003 l--9422/91 S3.00+ 0.00 Pergamon Press plc
Phytochemistry, Vol. 30, No. 2, pp. 415418, 1991 Printed in Great Britain.
THE AMINO ACID SEQUENCE OF FERREDOXIN HORDEUM VULGARE ISHAQ ABDEL-HAMID
FROM
TAKRURI
King Saud University, Abha Branch, College of Education, Chemistry Department, Abha, P.O. 944, Saudi Arabia (Received in revised form 25 June 1990)
Key Word Index-Hordeum oulgare; Gramineae; ferredoxin; amino acids; sequence.
Abstract-Ferredoxin from barley consists of a single polypeptide chain of 97 amino acids, four of which are cysteine. These residues, which bind the iron atoms of the active centre, are in identical positions to those of other ferredoxins. The primary structure shows considerable similarity with other plant ferredoxins.
INTRODUmION Ferredoxins are a group of iron-sulphur proteins which act as electron carriers in a number of different biochemical processes, including photosynthetic electron transport and nitrogen fixation. Several ferredoxins have been isolated and characterized from various plants, bacteria and algae [l, 23. The presence of different molecular species of plant-type ferredoxins in one organism has been reported in a variety of higher plants and algae [3, 41. Although several attempts have been made to determine whether the ferredoxin isoproteins have different biological functions, no conclusive evidence has been obtained [S, 61. Ferredoxins from plants and algae have a M, of ca 11000 and possess a 2Fe-2S active centre. Bacterial ferredoxins contain one or two 4Fe-4S active centres and their M,s range from 6000 to 15 000 depending on the bacterial source [7, 8-j. Ferredoxins in the oxidized state exhibit characteristic absorption spectra. They have negative oxidation reduction potentials and a characteristic electron spin resonance (ESR) signal in the reduced state [9, lo]. We describe the purification, characterization and the amino acid sequence of ferredoxin isolated from Hordeum uulgare and its relationship to various plant ferredoxins. RESULTSANDDISCUSSION Hordeurn vulgare ferredoxin was isolated in a high degree of purity with an absorption index A,20/A,,, =0.46. The homogenity of the protein was shown by isoelectric focusing. The protein migrated as a single band. The amino acid composition of the protein together with the CNBr fragments is shown in Table 1. The amino acid composition of the CNBr fragments shows good agreement with that of the finally derived sequence. The N-terminal residue of the protein was found to be Ala, while the C-terminal sequence of the protein was found to be Leu-Thr-Ala after digestion with carboxypeptidase A for different periods of time. As expected from the presence of a single methionine in the protein, CNBr cleavage produced two fragments which were purified by gel chromatography. The large
fragment (Xl) had the same N-terminal residue as the protein and a homoserine residue at the C-terminus. The small fragment (X2) had a C-terminal residue identical with that of the intact protein but it did not show a homoserine residue. The order of the fragments in the intact protein could be unequivocally established. The complete amino acids sequence of the protein is Table 1. Amino acid composition of ferredoxin of Hordeum uulgare and its CNBr fragments Total Protein Amino acid Asx Thr Ser H.% Glx Pro GUY Ala CYS Val Met Ile LRU Sr
Phe His f-Ys Arg Trp
A 10.44 5.11* 8.00’ 0 18.62 3.85 6.39 6.74 n.d. 7.50 0.42 4.29 7.80 3.54 0.79 1.66 4.59 0.84 0
B 10 5 8 0 19 4 6 7 4 8 1 4 8 4 1 2 5 :1)
Xl fragment A
X2 fragment
B
A
B
9.25
9
2 7 1 13 3 5 4 3 6 0 3 6 3 1 0 3 1 0
1.06 2.63* 1.35* 0 5.58 0.69 1.15 2.84 0.35 2.20 0 1.27 2.29 0.34 0 1.54 1.99 0 0
1
2.25* 6.83* 0.36 13.50 2.85 5.43 4.39 n.d. 6.50 0 3.22 6.29 2.71 0.79 0 3.42 0.77 0
3
1 0 6 1 1 3 1 2 0 1 2 1 0 2 2
The results are expressed as average residues per molecule (A) and the nearest integer(B). Cm-Cys (carboxymethylcysteine) was not determined after hydrolysis. Values for serine and threonine are extrapolated to zero time assuming first-order rate of destruction [20]. For valine and isoleucine maximal values (72 hr hydrolysis) were taken. Abbreviations: Hse, homoserine; Seq., values from sequence determination. 415
I. A.-H. TAKRURI
416
X1C3
Xl(‘2
XlCl +-----I F--------i ----v--=-v----T-~=-v---
XIT?
XlTl
XIC4 I
i
I
XlT3
,-_-c-_l
+-_-_-___------.
~
~.
..
. -
--v-v----
XIC6
XIC5
x1c4 ~_-_,
---
___-.
--I-
-T-~-----~------~~-v--
x11‘3 __.
~_.___
_.-+
_.~
-
XlT3H46
XIT3H4a H---A
+
XIT3H6
X1T3H5
--.-_,~~----
---
--_------
.___ ------
-----I ---
-------__-_
XZT2 _-__,+~~_-. ---_
_ --
-_-_
X2T3 --------I
+F --v-v--
-_
Fig 1. Amino acid sequence of Hordeum t&are ferredoxin. Xl and X2 represent the CNBr fragments; *indicates homoserine was identified as the derivative from CNBr cleavage. T, C and H represent pcptides obtained by tryptic, chymotryptic and thermolytic digestions, respectively. Arrows (+, -, -) represent automatic sequencing manual Rdman degradation and carboxypeptidase digestion respectively. Residues that were unambiguously identified are indicated with solid arrows. other with broken arrows.
Ferredoxin from barley
417
Table 2. Matrix of amino acid sequence differences of some plant ferredoxins
Hordeum m&are (Barley) T&cum aestivum (Wheat) Erassica nupus (Rape) Spinacia oleracea (Spinach) Sambucus nigra (Elder) Phytolacca americana (I) Phytolacca americana (II) Equisetum telmuteia (I) Equisetum telmateia (II)
1
2
0 3 17 20 18 30 31 32 38
20 21 19 32 34 32 42
3
4
5
6
7
0 24 21 24 31 34 44
0 21 30 31 39 45
0 29 31 38 40
0 23 35 43
0 39 47
89
0
shown in Fig. 1, together with the details of the overlapping sites and fragments which aided in the deduction of the sequence. The N-terminal 40 residues of the protein
were determined with an automated Beckman 890C protein sequencer. Phenylthiohydantion derivatives were identified as in ref. [l 11. A tryptic digestion of (Xl) CNBr fragment yielded five peptides (see Fig. 1) in accordance with the number expected from the three Lys and one Arg residues determined by amino acid analysis of the Xl fragment. The largest of these peptides (Tl) was purified by gel chromatography while the others were characterized by paper electrophoresis. When T3 peptide was digested using thermolysin, it was possible to separate 10 peptides by paper electrophoresis. The Xl fragment was also subjected to chymotryptic digestion and yielded eight peptides. All these peptides were completely sequenced and their order in the parent protein was established from overlap with the N-terminal 40 residues and from tryptic peptides. The X2 CNBr fragment yielded three peptides after digestion with trypsin. The order of these peptides (X2T1, X2T2, X2T3) in the parent fragment (X2) could be partially established from the following data. Peptide X2Tl had the same three N-terminal residues as those of fragment X2, the sequence of the C-terminal showed that two residues of peptide T3 were identical with those of fragment X2. Furthermore, all these peptides were overlapped by chymotryptic peptides which were produced from chymotryptical digestion of X2. The ferredoxin of Hordeum oulgare contains 91 amino acid residues, as compared with other plant/algal ferredoxins which contain 96-98 residues. No heterogeneity was observed during the determination of the amino acid sequence or the isoelectric focusing of the protein. The M, deduced from the sequence is 11200 including the active centre. Barley ferredoxin contains four cysteine residues which bind the two iron atoms of its active site and are identical in position to those of other ferredoxins. The number of amino acid differences that exist between this sequence and of other selected plant-type ferredoxins are given in Table 2. Triticum aestt’uum ferredoxin shows the greatest sequence similarity to it, with only three amino acid differences. EXPERIMENTAL
Materials. Ferredoxin was isolated and purified from the green leaves of barley as described in ref. [12]. Acrylamide and
0 29
0
N,hJ’-methylenebisacrylamide were from BDH. Trypsin (EC 3.4.21.4) (l-chloro4phenyl-l,3-L-tosylamidobutane-2-onetreated) and chymotrypsin (EC 3.4.21.1) were from Serva. Other chemicals and enzymes used were as described in ref. [12]. Methods. Curboxymethylation offerredoxin. Ferredoxin was denatured in 6 M guanidine hydrochloride, 1 M Tris-HCI pH 8.6 and carboxymethylated as described in ref. [13]. Carboxymethylated protein was desalted by passing it through a Sephadex G-10 column (2 x 20 cm) equilibrated with H,O. The eluted protein was subsequently lyophilized. Amino acid composition. Samples (2 mg) of carboxymethylated protein were hydrolysed in 6 M HCI at 110” in sealed evacuated tubes 24,48 and 72 hr. Samples of peptides were hydrolysed for 24 hr only. Duplicate hydrolyses were carried out for each hydrolysis time. Cleavage methods. 10 mg of the carboxymethylated protein were dissolved in 85% HCO,H and 8-fold excess (w/w) of CNBr was added. The mixt. was incubated at room temp. in the dark for 24 hr, diluted with 2 ml Hz0 then freeze-dried. Protein fragments resulting from CNBr cleavage were subjected to gel chromatography using a Sephadex G-50 column (1.5 x 130 cm) equilibrated with 85% HCO,H. Carboxypeptidase A or B was made soluble by method 1 of ref. [14] and digestion was with a 2% (w/w) ratio of enzyme to substrate for periods of up to 2 hr in N-ethyhnorpholine acetate buffer, pH 8.5. Digestion of CNBr fragments by trypsin and chymotrypsin was carried out as described in ref. [12]. The resulting peptides were applied to a Bio-Gel P-4 column (1.5 x 200 cm). Elution profiles were followed by measurement at A aso and Az06 and by N-terminal analysis using the dansyl technique [15]. Tryptic and chymotryptic peptides that remained impure after gel chromatography as well as the peptides resulting from digestion of XlT3 by thermolysin were subjected to HV paper electrophoresis at pH 6.5 (pyridine, HOAc and H,O in ratios of 25: 1: 225,by vol.) as in ref. [16]. Peptides were detected on paper using Cd/ninhydrin reagent [ 173.Tryptophan containing peptides were also identified by positive staining with Ehrlich’s reagent [18]. Amino acid sequence methods. N-Terminal residues of the protein, and peptides were determined by the dansyl Edman methods [14]. Pure peptides were sequenced by the (DABTIC) 4-dimethylaminoazobenzene-4’-isothiocyanate method [19]. The C-terminal amino acids, after carboxy peptidose release, were identified as their dansyl derivatives. Nomenclature. Peptides derived from digestion of the fragments or from a large peptide were numbered on the basis of their order within the parent fragment. The following abbreviations were used: X, CNBr fragments; T, tryptic peptides; C, chymotryptic peptides; H, thermolytic peptides.
418
I. A.-H.
Acknowledgements-1 would like to thank Prof. D. Boulter for giving me the privilege of using the facilities of the protein chemistry laboratory of the Biological Sciences Department at the University of Durham in the summer of 1986 and 1988. I also wish to thank Mr J. Giroy for his valuable technical assistance.
REFERENCES 1. Takruri, I. A. H. and Boulter, D. (1980) Biochem. J. 185,239. 2. Hase, T., Ymanashi, H. and Matsubara, H. (1982) J. Biothem. 91, 341. 3. Kimata, Y. and Hase, T. (1989) Plant Physiol. 89, 1193. 4. Sakihama, N. and Shin, M. (1987) Arch. Biochem. Biophys. 256,430. 5. Dutton, J. E., Rogers, L. J., Haslett, B. G., Takruri, I. A. H., Gleaves, J. T. and Boulter, D. (1980) J. Exp. Botany 31, 379.
6. Wada, K., Matsubara, H., Chain, R. K. and Arnon, D. I. (1981) Plant Cell Physiol. 22, 275. 7. Wada, K., Kagamiyana, H. and Matcubara, H. (1975) FEBS Letters 55, 102.
TAKRURI
8. Hall, D. O., Rao, K. K. and Cammack, R. (1975) Sci. Prog. 62, 285. 9. Glickson, J. D., Phillips, W. D., McDonald, C. C. and Poe, M. (1971) Biochem. Biophys. Res. Commun. 42, 271. 10. Cammack, R., Rao, K. K., Bargeron, C. P., Hutson, K. G., Andrew, P. W. and Rogers, L. J. (1977) Biochem. J. 168,205. 11. Haslett, B. G. and Boulter, D. (1976) Biochem. J. 153, 33. 12. Takruri, I. A. H. and Boulter, D. (1979) Biochem. J. 179,373. 13. Milne, P. R. and Wells, J. R. E. (1970) J. Biol. Chem. 245, 1566. 14. Ambler, R. P. (1972) Methods Enzymol. 25, 143. 15. Gray, W. R. and Hartley, B. S. (1963) B&hem. J. 89,378. 16. Thompson, E. W., Laycock, M. W., Ramshaw, J. A. M. and Boulter, D. (1970) Biochem. J. 117, 183. 17. Heilman, J., Barollier, J. and Watzke, E. (1957) HoppeSeyler’s Z. Physiol. Chem. 309, 219. 18. Easley, C. (1965) Biochim. Biophys. Acta 107, 386. 19. Chang, J. Y., Brauer, D. and Wittman-Liebold, B. (1978) FEBS Letters 93, 205. 20. Moore, S. and Stein, H. W. (1963) Methods Enzymol. 6,817.
Phytochemistry, Vol. 30, No. 2, pp. 415418, 1991 Printed in Great Britain.
THE AMINO ACID SEQUENCE OF FERREDOXIN HORDEUM VULGARE ISHAQ ABDEL-HAMID
FROM
TAKRURI
King Saud University, Abha Branch, College of Education, Chemistry Department, Abha, P.O. 944, Saudi Arabia (Received in revised form 25 June 1990)
Key Word Index-Hordeum oulgare; Gramineae; ferredoxin; amino acids; sequence.
Abstract-Ferredoxin from barley consists of a single polypeptide chain of 97 amino acids, four of which are cysteine. These residues, which bind the iron atoms of the active centre, are in identical positions to those of other ferredoxins. The primary structure shows considerable similarity with other plant ferredoxins.
INTRODUmION Ferredoxins are a group of iron-sulphur proteins which act as electron carriers in a number of different biochemical processes, including photosynthetic electron transport and nitrogen fixation. Several ferredoxins have been isolated and characterized from various plants, bacteria and algae [l, 23. The presence of different molecular species of plant-type ferredoxins in one organism has been reported in a variety of higher plants and algae [3, 41. Although several attempts have been made to determine whether the ferredoxin isoproteins have different biological functions, no conclusive evidence has been obtained [S, 61. Ferredoxins from plants and algae have a M, of ca 11000 and possess a 2Fe-2S active centre. Bacterial ferredoxins contain one or two 4Fe-4S active centres and their M,s range from 6000 to 15 000 depending on the bacterial source [7, 8-j. Ferredoxins in the oxidized state exhibit characteristic absorption spectra. They have negative oxidation reduction potentials and a characteristic electron spin resonance (ESR) signal in the reduced state [9, lo]. We describe the purification, characterization and the amino acid sequence of ferredoxin isolated from Hordeum uulgare and its relationship to various plant ferredoxins. RESULTSANDDISCUSSION Hordeurn vulgare ferredoxin was isolated in a high degree of purity with an absorption index A,20/A,,, =0.46. The homogenity of the protein was shown by isoelectric focusing. The protein migrated as a single band. The amino acid composition of the protein together with the CNBr fragments is shown in Table 1. The amino acid composition of the CNBr fragments shows good agreement with that of the finally derived sequence. The N-terminal residue of the protein was found to be Ala, while the C-terminal sequence of the protein was found to be Leu-Thr-Ala after digestion with carboxypeptidase A for different periods of time. As expected from the presence of a single methionine in the protein, CNBr cleavage produced two fragments which were purified by gel chromatography. The large
fragment (Xl) had the same N-terminal residue as the protein and a homoserine residue at the C-terminus. The small fragment (X2) had a C-terminal residue identical with that of the intact protein but it did not show a homoserine residue. The order of the fragments in the intact protein could be unequivocally established. The complete amino acids sequence of the protein is Table 1. Amino acid composition of ferredoxin of Hordeum uulgare and its CNBr fragments Total Protein Amino acid Asx Thr Ser H.% Glx Pro GUY Ala CYS Val Met Ile LRU Sr
Phe His f-Ys Arg Trp
A 10.44 5.11* 8.00’ 0 18.62 3.85 6.39 6.74 n.d. 7.50 0.42 4.29 7.80 3.54 0.79 1.66 4.59 0.84 0
B 10 5 8 0 19 4 6 7 4 8 1 4 8 4 1 2 5 :1)
Xl fragment A
X2 fragment
B
A
B
9.25
9
2 7 1 13 3 5 4 3 6 0 3 6 3 1 0 3 1 0
1.06 2.63* 1.35* 0 5.58 0.69 1.15 2.84 0.35 2.20 0 1.27 2.29 0.34 0 1.54 1.99 0 0
1
2.25* 6.83* 0.36 13.50 2.85 5.43 4.39 n.d. 6.50 0 3.22 6.29 2.71 0.79 0 3.42 0.77 0
3
1 0 6 1 1 3 1 2 0 1 2 1 0 2 2
The results are expressed as average residues per molecule (A) and the nearest integer(B). Cm-Cys (carboxymethylcysteine) was not determined after hydrolysis. Values for serine and threonine are extrapolated to zero time assuming first-order rate of destruction [20]. For valine and isoleucine maximal values (72 hr hydrolysis) were taken. Abbreviations: Hse, homoserine; Seq., values from sequence determination. 415
I. A.-H. TAKRURI
416
X1C3
Xl(‘2
XlCl +-----I F--------i ----v--=-v----T-~=-v---
XIT?
XlTl
XIC4 I
i
I
XlT3
,-_-c-_l
+-_-_-___------.
~
~.
..
. -
--v-v----
XIC6
XIC5
x1c4 ~_-_,
---
___-.
--I-
-T-~-----~------~~-v--
x11‘3 __.
~_.___
_.-+
_.~
-
XlT3H46
XIT3H4a H---A
+
XIT3H6
X1T3H5
--.-_,~~----
---
--_------
.___ ------
-----I ---
-------__-_
XZT2 _-__,+~~_-. ---_
_ --
-_-_
X2T3 --------I
+F --v-v--
-_
Fig 1. Amino acid sequence of Hordeum t&are ferredoxin. Xl and X2 represent the CNBr fragments; *indicates homoserine was identified as the derivative from CNBr cleavage. T, C and H represent pcptides obtained by tryptic, chymotryptic and thermolytic digestions, respectively. Arrows (+, -, -) represent automatic sequencing manual Rdman degradation and carboxypeptidase digestion respectively. Residues that were unambiguously identified are indicated with solid arrows. other with broken arrows.
Ferredoxin from barley
417
Table 2. Matrix of amino acid sequence differences of some plant ferredoxins
Hordeum m&are (Barley) T&cum aestivum (Wheat) Erassica nupus (Rape) Spinacia oleracea (Spinach) Sambucus nigra (Elder) Phytolacca americana (I) Phytolacca americana (II) Equisetum telmuteia (I) Equisetum telmateia (II)
1
2
0 3 17 20 18 30 31 32 38
20 21 19 32 34 32 42
3
4
5
6
7
0 24 21 24 31 34 44
0 21 30 31 39 45
0 29 31 38 40
0 23 35 43
0 39 47
89
0
shown in Fig. 1, together with the details of the overlapping sites and fragments which aided in the deduction of the sequence. The N-terminal 40 residues of the protein
were determined with an automated Beckman 890C protein sequencer. Phenylthiohydantion derivatives were identified as in ref. [l 11. A tryptic digestion of (Xl) CNBr fragment yielded five peptides (see Fig. 1) in accordance with the number expected from the three Lys and one Arg residues determined by amino acid analysis of the Xl fragment. The largest of these peptides (Tl) was purified by gel chromatography while the others were characterized by paper electrophoresis. When T3 peptide was digested using thermolysin, it was possible to separate 10 peptides by paper electrophoresis. The Xl fragment was also subjected to chymotryptic digestion and yielded eight peptides. All these peptides were completely sequenced and their order in the parent protein was established from overlap with the N-terminal 40 residues and from tryptic peptides. The X2 CNBr fragment yielded three peptides after digestion with trypsin. The order of these peptides (X2T1, X2T2, X2T3) in the parent fragment (X2) could be partially established from the following data. Peptide X2Tl had the same three N-terminal residues as those of fragment X2, the sequence of the C-terminal showed that two residues of peptide T3 were identical with those of fragment X2. Furthermore, all these peptides were overlapped by chymotryptic peptides which were produced from chymotryptical digestion of X2. The ferredoxin of Hordeum oulgare contains 91 amino acid residues, as compared with other plant/algal ferredoxins which contain 96-98 residues. No heterogeneity was observed during the determination of the amino acid sequence or the isoelectric focusing of the protein. The M, deduced from the sequence is 11200 including the active centre. Barley ferredoxin contains four cysteine residues which bind the two iron atoms of its active site and are identical in position to those of other ferredoxins. The number of amino acid differences that exist between this sequence and of other selected plant-type ferredoxins are given in Table 2. Triticum aestt’uum ferredoxin shows the greatest sequence similarity to it, with only three amino acid differences. EXPERIMENTAL
Materials. Ferredoxin was isolated and purified from the green leaves of barley as described in ref. [12]. Acrylamide and
0 29
0
N,hJ’-methylenebisacrylamide were from BDH. Trypsin (EC 3.4.21.4) (l-chloro4phenyl-l,3-L-tosylamidobutane-2-onetreated) and chymotrypsin (EC 3.4.21.1) were from Serva. Other chemicals and enzymes used were as described in ref. [12]. Methods. Curboxymethylation offerredoxin. Ferredoxin was denatured in 6 M guanidine hydrochloride, 1 M Tris-HCI pH 8.6 and carboxymethylated as described in ref. [13]. Carboxymethylated protein was desalted by passing it through a Sephadex G-10 column (2 x 20 cm) equilibrated with H,O. The eluted protein was subsequently lyophilized. Amino acid composition. Samples (2 mg) of carboxymethylated protein were hydrolysed in 6 M HCI at 110” in sealed evacuated tubes 24,48 and 72 hr. Samples of peptides were hydrolysed for 24 hr only. Duplicate hydrolyses were carried out for each hydrolysis time. Cleavage methods. 10 mg of the carboxymethylated protein were dissolved in 85% HCO,H and 8-fold excess (w/w) of CNBr was added. The mixt. was incubated at room temp. in the dark for 24 hr, diluted with 2 ml Hz0 then freeze-dried. Protein fragments resulting from CNBr cleavage were subjected to gel chromatography using a Sephadex G-50 column (1.5 x 130 cm) equilibrated with 85% HCO,H. Carboxypeptidase A or B was made soluble by method 1 of ref. [14] and digestion was with a 2% (w/w) ratio of enzyme to substrate for periods of up to 2 hr in N-ethyhnorpholine acetate buffer, pH 8.5. Digestion of CNBr fragments by trypsin and chymotrypsin was carried out as described in ref. [12]. The resulting peptides were applied to a Bio-Gel P-4 column (1.5 x 200 cm). Elution profiles were followed by measurement at A aso and Az06 and by N-terminal analysis using the dansyl technique [15]. Tryptic and chymotryptic peptides that remained impure after gel chromatography as well as the peptides resulting from digestion of XlT3 by thermolysin were subjected to HV paper electrophoresis at pH 6.5 (pyridine, HOAc and H,O in ratios of 25: 1: 225,by vol.) as in ref. [16]. Peptides were detected on paper using Cd/ninhydrin reagent [ 173.Tryptophan containing peptides were also identified by positive staining with Ehrlich’s reagent [18]. Amino acid sequence methods. N-Terminal residues of the protein, and peptides were determined by the dansyl Edman methods [14]. Pure peptides were sequenced by the (DABTIC) 4-dimethylaminoazobenzene-4’-isothiocyanate method [19]. The C-terminal amino acids, after carboxy peptidose release, were identified as their dansyl derivatives. Nomenclature. Peptides derived from digestion of the fragments or from a large peptide were numbered on the basis of their order within the parent fragment. The following abbreviations were used: X, CNBr fragments; T, tryptic peptides; C, chymotryptic peptides; H, thermolytic peptides.
418
I. A.-H.
Acknowledgements-1 would like to thank Prof. D. Boulter for giving me the privilege of using the facilities of the protein chemistry laboratory of the Biological Sciences Department at the University of Durham in the summer of 1986 and 1988. I also wish to thank Mr J. Giroy for his valuable technical assistance.
REFERENCES 1. Takruri, I. A. H. and Boulter, D. (1980) Biochem. J. 185,239. 2. Hase, T., Ymanashi, H. and Matsubara, H. (1982) J. Biothem. 91, 341. 3. Kimata, Y. and Hase, T. (1989) Plant Physiol. 89, 1193. 4. Sakihama, N. and Shin, M. (1987) Arch. Biochem. Biophys. 256,430. 5. Dutton, J. E., Rogers, L. J., Haslett, B. G., Takruri, I. A. H., Gleaves, J. T. and Boulter, D. (1980) J. Exp. Botany 31, 379.
6. Wada, K., Matsubara, H., Chain, R. K. and Arnon, D. I. (1981) Plant Cell Physiol. 22, 275. 7. Wada, K., Kagamiyana, H. and Matcubara, H. (1975) FEBS Letters 55, 102.
TAKRURI
8. Hall, D. O., Rao, K. K. and Cammack, R. (1975) Sci. Prog. 62, 285. 9. Glickson, J. D., Phillips, W. D., McDonald, C. C. and Poe, M. (1971) Biochem. Biophys. Res. Commun. 42, 271. 10. Cammack, R., Rao, K. K., Bargeron, C. P., Hutson, K. G., Andrew, P. W. and Rogers, L. J. (1977) Biochem. J. 168,205. 11. Haslett, B. G. and Boulter, D. (1976) Biochem. J. 153, 33. 12. Takruri, I. A. H. and Boulter, D. (1979) Biochem. J. 179,373. 13. Milne, P. R. and Wells, J. R. E. (1970) J. Biol. Chem. 245, 1566. 14. Ambler, R. P. (1972) Methods Enzymol. 25, 143. 15. Gray, W. R. and Hartley, B. S. (1963) B&hem. J. 89,378. 16. Thompson, E. W., Laycock, M. W., Ramshaw, J. A. M. and Boulter, D. (1970) Biochem. J. 117, 183. 17. Heilman, J., Barollier, J. and Watzke, E. (1957) HoppeSeyler’s Z. Physiol. Chem. 309, 219. 18. Easley, C. (1965) Biochim. Biophys. Acta 107, 386. 19. Chang, J. Y., Brauer, D. and Wittman-Liebold, B. (1978) FEBS Letters 93, 205. 20. Moore, S. and Stein, H. W. (1963) Methods Enzymol. 6,817.
