o x y m e t h y l groups n m n b e r of these esters was decreasing from eight to two a n d that each spot c o n t a i n e d only one ester. I n similar m a n n e r , esters c h r o m a t o g r a p h y (5-10 my) on silica c o l u m n gave seven fractions, each c o n t a i n i n g a n almost pure (95 p. cent) porp h y r i n i n t e r m e d i a t e whose spectral characteristics a n d c h r o m a t o g r a p h i c b e h a v i o u r were i d e n tical w i t h those of the c o r r e s p o n d i n g esters eluted from the p l a t e s ; these data are p r e s e n t e d i n tables II and III. 45
650
N. B r o u i l l e t , G. A r s e l i n D e C h a l e a u b o d e a u
The homogeneity of esters eluted from the plates or f r o m t h e c o l u m n w a s p r o v e d b y t h i n - l a y e r
a n d C. V o l l a n d .
c h r o m a t o g r , a p h y of e a c h s e e n i n f i g u r e 1.
solution
as it c a n
be
TABLE I I
Characteristics of the p o r p h y r i n e s t e r s obtained f r o m p o r p h y r i n s s y n t h e s i z e d by an acelIular extract of S a c c h a r o m y c e s c e r e v i s i a e . Esters of potpbyrins syl)lhesized by extract S Spots
i
2
I
t ti,o [cop~o t Prolo
7 IIIIMEjlIIMEIIXM --I----I t
E
3
A
5
6
0,53
0,64
0,~9
0,~0
0,29
0,78
1
407,? 406,51 403 ' 404
402
~5 40, 407,5
402
409
624,5
629 632i 628
624 - ~ 3 3
> for the esterifieation, e x t r a c t i o n a n d s e p a r a t i o n of the esters. CO0 ME n u m b e r was calculated as for the free carboxylic porp h y r i n s (Table I). (a) : Xe~¢it~tio. : 404 rim. TABLE III.
Absorption spectra of p o r p h g r i n e s t e r s in chloro[orm.
),m.~(nm)
Porpbyrinesters obtained from t}}e inclibatim~ mixture ]I _ _ _ _ ~
Standard porphyrines((,r s
[ t~I~00111,'~ 7 COOLIE 0 I]OOIIE 5 COOLIE ~ f~IiOIIE 3 COOLIE 2 CliIiiE i Uro-]ll~] f,opro-ME Proto-II}] Soret IV III II
407,5 406,5 405 503 502,5 502 536,5 537,5 536,5 573 572 571 (600) t602) ] ! 627 626 ~625,5 i (647,5)](646,5)
404 1402 405 501 502 504,5 537 534,5 539 570 569,5 573 (598,5)](597) 624 [623 625,5 !
409 507,5 542 577,5 631
407,5 402 502 500 [536,5 534 573 569,5 /(601) (598) 627 623
409 507 542,5 577,5 632
Fro. 1. - - Silica Gel thin-layer chromatogram of porphgrinesters. 7~
Solvent s y s t e m : b e n z e n e - e t h y l a e e t a t e - m e t h a n o l (42.5:13.5:0.75, v / v ) . M : m i x t u r e of esters o b t a i n e d b y esterification of t h e p o r p h y r i n s synthesized by a n aeellular extract S i n c u b a t e d 'with ALA. Urn, Cm and Pix correspond to p o r p h y r i n e s t e r s s t a n dard. Spots (1: Urn-ME; 2: 7-COO ME; 3 : 6-COO-ME ; 5 : Copro-ME ; 6 : 3 COO ME ; 7 : Proto-ME) correspond to esters o b t a i n e d f r o m siliea gel c o l u m n (see t h e text).
BIOCHIMIE, 1975, 57, n ° 5.
Characterization of porphgrins synthesized by yeast. The mass spectra of the five slo-~, m i g r a t i n g esters (1 to 5) revealed the p r e s e n c e of m o l e c u l a r peaks c o r r e s p o n d i n g to the following m o l e c u l a r weights : 942, 884, 826, 768 a n d 71'0 w h i c h are exactly those of p o r p h y r i n e s t e r s h a v i n g from eight to four c a r b o x y m e t h y l groups. The mass spectra of th e s u p p o s e d di- and t r i e a r b o x y l i c p o r p h y r i n e s ter ga\,e less reliable results, m a y be due to the i n s t a b i l i t y of these molecules.
651
the chromatoplates , varied w i t h the t e m p e r a t u r e : some increased (oeta-, tetra- and t r i c a r b o x y l i c porp h y r i n s ) w h e r e a s others decreased (penta- a n d dicarboxylic porphyrins).
3) Determination of incubation conditions for porphyrins biosynthesis.
c) Effect of incubation time : The acellular extract S was i n c u b a t e d for 2 to 24 h o u r s at 35 ~ a n d pH 7. Although the total a m o u n t of p o r p h y r i n s was regularly e n h a n c e d w i t h the i n c u b a t i o n time, the a m o u n t s rec.o.rded in table IV showed that the c o n t e n t of each p o r p h y r i n i n c r e a s e d or decreased a c c o r d i n g to their n,ature.
The aim of these e x p e r i m e n t s (carried out several times w i t h 3 to 5 ml of extract S a n d
d) Effect of oxygen : D u r i n g previous studies made in this l a b o r a t o r y [1], it was observed that,
TARLE IV.
Time dependence of porphgrins synthesis catalysed by an acellular extract of S a c c h a r m n y c e s cerevisiae. Percent distribution of porpbyrins {b) Time of incubation
Total amount of porphyrins (a)
3h
6,7
8h
25
24 h
50
(a) Expressed (b) Expressed matoplates. Incubation as and titration of
81:[6 coo. ooo. coo. : 11
9,5
40
15
7,5
13 15 2,6
in nanomoles of protoporphyrin/ml of incubated extract S. in percentage of total amount of porphyrins eluted from chroin table I and see for the extraction, separation porphyrin.
2,5 mM ALA) was to k n o w the best c o n d i t i o n s of ~incubation for o b t a i n i n g the largest a m o u n t of porphyrins. a) Effect of pH : T h e largest a m o u n t of total porp h y r i n s synthesized after an i n c u b a t i o n of 3 h at 35 ° was obtained at pH 7-7,2; the c o n t e n t Cat pH 6 a n d pH 8 being about 20 p e r c e n t of the m a x i m u m value. The chromatoplates s h o w e d that at pH 6,5 a n d 7,0, the seven p o r p h y r i n s ~'ere p r e s e n t i n i n c u b a t i o n mixtures but above pH 7,5 u r o p o r p h y t i n III was ahnost the only porphy, r i n p r e s e n t a n d very slight a m o u n t s of other p o r p h y r i n s are synthesized. b) Effect of temperature : The i n c u b a t i o n was c a r r i e d out at pH 7 d u r i n g 3 h o u r s at several t e m p e r a t u r e s b e t w e e n 25 ° a n d 50 ° . The total a m o u n t of s y n t h e s i z e d p o r p h y r i n s , expressed i n p r o t o p o r p h y r i n , regularly i n c r e a s e d w i t h temperature, a n d twice as high at 50`0 t h a n at 350 . I n fact, however, the q u a n t i t y of .each i n d i v i d u a l p o r p h y rin, as m e a s u r e d after s e p a r a t i o n a n d elution from
BIOCH1M1E, 1975, 57, n ° 5.
4
2
COOH COOIt
i n absence of oxygen, the acellular extract S did not synthesize p r o t o p o r p h y r i n , b u t synthesized c o p r o p o r p h y r i n . T h i n - l a y e r c h r o m a t o g r a p h y of p o r p h y r i n s confirmed this observation a n d showed the p r e s e n c e of octa- to t e t r a c a r b o x y l i c p o r p h y rins, w i t h p r e d o m i n a n t c o n t e n t of u r o - a n d coproporphyrin. F r o m these results, the best i n c u b a t i o n c~nditions ,as described i n Methods, have been chosen for the s t a n d a r d assay. In addition, these results have been used to modify, ~in some eases, the relative a m o u n t of the different p o r p h y r i n s synthesized. 4) Effect of heating and of enzymatic inhibitors on enzyme systems involved in protoporphyrin biosynthesis. I n these e x p e r i m e n t s , the extract S h a d been heated or i n c u b a t e d w i t h e n z y m a t i c i n h i b i t o r s (metal chelators, thiol reagents) before i n c u b a t i o n w i t h ALA, in order to k n o w if the b i o s y n t h e t i c p a t h w a y could be stopped at some point, w i t h 46
N. Brouillet, G. A r s e l i n De Chateaubodeau a n d C. Volland.
652
a c c u m u l a t i o n .of some i n t e r m e d i a t e s ; therefore, it w a s h o p e d to get some i n f o r m a t i o n about the sensitivity of the different e n z y m a t i c steps t o w a r d s these different effectors. a) Heating of extract S : W h e n the extract S was heated for 5 m i n u t e s to t e m p e r a t u r e s b e t w e e n 35 ° a n d 70 ° , the total a m o u n t of p o r p h y r i n s subsequently synthesized decreased w h e n temper a t u r e r e a c h e d 55 ° and, at 70 ° , no synthesis was observed. T h i n - l a y e r c h r o m a t o g r a p h y of these
150
x y l i c p o r p h y r i n s decreased w i t h i n c r e a s i n g conc e n t r a t i o n of i n h i b i t o r s , w h i l e that of the tetra- to d i c a r b o x y l i c p o r p h y r i n s increased. I d e n t i c a l e x p e r i m e n t s made w i t h n o n dialysed extract S showed that the p o r p h y N n - s y n t h e s i z i n g system was less inhibited' b y OP (OP 10-2M gave 65 p. cent i n h i b i t i o n ) t h a n i n dialysed extract. Similarly, 1.0-eMi EDTA i n h i b i t e d slightly this enzymatic system '(23 p. cent i n h i b i t i o n ) and, at low c o n c e n t r a t i o n , no activation was noted.
g
>I.--
>p> m
O
Studies on protoporphyrin biosynthetic pathway in Saccharomyces cerevisiae ; characterization of the tetrapyrrole intermediates. N. ]~ROUILLET (*), G. ARSEL1N-DE CHATEAUBODEAU (" *) a n d C. VOLLAND ( ' ) . Laboratoire d'Enzymologie, 96, Bd Raspail, 75006 Paris.
(23-10-1974). Summary. - - An acellular extract of the yeast, Saccharomgces cerevisiae, incubated with ALA (*), is able to synthesize protoporphyrin from this precursor. Several tetrapyrrole intermediates were extracted from the medium and purified by silica gel chromatography. The chromatographic behaviour and the spectral properties of the isolated seven free carboxylie porphyrins (and of the corresponding esters), show that each product has a different carboxyle number, varying from eight (uroporphyrin) to two (protoporphyrin). The identification of five of them (octa- to tetracarboxymethyl-porphyrinester) is confirmed by mass spectrometry. The effect of physical factors (temperature, pH, time) on the protoporphyrin biosynthesis system indicates that the enzymes catalysing the first steps of the pathway (ALA-)-Coproporphyrin) are more stable than those eatalysing the last steps (Coproporphyrin--> Protoporphyrin). Results obtained with some enzymatic inhibitors (EDTA, OP, pCMB) show the sensitivity of the ALA dehydratase to OP and to pCMB (confirming therefore its nature as a metallo- and sulfhydryl enzyme) and also of the overall porphyrin synthesis system to these three agents. INTRODUCTION. It has b e e n p r e v i o u s l y e s t a b l i s h e d [1] t h a t t h e aerohdcally g r o w n y e a s t Saccharomyces cerevisiae is c a p a b l e of c a t a l y s i n g p r o t o h a e m f o r m a t i o n f r o m s u c c i n y l CoA a n d g l y c i n e . T h i s s t u d y h a d d e m o n s tr.ated t h a t s e v e r a l p o r p h y r i n o g e n s w e r e s y n t h e s i z e d b y a y e a s t h o m o g e n a t e i n c u b a t e d w i t h ALA. Yet, in vivo, o n l y p r o t o p o r p h y r i n w a s e x t r a c t e d f r o m t h e cells. On t h e o t h e r h a n d , it m u s t be r e c a l l e d t h a t yeast, a n a e r o b i c a l l y g r o w n o r d u r i n g a d a p t a t i o n , e x c r e t e d c o p r o p o r p h y r i n o g e n in t h e m e d i u m [2, 3].
s e e m e d n e c e s s a r y to k n o w the n a t u r e of t h e t e t r a p y r r o l e m o l e c u l e s successiwely f o r m e d . H e n c e , t h i s p a p e r w i l l r e p o r t i s o l a t i o n a n d c h , a r a c t e r i z a t i o n of the i n t e r m e d i a t e s , w h i c h a r e a c c u m u l a t e d d u r i n g t h e i n c u b a t i o n of an a c e l h f l a r e x t r a c t of y e a s t w i t h ALA. I n f l u e n c e of s o m e p h y s i c a l f a c t o r s (pH, t e m p e r a t u r e , t i m e ) a n d of s o m e e n z y m a t i c i n h i b i t o r s w a s also s t u d i e d in o r d e r to o b t a i n m o r e i n f o r m a t i o n a b o u t the s e n s i t i v i t y of t h e d i f f e r e n t e n z y m a t i c steps to t h e s e e f f e c t o r s a n d e v e n t u a l l y to o b t a i n a l a r g e r a m o u n t of s o m e i n t e r m e d i a t e s due to t h e a c t i o n on t h e c o r r e s p o n d i n g e n z y m e s .
As a b e g i n n i n g t o w a r d the s t u d y of t h e e n z y m e s i n v o l v e d i n the p r o t o p o r p h y r i n s y n t h e s i s , it
M ATEI~IA'LS AND M E T H O D S .
(') Abbreviations. ALA : 5-aminolevulinic acid ; CO0 ME : carboxymethylester ; Copro-ME : coproporphyrin tetramethyle s t e r ; E D T A : ethylenediamine tetracetic a c i d ; O P : o rthophenanthroline ; pCMB : parachloromercuribenzoie a c i d ; Proto-ME : protoporphvrin dimethylester ; PBG : porphobilinogen ; Uro-ME :" uroporphyrin octamethylester. ALA dehydratase : EC 4.2.1.24.
ALA was purchased from Fluka ; Uro- and C o p r o p o r p h y r i n - e s t e r s ( i s o m e r s I a n d III) w e r e o b t a i n e d f r o m Sigma. E D T A d i s o d i u m salt w a s o b t a i n e d f r o m T o u z a r t and M a t i g n o n . F o l l o w i n g reagents were Merck products : benzene, ethyla c e t a t e , o r t h o p h e n a n t h r o l i n e , talc, s i l i c a gel p l a t e s F,25~ a n d s i l i c a gel 60 p o w d e r . O t h e r c h e m i c a l s were obtained fnom Prolabo.
(**) (*) Present adresses : (*) Universitd Paris VII, Tour 43, 2, place Jussieu ; 75221 Paris Cedex 05. ( ~ ) Laboratoire de Biochimie Biologie cellulaire ; Universitd Bordeaux II, 351, cours de la lib6ration, 33405 Talence.
REAGENTS.
Protoporphyrin dimethylester was from pig hemoglobin by the method by F a l k [4].
prepared described
648
N. Brouillet, G. Arselin De Chateaubodeau and C. Volland. METHODS.
The diploid w i l d s t r a i n Yeast F o a m of Saccharomyces cerevisiae was c u l t u r e d a e r o b i c a l l y using a complete m e d i u m (yeast extract (1 p. cent) a n d glucose (5 p. cent), a n d w a s harvested a c c o r d i n g to a previously described m e t h o d [1]. 1) Preparation of acellular extract S. The ceils (about 500 g w e t weight) s u s p e n d e d i n phosphate buffer 0,1 M, pH 7,2 were d i s r u p t e d w i t h a B r o n m i l l cell disintegrator (Braun), by aliquots of 20 g [1]. F o r large-scale p r e p a r a t i o n s of cell-free extract, the cells (2,5 Kg of cells made up to four liters w i t h phospha[e buffer), were disr u p t e d w i t h a refrigerated c o n t i n u o u s cell disintegrator (Dynonrill type KDL, W i l l y A. Bachofen, Basel, Switzerland) i n p r e s e n c e of one kilogram of glass heads (~ = ,0;5 ram) at 3000 r p m (flow rate : 60 m l / m i n ) . T h e acellular soluble .extract S was obtained from the b r o k e n cells s u s p e n s i o n by s p i n n i n g the s u p e r n a t a n t of a 1 5'0.0 g (15 rain) c e n t r i f u g a t i o n at 78 000 g for 1 hour. D e p e n d i n g u p o n the i n i t i a l a m o u n t of yeast ceils treated, from 1 to 6 h t e r s of the soluble .extract S (10 nag pr,oteins/ml) w e r e r e c o v e r e d ; aliquots of 10 or 2'00 ml were frozen a n d kept at - - 2,0o for several months, w i t h o u t loss of e n z y m a t i c activities. Before use a n d after thawing, they are, or not, dialysed overnight against 20 volumes of phosphate buffer ,0;1 M, pH 7,2, at 4 ° w i t h a gentle stirring. 2) Porphyrins synthesis. The acellular ,extract S was inctrbated aerobically at 35 ° i n the dark for 3 to 24 hours, in presence of 2,5 inM A,LA ; c h l o r a m p h e n i c o l (10 ~g/ml) w.as added w h e n i n c u b a t i o n lasted m o r e that 15 hours. After i n c u b a t i o n , the p o r p h y r i n s were extracted by 3N HCI a n d s p e c t r o p h o t o m e t r i e a l l y determ i n e d [13. The total a m o u n t of p o r p h y r i n s was expressed i n n a n o m o l e s of p r o t o p o r p h y r i n per ml of extract S, u s i n g EmM = 26.2 for the Soret b a n d at 4.07 n m [5]. 3) Separc~tion of porphyrins. The free p o r p h y r i n s w e r e e x t r a c t e d from the i n c u h a t i o n m e d i u m w i t h two volumes of ethylacetate-acetic acid (3:1, v/v), The p r e c i p i t a t e d p r o t e i n s w e r e centrifuged d o w n a n d the organic phase, c o n t a i n i n g the p o r p h y r i n s , was evaporated. The extracted p o r p h y r i n s a n d s t a n d a r d porp h y r i n s c o m m e r c i a l (proto-, cop'to-, a n d uroporp h y r i n ) , dissolved in c o n c e n t r a t e d a m m o n i a were
BIOCHIMIE, 1975, 57, n ° 5.
applied to a silica gel plate w h i c h w a s developed in the dark, using a 2,6-1utidin-water m i x t u r e (5:3', v/v) w i t h a r m n o n i a atmosphere [6]. M t e r development, the plate was d r i e d a n d the porp h y r i n s w e r e detected b y their red fluorescence in UV light (k ~ 366 rim). E l u t i o n of p o r p h y r i n s was realised as follows : the fluorescent spots w e r e s c r a p e d from the plates a n d the silica was s i m u l t a n e o u s l y v a c u u m - s u c k e d into the w i d e .end of a P a s t e u r pipet, the tip of w h i c h was closed w i t h glasswool ; the p o r p h y r i n s w e r e eluted from silica by 3N HC1 a n d spectrophotometTically identified a n d titrated. The following e x t i n c t i o n coeffi,cients w e r e used to calculate the c o n c e n t r a t i o n of each p o r p h y r i n : em~ = 262 ( p r o t o p o r p h y r i n ) for di- a n d tricarb o x y l i c p o r p h y r i n s ; em~ ~ 489 ( c o p r o p o r p h y r i n ) f.or tetra- a n d pentacarboxy~ic p o r p h y r i n s , a n d em~t = 541 ( u r o p o r p h y r i n ) for hexa-, hepta- a n d octacarboxylic p o r p h y r i n s (see table I for their respective km~). 4) Porphyrins esteri[ication and separation o[ esters. After i n c u b a t i o n of the e x t r a c t S d u r i n g 24 h o u r s as described above, the m e d i u m was adjusted to pH 4 w i t h glacial acetic acid. Talc was added (125 g for one liter of acellular ,extract S) whilst s t i r r i n g ; after centrifugation, the talc was w a s h e d twice with distilled w a t e r .and then d r i e d at 50 °. The p o r p h y r i n s were eluted fr,om the talc [7] w i t h the esterification m i x t u r e methanol-sulfuric acid (95:5, v / v ) ( 5 0 0 , ml for 125 g talc, 15 °, 15 h i n the dark) ; the esters were extracted with chloroform, i n presence of saturated s o d i u m acetate. The c h l o r o f o r m extract was w a s h e d several times w i t h distillated w a t e r a n d finally d r i e d w i t h a n h y d r o u s s o d i u m sulfate. About 65 per cent of the free in.trial p o r p h y r i n s were recovered as esters. F o r a n a l y t i c a l c h r o m a t o g r a p h y , - t h e esters dissolved i n c h l o r o f o r m were applied to a silica gel p l a t e ; they w e r e developed, i n the dark, w i t h a modified Doss [8] solvent as follows : benzeneethyl-acetate-methanol (42,5:13)5:0,75, v / v ) . The esters w e r e detected by the red fluorescence i n UV light a n d eluted w i t h the solvent m i x t u r e u s e d i n development, as described for the c a r b o x y l i e porphyrins. F o r p r e p a r a t i v e purpose, the esters dissoIved i n c h l o r o f o r m were separated on silica c o l u m n w i t h the same solvent as tbe one used for chromatoplates. A first c h r o m a t o g r a p h y on a small column (h = 18 cm, ¢ = 1,5 cm) allowed to e l i m i n a t e a
Characterization of porphyrins synthesized by yeast. b r o w n fraction w h i c h was a d s o r b e d on the top of the c o l u m n ; after c o n c e n t r a t i o n b y evaporation, the eluted m i x t u r e of esters w a s r e c h r o m a t o g r a p h e d on a higher c o l u m n (h = 50 c m ; ¢ = 3 cm) i n the same conditions. The elution was followed b y r e a d i n g the a b s o r p t i o n of each eluate at 405 n m a n d the p u r i t y of .each f r a c t i o n was tested b y t h i n - l a y e r c h r o m a t o g r a p h y . Only the eluates c o n t a i n i n g a single pure ester vcere collected a n d pooled ; after .evaporation, the ester was dissolved, i n a m i n i m a l a m o u n t of c h l o r o f o r m a n d kept at - - 2 0 ° i n the dark.
649
RESULTS. 1) Separation and identification of ]tee carboxylic porphyrins. T h i n - l a y e r c h r o m a t o g r a p h y (0,5-1 ~ g ) o f the porp h y r i n s synthesized b y acellular .extract S revealed the presence of seven p r o d u c t s (a to g) w i t h differ e n t Rf ; three of t h e m (a, e, g) migrate as s t a n d a r d u r o p o r p h y r i n III, c o p r o p o r p h y r i n III a n d protop o r p h y r i n IX. The 15near r e l a t i o n s h i p of Rf/Rf p,oto (this ratio was more const,ant t h a n the Rf itself) w i t h the c a r b o x y l i c groups n u m b e r o b t a i n e d for the porp h y r i n s a, e, a n d g [12] gave an estimation of the
The total a m o u n t of esters c o n t a i n e d i n chloroform solution were s p e c t r o p h o t o m e t r i c a l l y estima-
TABLE I.
Chromatographic and spectrophotometric data [or porphyrins synthesized by an acellular extract of Saccharomyces cerevisiae. Porphyrins synthesized by acellular extract I
Spots oi the chromatoplates
t
a I
[
• cOOHnumbo
).... (nm) Soret band
b I
1
i o,o4 i o,18 Ir_ . . 8. . . . . ~ _ _7,1 I 405
c
~4~
i
- -
d i - -
~e --i
f
Ig
' Standard porphyrins IUlrI° / Copro Proto III IX
i
1 o,34 !o,48 i0, l 0,ss 1 0,04 0,69 _6,1 _ 41 2,8 [. 2. . . 8 I 5,2 i 4 '!~ 2 4~-
403 - - 1402[ [ - [ -405 - - 1407 ~ - - 405 [ 402 i 407
The extract was incubated with 2.5 mM ALA, at 35 °, pH 7.2 for 3 h. Porphyrins were extracted and separated on silica gel chromatoplates (see methods for further details). The COOH number was calculated from the straight line obtained by plotting Rf,t:,,~oto against COOH number of the porphyrins a, e and g [11].
ted in micro- or m i l l i g r a m s of p r o t o p o r p h y r i n ester (en~.~~ = 171) ; after separation, each ester was estimated by using e,n~i ~ 171 for di- a n d t r i m e t h y l p.orphyrin ester, em~~ = 180 for t e t r a - a n d pentamethylester a n d em~t = 215 for hexa-, hepta- and octamethylester [5]. F l u o r e s c e n c e spectra of each ester was realized with a Safas r e c o r d i n g s p e c t r o p h o t o m e t e r equipped for fluorescence analysis. Mass spectra of esters wer~e realized w i t h a spectrometer AEI type MS9. 5) Other methods. P o r p h y r i n esters w e r e h y d r o l y s e d by 3N HC1 d u r i n g 24 hours, at 4 ~' in the dark. P r o t e i n s w e r e estimated by the m e t h o d L o w r y et al [9] w i t h lysozyme as a s t a n d a r d .
of
ALA dehydratase activity was m e a s u r e d by the method of Mauzerall [10] i n c o n d i t i o n s described by B u r n h a m [11] w i t h en,.~I of PBG given b y B u r n h a m : ~m.~ = 56 at ~ = 553 nm.
BIOCH1MIE, 1975, 57, n ° 5.
n u m b e r of c a r b o x y l i c groups for the other porp h y r i n s (table I). The spectral data of eluted porp h y r i n s confirmed their nature. 2) Separation and identification of porphyrin esters. T h i n - l a y e r c h r o m a t o g r a p h y of p o r p h y r i n esters showed also seven fluorescent spots. The relationship of Rf/Rf ~roto~m w i t h the n u m b e r of carboxymethyl groups was l i n e a r for file seven e s t e r s : this suggested that the c a r b o x y m e t h y l groups n m n b e r of these esters was decreasing from eight to two a n d that each spot c o n t a i n e d only one ester. I n similar m a n n e r , esters c h r o m a t o g r a p h y (5-10 my) on silica c o l u m n gave seven fractions, each c o n t a i n i n g a n almost pure (95 p. cent) porp h y r i n i n t e r m e d i a t e whose spectral characteristics a n d c h r o m a t o g r a p h i c b e h a v i o u r were i d e n tical w i t h those of the c o r r e s p o n d i n g esters eluted from the p l a t e s ; these data are p r e s e n t e d i n tables II and III. 45
650
N. B r o u i l l e t , G. A r s e l i n D e C h a l e a u b o d e a u
The homogeneity of esters eluted from the plates or f r o m t h e c o l u m n w a s p r o v e d b y t h i n - l a y e r
a n d C. V o l l a n d .
c h r o m a t o g r , a p h y of e a c h s e e n i n f i g u r e 1.
solution
as it c a n
be
TABLE I I
Characteristics of the p o r p h y r i n e s t e r s obtained f r o m p o r p h y r i n s s y n t h e s i z e d by an acelIular extract of S a c c h a r o m y c e s c e r e v i s i a e . Esters of potpbyrins syl)lhesized by extract S Spots
i
2
I
t ti,o [cop~o t Prolo
7 IIIIMEjlIIMEIIXM --I----I t
E
3
A
5
6
0,53
0,64
0,~9
0,~0
0,29
0,78
1
407,? 406,51 403 ' 404
402
~5 40, 407,5
402
409
624,5
629 632i 628
624 - ~ 3 3
> for the esterifieation, e x t r a c t i o n a n d s e p a r a t i o n of the esters. CO0 ME n u m b e r was calculated as for the free carboxylic porp h y r i n s (Table I). (a) : Xe~¢it~tio. : 404 rim. TABLE III.
Absorption spectra of p o r p h g r i n e s t e r s in chloro[orm.
),m.~(nm)
Porpbyrinesters obtained from t}}e inclibatim~ mixture ]I _ _ _ _ ~
Standard porphyrines((,r s
[ t~I~00111,'~ 7 COOLIE 0 I]OOIIE 5 COOLIE ~ f~IiOIIE 3 COOLIE 2 CliIiiE i Uro-]ll~] f,opro-ME Proto-II}] Soret IV III II
407,5 406,5 405 503 502,5 502 536,5 537,5 536,5 573 572 571 (600) t602) ] ! 627 626 ~625,5 i (647,5)](646,5)
404 1402 405 501 502 504,5 537 534,5 539 570 569,5 573 (598,5)](597) 624 [623 625,5 !
409 507,5 542 577,5 631
407,5 402 502 500 [536,5 534 573 569,5 /(601) (598) 627 623
409 507 542,5 577,5 632
Fro. 1. - - Silica Gel thin-layer chromatogram of porphgrinesters. 7~
Solvent s y s t e m : b e n z e n e - e t h y l a e e t a t e - m e t h a n o l (42.5:13.5:0.75, v / v ) . M : m i x t u r e of esters o b t a i n e d b y esterification of t h e p o r p h y r i n s synthesized by a n aeellular extract S i n c u b a t e d 'with ALA. Urn, Cm and Pix correspond to p o r p h y r i n e s t e r s s t a n dard. Spots (1: Urn-ME; 2: 7-COO ME; 3 : 6-COO-ME ; 5 : Copro-ME ; 6 : 3 COO ME ; 7 : Proto-ME) correspond to esters o b t a i n e d f r o m siliea gel c o l u m n (see t h e text).
BIOCHIMIE, 1975, 57, n ° 5.
Characterization of porphgrins synthesized by yeast. The mass spectra of the five slo-~, m i g r a t i n g esters (1 to 5) revealed the p r e s e n c e of m o l e c u l a r peaks c o r r e s p o n d i n g to the following m o l e c u l a r weights : 942, 884, 826, 768 a n d 71'0 w h i c h are exactly those of p o r p h y r i n e s t e r s h a v i n g from eight to four c a r b o x y m e t h y l groups. The mass spectra of th e s u p p o s e d di- and t r i e a r b o x y l i c p o r p h y r i n e s ter ga\,e less reliable results, m a y be due to the i n s t a b i l i t y of these molecules.
651
the chromatoplates , varied w i t h the t e m p e r a t u r e : some increased (oeta-, tetra- and t r i c a r b o x y l i c porp h y r i n s ) w h e r e a s others decreased (penta- a n d dicarboxylic porphyrins).
3) Determination of incubation conditions for porphyrins biosynthesis.
c) Effect of incubation time : The acellular extract S was i n c u b a t e d for 2 to 24 h o u r s at 35 ~ a n d pH 7. Although the total a m o u n t of p o r p h y r i n s was regularly e n h a n c e d w i t h the i n c u b a t i o n time, the a m o u n t s rec.o.rded in table IV showed that the c o n t e n t of each p o r p h y r i n i n c r e a s e d or decreased a c c o r d i n g to their n,ature.
The aim of these e x p e r i m e n t s (carried out several times w i t h 3 to 5 ml of extract S a n d
d) Effect of oxygen : D u r i n g previous studies made in this l a b o r a t o r y [1], it was observed that,
TARLE IV.
Time dependence of porphgrins synthesis catalysed by an acellular extract of S a c c h a r m n y c e s cerevisiae. Percent distribution of porpbyrins {b) Time of incubation
Total amount of porphyrins (a)
3h
6,7
8h
25
24 h
50
(a) Expressed (b) Expressed matoplates. Incubation as and titration of
81:[6 coo. ooo. coo. : 11
9,5
40
15
7,5
13 15 2,6
in nanomoles of protoporphyrin/ml of incubated extract S. in percentage of total amount of porphyrins eluted from chroin table I and see for the extraction, separation porphyrin.
2,5 mM ALA) was to k n o w the best c o n d i t i o n s of ~incubation for o b t a i n i n g the largest a m o u n t of porphyrins. a) Effect of pH : T h e largest a m o u n t of total porp h y r i n s synthesized after an i n c u b a t i o n of 3 h at 35 ° was obtained at pH 7-7,2; the c o n t e n t Cat pH 6 a n d pH 8 being about 20 p e r c e n t of the m a x i m u m value. The chromatoplates s h o w e d that at pH 6,5 a n d 7,0, the seven p o r p h y r i n s ~'ere p r e s e n t i n i n c u b a t i o n mixtures but above pH 7,5 u r o p o r p h y t i n III was ahnost the only porphy, r i n p r e s e n t a n d very slight a m o u n t s of other p o r p h y r i n s are synthesized. b) Effect of temperature : The i n c u b a t i o n was c a r r i e d out at pH 7 d u r i n g 3 h o u r s at several t e m p e r a t u r e s b e t w e e n 25 ° a n d 50 ° . The total a m o u n t of s y n t h e s i z e d p o r p h y r i n s , expressed i n p r o t o p o r p h y r i n , regularly i n c r e a s e d w i t h temperature, a n d twice as high at 50`0 t h a n at 350 . I n fact, however, the q u a n t i t y of .each i n d i v i d u a l p o r p h y rin, as m e a s u r e d after s e p a r a t i o n a n d elution from
BIOCH1M1E, 1975, 57, n ° 5.
4
2
COOH COOIt
i n absence of oxygen, the acellular extract S did not synthesize p r o t o p o r p h y r i n , b u t synthesized c o p r o p o r p h y r i n . T h i n - l a y e r c h r o m a t o g r a p h y of p o r p h y r i n s confirmed this observation a n d showed the p r e s e n c e of octa- to t e t r a c a r b o x y l i c p o r p h y rins, w i t h p r e d o m i n a n t c o n t e n t of u r o - a n d coproporphyrin. F r o m these results, the best i n c u b a t i o n c~nditions ,as described i n Methods, have been chosen for the s t a n d a r d assay. In addition, these results have been used to modify, ~in some eases, the relative a m o u n t of the different p o r p h y r i n s synthesized. 4) Effect of heating and of enzymatic inhibitors on enzyme systems involved in protoporphyrin biosynthesis. I n these e x p e r i m e n t s , the extract S h a d been heated or i n c u b a t e d w i t h e n z y m a t i c i n h i b i t o r s (metal chelators, thiol reagents) before i n c u b a t i o n w i t h ALA, in order to k n o w if the b i o s y n t h e t i c p a t h w a y could be stopped at some point, w i t h 46
N. Brouillet, G. A r s e l i n De Chateaubodeau a n d C. Volland.
652
a c c u m u l a t i o n .of some i n t e r m e d i a t e s ; therefore, it w a s h o p e d to get some i n f o r m a t i o n about the sensitivity of the different e n z y m a t i c steps t o w a r d s these different effectors. a) Heating of extract S : W h e n the extract S was heated for 5 m i n u t e s to t e m p e r a t u r e s b e t w e e n 35 ° a n d 70 ° , the total a m o u n t of p o r p h y r i n s subsequently synthesized decreased w h e n temper a t u r e r e a c h e d 55 ° and, at 70 ° , no synthesis was observed. T h i n - l a y e r c h r o m a t o g r a p h y of these
150
x y l i c p o r p h y r i n s decreased w i t h i n c r e a s i n g conc e n t r a t i o n of i n h i b i t o r s , w h i l e that of the tetra- to d i c a r b o x y l i c p o r p h y r i n s increased. I d e n t i c a l e x p e r i m e n t s made w i t h n o n dialysed extract S showed that the p o r p h y N n - s y n t h e s i z i n g system was less inhibited' b y OP (OP 10-2M gave 65 p. cent i n h i b i t i o n ) t h a n i n dialysed extract. Similarly, 1.0-eMi EDTA i n h i b i t e d slightly this enzymatic system '(23 p. cent i n h i b i t i o n ) and, at low c o n c e n t r a t i o n , no activation was noted.
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