Plant Physiol. (1986) 81, 603-6080032-0889/86/81/0603/06/$0 1.00/0

Neutral Peptidases in the Stroma of Pea Chloroplasts'Received for publication December 18, 1985 and in revised form February 27, 1986

XIANG-QIN LiU AND ANDRt T. JAGENDORF*Plant Biology Section, Cornell University, Ithaca, New York 14853

ABSTRACI

One endopeptidase (EPI) and at least three aminopeptidases (API,AP2, and AP3) were discovered in the stroma of chloroplasts isolatedfrom pea seedlings (Pisum sativum L.), and purified over 100-fold. EPIrequires added Mg2" or Ca2" for activity, may have an additional tightlybound metal atom, and is inhibited by sulfhydryl reagents but not byserine residue-directed inhibitors. It is reversibly inhibited by dithiothre-itol. Its specificity is for the bond between two adjacent Ala or Glyresidues. Its molecular mass is 93 kilodaltons, estimated on a gel filtrationcolumn. Aminopeptidase activities were detected with the aid of differentamino acyl-f-naphthylamides as substrates. They were resolved into atleast three individual proteins by gel filtration and DEAE-cellulosechromatography, having apparent molecular masses of 269,000 (API),84,000 (AP2), and 42,000 (AP3) daltons, respectively. Each has a uniquespecificity for substrates, with API hydrolyzing only the Prolyl-B-naph-thylamide. None of the APs require added divalent cations for activity,but the possibility of a tightly bound metal function was suggested inAP2 and AP3 (not API) from effects of inhibitors. A probable sulfhydrylresidue function was indicated for all three, from inhibition by p-hydrox-ymercuribenzoate and Zn2". All these peptidases had pH optima at 7.7.

Specific protein turnover or selective protein degradation hasbeen found to be an essential part of chloroplast development.In many cases there are reasons to think that the responsibleproteases are located inside the chloroplasts. Proteolytic activitywas postulated to be responsible for prolamellar body degrada-tion when etiolated leaves are put in the light (5); for the removalof damaged QB apoprotein, and therefore for its high turnoverrate (10, 15); for accelerated turnover of chloroplast-encoded 48and 34.5 kD polypeptides in thylakoids lacking PSII (1 1); andfor rapid degradation of NADPH:Pchlide reductase in barleyetioplasts once the plants are put into the light (9). During leafsenescence, extensive protein degradations occurring in appar-ently intact chloroplasts are explained by an internal set ofproteases (21). Chloroplast localization of peptidases was notedin wheat by Waters et al. (22). The best defined internal proteaseis the one in pea chloroplasts which processes ribulose 1,5-bis Pcarboxylase small subunit precursors imported from the cytosol;this was purified to a considerable degree ( 19).A thylakoid-located, ATP-dependent proteolytic activity was

found responsible for removal of a significant fraction of newlytranslated proteins in pea chloroplasts, perhaps those which aremature but unassembled into larger complexes (12, 14). On theother hand, an ATP-independent but Mg2+-dependent proteo-lytic activity seemed to be responsible for removal of newlytranslated proteins containing abnormal amino acids, and of

Supported in part by grant 83-CRCR- 1-1280 from the United StatesDepartment of Agriculture Competitive Research Grants Office, Photo-synthesis Program.

prematurely terminated proteins (13). In an effort to locate thespecific enzyme responsible for one or another of these activities,we have found and report here the occurrence of a Mg2-dependent soluble endopeptidase, and of three distinct amino-peptidases, in the stroma of pea chloroplasts.

MATERIALS AND METHODS

Intact chloroplasts were isolated from 8- to 10-d-old pea plants(Pisum sativum L., cv progress No. 9, from Agway Corp.), usinga Percoll gradient centrifugation method as described in Fish andJagendorf (8). Substrates for peptidase activity assays were allfrom Sigma Corporation.

Endopeptidase Activity Assay. In a standard assay, the reactionmixture (0.2 ml) contained 50 mM Hepes-KOH (pH 7.7), 8 mMMgC92, 0.5 mm substrate, and 0.1 unit of EP12 (endopeptidase1). The system was incubated at 37°C for 30 min. The reactionwas stopped by adding 50 gl of 5% SDS. The activity wasindicated by the release of free amino groups, measured eitherby fluorescence after adding OPA, or colorimetrically usingTNBS as described in (16) and (6), respectively. The substraterefers to succinyl-(Ala)3-nitroanilide, except in the substrate spec-ificity experiments (Table II). One unit of EPI was defined asthe amount which catalyzes the release of 1 ,umol of free aminogroup per min under the standard conditions.

Aminopeptidase Activity Assay. The standard assay mixture(0.2 ml) contained I mm substrate (one of various amino acidnaphthylamines, see below) and 0.1 unit ofAP in 50 mM Hepes-KOH (pH 7.7). Incubation was at 37°C for 30 min. The reactionwas stopped with 100 ,ul of 24% TCA. Activity was indicated bythe release of free naphthylamine which was measured by amethod described in (17). Briefly, a 300 ,ul sample was mixedwith 300 pl of 15 mm NaNO2 and incubated for 3 min at roomtemperature. Then 300 Iul of 43 mm ammonium sulphamate wasadded. After 2 min at room temperature, 600 pl of 2 mM N-( 1-naphthyl)-ethylenediamine hydrochloride was added, and thesample incubated at 37°C for 60 min. The A at 560 nm wasmeasured. One unit of AP was defined as the release of 1 gmolof free naphthylamine/min under standard conditions.

Purification of the Peptidases. Chloroplasts equivalent to 75mg of Chl were isolated from pea plants as in (8). These intactchloroplasts, after washing with an isotonic buffer (350 mMsorbitol in 50 mm Hepes-KOH buffer [pH 8.2]), were pelletedby centrifuging at 1,000g for 5 min. The pellet was resuspendedin 100 ml of 50 mm Hepes-KOH (pH 7.7). The lysate wascentrifuged at 27,000g for 30 min to pellet thylakoid membranes.The resulting supernatant was taken as stromal extract andpurified as described below.DEAE-Cellulose Column. The column (2.5 x 16 cm) was

2 Abbreviations: AP, aminopeptidase; EP, endopeptidase; NEM, N-ethylmaleimide; OPA, o-phthaldialdehyde; PHMB, p-hydroxymercuri-benzoate; TNBS, 2,4,6-trinitrobenzenesulfonic acid; PMSF, phenylme-thylsulfonyl fluoride.

603

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LIU AND JAGENDORF

washed and equilibrated with a buffer containing 50 mM Hepes-KOH (pH 7.7) and 50 mM NaCl. After sample loading, thecolumn was washed with 60 ml of the equilibration buffer.Elution was done with 300 ml of 50 mM Hepes-KOH buffer (pH7.7) containing a NaCl gradient of 50 to 300 mm at a flow rateof 2 ml/min. Three ml fractions were collected. The AP2 elutedat about 125 mm NaCl, and EPI at about 250 mm NaCl. Alloperations were done in a cold-room (about 4°C).Sephadex G-200 Column. The column (2 x 60 cm) was

preequilibrated with a buffer containing 50 mM Hepes-KOH (pH7.7) and 100 mm NaCl. A concentrated sample of 0.2 to 1.0 mlwas loaded onto the column. The same buffer was used forelution at a flow-rate of 0.3 ml/min, and 1.5 ml fractions werecollected.

Elastin-Cellulose Column. One-half g of finely powdered elas-tin (Sigma, 200 mesh) was added to a suspension of 25 mgcellulose in 300 ml of 50 mm acetate buffer (pH 4.5). Afterstirring for 30 min at room temperature, the material was packedinto a column and washed extensively with 50 mm Hepes-KOH(pH 7.7). The sample was loaded in 50 mM Hepes-KOH buffer(pH 7.7) and eluted by a NaCl gradient of 0 to 1 M in the samebuffer. All operations were done at 4°C.HPLC Column. An HPLC gel filtration column (TSK-G 3000-

SW, 7.5 x 300 mm, from LKB) was used. The column wasequilibrated with 100 mM NaH2PO4, 100 mm Na2HPO4, and100 mm NaCl (pH 7.0). After loading of20 to 70/4l concentratedsample, the column was eluted with the same buffer at a flow-rate of 0.1 ml/min, and 0.2 ml fractions were collected.

o -

j 50-

o, 450

-J140

0 20 30 40 50 60RETENTION TIME ( MIN )

FIG. 1. Mol wt estimation. All were done on the HPLC gel filtrationcolumn as described in "Materials and Methods." Mol wt standards (O)were pyruvate kinase (237 kD), aldolase (160 kD), serum albumin (68kD), egg albumin (45 kD), and Cyt C (1 1.7 kD).

SUCCINYL--ALA--ALA--ALA--NITROANILINE

(1(ENDOPEPTIDASE)

SUCCINYL--ALA + A

SUCCINYL--ALA--ALA +

ALA--ALA--NITROANILINE

ALA--NITROANILINE

RESULTS (AMINOPEPTIDASE)

Detection and Purification of EP1. When succinyl-(Ala)3-ni-troaniline was used as substrate, production of the yellow colorof free nitroaniline was detected in chloroplast homogenates onlywith added MgCl2 (data not shown). This activity was releasedonly upon breakage of the chloroplasts, indicating its intraorga-nellar location. A small amount of activity (about 10% of thetotal) was detected with unbroken chloroplasts from the Percollgradient; but this was the result of slow breakage of the chloro-plasts during the incubation. When the chloroplast homogenatewas separated into thylakoid membrane and stroma fractions,the activity was found exclusively in the stroma. The activity wasabolished by heating, indicating its protein nature. Starting withthis substrate, release of free nitroaniline actually required thecooperative action of the endopeptidase and an aminopeptidase(see below); both of these were soluble enzymes of the stroma.The sequence of a DEAE-cellulose column (ion exchange) and

Sephadex G-200 (gel filtration) column gave 75-fold purification(Table I) of the endopeptidase over the crude stroma extract.They removed almost all of ribulose 1 ,5-bis P carboxylase proteinwhich accounted for more than 80% of the total protein in thestroma extracts. Further purification was achieved on a presumedaffinity column containing elastin and cellulose. The enzymefraction collected from this column, although over 100-fold

Table I. Purification ofEPIDetails for each purification step are described in "Materials and

Methods."

Step Protein Activity Spctvitc Recovery Purification

mg units unit/mg % foldStroma 747 1495 2.0 100 (1)DEAE-cellulose 62 960 15.5 64 7.8Sephadex G-200 17.7 820 46.3 55 23DEAE-cellulose 3.4 510 150 34 75Elastin-cellulose 0.9 195 217 13 109

SUCCINYL--ALA

SUCCINYL- -ALA- -ALA

2 NITROANILINE

3 ALA

FIG. 2. Scheme for the hydrolysis of Succinyl-(Ala)3-nitroaniline bythe peptidases.

purified, was not pure, since 3 to 5 bands were seen on a SDSpolyacrylamide gel (data not shown). The HPLC gel filtrationcolumn, though effective in the purification of AP2 (see below),resulted in extensive denaturation ofEP 1. However, this column(see "Materials and Methods") could be used to estimate the molwt of EPI, as 97 kD (Fig. 1). This estimate was confirmed usinga Sephadex G-200 column (data not shown).

Characterization of EP1. The site of cleavage of succinyl-(Ala)3-nitroaniline was determined by analyzing the ratio ofproducts formed. The substrate was incubated with the purifiedEPI and an excess of partially purified AP2 (see below). Thereaction products were analyzed for free Ala colorimetricallyusing TNBS (see "Materials and Methods"), and a minor contri-bution from nitroaniline (estimated from its A at 410 nm) was

subtracted. The ratio of Ala to nitroaniline, measured in thisway, was 1.5 to 1. This ratio indicates that the EPI is able tocleave only between two internal Ala residues, with neither ofthem in either the N-terminal or carboxy-terminal position (seeFig. 2 for an illustration of the way in which the predictedstoichiometry occurs). This conclusion was further supported byanalysis of the cleavage products using TLC (data not shown).Seventeen other peptides were also tested as substrate for EP1.Among them, only two were cleaved by the enzyme (Table II).The presence of either Mg2" or Ca2" was essential for EP1

activity (Fig. 3A), with saturation occurring at 4 mM. Replottingthe data indicate a Km for MgCl2 of 0.64 mm (Fig. 3B). Surpris-ingly, other divalent cations not only failed to permit activity bythemselves, but also strongly inhibited the Mg2+-stimulated EP1activity (Table III). Among them, Zn2+ was the most inhibitory,

i I

_ API:269 KD

'-,° EPI:97KD- o' AP2:84 KD

o,q& AP3 42 KD

_~~~~~~~~~~~~~~~-o

fo[)r|XXWL

604 Plant Physiol. Vol. 81, 1986

r, r,

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CHLOROPLAST PEPTIDASES

Table II. Cleavage ofPeptides by EPJAll peptides were prepared as 100 mm stock solutions in DMSO, and

assayed under standard conditions (see "Materials and Methods"). Rel-ative activity of 100 is equivalent to 217 units/mg protein. Abbreviations:-NA, -p-nitroanilide; -MCA, -7-amino-4-methyl-coumarin; CBZ-, N-carbobenzoxy; BOC-, N-tert-butoxycarbonyl.

Substrate Relative ActivitySuccinyl-Ala-Ala-Ala-NA 100CBZ-Gly-Pro-Gly-Gly-Pro-Ala 293Succinyl-Ala-Ala-Pro-Phe-NA 14Succinyl-Ala-Pro-Ala-MCA 0Succinyl-Ala-Ala-Phe-MCA 0Benzoyl-Phe-Val-Arg-NA 0Benzoyl-Pro-Phe-Arg-NA 0BOC-Glu-Glu-Leu-methyl ester 0BOC-Ala-Tyr-Met-Asp-Phe-amide 0CBZ-Val-Gly-Gly-Benzyl ester 0CBZ-Pro-Leu-Gly-amide 0CBZ-Phe-Ala-Alanine 0CBZ-Ile-Gly-Glycine 0CBZ-Val-Tyr-methyl ester 0CBZ-Val-Leu-methyl ester 0CBZ-Met-Gly-ethyl ester 0CBZ-Ile-Leucine 0CBZ-Phe-Methionine 0

z

w

0

cr: 180

E

120z

, 60!-0

0 2 3 4 5 6 7 8 9CONCENTRATION (mM)

FIG. 3. A, Dependence of EP I activity on Mg2+ or Ca2+. Assays weredone under the standard condition (see "Materials and Methods"), exceptthat the 8 mM MgCl2 was replaced with MgCl2 (A) or CaCI2 (E) at theindicated concentrations. B, Measurement of Km for MgCl2. Activity ofEP 1 was measured in the presence of different concentrations of MgCl2.Reciprocal of the activity was plotted against reciprocal of MgCl2 con-centration.

while Mn2" was the least. Fifty % inhibitions occurred at 6 ,uMZn2+ and 10 jAM Cu2+ (data not shown). Neither NaCl nor KCI,at up to 100 mm, had any effect on the activity.DTT was inhibitory to EPI activity, with 50% inhibition at

about 1 mM (Fig. 4).This inhibition was partially relieved byglutathione in its oxidized form, within 5 min after addition(data not shown). The inhibition was also partially relieved whenDTT was removed by dialysis.

Buffers, containing a mixture of 50 mm sodium acetate and100 mm Hepes, were titrated with KOH to a series ofpH valuesranging from 5.5 to 8.2. When EPI was assayed in these buffers,a sharp pH optimum at 7.7 was observed (Fig. 5).The sulfhiydryl-modifying reagents NEM and PHMB strongly

inhibited EPI activity (Table III). The heavy metal chelators1, 10-phenanthroline and 2',2'-dipyridyl inhibited the activity

605

Table III. Effects ofInhibitors on the PeptidasesAssays were done under the standard condition (see "Materials and

Methods"), except that inhibitors were included in the reaction mixturesat the indicated concentrations. When the metal chelators EDTA, 1,10-(or 1,7-)phenanthroline, and 2',2'-dipyridyl were used with EPI, extraMgCl2 was added to keep the free Mg2' concentration at 8 mM.

InhibitionInhibitor Concentration

EPI API AP2 AP3

NEM 5 mM 85 0 9 5PHMB 1 mM 100 100 100 100Bestatin 100 FM 0 0 90 0Leupeptin 150 ,g/ml 12 0 0 0Pepstatin 100 gg/ml 26 0 0 0PMSF 2 mM 0 0 0 0ZnCl2 15 AM 100 90 92 98CuSO4 500,gM 100 0 88 100MnCI2 500,FM 10 3 5 0FeSO4 500 Mm 1 5 2 5 5EDTA 2 mM 65 0 0 01,10-Phenanthroline 2 mM 100 10 100 1001,7-Phenanthroline 2 mM 14 5 60 342',2'-Dipyridyl 10 mm 95 5 50 75

Z 160 \0

0~~~~~~~X- 120O-\

80-z

0

>- 40> ^s0-A

0IIII

O 2DTT (mM)

3 4

FIG. 4. Inhibition of EPI by DTT. Assays were done under thestandard condition (see "Materials and Methods"), except that DTT waspresent in the reaction mixture at the indicated concentrations.

WIU-10z

w 8CI--

>- 0r 6t> cys E 4C

z 2C

-uu

0// a

0 4 6 8 10pH

FIG. 5. Effect of pH on EPl and AP2 activities. Assays were doneunder the standard condition (see "Materials and Methods"), except thatthe standard buffer (50 mM Hepes-KOH [pH 7.7]) was replaced by aseries of assay buffers. The assay buffers contained 50 mm acetic acidand 100 mm Hepes titrated to the indicated pH values with KOH.Relative activity of 100 for EPI (A) and AP2 (0) is 165 and 850 units/mg protein, respectively.

(A)

-a~~~~~~~~~0-/0 a~~~2

12

3 ~~~4-2 0 2 4 6

MgCL2 (MM-')

/;U4*,

_

3

^

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606 LIU AND JAGENDORF

even in the presence of 8 mm free Mg2+. The nonchelating analog1,7-phenanthroline, the serine protease inhibitor PMSF, and thevacuole protease inhibitor leupeptin all had little or no effect onEPI activity.Aminopeptidase Detection and Purification. Crude stromal

extracts were assayed for aminopeptidase activities, using differ-ent amino acyl-,B-naphthylamides as substrates. Activities fordifferent substrates ranged from most active with Met-NA toleast active with Pro-NA (Table IV). Activities for Asp-NA, Glu-NA, Cys-NA, His-NA, Val-NA, and Ile-NA were undetectableunder the conditions used. All these activities were absent in thethylakoid membrane fractions, and were not accessible withunbroken chloroplasts (data not shown).A crude stromal extract was run through a DEAE-cellulose

column as described in "Materials and Methods." Aminopepti-dase activities appeared in three peaks designated as API, AP2,and AP3, respectively (Fig. 6). Each peak was collected separatelyand run through an HPLC gel filtration column (see "Materialsand Methods"); none of them was resolved into separate peaksactive with individual amino acid adducts (data not shown).Their apparent mol wt were estimated on the same columncalibrated with mol wt standards (Fig. 1). API was the biggest(269 kD), followed by AP2 (84 kD) and AP3 (42 kD).

Table IV. Detection ofAminopeptidase Activities in StromaaCrude stroma extracts were assayed under the standard condition (see

"Materials and Methods") for each substrate listed. Abbreviation: NAfor ,B-naphthylamide.

Substrate Activity

units/mg Chl equivalentMet-NA 85Phe-NA 76Leu-NA 69Ala-NA 67Trp-NA 60Gly-NA 34Lys-NA 27Tyr-NA 1 7Arg-NA 9Ser-NA 4Pro-NA 3

a No activity detected with naphthylamides of Asp, Glu, Cys, His, Val,or Ile.

Z ALA GLY TRP LEU TYR0 PRO ARG LYS MET PHE0100 0

U- 90 f

cr 80-w

70I-0%l

50 I420

/0

La.0 o

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20FRACTION NUMBER

FIG. 6. Resolution of the aminopeptidases. Stromal extract was runon a DEAE-cellulose column as described in "Materials and Methods."Each fraction was assayed for its activity against the indicated substratesunder the standard condition (see "Materials and Methods").

Plant Physiol. Vol. 81, 1986

Table V. Specificity ofAminopeptidasesThe three aminopeptidases (API, AP2, AP3) were obtained by col-

lecting the three individual activity peaks from the HPLC gel filtrationcolumn. Each of them was assayed for all of the substrates listed, usingthe standard conditions (see "Materials and Methods"). Relative activityof 100 for APl, AP2, and AP3 was equivalent to 25, 500, and 615 units/mg protein, respectively. None of the three aminopeptidases were activeon naphthylamides (-NA) of Asp, Glu, Cys, His, Val, or Ile.

Relative ActivitySubstrate

API AP2 AP3

Pro-NA 100 0 0Ala-NA 0 100 0Gly-NA 0 80 0Lys-NA 0 40 0Arg-NA 0 10 0Met-NA 0 0 100Phe-NA 0 0 89Leu-NA 0 0 79Trp-NA 0 0 71Tyr-NA 0 0 20

Each of the three aminopeptidases was collected from theHPLC column and assayed for its activity with different sub-strates (Table V). APl was specific for Pro-NA; AP2 hydrolyzedonly Ala-NA, Gly-NA, Lys-NA, and Arg-NA; AP3 was activewith Met-NA, Phe-NA, Leu-NA, Trp-NA, and Tyr-NA. As inthe crude extract, Asp-NA, Glu-NA, Val-NA, Ile-NA, Cys-NA,and His-NA were not hydrolyzed by any ofthe separate enzymes.Each of the enzymes was partially purified by the sequence of

DEAE-cellulose and HPLC gel filtration steps. Details of thepurification procedure for AP2 are shown in Table VI. Specificactivity ofAP2 was increased more than 100-fold by the isolationprocedures used. The enzyme, however, is not yet homogeneous,since SDS gel electrophoresis revealed several protein bands stillpresent.

Characterization of the Aminopeptidases. The aminopepti-dases were assayed for their pH-optima. Assay buffers contained50 mM acetic acid, 100 mm Hepes, and 50 mM Tris, titrated withKOH to a series of pH values ranging from 5.0 to 8.9. They allshow maximal activity at pH 7.7, as exemplified by the pH curveofAP2 (Fig. 5).None of the aminopeptidase activities required the addition of

divalent cations, consistent with the observation (Table III) thatEDTA had no effect on their activities. The pattern of effectiveinhibitors (Table III) varied for each aminopeptidase. Hydrolysisby AP2 and AP3 were strongly inhibited by 2 mm 1, 10-phenan-throline and 10 mM 2',2'-dipyridyl, both strong heavy metalchelators, while the nonchelating analog, 1,7-phenanthroline,inhibited much less. APl was unaffected by these chelators, andalso by Cu2" which inhibited AP2 and AP3. All three wereinhibited by Zn2+ and by PHMB, a strong sulfhydryl reagent,but not by the weaker reagent NEM. AP2 was unique in beinginhibited by bestatin, an inhibitor for aminopeptidases of mam-malian cells (1).

DISCUSSION

Despite the well-documented specific protein turnover in chlo-roplast biogenesis (5, 9-15, 19, 21), relatively little has beenknown about the chloroplast proteases responsible for theseturnovers. Efforts in detecting and studying chloroplast proteaseswere made difficult by the proteases' high specificity and lowactivity, and by contaminating proteases from vacuoles or othersources (20). By starting with washed intact chloroplasts we wereable to get around these problems. The peptidases described herehave a slightly basic pH optimum, whereas vacuole proteases

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CHLOROPLAST PEPTIDASES

Table VI. Purification ofAP2Details for each purification step are described in "Materials and Methods."

Step Protein Activity Specific Activity Recovery Purificationmg units unit/mg % -fold

Stroma 747 4953 6.6 100 (1)DEAE-cellulose 125 3265 26.1 66 3.9Sephadex G-200 39 3285 84.2 66 12.7DEAE-cellulose 11 2638 324 53 36.2HPLC gel filtration 1.2 897 747 18 113

have acidic pH optima. Furthermore, isolated fractions enrichedin mitochondria and nuclei showed little or no peptidase activi-ties (data not shown).The neutral, divalent cation-requiring endopeptidase is a pre-

sumptive protease, although not a serine protease as indicatedby failure of PMSF or leupeptin to inhibit. Its specificity, asdeduced from the range of synthetic peptides tested, is for thepeptide bond between at lest two consecutive Ala or Gly residuesflanked by short chain amino acids. Thus, there was no activitywith succinyl-ala-ala-phe-MCA, and rather low activity withsuccinyl-ala-ala-pro-phe-NA.

Behavior of the enzyme on DEAE-cellulose and Sephadex G-200 columns was not changed by the presence or absence ofMg2" (data not shown), indicating that extensive changes insurface charge or molecular size were not caused by bindingdivalent cation which is probably needed for catalysis. In additionto the added divalent cation, the enzyme probably requires amore tightly bound heavy metal atom. This is suggested by thestrong inhibition by the heavy metal chelators 1, 10-phenanthro-line, 2',2'-dipyridyl and EDTA even when adequate levels offree Mg2" were still present; and further confirmed by ineffec-tiveness of 1,7-phenanthroline, a nonchelating analog of 1,10-phenanthroline.The most unusual aspect of this peptidase is its reversible

inhibition by DTT. This suggests, of course, that in vivo it mightbe more active in the dark than in the light, since a number ofstroma enzymes are reduced by the thylakoids and/or thiore-doxin in the light (2). Regulatory aspects of this endopeptidaseare yet to be investigated.EP1 characteristics are different from those of the processing

protease characterized by Robinson et al. (19), which had a molwt of 180 kD, a pH optimum at 9.0, and was insensitive to thiolprotease inhibitors. On the other hand, EPl may be the enzymewhich degrades immature or abnormal polypeptides in thestroma (as, for instance, those containing amino acid analogs;see Ref. 13). This degradation process, like the EPI activity,required the presence of free Mg2", was inhibited by PHMB or1, 10-phenanthroline, and was resistant to PMSF or leupeptin(13). Furthermore, similar levels of EPl activity were detectedin chloroplasts isolated from pea plants of different ages (datanot shown), indicating that its physiological function is probablynot restricted to the senescent condition.Of the three stromal aminopeptidases defined here, API was

actually an iminopeptidase, since it was active only on Pro-NA.Its large molecular size (269 kD) is reminiscent of the even larger(400 kD) iminopeptidase found by Waters and Dalling (23) inwheat primary leaves.AP2 was apparently specific for short chain amino acids (Gly-

NA, Ala-NA), and surprisingly, also for basic amino acids (Lys-NA, Arg-NA). These activities comigrated as a single band onboth isoelectric focusing gels and nondenaturing polyacrylamidegels (data not shown), so it seems possible that they belong to asingle protein.Although AP3 was specific for the long chain, hydrophobic

amino acid Leu-NA, it failed to cleave the rather similar Val-

NA and Ile-NA substrates. It was also active on Trp-NA andTyr-NA which are quite different structurally from Leu-NA.

All of the aminopeptidases are probably sulfhydryl enzymes,since they are strongly inhibited by PHMB and heavy metals.AP2 and AP3 may have a bound heavy metal functioning incatalysis, since they are inhibited by 1,10-phenanthroline and2',2'-dipyridyl. The evidence, however, is not conclusive, sincea nonchelating analog, 1,7-phenanthroline, also inhibited theiractivities, although to a lower extent. The pH optima (pH 7.7)of the aminopeptidases are both consistent with their stromallocation, and helpful in explaining the observation that degra-dation of ribulose 1,5-bis P carboxylase protein by isolatedchloroplasts (at pH 4.5-5.0) could be detected by SDS-PAGE,but not by measuring the release of free amino acids ( 18).Aminopeptidases were detected in pea seeds and in pea seed-

lings (3, 7), and it seems probable that one or more of theenzymes described here should have been present in the crudeextracts. Subcellular locations of the peptidases were not deter-mined in the earlier work. The pea seedling contained twoaminopeptidases, one sensitive to PHMB but not Zn2+, and theother vice-versa. The activities separated here appear to be sen-sitive to both of these reagents, and so may not be identical tothe ones in the crude extract. Wheat chloroplasts were demon-strated [22] to contain a Leucyl-Tyrosyl dipeptidase (not lookedfor in the current series) and neutral aminopeptidase activities,probably analogous to the ones described here.No carboxypeptidase activity was detected in pea chloroplasts,

using N-carbobenzoxyl derivatives of different amino acids assubstrates (data not shown). In an analogous finding, carboxy-peptidases in wheat leaves were found to be located exclusivelyin vacuoles (22).

LITERATURE CITED

1. BOTBOL V, OA SCORNIK 1978 Degradation of abnormal proteins in intactmouse reticulocytes: accumulation of intermediates in the presence of bes-tatin. Proc Natl Acad Sci USA 70: 1554-1558

2. BUCHANAN BB 1980 Role of light in the regulation of chloroplast enzymes.Annu Rev Plant Physiol 31: 341-374

3. CALDWELL JB, LG SPARROW 1980 Purification and characterization of anunusual aminopeptidase from pea seeds. Aust J Plant Physiol 7: 131-140

4. COLLIER MD, DR MURRAY 1977 Leucyl-fi-naphthylamidase activities in de-veloping seeds and seedlings of Pisum sativum. Aust J Plant Physiol 4: 571-582

5. DEHESH K, K APEL 1983 The function of proteases during the light-dependenttransformation of etioplasts to chloroplasts in barley. Planta 157: 381-383

6. DIELDS R 1979 The measurement of amino groups in proteins and peptides.Biochem J 124: 581-590

7. ELLEMAN TC 1974 Aminopeptidases of pea. Biochem J 141: 113-1188. FISH L, AT JAGENDORF 1982 High rates of protein synthesis by isolated

chloroplasts. Plant Physiol 70: 1107-11149. HAUSER I, K DEHESH, K APEL 1984 The proteolytic degradation in vitro of

the NADPH-protochlorophylide oxidoreductase of barley (Hordeum vulgareL.). Arch Biochem Biophys 228: 577-586

10. KYLE DJ, I OHAD, CJ ARNTZEN 1984 Membrane protein damage and repair:selective loss of a quinone-protein function in chloroplast membranes. ProcNatl Acad Sci USA 81: 4070-4074

11. LETo KJ, E BELL, L MCINTOSH 1985 Nuclear mutation leads to an acceleratedturnover of chloroplast-encoded 48KD and 34.5KD polypeptides in thyla-koids lacking photosystem II. EMBO J 4: 1645-1653

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