Isolation, characterization and partial amino acid sequence of a chloroplast-localized porphobilinogen deaminase from pea (Pisum sativum L.)

Biochimica et Biophysiea Acre, ]0760991) 29-36

© 1991ElsevierSci~mc~PublishersB.V.(BiomedicalDivision)0167-4838/91/$03,50 ADONIS 0|67483891000620

29

BBAPRO33791

Isolation, characterization and partial amino acid sequence of a chloroplast-localized porphobilinogen deaminase from pea ( Pisum satioum L.) Anthony J. Spano and Michael P. Timko Dtpartment o/Biology. Universityof Virginia. Chadotte~ville. 7A fU.S.A.)

(Received27June1990} Keywords: porphobilinogcndeamin~e;Chloroplast:Cltlotophyllbiosynthesis;(P~sum) Porphobilinogen dearninase catalyzes the condensation of four porphobilinogen monopytrole units into hydroxymelhylbilane, a linear tetrapynele necessary for the formation of cldurophyll and heine in hillher plant cells. We report file puxilieation to homogeneity of a cblorOl~-'t.localized form o| the enzyme from pea (Pisum ~ t i v u m L) by a novel pmilicafion scheme incolvinlgdye-ligend aff'mity chromatography. The pufilied chloroplast porphobilinogen deaminase consists of a single polypeptide with a relative molecular mass of 36-45 kDa as determined by slze-excluslon chromatography and sodium ~deeyl sulfate polyacfflamide gel electrephoresis, The isoeleetrlc point of the protein is acidic. The activity of the enzyme shows dllferant levels o! sensitivity to divalent cations and is most sensitive to Fe z4". The amino terminus of pea enzyme has been oblained by microsequeneing and determined to bear little similarity to the amino acid sequences of pu~pbubilinogendeaminases purified from other organisms. Polyclonal anti~ra elicited against the purified proiein has been used to examine the abumlance and cellular distribution of the enzyme. Introduction Tetrapyrroles and their derivatives, notably the homes, sirohemes and chlorophylls, play an important role in the growth and differentiation of higher plant cells. Home is present in all cell types, where it functions as the prosthetic group of integral membrane components (e.g., cytochromes of plastidic and mitochondtial electron transport chains) as well as numerous soluble proteins (e.g., catalase, pvroxidase, nitrite and sulfite reductases). In contrast, the chlorophylls are restrictvd to the chloroplast thylakoid membranes in photosynthetic cells where they serve as chromophores in the Photosystem I and Photosystem II reaction centers and light-harvesting apparatus. In all organisms studied thus far the first committed step in tetrapyrrole formation is the synthesis of 8aminolevulinic acid (ALA) [1,2]. In some prokatyotes and most eukaryotes the formation of ALA is mediated

Abbv.-.viations:ALA, 8-aminolevaliaicacid; SDS.PAGE,~tium dodecyl sulphate polyacrylaroide gel electrophoresis" PBG, porphobilir~ogen. Correspondence:M.P.Timk¢~Departmentof Biology,University of Virginia,Charlottesville,VA 22901,U.S.A.

by the soluble enzyme ALA synthase, in yeast and animal cells, ALA synthase has been demonstrated to be a nuckar-encoded, mitochondria-localized activity [1,2]. In higher plants and green algae the formation of ALA is mediated by a series of plastid-localized en. zymatic activities involving novel tRNA intermediates [1]. Two molecules of ALA are subsequently converted imo one mol~ule of porphobilinogen (PBG) by the activity of a porphobilinogen synthasc. Four monopyr. role PBG units are then condensed into a linear tetra, pyrrole, termed hydroxymethylbilane, by the enzyme porphobifinogen deaminase (hydroxymethylbilane syn* thase, EC 4.3.1.8) [3]. Hydroxymethylbilane is cyclizcd and isomerized to yield uroporphorinogen IIl, the precursor of all biologically active homes and chlorophylls. The biochemical and kinetic properties of porphobb [inogen deaminases from a variety of prokaryotic and eukaryotic organisms have been reported [4,27,5]. The exact sequence of steps involved in the assembly of the linear hydroxymethylbilane on the porphobilinogen deaminase backbone is also known [6-8]. Additionally, it has now been demonstrated that the assembly of hydroxymethylbilane on the porphobilinogen deaminase backbone involves a dipyrromethane ~factor [7,8]. Cloned cDNAs or ganomic sequences encoding porphobllinogen deaminase have been isolated and characterized from a variety of organisms including E.

30

cell [9], Eugtena [10], yeast [11] and animal cells [1233]. As a consequence of its involvement in certain genetic porphyrias, considerable effort has been devoted toward understanding the molecular genetic mechanisms active in coatroll;,ng the synthesis and activity of porphobilinogen deaminas¢ in mammalian cells {13]. Little informatioo is presently available on the synthesis and regulation of ~!his protein in higher plants. it has been previously suggested that duplicate pathways for tctrapyrrole synthesis exist in higher plant cells, namely ~ chloroplast-localized pathway for the synthesis of chlorophyll and plastid heme and an extraplastidic patbway for heroe synthesis [14,15]. The extent of this duplication and its significance are unknown. These dual pathways, however, must be coordinated and integrated since they share a common pool of biosynthetic intermediates [2]. As a step toward understanding the molecular genetic control of tetrapyrrote formatioo in plant cells, we have examined the chloroplast-localized porphobiiinogen deaminase activity from pea, (Pisum sativum L.). We describe a novel and efficient procedure for the purifi. cation of porphobilinogen deaminase from plant tissue. The properties of the purified plastid-localized enzyme and its developmental and cell.specific distribution are discussed. Materials e~l Methods

P~ant materials and growth Pea seeds (Pisum sativum L. vat. Progress No. 9) (obtained from Burpee Seed Company) were imbibed in distilled water overnight at room temperature and planted in moistened vermiculite. Plants used for enzyme isolation were grown at 28°C undr 16 h of illumination for 10-14 days prior to harvesting of leaves. Plants used in greening studies were grown in total darkness at 28<)C for 10-14 days. Etiolated leaf tissues were harvested from a portion of the plants, immediately frozen in fiquid nitrogen and stored at - 8 0 ° C . The remainder of the plants were transferred to a separate g r o ~ h chamber and illuminated at 4000 hix from a bank of coobwhite fluorescent tubes for the times indicated. All manipulations of dark-~o~n plants were performed under green safclights |16]. Kc~t ti.~sue was collected from 10-14-day-old greenhouse grown plants or the actively growing terminal portions of radicles of 3-4,day-old seedlings germinated on moistened blotting paper. Chloroplast isolation and purification of porphobilinogen deaminase Structurally intact, functional chloroplasts were isolated from pea leaf tissue as described previously 117]. Briefly, leaf material was homogenized in batches of 400-500 g in 800 ml of ice cold isolation buffer (0.33 M

sorbitol, 50 mM Hepes-KOH, pH 7.5, 5 mM sodium ascorbate, 2 mM Na2-EDT,~ 1 mM MgCI 2 and 1 mM MnCI2) in a Waring blender at low speed for 5 s. Following filtration through four layers of miracloth, chloroplasts were collected from the filtrate by centrifugation at 4300 × g for 5 rain. The pelleted plastids were resuspended in the a~ove izolatioo buffer and intact plastids purified by sedimentation through Percoil density gradients [1"1]. The band corresponding to intact plastids was carefully removed from the gradients, diluted with 5 vols. of isolation buffer and the plastids were collected by centrifugation as described above, The purified, washed chloroplasts were held at 4°C until further use. Unless specified, all subsequent purification step were performed at 0 - 4 ° C . Percolbpurified chloroplast~ isolated from about 0.5-1.5 kg of leaf tissue were resuspended 50-150 mi of buffer containing 100 mM Tris-HCI (pH 8.0), 5 mM Naz-EDTA, 10 mM benzamidine~HCl and I m g / m l PMSF. Plastids were incubated on ice for 30 rain to allow for gentle lysis. The plastid lysate was centrifuged for 15 rain at 20000 × g. The resulting supernatant was carefully removed and divided into 5 ml aliqnots. Individual aliquots were heated for 10-12 rain at 60°C and then rapidly chilled to 4 ° C by immersina in an ice-water bath. The heat-treated lysate was clarified by centrifugation at 1 0 0 0 0 x g for 10 nfin and the resulting supemamnt concentrated to a final volume of 2 - 3 ml in an Amieoo concentrator equipped with a PM 10 membrane. The concentrated extract (2-3 ml) was clarified by centrifugation for 10 rain at 10000× 8 and applied immediately to a 1.5 × 100 cm column of Sephadex G-100 (120 mesh) equilibrated in 10 mM Tris-HCl (pH 8.0) and 50 mM NaCI. TI',~ flow rate through the column was maintained at 20-30 m l / h throughout the run. Fractions during from the Sephadex column were colleeted and those with the highest enzymatic activity were pooled and dialyzed overnight against 10 mM "fris-HCl (pH 8.0). The dialyzate was applied to a I × 10 cm Reactive Red 120 Sepharose column equilibrated in 10 mM Tris-HCI (pH 8.0) buffer at a flow rate of 10-15 ml/h. "lhe column was washed with 10-15 ml of the equilibration buffer and the enzyme was eluxed using a linear gradient from 0.0 to 1.5 M NaCI in the same buffer (total eel. 30 mi). To locate enzyme activity, aliquots were taken from the column fractions colleeted~ diluted 4-8-fold in assay buffer and porphobilinogen deaminase ae).ivity determined as described below. The most active fractions were pooled and used in the studies described in the text.

Preparation of tissue extracts ];or enzyme activity measurements during development Approx. 0.5 g fresh weight of leaf or root tissue were ground in a glass homogenizer at 4 ° C in 2 ml of buffer

31 containing 100 mM Tris-HC! (pH 8.0). The extract was cl~.rified by centrifugation for 5 rain at 12000 × g and aliquots of the supernatant were taken for enzyme assay and protein determination.

Analytical methods Porphobi!inogen deaminase activity was assayed according to the method of Williams, et al. [18]. Protein content was determined by either the Bradford procedure [19], the BCA method [20] or spcctrophotometrirally by reading Azs o. Denaturing polyacrylamtde gel electrophoresis and isoetectric focusing Denaturing SDS-PAGE was performed on 12.5% (w,/v) polyacrylamid¢ gels [21] with minor modifications [22]. Electrophoresis was carried out at room temperature at 100 V during migration through the stacking g~l (1 cm) and 175 V through the resolving gel (10 cm). Protein bands w~c visaalized by staining with silver i33l. Isoelectric focusing was carried out in 4~ (w/v) acrylamide gels (110 mm long) containing 5% (v/v) Ampholine (Ampholyte pH 3-10; Sigma) cast on Gelbond according to the manufacturer's instructions (Pharmacia-LKB). Gels were prefocused at 500 V for 15 min prior 1o loading samples and protein samples were run at 1000 V for 30 rain followed by 2000 V for 30 rain. Throughout the run the apparatus was maintained at 8 ° C Samples were applied to a small paper tab placed on the surface of the gel at positions near the cathodic and anodie ends of the gel. In all cases, samples containing standard proteins or the porphobilinogen deaminase migrated to identical positions in the gel when loaded at either end, indicating a migration to isoclectrie equilibrium had occurred. The locatinn of the porphobilinngen deaminas¢ in the gel was located by in situ staining of the enzyme as previously described [5]. The isoeleetric point was calculated by slicing a gel strip into 5 mm portions, incubating ',he slices in 2 ml of 10

mM KCI at 25°C ff]r 2 h with constant shaking and measuring the pH of each solution,

Amino acid sequence analysis Purified porphobiiinogen deaminas¢ was electro. blotted from 15~ (w/v) SDS-polyac~lamide gels (1,5 mm thickness) in Immobilon-PVDF membranes (Millipore Corp., Bedford, MA), Transfer was allowed to proceed to completion for 3 h at 90 V (about 400 mA). Microscquence analysis (automafed Edman degradation) was performed directly from Immobilon strips i231. Preparation of po(yclonal antibodies and Western blot analysis Polyclonal antibodies were raised in guinea pigs using SDS-denatured protein as antigen. Purified porphobilinogen dcaminase was electrophoresed on either 12% (w/v) or 15~ (w/v) non-denaturing polyacrylamide gels run in the Tris-glycine buffer system of Anderson and Desnick [5]. Gel strips containing the pure enzyme (approx. 10-15 tLg) were equilibrated at room temperalure in 125 mM Tds-HCI (pl-I 6.8) for 30-60 rain and then in the same buffer containing 0.2~ (w/v) SDS for an additional 3 0 60 rain. The gel slices were crushed and mixed with either complete Freund's adjuvant (1 : 2 v/v) (first injection) or incomplete adjuvant (1 : 2 v/v) (subsequent three injections). Animals were injected every 10 days and the scra was tested for immunereactivity after 4-6 weeks. Preimmune sera collected prior to injection showed no immunoreactivity against pure pea porphobilinogen deaminasc or against any crude plant extracts. Wcstcru blot analysis was camcd out as pTeviously described [22]. Immtmoreactive proteins were visuafized

by using an alkaline phospbatase-conjugated goat antiguinea pig second antibody [24]. Extracts used for Western blots were prepared from etiolated leaf, greening leaf or root tissue by grinding approx. 2 g of tissue in 5 mt of a 10 mM NH4HCO 3

TABLE I Puri/icolion~f lhe pea chtoroptastporphobitinogendeamina.re Step

Volun~ (ml)

l~otein (mg/ml)"

Total activily,

Specific activity

(units)'~

(units/ring)

Purification (-fold)

Recovery (perCent)

Crude ~tracl Heamcl

170

12.00

10606

S.2

extract Sephadex

1"/0

6,38

9869

9.1

1.7

93

(3-100

18

1.48

5744

215,6

41,1

53

0.06

2723

4880.0

934.0

25

R~ti~e ted 120 Scpharose

9.3

Eslin~atcd by absorbance at 280 nm. b One unlt is defined as the formatioh o~ 1 nine1 of ~;roporphyrin t per h.

tO0

32 (pH 7.7) buffer at 4 ° C until the tissue was completely disrupted. The extract was centrifuged for 20 rain at 100O0×g at 4 ° C and the snpernatant fluid was removed to a clean tube, frozen in fiquid nitrogen and lyophyllized to a powder. The powder was resuspended in water and aliqnots were used for protein determination and SDS-PAGE. Ile~ulis ~

Discussion

Isolation of o chloroplast-localizedporphobitinogen deamiA chloroplast-localized form of porphobilinogan deaminas¢ has been purified to homogeneity from pea (Pisum ~ativum L.) leaf tissue. The results of a typical purification are presented in Table !. In the course of our investigation, we found that the pea porphobilinogun deaminase binds tightly to Reactive Red t20 Sepharose. The enzyme can be eluted efficiently from this dye-figa~d affinity matrix at a relatively high salt concentration, thus effocting a substantial purification when compared with the enzyme obtained after the Sephadex (3-100 step (Fig. 1). The specific activity of the pure enzyme from pea chlorop!:-~. ~ is comparable to that of the enzyme isolated from Euglena PSI and is substantially higher than those reported for enzymes purified from other sources [see ReL 18 and references therein]. Under our isolation conditions porpobilinogen deaminase activity in crude plastid lysate is stable for at least 2 weeks when frozen at - 2 0 ° C . The highly purified porphobilinogen deaminase, however, is relatively unstable and its activity decays after several days when stored at either 4 ° C or - 2 0 ° C . While we have not undertaken a systematic survey of conditions aimed at stabilizing the purified enzyme's activity, we have observed that the addition of reductant (dithiothreitol, 1.0 mM), altering pH (over a range of pH 6.0-8.0), or cold o.lo

2,1~

o.~e

~ ,20

D,02

OAO

z

o.oo : . -

2

4

§

:

= - ~" ooc.

;B 10 12 14 1E 1B 2U 22 2# E'5 28 10 32

Fig. 1. Chromatography of pea e . . h l o ~ p l a s t porphobilinage, dcaminasc (PBOD) on Reactive Red 120 Scphazose. Pooled and dialyzed peak fractions obtained after Sephadex O-100 chromatography were applied to Ihc dyc-ligand affi~ty ¢olanm and PBOD activity eluted wilh

a linear gradient of NaCI. The bulk oi PBGD activitydales belweea 0,6-0+8 M NaCL Proteincontent(O)and PBGD activity(,,) (A~/50 Id/hr) in the individual |factions (1 ml eat]i) were measured as describedin the Materialand Methods.

storage ( - 2 0 ° ( ? ) in the presence of 30~ (v/v) glycerol does not prevent loss of activity. Factors, such as low protein concentration, that result during the final purification steps may contribute to the loss of activity of the highly purified enzyme preparation. Both the pea chloroplast and E. colt porphobitinogen deaminases (dam not shown) can be elated from Reactive Red 120 Sepharose by washing with 100-200 ~tM PBG in 10 mM Tris-HCi (pH 8.0). A pyrrole binring site common to both enzymes may be involved in the interaction of these proteins with the dye-figand affinity matrix, Alternatively, a PBG-induced conformational change in these two proteins may be the basis for their identical eintion properties from this column. Binding and elation of porphobifinogen deaminase on Reactive Red 120 Sepharose appears to occur in a manner consistent with a substrata-protein interaction. Our ~¢sults are in marked contrast to previous attemWs to purify porphobilinogen deaminase using PBG-linked affimty chromatography where elation with PBG was observed to be nonspecific [18). The purification of porphobilinogen deaminase suitable for kinetic analyses by Reactive Red 120 Sepharose chromatography using PBG containing buffers requires the subscqu:nt removal of residual PBG prior to assay. We have found thai removal of PBG can be achieved by multiple centrifugations using a Centricon PM 10 device. If should also be possible to utilize various s u b stratus (e.g., analogs or chemically modified derivatives of PBG) with this matrix to probe conformational inter. actions in porphobilinogen deaminases isolated from various sources. For routine purposes, we prefer to use NaCI gradients to elute the enzyme

Properties of the purified pea chloroplasz poFphob~.!inogen deaminase The enzyme preparation pooled from the most active fractions dated from the Reactive Red 120 Scpharose column consists of a single silver-stainlng protein band of M, approx. 45000 (Fig. 2B and C). This molecular weight estimate is similar to that reported previously by Castelfranco et aL [25] for u covalent [I'tC]PBG-PBGD complex in pea chloroplasl lysates. The reproducibly smaller size estimate (M, = 36000) we obtained for the native form of the enzyme during chromatography on Sephadex O-100 (Fig, 2A) is likely an underestimate of its molecular mass and is probably the result of sfightly anomalous migration under our experimental conditions. Our data are consistent with the purified native protein existing as a monomer in rive. Isoelectric focusing of the purified enzyme or crude plastid lysates followed by in situ detection of porphobilinogen deaminase activity gave only one detectable band which migrates to a position correspmtding to a p l of 4 . 4 :E 0.2 (n ~ 3 + S.E.). The isocloctri¢ point for the pea porphobilinogen deaminase is similar

33 4.90

B

A

4.80

I-

,o /¢

4.70 0D

4.6D 4.50 -

,?, 0'5 O

q

•..,

4.40~:

PBGD ~

4.304,20-

ili:~:

4.104.00 57

7'7

8'7

9'? 1C'7 117

o.o o:2 o:4 o16 o:8

ELUTION VOLUME(ml)

U,

Fig. 2. Molecular we~ghl determination of the purified pea chloroplast porphobilinogen dcamiaase (PBGD). Panel A shows the chromatographic profile of PBGD oa Sephadex-G-[Q0against slal~xl, pxoteins. PBGD was loaded as a concentrated hcat-trc.ated fraction from plastic tysatcs (see Matez~als and Methods). Molecular mass staadanJs are the Following: t~viae serum albumin (BSA), 66 kDa; ovalbamin (OA), 45 kDa; carbonic anhydrase (CA), 29 kDa; cy~ocluomec (cyt c), 12.4 kDa. Highly purified PBGD was fractionated by SDS-PAGE as described in the Materials and Methods, The Mr was delcrmincd relative to the migration of standard pwt¢ins of known Mr (Pang| B). [n addition to the standard proteins indicated in Panel A, the following proteins were used: glyceraldehyde-3-phosphatedehydtogenase (G3PD}, 36,5 kDa; trypsin iah/,bitor (T]), 21 kDa; and lactalbemin (LA), 14 kDa. 'The gD~-PAGE p[ofiLeof the puttied PBGD following silver staining is shown in Panel C. The sample comamed O,5.ggprotein from Ihe puoted, dialyzed peak fraction taken ahcr Reactive Red 120 Sepharos¢ chromatography.

to that reported for the porphobilinogen deaminase from wheat germ [4] and both planbderived enzymes are more acidic than the crythrocyte enzyme ( p / = 6.26.6) [5l. Collectively our observations indicate that only a single molecular form of porphohilinogen deaminase is present within the pea chloroplast. We cannot, however, rule out that multiple forms of the enzyme exist which arc not resolved under our present fractionation condilions,

Effects of pH and divalent metal ions on enzymatic activity We have examined several biochemical character. istics of the purified pea chloroplast enzyme. The purified enzyme exhibits a broad pH optimum for catalyzing the condensation of 4 tool PBG to 1 mol uroporphyrinogen l and 4 tool ammonia. The highest detectable activity lies in a range [rum pH 7.9-8.2 (Fig. 3). This is consistent with the reported optima for other plant [26] and mammalian [5] enz?mes. The purified pea enzyme is sensitive to NH 2 ions with concentrations of ammonium acetate greater than 10 mM resulting in a 30% inhibition of enzyme activity compared to the untreated control. Similar results have been observed for enzyme preparations from wheat germ [26] and soybean callus [27]. Sodium acetate at 100 mM had no effect on enzyme activity, nor did NaCI

up to concentrations of 300 raM. Concentrations of NaCI greater than 300 mM resulted in a progressive inhibition of enzyme activity with a 50% inhibition of the control (without NaCI) at a concentration of 600 mM. The effect of various divalent metal ions on the activity of the purified pea porphobilinogen deaminaa¢ 0~70, 9.60'

KPhQs ~ . * BO~te *

0~SO.

~.

* •

e

a

~ o *

0.30,

0,20' 0.10-

0.00. - -

,

s.50 e,ao ~.,~0 7.ha ?.50 e.00 e,so ~.00 9,50 pH

Fig. 3. Eff~t of pH an pea chloroplast porphobilmogea deamJnase (PBGD) activi~+ The: enzymatic a¢livity of the highly purified pea chloroplast PBGD was assayed at various pHs as described in Materials and Methods. Either 50 mM pulassium phosphate (KPhos o. ~,) or 50 mM sodium borato (Borate O) adjusted to the indicated pH was ased in these experin~nts. Ezym¢ activity is expressed in terms o[" A~,~/50 p] dia]ysate/b. The b~t fit curve through the data points was generated using po]ynornial regression analysis or Sigma Plot Version 3.1 (Jandd Scientific).

34 TABLE |l Inhibitory effects of carious d~valem metal ions o,I partfi¢d pea porphobilinogen deaminaseaetivily Compound CaCI2 CoCI2 CuSO,=

Cou~eatratlon traM) 0.5 0.5 0.5

Percentiohibilion of activity = 8.5± 3.0 6g.8± 8.5 11.65:6.0

FeSO4

0.5

1~7,6:1:3.6

MgCI2 MnCI2

25.0 0.5 25.0 0.5

30.9+ 9.8 36.3+_ 2.1 94.9~ 2,3 62.8± 11.3

ZnCI2

Pel~eenlinhibitionaf enzymeacfivily was calculated rclalive lo a control sample to which no metal cofaclOts were added. Dala rcprcscnt the mean±standard etmr rot three experiments,except for FcSO,whichwas repeatedonce. was also tested (Table 11). We observed that Coz+, Mn~+, Fe 2÷ and Zn 2÷ were all strong inhibitors of enzyme activity, but that Ca 2" and Mg 2+ were only weakly inhibitory at physiological concentrations [281. This is in contrast to the human erythrocyte enzyme which shows a strong inhibition by Mg ~+ [5,341. We also observed that Mn2+ can inhibit the pea enzyme at submillimolar concentrations. These coneentrations of Mn2÷ approach those thought to be present with the chloroplast stroma [G. Berkowitz, personal communication] suggesting a possible role for this divolent metal ion in the control of metabolite flux through the heme pathway under physiological conditions. The physiological relevance of Co2- and Fe 2÷ are not as clear, since these ions are probably not be present in an uncomplexed form in these relatively high •:oneentrations in viva. Sequence of the amino terminus of the pea chloroplast porphobilinogen deaminase The purified pea chloroplast porphobilinogen deaminase was subjected to protein mierosequenee analysis. The protein was not blocked at its amino terminus and the following primary amino acid sequence data was obtained: Ser-Leu-Ala-Val-Glu-Gln-GIn-Thr-(GIn,Cys)-GIn-(Asp,Gln)-X-Thr -hla-Gly At residues 9 and 11 it was not possible to distinguish between the two amino acids shown in the oligopeptide sequence. Also, no assignment could be made for residue number 12 (indicated with an 'X' in the sequence). Comparison of the amino acid sequence of the pea chloroplast porphobilinogen demainase amino terminus with those reported from other organisms [911,13,29,30] did not reveal any significant similarities. It is interesting to note., however, that the determined

amino terminal residue (i.e., Ser) of the mature porphohilinogen deaminase polypeptide from pea is identical to that predicted for the mature Euglena enzyme [10]. From pair-wise comparisons, we and others [10] note the presence of considerable sequence heterogeneity among porphobilinogen deaminases from various organisms, particularly in the amino terminal portions of the polypcptide. Several short highly conserved domains are also present (see Ref. 10). Our data suggests that some sequence similarity must exist between the pea and E. colt enzymes, since both behave similarly during chromatography and elution from the reactive Red 120 Sepharose. This will only be resolved when the complete sequence for the pea enzyme becomes available. Smith 131] has also reported that antisera directed against the Eugtena porphobilinogan dcaminase shows a general immunocross-reaetivity with enzymes from several higher plant species including pea. Western blot analysis of the celhdar distribution and light.regulated accumulation of pea chloroplast phorphobtlinogen deaminase Polyclonal antisera generated against the purified pea porphnhilinogen deaminase detects a single polypeptide in soluble extracts of Percoll gradient-purified chloroplasts isolated from greenhouse-grown plants (Fig. 4). The antibody also detects only a single polypepfide band of apparently identical M~ in unfraetionated soluble extracts from leaves of greenhouse-grown plants (Fig. 4A). A faint irnmunoreaetive band was observed in soluble extracts of stem tissue using total protein concentrations equivalent to that in leaf extracts, in contrast, no immunoreaetive band was observed in extracts prepared from either the roots of 10-14-day-old greenhouse-grown plants, or elongating radicles of 3-4day-old seedlings even when protein concentrations up to 4-times higher than that present in leaf extracts were used (not shown). We could not detect porphobilinogen deaminase activity in extracts from 10-14-day-old pea roots, consistent with the lack of immunoreaetivity of the anti-PBGD antiserum against these extract. In younger tissues, however, low but detectable levels of porphobilinogen deaminase activity (approx. 0.19 n m d / m g protein per h) were found in these tissues. Therefore, the observed lack of immnnoreactivity in our root preparations may result from the inability of our antibody to detect the small amounts of enzyme protein present in these cells. Alternatively, our antibody might be unable to recognize the root-localized form of the enzyme. It has been reported [31] that almost all of the porphobilinogen dcaminase activity is localized exclusively in the chloroplast fraction in pea and Arum cells, even in non-photosynthetic tissue. While the present studies agree well in the case of pea leaf tissue, the basis for the discrepancy concerning root tissue to not clear,

35

A

B HOURS IN L[GH] a-

a

5

10

25

48

66-

4529-

21-

14-

Fig. 4, Western blo:. analyses of po~hobilinogen deaminase (PBGD) in various tissues and in etiolated and greening leaves from pea. Tile presence of PBGD in various tissue extracts is shown in Panel A. Tissues samples were extracted, fractiuaated by SDS-PAOE and blotted to n i t r o ~ l h i l ~ lor Western ana;ysis as described in the Material and Methods. A .,singleimmunoreacfiv¢ band similar to that observed in purified enzyme prepasatiooS (ENZYME) are found in NH4HCO J extracts of Pcrcoll-purified chloroplasts (PLASTID) or whole leaf (LEAF) extracts. Protein conccnlralio~ for the samples shown are 0.08, 0.50, 50 and 190 //8, respectively, Molecular mass standards are the following: bovine serum albumin, 66 kDa; ovalburain` 45 kDa: carbonic anhydras¢, 29 kDa; soybean trypsin inhibitor. 21 kDa; and laehalhumin, 14 kDa. In panel B the effc~t of light on ~he levels of immenodmectablc PBGD in leaf extracts is shown. Extracts prepared from ¢tinlated leaves and leaves illuminated for the lengths of time indicated were tracfionated, blotled and probed with antiPIKID sera as des¢~ihed in the Malenals and Methods. All samples contained 150 pg total protein. The position of the irnmunoreaetive hand corresponding zo PBGD is indieatd.

levels due to de nova synthesis. Under our experimental conditions we are able to detect as little as a 2-fold increase in porphobilinogen deaminase levels. While we can not rule out small changes in enzyme protein levels during greening, our data suggests that the increase in porphobilinogen deaminase activity that occurs can not solely be due to increased accumulation of the enzyme, but may also reflect a several-fold increase in enzyme activity, The results of this study contribute to the growing body of information on the structure of proteins involved in chlorophyll and heme synthesis in higher plant cells. This information will be useful in the design of future experiments to examine the molecular genetic mechanisms controlling tetrapyrrole metabolism in plant calls. They should allow us to resolve more thoroughly the cellular distribution of tetrapyrrole biosynthetic enzymes and the relative contributions of the plastidic and extraehloroplastic pathways to cellular home and chlorophyll formation.

Acknowledgments We would like to thank Dr. J. Fox at the University of Virginia Protein Sequencing Facility for conducting the protein mierosequenee analysis. This work was supported in part by an NSF grant (DCB-8711095) awarded to M.P.T. References 1 Kannangara, C.G., Gnugh. S.P,, Bruyant, P., H o ~ c r , J.K., K ~ . A. and Wettstein,D. (1988) TIBS t3, 139-143, 2 Castelfranco, P.A. and Beale, S.L (19831 in Amtnal Review of Plant Physiology, Voh 34 (Bnggs, W.R~ Jnncs, R.L and Wulbot,

but may reflect differences in the age of the tissue used, the precise method of preparation of the tissue extracts or antibody specificity. We also examined the effect of light on the levels and activity of chloroplast porphobilinogen deaminase. Porphobilinogen deaminase activity in leaf extracts increased approximately 6-fold during the fast 48 h of light.induced development from 0.5 nmol/mg protein per h in etiolated leaf extracts to 3.1 nmol/mg protein per h in 48 h greened material. Fig. 4B shows the results of a typical Western blot analysis of chloroplast porphobilinogen deaminase levels in pea leaves during greening. No significant change in the level of immunoreactive porphobilinogen deaminase was observed when extracts prepared from etiolated leaves (Fig. 4B, "0 h') were compared with. identically prepared extracts obtained from leaf tissues illuminated for 5, 10, 23 or 48 h. Smith [32] has previously reported a 3-fold increase in porphobilinogen deaminase activity upon illumination of etiolated pea leaf tissue for 60 h and suggested that increased enzyme activity results from increased enzyme

V., cds,), pp, 241-278, Annual Reviews. California. 3 nattershy, A.R. (1988) J. Natural Prod. 51, 629~642. 4 Higuchi, M. and Bugomd, L. (1975) Ann. N.Y. Acad. Sci. 244, 401-417. 5 AndersOn, P.M. and I)esnick, RJ. (1980) L Biol. Chore, 255, 1993-1999, 6 Hart, G.J., Miller, A.D. and Battersby, A.R. (1988) Binchem. J. 252, 9119-912.. 7 Warren, M.J. and Jordan, P.M. (1938) Biochemistry 27, 9020-9030. 8 Jordan, P.M. and Warren. MJ. (19871 FEBS Letters 22S, 87-92. 9 Themes, S.D. and Jordan, P.M. (1986) NAR 14. 6215-6225. l0 S'harif, A.L., Smith, A.G. and Abell, C. (19891 Ear. J. Biochem. 184, 353-359. It Gellerfors, P.L, Saltzgaher-Malter, J. and Do=81~, M.G. (19'd6) Biochem. J. 240, 673-676. 12 Raich. N,. Romeo, P.H., Dabart, A., Beaupain, D., Cohen-Solal, M. and Goosseas, M. 0986) Nucleic Acids Res. 14, 5955-5968. 13 Ch~tien` S., Dabast, A,, Beaupain, D., Raich, N., Grandchamp, B., Rosa, J., Goossens, M. and Romeo, P.H. (1988) Proe. Nail. Acad. Sci. USA 85, 6-tO. 14 Jacobs, /.M., lacobs, N,J. and De Maggio, A,F_ (1982) Arch. Bicchem. Biophys. 218, 233-239, t5 Little, H.N. and Jones, O.T.G. {1976) Biochem. J. 156. 309=314. 1.6 Schif|, J.A. (1972) Methods Enzymol. 24, 321-322. 17 Bartlett, S.G., Gressman, A,IL and Chua, N.H, 11982) in Methods in Chloroplast Molecular Biology (Edelmnn, M., Hallick, R.B. and

36 Chua, N+H., cos+), pp. I08L-1091, Elsevier Biomedical Pros, ArrL~lerdatrl. 18 Williaras, D+C+. Morgan. G.S.. McDonald, E. and Battersby, A+R. (1981) Biochem. J. 193, 301-310, ]9 Brantford, M.M, (1976) Anal. Biochem+ 72, 248-254. 20 Smith, P.K., Kxolm, P,-I., Hermanson, G.T., Mallia, A-K+,Garmer, F.H.+ P t ~ n o , M+D., Fujimolo, E+K+, Gc~ke+ N,M., Olson, B.J+ and Klenk. D.C. (1985) Anal+ Biochem, 150, 76-85. 21 LaemmlJ. U.K. (1970) Nature 227, 680-685. 22 Spano, AJ. and Schiff, J.A. (1987} Biochim. Biophys. Acta 394. 484-498. "~3 Matsmlaira, P. (19871 J. BioL Chem. 262, 10035-10038. 24 Miel~ndoff, R.C., Percy, C. and Young, R.A. (198"/) Methods En~mol. 152. 458--469. 25 CasteIfran¢o, P.A., "[hayer, S.S., Wilkinson, J.Q. and ik, nner, B.A. 0958) Ap~h* Biochem. Biophys. 266, 219-226. 26 Frydman, R.B. and Frydman, B. (1970) Arch. Biochira. Biophys. 136, 193-202.

2"/ 1.3ambias, E.B+ end D~I, C. and Bizttle. A. (1970) Biochim. Biophys. Acla 220, 552 559. 28 PorliS, A+IL and HeiSt, H.W. (39?6) Biochin'L Binphys. Acta 449+ 434-446. 29 Sv.ubnicer. A.C., Picat, C. and Grandcbamp, B. (1988) NAR lh, 3102. 30 Grandchamp, B., De Vcmeuil, H., Beaumont, C., Chrelien, S.~ Walte¢. O. and Nordmann, Y+ ([987} Eur. J. Biochem. 162, 105110. 31 Smith, A, (1988) Biochem. J. 249, 423-428. 32 Smith, A. (1986) in Rcgulalion of Chloroplast Diffcfcmialiort (Akoyano~lou, G. and Seager. H., eds.), pp+ 49r54+ Alan R. Lie.s, N~'w York. 33 Blum. H , Beier, H. and Gross, H.J. (198"]) El~tmphor~i~ 8, 93 99+ 34 F~l.man+ R+B. and Fein.stein+ G+ (19/4) Bioehim. Biophys. Acta 350, 3.58-373.