In vitro activation of δ-aminolevulinate dehydratase from far-red irradiated radish (Raphanus sativus L.) seedlings by thioredoxin f

Plant Science Letters, 32 (1983) 253--259 Elsevier Scientific Publishers Ireland Ltd.

253

IN V I T R O A C T I V A T I O N OF 5-AMINOLEVULINATE DEHYDRATASE FROM FAR-RED IRRADIATED R A D I S H (RAPHANUS SATIVUS L.) SEEDLINGS BY THIOREDOXIN f

A.P. BALANGE a and C. LAMBERT b aLaboratoire de Photobiologie, Laboratoire associd au CNRS (LA 203) B.P. 67-76130 Mont Saint Aignan and b U.E.R. Scientifique de Luminy, Ddpartement de Biologie Moldculaire et Cellulaire, Biochimie Fonctionneile des Plantes, 13288 Marseille Cedex 2 (Fraace) (Received November 30th, 1982) (Revision received June 13th, 1983) (Accepted June 18th, 1 9 8 3 )

SUMMARY

5-Aminolevulinate dehydratase (EC 4.2.1.24) has been f o u n d to be activated in vitro by dithiotreitol (DTT) and factors isolated from radish cotyledons grown under continuous far-red (FR) light. Cross experiments, between fructose 1-6 bisphosphatase (FBPase) system, and 5-aminolevulinate dehydratase, show t h a t these factors are functionally identical to thioredoxin f. •

Key words: Raphanus sativus -- 5 -Aminolevulinate dehydratase -- Fructose 1-6 bisphosphatase -- Thioredoxin -- Activation INTRODUCTION

A L A D transforms 2 8 ~aminolevulinic acids into one porphobilinogen (PBG, monopyrrole) and is the firstenzyme detectable in higher plants of tetrapyrrole biosynthesis [1]. W e have previously shown that A L A D activity is under phytochrome control [2]. After a cytoplasmic synthesis, A L A D is translocated into etioplasts [3 ]. Chloroplast enzymes are regulated in the light by a redox-system involving thioredoxins. Most of these enzymes are activated by reduction. Only glucose-6-phosphate dehydrogenase and phosphofructokinase are deactivated Abbreviations A L A D , 6-aminolevulinic acid dehydratase; DTT, dithiotreitol;FBPase, fructose i-6 bisphosphatase; FR, standard far-red light (k _~ 720 nm); M D H , malate dehydrogenase; PBG, porphabilinogen. 0304-4211/83/$03.00 © 1983 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

254

in the light [4]. The thioredoxin system has been shown to regulate metabolic pathways directly dependent on light for activity, through key enzymes such as FBPase, [5], or NADP-linked malate dehydrogenase (NADP-MDH), [6], but also enzymes belonging to pathways indirectly related to light such as phenyl alanine ammonia lyase [7] or 5-aminolevulinate synthetase [8]. We describe here the effect of this activation system on ALAD activity in vitro an d compare it with activation of the well-known FBPase system. M A T E R I A L S AND METHODS

Radish seeds (Raphanus sativus L. cv. Longue Rave Saumon6e) are sown as described previously [2], under continuous FR light. Standard FR, equivalent to monochromatic irradiation at 720 nm is obtained with incandescent lights Osram Linestra and plexiglass filters (RShm and Haas, Blue no. 627, Red no. 501). Light energy is 3500 erg. cm -2 s -1 (ISY radiameter). Spinach leaves (Spinacia oleracea) are purchased at local markets.

Purification of ALAD from etioplasts Fifty grams fresh wt. of radish seedlings, that were grown for 120 h from sowing under continuous FR light, are ground with 150 ml Tris--HC1 buffer 100 mM pH 9, containing D-mannitol 600 mM, ~-mercaptoethanol 2 mM, MgC12 10 raM, 3 X 5 s in a Waring blendor, fitted with a special razor blade bucket [9]. Brei is filtered through cheese cloth and the filtrate centrifuged at 100 × g for 15 min (Beckman J-21 B, Rotor JA-20). The supernatant is centrifuged at 1000 × g for 15 min. The pellet, which is enriched in etioplasts, is carefully resuspended in 20 ml buffer and centrifuged again at 1000 X g for 15 min. The purified etioplasts are disrupted by osmotic shock in 20 ml Tris--HC1 buffer 10 mM pH 8.7, containing MgCI: 10 mM, and centrifuged at 30 000 × g for 15 min. The supernatant contains ALAD in the soluble fraction from etioplasts (stroma). All these operations take place under dim green safelight at 2°C. Stromal fractions ( 2 0 - 3 0 ml) are purified by Sephadex G-50 gel-sieving and DEAE-cellulose chromatography (Trisacryl, Pharmindustrie IBF, Vflleneuve la Garenne, France) on columns equilibrated with Tris--HC1 10 mM, MgC12 10 mM, pH 8.7. The column is rinsed with the same buffer containing 50 mM NaC1. ALAD is eluted with a linear NaC1 gradient (50-350 mM, pH 8.7,180 ml).

Purification of thioredoxin f and FBPase Thioredoxin f is purified either from 100 g fresh wt. radish seedlings grown 120 h under continuous FR light, or from spinach leaves, using heattreatment (60°C, 4 min) to destroy enzymatic material, Sephadex G-50 gel-

255 sieving, [NH412 SO4 precipitation (30--90%) and DEAE-cellulose chromatography as described in Ref. 10. For preliminary experiments with an impure preparation of thioredoxin, DEAE,cellulose chromatography was omitted. Thioredoxin m was a gift from Dr. Jacquot (Universit~ d'Orsay). FBPase was partly purified according to Buchanan et al. [5] by acid precipitation, [NH412 SO4 precipitation and Sephadex G-50 gel-sieving.

Determination of enzyrnatic activities ALAD activity was determined from the a m o u n t of PBG formed [2], corrected for conversion o f PBG to porphyrin by measuring porphyrin [11]. The correction necessary for porphyrir~ formation from PBG was not more than 10%. Results are expressed in nM PBG formed per rain. FBPase activity is measured by the release of inorganic phosphorus (Pi) from fructose 1.6 bisphosphate [5]. Pi concentration is estimated according to Fiske and Subba-Row [12]. Results are the mean value o f 4 independent experiments. RESULTS AND DISCUSSION ALAD activity is increased in vitro after preincubation with thioredoxins and DTT, a non-physiological reducdant used for activation studies of m a n y enzymes [5 ]. After preincubation for 15 min in presence of thioredoxins and DTT without substrate, ALAD activity is enhanced (Fig. la). Thioredoxins are inactive when DTT is omitted, but increasing amounts of reductant up to 12 mM lead to a 2-fold increase in ALAD activity. Higher concentrations of DTT yield lcwered enzymatic activities (Fig. lb). The effect of pH during preincubation is shown in Fig. l c . ALAD activity is only detected between pH 7 and 9 in controls and high activation ratios are observed for pH 8.5--9, as reported also for other enzymes regulated by thioredoxin system [14]. The effect o f Mg2÷ is shown in Fig. l d . At low Mg2÷ concentration, e n z y m e activity is low, either with or without reductant. At high Mg2÷ concentration (above 20 raM), ALAD activity is high and no longer dependent on the presence of reductants. Thus, the highest activation ratios are measured for Mg2÷ concentrations ranging around 10 mM. The same observation of ion effect on FBPase activity was also previously reported [15]. These results indicate that ALAD activity may be increased by thioredoxins and DTT in vitro, as other molecules directly or indirectly regulated by light. For subsequent experiments, we used the following procedure for activation studies: ALAD is preincubated for 30 min in the presence of DTT 10 raM, Mg2÷ 10 mM, and thioredoxins, pH 8.7. Then substrate is added (5-ALA 16 raM, Tris--HC1 106 mM, pH 8.7) and aliquots are taken to measure PBG concentration formed from 5 -ALA vs. time. Activation ratio is expressed as percentage increase between ALAD activity with DTT and thioredoxins, and non-activated ALAD, incubated in the same way with buffer lacking activators.

256 %

T

10(

100.

=r ImO

3=0

5=0

-"

7=0 mn

1=0

"

2ZO

115

I

mM

U

a

%

%

..J

20C

20

.E

pH U

®

,%

100

7~

'8.'5

'

15

I

2115 • mM

Fig. 1. Conditions for ALAD activation by DTT and a crude preparation of thioredoxins. ALAD was partly purified from stromal fraction from etioplasts after Sephadex G-50 gel-sieving chromatography. Thioredoxins were partly purified (see Materials and Methods) except that DEAE-cellulose chromatography.was omitted. The complete mixture for activation contains: Tris--HC1 buffer pH 8.7 100 raM, MgCl2 10 raM, ALAD preparation 0.2 mg. DTT 10 mM and thioredoxins 0.05 mg are replaced by buffer in controls. After 30 min preincubation, 5 -ALA 16 mM in Tris--HC1 100 mM pH 8.7 was added. Activation is the ratio between activated and non,activated ALAD. Effect of varying: (a) preincubation time, (b) DTT concentration, (c) pH during preincubation time. pH was readjusted to 8.7 for tneasurement of ALAD activity. (d) Effect of varying Mg~+ concentration. Bars represent S . D . n = 4 It has been p r e v i o u s l y s h o w n t h a t t h i o r e d o x i n s are p r e s e n t in etiolated seedlings [ 1 6 ] a n d it is o f i m p o r t a n c e t o d e t e r m i n e if o u r c r u d e p r e p a r a t i o n really c o n t a i n s t h i o r e d o x i n s . We h a v e purified t h i o r e d o x i n f f r o m radish c o t y l e d o n s , a n d f r o m s p i n a c h leaves, a n d c h e c k e d their activity o n b o t h A L A D or FBPase.

257 TABLE I A C T I V A T I O N O F A L A D F R O M R A D I S H A N D FBPase F R O M S P I N A C H L E A V E S D T T A N D T H I O R E D O X I N f I S O L A T E D F R O M R A D I S H O R SPINACH.

BY

A L A D activity was determined as in Fig. 1. FBPase was partly purified according to Materials and Methods. The complete mixture for activation of FBPase contains Tris-HCI buffer p H 8 100 raM, MgCI~ 1.6 raM, D T r 10 m M , thioredoxin f 0.050 mg, FBPase preparation 0.060 m g in a volume of 0.5 ml. After 15 rain preincubation 0.05 ml of F B P 60 m M is added, and reaction is stopped after 15 min by adding 2 ml of mixture for Pi analysis.

ALAD from radish (nM PBG/min) FBPase from spinach (nM Pi/min)

Spinach thioredoxin f

Radish thioredoxin f

--DTT

--DTT

3.4 68 .

+ DTT

4.6 386

3.2 67

+ DTT

4.9 804

Table I indicates t h a t both thioredoxin preparations are efficacious on ALAD activity in the presence o f DTT. As it can be seen, spinach FBPase is activated by thioredoxins from spinach, as expected, and also by thioredoxins from F R irradiated radish seedlings. FBPase activation rates are generally quite different with thioredoxins from spinach (--~6) or from radish (~-12). This observation cannot be actually explained as protein concentration was identical in both cases. Furthermore, the activation ratio of ALAD from radish cotyledons is constant with these preparations (--~1.5). Two main thioredoxin groups are found in higher plants: thioredoxin f regulates all enzymes, except NADP-dependent MDH which is activated by thioredoxin m. Figure 2 shows that, in the presende of DTT, thioredoxin f from radish cotyledons is more efficacious than thioredoxin m for the same enzyme concentration. It can be observed too, that DTT alone increases 1.3fold ALAD activity, as generally reported [ 17 ]. Thus with thioredoxin f and DTT, the ratio of ALAD activation is around 2. Activation ratios for enzymes t h a t are directly switched on or o f f by light, like FBPase-or NADP-dependent MDH are higher [ 4 ]. Lower ratios are observed for enzymes indirectly affected by light (2--3, Refs. 7 and 8). We think that ALAD belongs to this group with a low activation capacity. We have previously observed that ALAD activity may be detected in etioplasts purified from cotyledons grown 96 or 120 h from sowing under continuous FR light. If the enzyme is purified through DEAE-cellulose chromatography, 2 peaks of activity are eluted by a linear NaC1 gradient [18]. Fractions corresponding t o each peak were pooled and checked for activation by thioredoxin f (Fig. 3). It can be observed that only the rapidly eluted ALAD fraction at 0.11 M NaCI concentration have an activation ratio o f 2.5. On the contrary, ALAD eluted at 0.15 M NaC1 concentration cannot

258 %

,>

Thioredoxin f

a

<{

.

Thioredoxin m "'0 . . . . ~' "

<.E

1

~oo.

,, ,~

Protein added

c

1%

2=0

4%

310

g.lO "6

Fig. 2. Effect o f increasing a m o u n t s o f t h i o r e d o x i n f or m on A L A D activity. T h i o r e d o x i n f were prepared as described in Materials and Methods. A L A D 0.2 mg was partly purified as in Fig. 1. Bars represent S. D. n = 4.

Increase %,

ALAD

in activity

.'T" i

200.

i |

I

',

n

e-

E o.

.-r-

0.3

C

>

t'"~:

0.15

o

/....

<

<

.,J

Lo

.-- ..........

,. . . . . . . t

.... r 110 i

P

NaCI 0

L 50 3~)

5zO

7ZO

//1810

mr-:

Elution Volume Fig. 3. DEAE-cellulose c h r o m a t o g r a p h y : elution profile o f A L A D purified f r o m etioplasts o f F R irradiated seedlings g r o w n 120 h f r o m sowing. E n z y m a t i c activity ( = ); NaCl gradient ( . . . . . . ). U p p e r part: fractions are separately p o o l e d and assayed as in Fig. 1. T h i o r e d o x i n f : 0 . 0 3 mg. C o n t r o l (v); A c t i v a t e d A L A D ( . . . . . . ).

259

be activated as previously reported [ 1 8 ] , these fractions have different molecular characteristics. The first eluted enzyme represents different agregation states, and activation m a y be the result of some u n k n o w n structural changes, probably reassociation of less or inactive subunits. Thus NADP glyceraldehyde-3-phosphate dehydrogenase activity is controlled b y dissociation leading to more active monomers [19]. A similar process might be expected for ALAD control of activity. On the other hand, the second eluted peak is composed of a stable molecule, 245 000 in mol. wt., which cannot be activated. At this time, we assume that thioredoxin f action is to give a constant structure to ALAD molecules, with a high activity. This report describes activation of ALAD b y thioredoxin system. It will be of importance to k n o w if the process observed in vitro," also operates in vivo under continuous F R light and involves p h y t o c h r o m e . REFERENCES 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

C.A. Rebeiz and P.A. Castelfranco, Annu. Rev. Plant Physiol., 24 (1973) 129. A.P. Balang~ and P. Rollin, Physiol. Veg., 17 (1979) 153. A.P. Balang~ and C. Lambert, Phytochemistry, 19 (1980) 2541. B.B. Buchanan, in: D.E. Atkinson and C.F. Fox (Eds.), Modulation of Protein Function Academic Press, New York, 1979, 345. B.B. Buchanan, P. Schiirmann and P.P. Kalberer, J. Biol. Chem., 246, (19) (1971) 5952. J.P. Jacquot, J. Vidal and P. Gadal, FEBS Lett., 71 (1976) 223. A.N. Nishizawa, R.A. Wolosiuk and B.B. Buchanan, Planta, 145 (1979) 7. J.D. Clement-Metral, FEBS Lett., 101 (1979) 116. C. Kannangara, S.P. Gough, B. Hansen, J.N. Rasmussen and D.J. Simpson, Carlsberg Res. Commun., 42 (1977) 431. R.A. Wolosiuk, B.B. Buchanan and N.A. Crawford, FEBS Lett., 81, (2) (1977) 253. C. Rimington, Biochem. J., 75 (1960) 620. C.H. Fiske and Y. Subba-Row, J. Biol. Chem., 66 (1926) 375. O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, J. Biol. Chem., 193 (i 951 ) 265. V.D. Breazeale, B.B. Buchanan and R.A. Wolosiuk, Z. Natiirforsch.,33 (1978) 521. P. Schiirmann and R.A. Wolosiuk, Biochim. Biophys, Acta., 522 (1978) 130. N.A. Crawford, B.C. Yee and B.B. Buchanan, Plant Sci. Lett., 22 (4) (1981) 317. D. Shemin, in: S.P. Colowick, N.O. Kaplan (Eds.), Methods in Enzymology, Academic Press, N e w York, 5 (1962). 883. A.P. Balang~ and C. Lambert, Plant Sci. Lett., 32 (1983) in press. O. Wara-Aswapati, R.J. Kemble, J.W. Bradbeer, Plant Physiol., 66 (1980) 341.