Amino acid sequence of spinach chloroplast fructose-1,6-bisphosphatase

ARCHIVES

OF BIOCHEMISTRY

AND

BIOPHYSICS

Vol. 279, No. 1, May 15, pp. 151-157,199O

Amino Acid Sequence of Spinach Chloroplast Fructose-l ,6-bisphosphatase’72 Frank

Marcus”

and Peter B. Harrsch4

Department of Biological Chemistry and Structure, University of Health Sciences/The Chicago Medical School, North Chicago, Illinois 60064, and Chiron Research Laboratories, Chiron Corporation, Emeryuille, California 94608

Received November

:3, 1989

The amino acid sequence of the spinach chloroplast fructose- 1,6-bisphosphatase (FBPase) subunit has been determined. Placement of the 358 residues in the polypeptide chain was based on automated Edman degradation of the intact protein and of peptides obtained by enzymatic or chemical cleavage. The sequence of spinach chloroplast FBPase shows clear homology (ca. 40%) to gluconeogenic (mammalian, yeast, and Escherichia coli) fructose- 1,6-bisphosphatases and 80% homology with the wheat chloroplast enzyme. The two chloroplast enzymes show near the middle of the structure a unique sequence insert probably involved in light-dependent regulation of the chloroplast FBPase enzyme activity. This sequence insert contains two cysteines separated by only 4 amino acid residues, a characteristic feature of some enzymes containing redox-active cysteines. The recent X-ray crystallographic resolution of pig kidney FBPase (H. Ke, C. M. Thorpe, B. A. Seaton, F. Marcus, and W. N. Lipscomb, 1989, Proc. Natl. Acad. Sci. USA 86,1475-1479) has allowed the discussion of the amino acid sequence of spinach chloroplast FBPase in structural terms. It is to be noted that most of pig kidney FBPase residues shown to be either at (or close to) the sugar bisphosphate binding site or located at the negatively charged metal binding pocket are con’ This work was supported in part by grants from the National Institutes of Health (DK 265fi4) and the U.S. Department of Agriculture (87-CRCR-l-2395 and 88-37262-4140). Part of this work was taken from the Ph.D. thesis suhmitted by P.B.H. to the Graduate School of the LJniversity of Health Sciences/The Chicago Medical School. ’ Portions of this paper, including the amino acid sequence data of all peptides used for establishing the sequence of spinach chloroplast fructose-1,6-hisphosphatase (Tables II and III) are presented as a Miniprint Supplement. ” Formerly at the University of Health Sciences/The Chicago Medical School. To whom correspondence should he addressed at Chiron Research Laboratories, Chiron Corporation, 4560 Horton Street, Emeryville, CA 94608. 4 Present address: Sterling Research Group, Sterling Drug, Inc., Great Valley Parkway, Malvern, PA 19355. 0003.9861/90

Copyright All

rights

$3.00 (~1 1990 hy Academic Press, &reproduction in any form

served in the chloroplast enzyme. The unique chloroplast FBPase insert presumably involved in light-dependent activation of the enzyme via a thioredoxinlinked mechanism can be accommodated in the surface of the FBPase molecule. @I 1990 Academic Press. Inc.

Fructose-1,6-bisphosphatase (FBPase)” catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6phosphate and inorganic phosphate. In green plant leaves, the enzyme exists as cytosolic and plastid isozymes (1). Leaf cytosolic FBPase participates in the pathway of sucrose biosynthesis and the enzyme has characteristics which are typical of gluconeogenic FBPases (2,3). The complete amino acid sequence of six gluconeogenic FBPases has been reported. These include pig kidney (4), sheep liver (5), rat liver (6), yeast Saccharomyces cerevisiae (7), yeast Schizosaccharomyces pombe (7), and Escherichia coli (8) FBPases. In contrast, chloroplast FBPase which is an essential enzyme in carbon dioxide fixation into sugars (9) has unique characteristics. These include insensitivity to AMP inhibition (10) and light-dependent activation via a ferredoxin/ thioredoxin system (11). The uniqueness of the chloroplast enzyme led us and others to believe (although not stated) that the chloroplast FBPase was structurally distinct from gluconeogenic FBPases. In 1985, however, we showed that strong sequence similarity exists between chloroplast and mammalian gluconeogenic FBPases (12). With this background, we continued our sequence analysis of the FBPase from spinach leaf chloroplasts. We have previously completed (13) sequence data for about 75% of the primary structure. In this paper, the entire sequence of the 358 amino acids of spinach chloroplast FBPase is presented and discussed. This is the sec’ Abbreviations used: FBPase, fructose-1,6-bisphosphatase; trifluoroacetic acid; CM, carhoxymethyl; PE, pyridylethyl.

TFA, 151

Inc. reserved.

152

MARCUS

SCh WCh PK SC EC SCh WCh PK SC EC

AND

HARRSCH SO

20

10 AAVGEA-A-TQTKARTRSKYEIETLTGWLLK-Q .V.DT.S.PAPAA..K..S.DMI...T....-. TDPAAFDTN.V...RFVME-. PTLVNGPRRDSTEGFDTD.I..PRFIIEH.

GEFIVQ-K

K.. 40 PMAGV-IDAELTIVLSSISLACKQlASLVQR EQE..-..N.M........T....V...... GRKAR-GTG.ll.PL.N.LCT.V.A.STA.RK LlKQFKNATGDF.L..NALQF.F.FVSHTIR. lJHEFSHATG...AL..A.K.GA.I.HRDIN.

SO

60

70 AGISNLTGIPGAVNIQGEDPKKLDVVSNEV .P......V...T.V..........I.... .AH.Y..A.ST.VT.DQV.....L..DL :;Lv.. V.LA..S.FT.DQ......LGD.l . . LVDIL.AS..E.V...V.Q...LFA..K

00

90

SCh WCh PK SC EC

100 FSSCLRSSGRTGIIASEEEDVPVAVEEBYS ..N...W.... .v................. VINV.K..FA.CVLVT..DKNAII..PEKR . 1NAH.A. GIIKVLV...QEDLIVF-PTNT LKAA. KARDIVAG.......EI.VF.GCEN

110

120

SCh WCh PK SC EC

130 GNVIVVFDPLDGSSNIDAAVSTGSIFGIVS . . . . . . . . . . . . . .K.V.C...........CL..I.T.....R . S.A. CC..I.....L..G..V.T.AS.FR AK.V.Lll..........VN..V.T..B..S..R

140

150

SCh WCh PK SC EC SCh WCh PK SC EC

.

.

.

.

.

.

160 P-NDECIVDSDHDDESPLSAEEQRCVVNVCQ . -6.. .HIG---..AT-.DEVT.H.I..... STDEP-.E-K.A--.------------. K-. L-LP-DSSGTI-N.V--.------------R RVTPVGT.VTE-E.F--.------------.

SCh WCh PK SC EC

190 PGDNLLAAGVCMVSSSVIFVLTIGKGVVAF . . S . . . . . . . . . . . . . . . ..R..V.... AL.G.ATML..A!lVN..NC. C.KEMV..CAA..G..THL...L.D..DG. ..NKPV..C.VV.G..TML.Y.l.C..N..

SCh WCh PK SC EC

220 TLDPMYGEFVLTSEKIQIPKAGKIYSFNEG . . . . . . . . . . . . cl..v....s......... tl.. . AI...I.VDRNVK.K.K.S...I... . . . TNL...I..HPNLR..PQKA...I... C.CQ.RMRF.EK..T..I... . Y..S.L.V.

SCh WCh PK SC EC

250 NYKHWPDKLKKVMDDLKEP-GE-SQKPYSSRY D . . . . . . . .S....-.T-.G....AK. . .AL. EFDPAITE.IERK.F.-PD-NSA..GA.. YA. NETIRTFIEKV.Q.PADNNN..F.A.. . TLV. . . IKF.NGV...IKFCP.E-DKSTNR..T...

SCh WCh PK SC EC

.

.

510 LRLLVECAPMSFIVEQAGGKGS-DGHQRILD . . . . . . . . . . . . . A........-..... AVVtl.K...LAT-T.KEAV.. . . . . . ..N.. A.LH......AVN.RQE.... . . . . . . AF.. A.LA......A.-..KE.... . . . . . ..N..

SCh WCh PK SC EC

540 350 IQPTEIHQRVPLYIGSVEEVEKLEKYLA A V. . . . . . . V. . . . . . . .n.. IIL..P.D.TE.LEIYQKHA(SSS) Il.. . .A. .v.. DKSSIWL..SQ.ID.FLDHIGKSQ~S47) LV.SH.. . I.ETL...RSFFV.NDNM..DV.RFIREFPDAO

.

.

.

..m

.

.

170

100

200

210

.

.

.

.

.

.

T...V.

240

250

260

270

200 290 IGSLVGDFHRTLLYGGIVGYPRDAKSKNQK . . . . . . . . . ..M......... V..M.A.V.... V.. . .Ftl.. .LFA..C.K..PN.. v..n. A.V.. . FV.. K....L.. . . . . . A . . ..N..

SCh WCh PK SC EC

.

300 8.0 . ANK..PK.. ST.

.

.

.

530 v..

V.

.

SNPD..

520

.

.

. F .

(358) 8 SE (358)

SPINACH

CHLOROPLAST

FRUCTOSE-1,6-BISPHOSPHATASE

ond chloroplast FBPase structure to become available, since a recent report (14)) has disclosed the nucleotide sequence encoding wheat leaf chloroplast FBPase and its deduced amino acid sequence. Comparisons made herein between the two chloroplast enzymes and other known FBPases should contribute toward understanding structure-function relationships in this important group of enzymes. MATERIALS

AND

153

eluted with a linear gradient from 0 to 30% acetonitrile in 100 mM Na phosphate (pH 6.3) over a period of 70 min at a flow rate of 0.7 ml/ min. The HPLC elution pattern revealed one major symmetrical peak eluting at about 36 min and a broad peak eluting at about 40 min. The fraction containing the major symmetrical peak (designated T-ins) was desalted by gel centrifugation chromatography (17) using a l-ml volume column of Sephadex G-25 equilibrated in 10 mM N-ethylmorpholine acetate (pH 8.5). The desalted peptide was lyophilized and stored at -20°C prior to sequence analysis. Tryptic digestion of PE-chloroplast FBPase was performed as described for the CM-enzyme (15).

METHODS

Several t,echniques used in this paper have been previously reported. These include spinach chloroplast FBPase purification and assay (15), S-carboxymethylation (4), and S-pyridylethylation (13). Other techniques are detailed below. CNHr cleauajie. Cleavage of S-carboxymethylated chloroplast FBPase with CNBr (4 mg/mg of protein) was performed in 70% formic acid. After reaction in the dark for 20-24 h, the solution was diluted lo-fold with water and lyophilized. The dried mixture of CNBr fragments was suspended in 3 ml of 30% acetic acid. The soluble fraction was separated by centrifugation and used for the HPLC separation of the soluble CNBr fragments. Reversed-phase HPLC of CNBr fragments was performed as previously described (12). The insoluble pellet was washed twice with 30% acetic acid, and this insoluble fraction was used as such for peptide sequencing. Partial cleavage at Asp of the above mixture of soluble CNBr fragments was performed essentially as described by Tarr (16). Heating in O.lC; TFA was carried out at 100°C under N, for 270 min. The resulting peptides were separated by HPLC as previously described for tryp tic peptides (16). Digestion of S-pyridylethylated Digestion with chymottypsin. chloroplast FBPase with chymotrypsin was performed for 24 h at 22°C in 50 mhf N-ethylmorpholine acetat,e buffer (pH 8.5) at a 5O:l ratio of FBPase to chymotrypsin. The resulting peptides were separated by HPIK as previously described for tryptic peptides (15). Digestion of S-pyridylethylated chloroplast Iligestion with pepsin. FBPase with pepsin was performed as previously described (13). In some experiments, reduction of FRPase with dithiothreitol was omitted and S-pyridylethylation was carried out with native enzyme unfolded in 6 M guanidine hydrochloride. This enzyme is herein referred to as PE-oxidized FBPase. Peptides were separated by HPLC as previously described for tryptic peptides. Digestion of SUig&ion lcith Staphylococcus aureus VX protease. pyridylethylated chloroplast FBPase with S. CZU~EUS protease was performed at 24 h at 37°C in 100 mM potassium phosphate (pH 7.8) in the presence of 1 M urea at a 5O:l ratio of FBPase to protease. The resulting peptides were separated by HPLC as previously described for the tryptic peptides (15). Digestion of S-carboxymethylated chloroIligestiun with trypsin. plast FBPase with trypsin was performed as previously described (15). This treatment generated a large number of tryptic fragments which were soluble at pH 4. These peptides were separated by HPLC as described earlier (12, 15). The pH 4 insoluble material was dissolved in 0.1 M N-ethylmorpholine acetate (pH 8.5) and sonicated intermittently for 15 min. The solubilized peptides were separated by reversedphase HPLC using a Supelco LC3DP phenyl column (4.6 X 250 mm) equilibrated with 100 mM Na phosphate (pH 6.3). The peptides were

RESULTS

AND

DISCUSSION

The complete amino acid sequence of spinach chloroplast FBPase is shown in Fig. 1, top line. The protein contains 358 amino acid residues, with alanine residues at both free ends. Its subunit molecular weight was calculated to be 39,164 which is about 10% lower than the value (ca. 44,000) estimated by polyacrylamide gel electrophoresis (15). The entire sequence was obtained by analysis of the intact protein and of 29 peptides obtained by enzymatic or chemical cleavage. Sequence data for several of the above peptides have been previously reported (12, 13, 15, 19). Useful fragments were obtained by enzymatic cleavage with trypsin, chymotrypsin, pepsin, and S. aureus protease. Additional fragments were obtained by chemical cleavage with cyanogen bromide, cleavage which in some instances was followed by acid cleavage. All cleavage points were overlapped and several of the overlaps were established more than once. The recently published nucleotide sequence and deduced amino acid sequence of the wheat chloroplast FBPase gene (14) has allowed the confirmation of our peptide order, including those regions which contained only 1 or 2 amino acid overlaps. Unless otherwise stated, peptides were purified by HPLC on a Bio-Rad RP-304 column with a trifluoroacetic acid/acetonitrile based solvent system and peptide sequences were determined by automated Edman degradation. Although the properties of chloroplast FBPases are clearly distinct from those of gluconeogenic FBPases, the amino acid sequence of the chloroplast enzyme shows characteristics common to all FBPases. The sequence homologies allow an alignment of all known FBPases and as an example, Fig. 1 shows an alignment of the sequence of spinach chloroplast FBPase and four other FBPases. These include wheat leaf chloroplast (14), pig kidney (18), yeast S. cereuisiae (7), and E. coli (8) FBPases. The comparison between the spinach and wheat chloroplast FBPases shows an 80% homology between the two enzymes. The wheat chloroplast enzyme

FIG. 1. Amino acid sequence alignment of five FBPases. Amino acids are indicated by the single letter code. Only the sequence of spinach chloroplast (SCh) FBPase is given in full. Other sequences are for wheat chloroplast (WCh, Ref. (14)), pig kidney (PK, Ref. (18)), Saccharomycc,s wrwisiae (SC, Ref. (7)), and Eschrrichia coli (EC, Ref. (8)) FBPases. The numbers indicated above some residues correspond to their location in the amino acid sequence of spinach chloroplast FBPase. Dots indicate identity with the residue in the spinach chloroplast enzyme and dashes indicate deletions. The number in parenthesis at the end of each sequence denotes the number of the last residue.

154

MARCUS

AND

FIG. 2. A plot of (Y-carbon atoms in the pig kidney FBPase Cl subunit. This figure is a modification of Fig. 3 in Ref. (22). The N and C termini are represented by the letters N and C, respectively. Assuming a similar conformation for the spinach chloroplast FBPase subunit, the arrow signals the area into which the chloroplast insert has to be accommodated.

also shows the unique sequence insert (residues 165 179) presumably involved in light-dependent regulation of the chloroplast enzyme activity. In both enzymes the sequence insert contains two cysteines separated by only four other amino acid residues, a characteristic feature of at least three other enzymes containing redox-active cysteines (13). This unique region is not present in pig kidney (18), sheep liver (5), rat liver (6), yeast S. cereuisiae (7), yeast S. pombe (7), and E. coli (8) FBPases; all of which are gluconeogenic FBPases. Three of these sequences, pig kidney, S. cerevisiae, and E. coli FBPases, are included in Fig. 1 as prototypes of mammalian, yeast, and prokaryotic FBPases. The sequence insert is not a characteristic of all plant FBPases since it is absent in the gluconeogenic spinach cytosolic FBPase isozyme (U. Ladror and F. Marcus, unpublished results). The sequence alignment of Fig. 1 reveals that the identity between chloroplast FBPase and the three selected prototype FBPases is about 40%, a value which is very close to the 41-43% identity exhibited between the eukaryotic (pig kidney and yeast) and the prokaryotic E. coli FBPase. Thus, no conclusions can be made here as far as the evolutionary origin of chloroplast FBPase. The sequence of wheat chloroplast FBPase, which was deduced from the sequence of the gene and was published while our work was in progress (14), includes a

HARRSCH

presequence transit peptide (not shown in Fig. 1). The transit peptide is necessary for directing FBPase, a nuclear-encoded protein (20, 21), into the chloroplast. Our data on the direct amino acid sequencing of the mature protein demonstrate that Raines et al. (14) were correct in suggesting a cleavage site between methionine51 and alaninein their nucleotide derived sequence. Alaninein their nomenclature corresponds to Ala-l of the spinach chloroplast enzyme. It is also of interest to note that all seven Cys residues are conserved in both plant FBPases, but none of the cysteines is conserved in all FBPases. The recent elucidation of the molecular structure of pig kidney FBPase at 2.8 A resolution (22), the first established three-dimensional structure of an FBPase, allows some additional discussion of the structure of the spinach chloroplast enzyme. This discussion is possible because it would appear likely that these two isozymes which exhibit 42% sequence identity plus 18% of conservative replacements will probably exhibit similarities in the folding of the polypeptide chain. An analysis of chloroplast FBPase residue conservation using the elements of secondary structure of pig kidney FBPase (Fig. 2; Ref. (22)) illustrates that some FBPase regions show a higher sequence homology than the average of 42%. These correspond to the region covering from the beginning of sheet B3 to the end of sheet B4 which shows 78% of sequence identity, and the region beginning in sheet BlO and ending in helix H7 which shows 64% of sequence identity. As expected, the sequence conservation is even

TABLE

I

Equivalency of Some Functional Residues in Pig Kidney and Spinach Chloroplast Fructose-1,6-bisphosphatase Pig kidney FBPase residue”

Equivalent in spinach chloroplast FBPase

Residues very near or interacting with Fru-2,6-P,

Asn-125 Asn-212 Tyr-215 Arg-243 Tyr-244 Gly-246 Ser.247 Met-248 Tyr-264 Lys-269 Lys-274

Asn-I 35 Asn-238 Asn-241 Arg-269 Tyr-270 Gly-272 Ser.273 Leu-274 Tyr-290 Lys-295 Lys-300

Residues in the negatively charged pocket close to the Fru-2,6-P* site

Lys-71 Glu-97 Glu-98 Asp-118 Asp-121 Arg-276 Glu-280

Lys-81 Glu-107 Glu-108 Asp-128 Asp-131 Arg-302 Glu-306

Structural

“Data

location

from Ref. (22).

SPINACH

CHLOROPLAST

155

FRUCTOSE-1,6-BISPHOSPHATASE

higher in residues presumably located in the active site region of the enzyme (22). Indeed, 15 of 17 pig kidney FBPase residues located either at or close to the sugar bisphosphate site or located at the negatively charged pocket presumed to be the catalytic metal binding site are conserved in spinach chloroplast FBPase (Table I). Structural data on the pig kidney FBPase-fructose 2,6bisphosphate complex at 2.8 A resolution (H. M. Ke, C. M. Thorpe, B. A. Seaton, W. N. Lipscomb, and F. Marcus, unpublished results) demonstrate that the interacting residues are conserved in all the known FBPases. These findings further support the notion that fructose 2,6-bisphosphate binds at the enzyme’s active site. Finally, the question of location of the unique extra segment of chloroplast FBPases can also be preliminarily answered using the determined X-ray structure of pig kidney FBPase (22). Indeed, the extra segment of chloroplast FBPase which inserts between Leu-153 and Gln154 of pig kidney FBPase can be accommodated in the molecular structure of the mammalian enzyme. This region (shown by an arrow in Fig. 2) is exposed to the solvent and is not involved in subunit interactions. Thus, it could interact with thioredoxin as required for lightdependent activation of chloroplast FBPase. Crystallographic data obtained with thioredoxin from E. coli and bacteriophage T4 indicate that the thioredoxin active site -Cys-Gly-Pro-Cysis located in a protrusion exposed to the solvent (23). ACKNOWLEDGMENTS

2. Zimmerman, G., Kelly, G. ,J., and Latzko, E. (1976) J. Biol. Chem. 253,5952-5956. 7I Harhron, S., Foyer, C., and Walker, phys. 212,237-246.

D. (1981) Arch. Biochem. Bio-

4. Marcus, F., Edelstein, I., Saidel, L. J., Keim, P. S., and Heinrikson, R. L. (1981) Arch. Biochem. Biophys. 209,687-696. 5. Fisher, W. K., and Thompson, 36,235-250.

E. 0. P. (1983) Aust. J. Biol. Sci.

6. El-Maghrahi, M. R., Pilkis, J., Marker, A. J., Colosia, A. D., DAngelo, G., Fraser, B. A., and Pilkis, S. J. (1988) Proc. Natl. Acad. Sci. USA 85,8430-8434. 7. Rogers, D. T., Hiller, E., Mitsock, Chem. 263,6051-6057.

L., and Orr, E. (1988) J. Aiol.

8. Hamilton, W. D. O., Harrison, Nucleic Acids Res. 16,8707.

D. A., and Dyer,

9. Halliwell, B. (1981) Chloroplast Univ. Press, New York.

Metabolism,

10. Preiss, J., Biggs, M. L., and Greenberg, 242,2292-2294. 11. Buchanan, B. B., Schurmann, Biol. Chem. 246,5952-5959.

T. A. (1988)

pp. 66-88, Oxford

E. (1967) J. Biol. Chem.

P., and Kalherer,

P. P. (1971) J.

12. Harrsch, P. B., Kim, Y., Fox, J. L., and Marcus, F. (1985) Biochem. Biophys. Res. Commun. 133,520-526. 13. Marcus, F., Moherly, L., and Latshaw, Acad. Ski. CJSA 85,5379-5383.

S. P. (1988) Proc. Natl.

14. Raines, C. A., Lloyd, d. C., Longstaff, M., Bradley, T. (1988) Nucleic Acids Res. 16,7931-7942. 15. Marcus, F., Harrsch, P. B., Moberly, L., Edelstein, shaw, S. P. (1987) Biochemistry 26,7029-7035.

D., and Dyer, I., and Lat-

16. Tarr, G. E. (1986) in Methods of Protein Microcharacterization (Shively, J. E., Ed.), pp. 154-194, Humana, Clifton, NJ. 17. Andersen, l-8.

K. B., and Vaughan,

M. E. (1982) J. Chromatogr.

240,

We thank Stephen I’. Latshaw and Lorraine Moherly for technical assistance, and Ms. Mildred M. Churich for excellent secretarial assistance. We also thank Drs. William N. Lipscomh and Hengming Ke (Harvard IJniversity) for helpful discussions with regard to the putative location of the chloroplast fructose-1,6-hisphosphatase insert in the structure of the pig kidney enzyme (Ref. (22)).

20. Chua, N.-H., and Schmidt, G. W. (1979) J. Cell Riol. 81, 461m483.

REFERENCES

21. Chueca, A., Lazaro, J. J., and Lopez-Gorge, iol. 75,539%541.

1. Kelly, G. J., Zimmerman, G., and Latzko, E. (1982) in Methods in Enzymology (Wood, W. A., Ed.), Vol. 90, pp. 371-378, Academic Press, San Diego.

18. Marcus, F., Edelstein, I., Reardon, I., and Heinrikson, Proc. Natl. Acad. Sci. lJSA 79, 7161-7165. 19. Marcus, F., and Fickenscher, 117-121.

R. L. (1982)

K. (1988) Arch. Biol. Med. Exp. 21,

J. (1984) Plant Phys-

22. Ke, H. M., Thorpe, C. M., Seaton, B. A., Marcus, F., and Lipscomh, W. N. (1989) Proc. Natl. Acad. Sci. USA 86,1475-1479. 23. Holmgren,

A. (1981) Trends Biochem. Sci. 6,26-29.

156

MARCUS

AND

HARRSCH

SPINACH Table

Ill.

CHLOROPLAST Pepbdes

used

for estabhshina

157

FRUCTOSE-WBISPHOSPHATASE the amino

acid

secruence

of winach

Dessg”at,o” T-PE3

chloroplast

and Source Tryptlc

fragment

of PE-FWase

TryP!lC fragment

0, CM-Fwase

CN&

f;agment

C5ymOtryptlC Tryptlc

Of CwFBPaSe peptIde

fragment

CNBr fragment Trypflc CNBr PeptIde

fragment fragment

Peptlde Tryptlc CNBr

fragmen!

Trypt~c fragmen:

12 I” re,

II in Fig

from PE-Fwase

(eiJtes

from CM~FBPase 01 CWFBPase

of CM-FBPase

(peak

from PE-oudaed CNBr

from CM-FBPase from CwFBPase from CM-FBPase born PE-ox,dued

Tryptlc ‘ragmen: 21 mn,

from CM-Fwae

at 41 ml”)

?A ref 12) 2 I” 15)

III I” Fig

fragments FBPase fragments (peptlde

15)

,A ref 12)

t i” re, 15)

/ I” Fig

(peptIde

CN&

Peptic fragment

(peptIde

(Peak

from CM-FBPase

from ac,d heated fragment

at 115 men)

(peptide (peak

from and heated

Peptic fragment

(elutes

,A re, 12)

(elutes

at 66 r”l”)

(&es

at 63 m,“)

(ebtes

at 60 “,I”)

,O I” re,

15)

(peak ” in FIQ ,A, re, 12) (peptide FBPase

9 I” ref (ebtes

15)

at 51 m,“)

(FIQ 1 B I” r.4 12. peak

eluting

a,