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Studies of the multiple forms of ferredoxin-NADP oxidoreductase from spinach
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 194, No. 2, May, pp. 593-599, 1979
Studies
of the Multiple Forms of Ferredoxin-NADP Oxidoreductase From Spinach]
WILLIAM Department
L. ELLEFSON of Biochemistry,
Purdue
AND DAVID University,
W. KROGMANN
West Lafayette,
Indiana
47907
Received October 3, 1978; revised December 8, 1978 Six different forms of ferredoxin-NADP oxidoreductase from spinach were separated by isoelectric focusing and were found to differ in their specific activities in various assay systems and in their affinity for NADPH. The forms of the enzyme were found to be interconvertible when the enzyme was stored in the cold.
Recently Gozzer et al. reported the existence of five active ionic forms of ferredoxin-NADP oxidoreductase isolated from spinach leaves (1). These forms, identified by isoelectric focusing, were present at all stages of purification and were found to have their isoelectric points between pH 4.8 and pH 6. Gozzer et al. found that purification of the reductase, either in the presence of the reducing agent dithiothreitol or of serine protease inhibitor phenylmethylsulfonyl fluoride, had no effect on the ionic form distribution. Examination of the amino acid composition and diaphorase activities of some of the forms gave no significant differences among the forms. Studies in our laboratory with multiple forms of reductase isolated through the use of isoelectric focusing confirm the results obtained by these authors and extend their results to include: (a) a comparison of the relative specific activities of these forms in four different reductase assays; and (b) a documentation of the interconversion among these forms. Preliminary results are also presented which suggest that differences in the oxidation-reduction state among the forms are responsible for their appearance. MATERIALS
AND METHODS
Spinach chloroplasts were prepared from deveined leaves in 0.4 M sucrose containing 0.05 M NaCl by a 1 This work was supported by NSF Grant PMC01956 A02. 593
method similar to that. of Jagendorf and Avron (2). Chloroplasts prepared for enzyme purification were isolated from 10 kg of washed leaves with the aid of a Szent-Georgyi and Blum continuous flow apparatus used with a refrigerated Sorval centrifuge. Chloroplast fragments were prepared from whole chloroplasts by resuspension in 60 ml of 0.01 M Tris-Me? buffer, pH 7.5 and stirred overnight at 0°C. The suspension was centrifuged at 18,000 g for 15 min to collect the swollen plastids. The pellet was washed twice with 0.01 M Tris-Mes buffer, pH 7.5, and finally resuspended in the same buffer to give a chlorophyll concentration of 0.15 mg/ml. Chlorophyll concentrations were determined by the method of Arnon (3). During the early stages of purification, protein was determined by the method of Warburg and Christian (4). Protein concentrations in the latter stages of purification were based on published molecular weights and millimolar extinction coefficients. An extinction coefficient for ferredoxin-NADP oxidoreductase at 456 nm of 10.74 mM-’ cm-’ and an assumed molecular weight of 40,000 d were used to determine protein concentrations for the reductase (5). Concentrations of ferredoxin were determined using an extinction coefficient of 9.7 tnM-’ cm-l at 420 nm and a molecular weight of 11,500 d (6). The enzyme ferredoxin-NADP oxidoreductase was prepared in a manner similar to that of Borchert and Wessels (7), except that spinach chloroplasts were used as the starting material. The purity criteria were those described by Shin (8). Ferredoxin was purified as described by Arnon and Buchanan (6). Pure fractions had a ratio of A 420.,/A,,, nmgreater than 0.4. Approximately 35 mg purified ferredoxin-NADP oxidoreductase, which had been freed of most ions by ’ Abbreviations used: MES, 2-(N-Morpholino)ethanesulfonic acid; TCA, trichloroacetic acid; DCIP, dichlorophenol-indophenol; TCIP, trichlorophenol-indophenol; SDS, sodium dodecyl sulfate. 0003.9861/79/060593-07$02.00/O Copyright 0 19’79by Academic Press, Inc. All rights of reproduction in any form reserved.
594
ELLEFSON
AND
dialysis prior to lyophilization, was resuspended in a small volume of cold distilled water. The suspension was centrifuged at 10,OOOg for 10 min to remove any insoluble material and subsequently used as a sample for isoelectric focusing experiments and the preparation of each of the multiple reductase forms. Columns were prepared as outlined in the LKB Isoelectric Focusing Column Instruction Manual. The initial isoelectric focusing of purified reductase was conducted on a sucrose density gradient containing either 2 or 4% ampholyte (pH 5-7 range) with the anode at the bottom. During focusing, the wattage was kept between 2-3 Wand focusing was carried out for 36-75 h. Fractions of 15 drops were collected at an emptying rate of 1 drop/8 s. The pH of every fifth fraction was measured at 4°C. Diaphorase activity and, in some cases, absorbance at 456 nm, were measured spectrophotometrically. Further resolution of the multiple forms required additional isoelectric focusing columns. The major multiple forms were prepared by refocusing the fractions contained between pH 5-6. For this a 2% initial ampholyte concentration was required. To resolve the minor multiple forms, a pH span of 0.5 units was used to refocus after focusing initially with a 4% concentration ofampholytes (pH 5-7). For the more acidic forms, fractions focusing between pH 4.9-5.5 were pooled and refocused. For the less acidic forms, fractions focusing between pH 5.51-6.1 were pooled and subsequently refocused. To remove the ampholytes from the protein fractions, the fractions containing the multiple form of interest were pooled and loaded on a small DEAEcellulose column (1.0 x 3.0 cm) equilibrated with 0.01 M (NH,)HCO,,. The sample was washed with 10 ml of equilibration buffer to remove the sucrose and then with 10 ml of 2 N NaCl in ammonium bicarbonate buffer. The peak fractions were pooled and then loaded on a Sephadex G-50 fine column equilibrated with the same buffer. Fractions having diaphorase activity and a ratio of A2ROnm/A26,,nm of greater than one were pooled and lyophilized. The dried samples were used as a source of pure multiple forms for further study. Analytical gel electrophoresis was carried out according to the method of Righetti and Drysdale on 4% acrylamide gels containing 2% ampholyte (9). The gels were stained for protein by the method of Fairbanks et al. after removal of ampholytes with 5% TCA (10). The gels were scanned at 550 nm using a Gilford spectrophotometer with a linear transport device and an x-y recorder. Gels stained for activity were immersed for 20 min in 1 mM nitroblue tetrazolium dissolved in 0.1 M Tris-Cl buffer, pH 8.7, rinsed several times with water and then incubated in 0.5 InM NADPH for 20-35 min thereafter. The location of active reductase was noted by the appearance of an insoluble purple formazan band on the gel. These gels were scanned at 540 nm.
KROGMANN Performic acid oxidation of reductase was performed by the methods of Hirs (11). Oxidation by the peroxy acid was terminated by dilution of the reaction mixture with 10 vol of water followed by lyophilization to dryness. The powder was resuspended in another 10 vol of water and lyophilization repeated. The residue was resuspended in 0.01 M Tris-Cl, pH 7.5 and dialyzed against 1 liter of the same buffer to remove the FAD. The colorless protein was used for some of the gel electrofocusing experiments described below. NADP photoreduction assays were performed according to the method of Shin et al. (8). In a 3.0 ml total volume, the assays contained 1000 pmol of Tris-Cl, pH 7.8, 10 pmol of sodium ascorbate, 0.2 krnol of DCIP, 1 pmol of NADP, 9.3 nmol of ferredoxin, 0.68 nmol of ferredoxin-NADP oxidoreductase, and chloroplast fragments equivalent to 60 pg of chlorophyll. After initial absorbance readings were made at 340 nm, the reaction was started by illumination with a tungsten spotlight filtered through a clear glass water tank. The increase in absorbance at 340 nm was measured spectrophotometrically after 4 min of illumination at a saturating light intensity. Rates of NADP reduction were calculated using a millimolar extinction coefficient of 6.21 for NADPH. Cytochrome c reductase activity was measured by the method of Shin (8). The assays contained in a total volume of 3.0 ml: 10 prnol of Tris-Cl buffer, pH 7.8, 100 pmol of cytochrome c, 0.2 pmol of NADPH, 9.3 nmol of ferredoxin, and 5.6 x 10 ,1 nmol of reductase. Reactions were initiated by the addition offerredoxinNADP oxidoreductase. The increase in absorbance at 550 nm was measured 60 s thereafter. A millimolar extinction coefficient of 18.5 for cytochrome c (reduced minus oxidized) was used to calculate these rates (12). Transhydrogenase was measured fluorimetrically in an Eppendorf photometer Model 1100 equipped with a fluorescence attachment. The rate of the reaction was monitored using a Sargent SLRG recorder. The excitation beam was passed through a 313. to 360.nm band pass filter and the emission beam through a 400. to 300~nm cutoff filter. In this way the disappearance of NADPH fluoresence could be followed. Assay mixtures contained in a 3.0 ml total volume: 20 pmol of Tris-Cl, pH 7.8; 0.2 pmol of NADPH; 0.12 pmol of thio-NADP (a nonfluorescent acceptor), and approximately 1.8 pg of reductase to start the reaction. Diaphorase activity was measured by essentially the same method as employed by Avron and Jagendorf (13). Assays contained 256 pmol of Tris-Cl, pH 7.5, 0.2 pmol of NADPH, 0.09 pmol of TCIP and 2.2 pg of flavoprotein in a total volume of 3.0 ml. Assays were initiated by the addition of reductase and the decrease in absorbance at 620 nm was measured after 0.5 and 1 min. Rates of TCIP reduction were calculated using a millimolar extinction coefficient of 25.4 for the oxidized dye at pH 7.5. Lineweaver-Burke plots were prepared by a
MULTIPLE
FORMS OF FERREDOXIN-NADP
Hewlett-Packard computer Model 9825A equipped with plotter from data obtained for cytochrome c reductase and diaphorase activities of reductase at variable NADPH concentrations. Values for K,,,,,,,, and V in each case were calculated using a linear regression treatment of the data by the computer. The individual forms of reductase were dansylated (14) and the N-terminal residue was determined (15). All forms showed a lysine at the N-terminal position. The amino acid composition of forms B, C, and E were determined (16) and were indistinguishable. Antibodies to purified ferredoxin-NADP oxidoreductase which had not been resolved into the individual forms were elicited in rabbits according to the procedure of Keister et al. (17). Immunodiffusion of this antiserum against individual forms of the enzyme gave a single preciptin line. NADPH, nitroblue tetrazolium, Coomassie blue R, Mes, and Trizma Base were purchased from Sigma Chemical Company. TCIP, acrylamide, and NJmethylenebisacrylamide were bought from Eastman Kodak. Ampholytes, pH 5-7 and pH 3.5-10, were obtained from either LKB or Brinkmann. NADP was purchased from Calbiochem. Hydroxylapatite HTP was purchased as a powder from Bio-Rad Laboratories. Whatman DEAE-52 was used for ion-exchange chromatography while Sephadex G-100 was used for gel filtration. All other chemicals were reagent grade quality. RESULTS
When purified reductase from spinach chloroplasts was subjected to column isoelectric focusing between pH 5 and 6, six peaks of diaphorase activity were recovered (Fig. 1). The peaks and their corresponding forms have been designated A, A’, B, C, D, and E on going from the most basic to the most acid end of the elution pattern following the designation by Gozzer et al. In addition to the five peaks reported by Gozzer et al. (l), we also recover a minor peak A’ having an isoelectric point of pH 5.97 at 4°C. The relative amounts of enzyme in each form are slightly different from that reported by Gozzer et al., but peak C is the predominant species as reported by Gozzer et al. Activity
Studies
The enzyme ferredoxin-NADP oxidoreductase catalyzes a variety of oxidationreduction reactions among which are NADP photoreduction, transhydrogenase, ferre-
OXIDOREDUCTASE
Fl?ACTION
595
NUMBER
FIG. 1. Preparative isoelectric focusing column focused between pH 5 and 6. Fractions focusing between pH 5 and 6 from a pH 5-7 column were pooled and refocused for 48 h at 6°C. Column construction, operation, elution, and analysis were performed as specified under Materials and Methods.
doxin-dependent cytochrome c reductase, and diaphorase. When the various forms of reductase were compared for their ability to catalyze these reactions, the results in Table I were obtained. All forms examined catalyzed similar rates of NADP photoreduction when supplemented with an artificial electron donor, chloroplast fragments, ferredoxin, NADP, and light. When diaphorase activities were compared, all but the most acidic form E catalyzed nearly identical rates of indophenol reduction. Rates of transhydrogenase activity were highest with the two most basic forms A and A’ and slightly lower, i.e., 20% in the case of forms B and C. However, the most acidic form E was only one-half as active as the most basic form A. Examination of the rates of ferredoxin-dependent cytochrome c reduction provided the most interesting comparison. As the isoelectric point of the form decreased, the specific activity fell in a parallel manner. Most notably, the rate of cytochrome c reduction by the most acidic form E was only 33% of the maximal rate seen with form A: similarly acidic form D was only 50% as active as form A. Although the most acidic form E was able to catalyze NADP photoreduction at a rate identical to that of the other forms, it was not capable of transferring electrons as efficiently in the other direction-away from reduced NADP. Some support for this observation is pre-
596
ELLEFSON
AND KROGMANN TABLE
RATES OF REDUCTION
OF VARIOUS
Form
NADP
A A’ B C D E
1.V 1.5 1.9 1.7 1.9
ACCEPTORS
I
BY MULTIPLE
Trichlorophenolindophenol 24 22 26 26 25 15
FORMS
OF FERREDOXIN-NADP
Cytochrome c
REDUCTASE”
Thio-NADP (transhydrogenase)
84 75 60 49 41 30
12 11 9 9 6
a NADP photoreduction, cytochrome e reductase, transhydrogenase, and diaphorase assays were performed and specific activities calculated as described under Materials and Methods. For the calculation of specific activities reported here, results obtained over a threefold concentration range of reductase forms were averaged except those reported for NADP photoreduction. b Rates expressed as pmol acceptor reducedimg enzyme/min.
sented in Table II, where the effect of varying NADPH concentration on diaphorase activity catalyzed by the various reductase forms is compared. The KNAoPHdetermined for form E was found to be approximately twofold greater than that determined for the other forms. Thus the correlation between the K,,,,, constants for the various reductase forms and their observed diaphorase specific activities is apparent.
Interconversion
of Forms
In order to establish the identity of the reductase forms observed on the isoelectric focusing column, refocusing experiments were carried out. When purified form B was refocused on gels at various times after isolation and stained for activity, the results in Fig. 2 were obtained. Freshly isolated form B was found to refocus to give essentially one peak of activity belonging to form B. Storage of form B for 4 days at 4°C prior to refocusing gave a gel pattern in which TABLE II form B still predominates but other peaks SPECIFIC ACTIVITIES AND K,,,,, FOR MULTIPLE are beginning to emerge. After 11 days FORMS OF FERREDOXIN-NADP REDUCTASE storage, the activity trace obtained for form CATALYZING DIAPHORASE ACTIVITY” B now clearly shows the presence of other reductase forms, i.e., A’, C, D, and E. These K NADPH storage-generated forms were identical to Form (PM) those found in the unresolved reductase. The removal of ampholytes from form B 12.2 A accelerated the interconversion process so A’ 10.4 that within 2 days, the activity scan showed B 12.6 15.5 the same pattern of multiple forms as that C D 11.1 found for the 11-day, ampholyte-containing 24.3 E form B. Furthermore, the results observed with form B were noticed with all other u Diaphorase activity of each form was measured as forms of reductase. described under Materials and Methods except the rate In an attempt to understand the physical of TCIP reduction was recorded 15 s after reductase interrelationship among the reductase addition. Each assay contained besides the normal rehave been agents, variable concentrations of NADPH, and 2 pg forms, several experiments conducted. of each form. K,,,,, for each form was determined Gel electrofocusing of the unresolved refrom a Lineweaver-Burke plot of the data as specified under Materials and Methods. ductase treated with 6 M urea and 5 mM
MULTIPLE
FORMS OF FERREDOXIN-NADP
dithioerythritol on 6 M urea-containing gels gave five bands when stained for protein. The same results were found by Gozzer et al. (1). Immunodiffusion of an antiserum to the unresolved reductase against each of the six forms failed to reveal any antigenie differences among the forms. Preliminary evidence to support the premise that oxidation-reduction differences might be a major cause of these multiple forms is presented in Fig. 3. Here unresolved reductase was treated with the strong oxidant, performic acid, and then focused on isoelectric focusing gels. Instead of the normal pattern of five to six bands, only two bands were observed after staining for protein. The two remaining bands might be the result of partial oxidation of some amino acid, e.g., cysteine, methionine, or tryptophan which are known to be affected by this treatment (18). If cysteine oxidation is involved in the formation of the multiple forms, it is not a simple phenomenon since treatment with mercaptoethanol or isoelectric focusing in
0.30
I
I
I
I
I
0.15
$ A?
-1 0
,
I
I
I
I
2
3
4
cm of gel FIG. 2. Conversion of form B to other multiple forms: time study. Samples consisting of 2 pg of form B were subjected to isoelectric focusing on pH 5-7 gels at various times after purification. Gels were stained for activity and scanned as described under Materials and Methods. Storage conditions were at 4°C. Fresh form B (-1, I-day-old form B (- - -), and 11-day-old form B (- - -1.
OXIDOREDUCTASE
597
0.6
t
03
,o 2 0 cm of gel
FIG. 3. Electrofocusing of reductase after per-formic acid oxidation. Ferredoxin-NADP reductase was oxidized with performic acid and treated as described under Materials and Methods. A sample consisting of 10 pg of performic acid-oxidized reductase was electrofocused on pH 3.5-10 gels and stained for protein as described under Materials and Methods.
its presence does not alter the observed pattern on gels or columns. DISCUSSION
The results obtained in our laboratory agree well with those observed by Gozzer et al. (1) that, within the limits of the physical methods employed, i.e., N-terminal analysis, molecular weight analysis by SDS-gel electrophoresis and gel filtration, amino acid composition, and immunodiffusion, the multiple forms of reductase are indistinguishable except by isoelectric focusing. However, a comparison of the specific activities of each of the forms in four reductase dependent activities does reveal some differences. Although all forms catalyze a similar rate of NADP photoreduction, form E catalyzed a significantly lower rate of diaphorase, transhydrogenase, and ferredoxin-dependent cytochrome c reductase activity. This lower activity found for form E in the diaphorase, transhydrogenase, and the cytochrome c reductase assays is the result of a higher K,,,,, of this form. Initial velocity experiments conducted by several investigators all suggest an identical reaction mechanism for these three activitiesthe first steps of which are the binding of NADPH by the enzyme, reduction of the enzyme-bound FAD, and release of NADP prior to binding of the reducible substrate
598
ELLEFSONANDKROGMANN
(5, 19, 20). Therefore, the correlation between diaphorase specific activity and K NADPHfound for form E should affect its performance in these other reactions, which appears to be the case. The cause of this lowered affinity for NADPH by form E is not known at this time. Davis and San Pietro studied the interconversion between high and low activity forms of ferredoxin-NADP oxidoreductase and attributed these interconversions to a modification of a sulfhydryl group within the enzyme (21). This is consistent with results presented here which suggest that modifications of sulfhydryl groups play a role in the interconversion of multiple forms of this enzyme. The decline in ferredoxin-dependent cytochrome c reductase activity with decreasing isoelectric point of the reductase form provides another kinetic distinction among the forms. Preliminary experiments reveal that the KNADpH determined for forms D and A (50% difference in specific activity) in the cytochrome c reductase activity are identical within error. Thus it would appear that the differences in specific activity observed are due to differences in the affinity for the ferredoxin-cytochrome c acceptor. Although Gozzer et al. (1) have reported no difference in affinity for ferredoxin by the various reductase forms, the true acceptor for this activity is a cytochrome c-ferredoxin complex (22) which may have a different attraction for the reductase forms. Refocusing experiments with each of the reductase forms gave multiple peaks, representative of those found for the unresolved reductase, after storage at 4°C. The time interval required for maximum interconversion was accelerated fivefold by the removal of ampholytes from the sample. Although the original form still predominated, other more basic and acidic forms were generated. Several attempts to prevent this interconversion were unsuccessful. From the data and experiments described above, several conclusions can be made concerning the process of reductase interconversion. First deamidation can be ruled out as the sole cause of these forms because only more acidic species would be formed and this is not the case. Likewise, simple
conformational equilibrium, which would be eliminated by prior treatment of reductase with 6 M urea and 5 mM dithioerythritol followed by electrofocusing on 6 M ureacontaining gels, did not give a simplified gel pattern. The possibility of oxidation-reduction differences among the forms has the best support at the present time. Treatment of reductase with performic acid would be expected to oxidize all sulfur-containing amino acids and other readily oxidizable groups to a uniform oxidation state. Isoelectric focusing after this treatment reduced the gel pattern from five to six bands to two bands. The remaining two forms found after treatment with performic acid may represent two different oxidized products or the forms might reflect accessibility differences of a labile group to oxidation. Further experimentation will be required to answer these questions. The meaning of the multiple forms of ferredoxin-NADP reductase is obscure. All reasonable efforts to suppress the appearance of multiple forms of the enzyme from appearing on refocusing of a single form have failed. For example, inclusion of excess P-mercaptoethanol does not prevent the conversion of one form into several forms. Our failure to gain chemical control over formation of these isozymes suggests that the process is not physiologically significant. However, Ortega et al. (23) have found that activity of ferredoxin-NADP reductase ofNostoc muscorum can be lowered by exposure to NADPH and they suggest that this is a physiological regulation achieved by reduction of the enzyme. It will be of interest to learn if this kind of regulation occurs with the spinach enzyme and if it can be related to the multiple forms of the enzyme described in this paper. REFERENCES 1. GOZZER, C., ZANETTI, G. A., MINCHIOTTI, Biochim. Biophys.
G., GALLIANO, M., SACCHI, L., AND CURTI, B. (1977) Acta 485, 278-290.
2. JAGENDORF, A. T., AND AVRON, M. (1958) J. Biol. Chem. 231,2’7-290. 3. ARNON, D. I. (1949) Plant Physiol. 24, l-15. 4. WARBURG, them.
O., AND CHRISTIAN, Z. 310,384~389.
W. (1941)
Bio-
MULTIPLE
FORMS
OF FERREDOXIN-NADP
5. ZANETTI, G., AND FORTI, G. (1966)J. Biol. Chem. 241, 279-285. 6. ARNON, D. I., AND BUCHANAN, B. (1971)in Methods in Enzymology (San Pietro, A., ed.), Vol. 23, pp. 413-440, Academic Press, New York. 7. BORCHERT, M. T., AND WESSELS, J. S. C. (1970) Biochim. Biophys. Acta 197, 78-83. 8. SHIN, M. (1971) in Methods in Enzymology (San Pietro, A., ed.), Vol. 23, pp. 440-447, Academic Press, New York. 9. RIGHETTI, P., AND DRYSDALE, J. W. (1971) Biochim. Biophys. Acta 236, 17-28. 10. FAIRBANKS, G., STECK, T. L., AND WALLACH, D. F. H. (1971) Biochem,istry 10, 2606-2617. 11. HIRS, C. H. W. (1967) in Methods in Enzymology (Hirs, C. H. W., ed.), Vol. 11, pp. 59-62, Academic Press, New York. 12. NELSON, N., AND NEUMANN, J. (1969) J. Biol. Chem. 244, 1926-1931. 13. AVRON, M., AND JAGENDORF, A. T. (1956) Arch. Biochem. Biophys. 65, 475-490. 14. GROS, C., AND LABOUESSE, B. (1969) Eur. J. Biothem. 7, 463-470. 15. GRAY, W. (1972) in Methods in Enzymology (Hirs, C. H. W., and Timashoff, S. N., eds.), Vol. 25,
16.
17.
18.
19. 20.
21. 22. 23.
OXIDOEEDUCTASE
599
Pt. B, pp. 121-138, Academic Press, New York. MOORE, S., AND STEIN, W. (1963) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 6, pp. 819-830, Academic Press, New York. KEISTER, D. L., SAN PIETRO, A., AND STOLZENBACH, F. E. (1960) Arch. Biochem. Biophys. 98, 235-241. MEANS, G. E., AND FEENEY, R. E. (1971) Chemical Modification of Proteins, pp. 160-162, Holden-Day, San Francisco. NAKAMURA, S., ,~ND KIMURA, T. (1971) J. Biol. Chem. 246, 6235-6241. SHIN, M. (1968) in Flavins and Flavoproteins, Proc. of 2nd Conference on Flavins and Flavoproteins (Yagi, K., ed), p. 1, Univ. Park Press, Baltimore. DAVIS, D. J., AND SAN PIETRO, A. (1977) Adz. Biochem. Biophys. 184, 572-577. DAVIS, D. J., AND SAN PIETRO, A. (1977) Arch. Biochem. Biophys. 182, 266-272. ORTEGA, T., RIVAS, J., CARDENAS, J., AND LoSADA, M. (1971) Biochem. Biophys. Res. Commun. 78, 185-193.
Studies
of the Multiple Forms of Ferredoxin-NADP Oxidoreductase From Spinach]
WILLIAM Department
L. ELLEFSON of Biochemistry,
Purdue
AND DAVID University,
W. KROGMANN
West Lafayette,
Indiana
47907
Received October 3, 1978; revised December 8, 1978 Six different forms of ferredoxin-NADP oxidoreductase from spinach were separated by isoelectric focusing and were found to differ in their specific activities in various assay systems and in their affinity for NADPH. The forms of the enzyme were found to be interconvertible when the enzyme was stored in the cold.
Recently Gozzer et al. reported the existence of five active ionic forms of ferredoxin-NADP oxidoreductase isolated from spinach leaves (1). These forms, identified by isoelectric focusing, were present at all stages of purification and were found to have their isoelectric points between pH 4.8 and pH 6. Gozzer et al. found that purification of the reductase, either in the presence of the reducing agent dithiothreitol or of serine protease inhibitor phenylmethylsulfonyl fluoride, had no effect on the ionic form distribution. Examination of the amino acid composition and diaphorase activities of some of the forms gave no significant differences among the forms. Studies in our laboratory with multiple forms of reductase isolated through the use of isoelectric focusing confirm the results obtained by these authors and extend their results to include: (a) a comparison of the relative specific activities of these forms in four different reductase assays; and (b) a documentation of the interconversion among these forms. Preliminary results are also presented which suggest that differences in the oxidation-reduction state among the forms are responsible for their appearance. MATERIALS
AND METHODS
Spinach chloroplasts were prepared from deveined leaves in 0.4 M sucrose containing 0.05 M NaCl by a 1 This work was supported by NSF Grant PMC01956 A02. 593
method similar to that. of Jagendorf and Avron (2). Chloroplasts prepared for enzyme purification were isolated from 10 kg of washed leaves with the aid of a Szent-Georgyi and Blum continuous flow apparatus used with a refrigerated Sorval centrifuge. Chloroplast fragments were prepared from whole chloroplasts by resuspension in 60 ml of 0.01 M Tris-Me? buffer, pH 7.5 and stirred overnight at 0°C. The suspension was centrifuged at 18,000 g for 15 min to collect the swollen plastids. The pellet was washed twice with 0.01 M Tris-Mes buffer, pH 7.5, and finally resuspended in the same buffer to give a chlorophyll concentration of 0.15 mg/ml. Chlorophyll concentrations were determined by the method of Arnon (3). During the early stages of purification, protein was determined by the method of Warburg and Christian (4). Protein concentrations in the latter stages of purification were based on published molecular weights and millimolar extinction coefficients. An extinction coefficient for ferredoxin-NADP oxidoreductase at 456 nm of 10.74 mM-’ cm-’ and an assumed molecular weight of 40,000 d were used to determine protein concentrations for the reductase (5). Concentrations of ferredoxin were determined using an extinction coefficient of 9.7 tnM-’ cm-l at 420 nm and a molecular weight of 11,500 d (6). The enzyme ferredoxin-NADP oxidoreductase was prepared in a manner similar to that of Borchert and Wessels (7), except that spinach chloroplasts were used as the starting material. The purity criteria were those described by Shin (8). Ferredoxin was purified as described by Arnon and Buchanan (6). Pure fractions had a ratio of A 420.,/A,,, nmgreater than 0.4. Approximately 35 mg purified ferredoxin-NADP oxidoreductase, which had been freed of most ions by ’ Abbreviations used: MES, 2-(N-Morpholino)ethanesulfonic acid; TCA, trichloroacetic acid; DCIP, dichlorophenol-indophenol; TCIP, trichlorophenol-indophenol; SDS, sodium dodecyl sulfate. 0003.9861/79/060593-07$02.00/O Copyright 0 19’79by Academic Press, Inc. All rights of reproduction in any form reserved.
594
ELLEFSON
AND
dialysis prior to lyophilization, was resuspended in a small volume of cold distilled water. The suspension was centrifuged at 10,OOOg for 10 min to remove any insoluble material and subsequently used as a sample for isoelectric focusing experiments and the preparation of each of the multiple reductase forms. Columns were prepared as outlined in the LKB Isoelectric Focusing Column Instruction Manual. The initial isoelectric focusing of purified reductase was conducted on a sucrose density gradient containing either 2 or 4% ampholyte (pH 5-7 range) with the anode at the bottom. During focusing, the wattage was kept between 2-3 Wand focusing was carried out for 36-75 h. Fractions of 15 drops were collected at an emptying rate of 1 drop/8 s. The pH of every fifth fraction was measured at 4°C. Diaphorase activity and, in some cases, absorbance at 456 nm, were measured spectrophotometrically. Further resolution of the multiple forms required additional isoelectric focusing columns. The major multiple forms were prepared by refocusing the fractions contained between pH 5-6. For this a 2% initial ampholyte concentration was required. To resolve the minor multiple forms, a pH span of 0.5 units was used to refocus after focusing initially with a 4% concentration ofampholytes (pH 5-7). For the more acidic forms, fractions focusing between pH 4.9-5.5 were pooled and refocused. For the less acidic forms, fractions focusing between pH 5.51-6.1 were pooled and subsequently refocused. To remove the ampholytes from the protein fractions, the fractions containing the multiple form of interest were pooled and loaded on a small DEAEcellulose column (1.0 x 3.0 cm) equilibrated with 0.01 M (NH,)HCO,,. The sample was washed with 10 ml of equilibration buffer to remove the sucrose and then with 10 ml of 2 N NaCl in ammonium bicarbonate buffer. The peak fractions were pooled and then loaded on a Sephadex G-50 fine column equilibrated with the same buffer. Fractions having diaphorase activity and a ratio of A2ROnm/A26,,nm of greater than one were pooled and lyophilized. The dried samples were used as a source of pure multiple forms for further study. Analytical gel electrophoresis was carried out according to the method of Righetti and Drysdale on 4% acrylamide gels containing 2% ampholyte (9). The gels were stained for protein by the method of Fairbanks et al. after removal of ampholytes with 5% TCA (10). The gels were scanned at 550 nm using a Gilford spectrophotometer with a linear transport device and an x-y recorder. Gels stained for activity were immersed for 20 min in 1 mM nitroblue tetrazolium dissolved in 0.1 M Tris-Cl buffer, pH 8.7, rinsed several times with water and then incubated in 0.5 InM NADPH for 20-35 min thereafter. The location of active reductase was noted by the appearance of an insoluble purple formazan band on the gel. These gels were scanned at 540 nm.
KROGMANN Performic acid oxidation of reductase was performed by the methods of Hirs (11). Oxidation by the peroxy acid was terminated by dilution of the reaction mixture with 10 vol of water followed by lyophilization to dryness. The powder was resuspended in another 10 vol of water and lyophilization repeated. The residue was resuspended in 0.01 M Tris-Cl, pH 7.5 and dialyzed against 1 liter of the same buffer to remove the FAD. The colorless protein was used for some of the gel electrofocusing experiments described below. NADP photoreduction assays were performed according to the method of Shin et al. (8). In a 3.0 ml total volume, the assays contained 1000 pmol of Tris-Cl, pH 7.8, 10 pmol of sodium ascorbate, 0.2 krnol of DCIP, 1 pmol of NADP, 9.3 nmol of ferredoxin, 0.68 nmol of ferredoxin-NADP oxidoreductase, and chloroplast fragments equivalent to 60 pg of chlorophyll. After initial absorbance readings were made at 340 nm, the reaction was started by illumination with a tungsten spotlight filtered through a clear glass water tank. The increase in absorbance at 340 nm was measured spectrophotometrically after 4 min of illumination at a saturating light intensity. Rates of NADP reduction were calculated using a millimolar extinction coefficient of 6.21 for NADPH. Cytochrome c reductase activity was measured by the method of Shin (8). The assays contained in a total volume of 3.0 ml: 10 prnol of Tris-Cl buffer, pH 7.8, 100 pmol of cytochrome c, 0.2 pmol of NADPH, 9.3 nmol of ferredoxin, and 5.6 x 10 ,1 nmol of reductase. Reactions were initiated by the addition offerredoxinNADP oxidoreductase. The increase in absorbance at 550 nm was measured 60 s thereafter. A millimolar extinction coefficient of 18.5 for cytochrome c (reduced minus oxidized) was used to calculate these rates (12). Transhydrogenase was measured fluorimetrically in an Eppendorf photometer Model 1100 equipped with a fluorescence attachment. The rate of the reaction was monitored using a Sargent SLRG recorder. The excitation beam was passed through a 313. to 360.nm band pass filter and the emission beam through a 400. to 300~nm cutoff filter. In this way the disappearance of NADPH fluoresence could be followed. Assay mixtures contained in a 3.0 ml total volume: 20 pmol of Tris-Cl, pH 7.8; 0.2 pmol of NADPH; 0.12 pmol of thio-NADP (a nonfluorescent acceptor), and approximately 1.8 pg of reductase to start the reaction. Diaphorase activity was measured by essentially the same method as employed by Avron and Jagendorf (13). Assays contained 256 pmol of Tris-Cl, pH 7.5, 0.2 pmol of NADPH, 0.09 pmol of TCIP and 2.2 pg of flavoprotein in a total volume of 3.0 ml. Assays were initiated by the addition of reductase and the decrease in absorbance at 620 nm was measured after 0.5 and 1 min. Rates of TCIP reduction were calculated using a millimolar extinction coefficient of 25.4 for the oxidized dye at pH 7.5. Lineweaver-Burke plots were prepared by a
MULTIPLE
FORMS OF FERREDOXIN-NADP
Hewlett-Packard computer Model 9825A equipped with plotter from data obtained for cytochrome c reductase and diaphorase activities of reductase at variable NADPH concentrations. Values for K,,,,,,,, and V in each case were calculated using a linear regression treatment of the data by the computer. The individual forms of reductase were dansylated (14) and the N-terminal residue was determined (15). All forms showed a lysine at the N-terminal position. The amino acid composition of forms B, C, and E were determined (16) and were indistinguishable. Antibodies to purified ferredoxin-NADP oxidoreductase which had not been resolved into the individual forms were elicited in rabbits according to the procedure of Keister et al. (17). Immunodiffusion of this antiserum against individual forms of the enzyme gave a single preciptin line. NADPH, nitroblue tetrazolium, Coomassie blue R, Mes, and Trizma Base were purchased from Sigma Chemical Company. TCIP, acrylamide, and NJmethylenebisacrylamide were bought from Eastman Kodak. Ampholytes, pH 5-7 and pH 3.5-10, were obtained from either LKB or Brinkmann. NADP was purchased from Calbiochem. Hydroxylapatite HTP was purchased as a powder from Bio-Rad Laboratories. Whatman DEAE-52 was used for ion-exchange chromatography while Sephadex G-100 was used for gel filtration. All other chemicals were reagent grade quality. RESULTS
When purified reductase from spinach chloroplasts was subjected to column isoelectric focusing between pH 5 and 6, six peaks of diaphorase activity were recovered (Fig. 1). The peaks and their corresponding forms have been designated A, A’, B, C, D, and E on going from the most basic to the most acid end of the elution pattern following the designation by Gozzer et al. In addition to the five peaks reported by Gozzer et al. (l), we also recover a minor peak A’ having an isoelectric point of pH 5.97 at 4°C. The relative amounts of enzyme in each form are slightly different from that reported by Gozzer et al., but peak C is the predominant species as reported by Gozzer et al. Activity
Studies
The enzyme ferredoxin-NADP oxidoreductase catalyzes a variety of oxidationreduction reactions among which are NADP photoreduction, transhydrogenase, ferre-
OXIDOREDUCTASE
Fl?ACTION
595
NUMBER
FIG. 1. Preparative isoelectric focusing column focused between pH 5 and 6. Fractions focusing between pH 5 and 6 from a pH 5-7 column were pooled and refocused for 48 h at 6°C. Column construction, operation, elution, and analysis were performed as specified under Materials and Methods.
doxin-dependent cytochrome c reductase, and diaphorase. When the various forms of reductase were compared for their ability to catalyze these reactions, the results in Table I were obtained. All forms examined catalyzed similar rates of NADP photoreduction when supplemented with an artificial electron donor, chloroplast fragments, ferredoxin, NADP, and light. When diaphorase activities were compared, all but the most acidic form E catalyzed nearly identical rates of indophenol reduction. Rates of transhydrogenase activity were highest with the two most basic forms A and A’ and slightly lower, i.e., 20% in the case of forms B and C. However, the most acidic form E was only one-half as active as the most basic form A. Examination of the rates of ferredoxin-dependent cytochrome c reduction provided the most interesting comparison. As the isoelectric point of the form decreased, the specific activity fell in a parallel manner. Most notably, the rate of cytochrome c reduction by the most acidic form E was only 33% of the maximal rate seen with form A: similarly acidic form D was only 50% as active as form A. Although the most acidic form E was able to catalyze NADP photoreduction at a rate identical to that of the other forms, it was not capable of transferring electrons as efficiently in the other direction-away from reduced NADP. Some support for this observation is pre-
596
ELLEFSON
AND KROGMANN TABLE
RATES OF REDUCTION
OF VARIOUS
Form
NADP
A A’ B C D E
1.V 1.5 1.9 1.7 1.9
ACCEPTORS
I
BY MULTIPLE
Trichlorophenolindophenol 24 22 26 26 25 15
FORMS
OF FERREDOXIN-NADP
Cytochrome c
REDUCTASE”
Thio-NADP (transhydrogenase)
84 75 60 49 41 30
12 11 9 9 6
a NADP photoreduction, cytochrome e reductase, transhydrogenase, and diaphorase assays were performed and specific activities calculated as described under Materials and Methods. For the calculation of specific activities reported here, results obtained over a threefold concentration range of reductase forms were averaged except those reported for NADP photoreduction. b Rates expressed as pmol acceptor reducedimg enzyme/min.
sented in Table II, where the effect of varying NADPH concentration on diaphorase activity catalyzed by the various reductase forms is compared. The KNAoPHdetermined for form E was found to be approximately twofold greater than that determined for the other forms. Thus the correlation between the K,,,,, constants for the various reductase forms and their observed diaphorase specific activities is apparent.
Interconversion
of Forms
In order to establish the identity of the reductase forms observed on the isoelectric focusing column, refocusing experiments were carried out. When purified form B was refocused on gels at various times after isolation and stained for activity, the results in Fig. 2 were obtained. Freshly isolated form B was found to refocus to give essentially one peak of activity belonging to form B. Storage of form B for 4 days at 4°C prior to refocusing gave a gel pattern in which TABLE II form B still predominates but other peaks SPECIFIC ACTIVITIES AND K,,,,, FOR MULTIPLE are beginning to emerge. After 11 days FORMS OF FERREDOXIN-NADP REDUCTASE storage, the activity trace obtained for form CATALYZING DIAPHORASE ACTIVITY” B now clearly shows the presence of other reductase forms, i.e., A’, C, D, and E. These K NADPH storage-generated forms were identical to Form (PM) those found in the unresolved reductase. The removal of ampholytes from form B 12.2 A accelerated the interconversion process so A’ 10.4 that within 2 days, the activity scan showed B 12.6 15.5 the same pattern of multiple forms as that C D 11.1 found for the 11-day, ampholyte-containing 24.3 E form B. Furthermore, the results observed with form B were noticed with all other u Diaphorase activity of each form was measured as forms of reductase. described under Materials and Methods except the rate In an attempt to understand the physical of TCIP reduction was recorded 15 s after reductase interrelationship among the reductase addition. Each assay contained besides the normal rehave been agents, variable concentrations of NADPH, and 2 pg forms, several experiments conducted. of each form. K,,,,, for each form was determined Gel electrofocusing of the unresolved refrom a Lineweaver-Burke plot of the data as specified under Materials and Methods. ductase treated with 6 M urea and 5 mM
MULTIPLE
FORMS OF FERREDOXIN-NADP
dithioerythritol on 6 M urea-containing gels gave five bands when stained for protein. The same results were found by Gozzer et al. (1). Immunodiffusion of an antiserum to the unresolved reductase against each of the six forms failed to reveal any antigenie differences among the forms. Preliminary evidence to support the premise that oxidation-reduction differences might be a major cause of these multiple forms is presented in Fig. 3. Here unresolved reductase was treated with the strong oxidant, performic acid, and then focused on isoelectric focusing gels. Instead of the normal pattern of five to six bands, only two bands were observed after staining for protein. The two remaining bands might be the result of partial oxidation of some amino acid, e.g., cysteine, methionine, or tryptophan which are known to be affected by this treatment (18). If cysteine oxidation is involved in the formation of the multiple forms, it is not a simple phenomenon since treatment with mercaptoethanol or isoelectric focusing in
0.30
I
I
I
I
I
0.15
$ A?
-1 0
,
I
I
I
I
2
3
4
cm of gel FIG. 2. Conversion of form B to other multiple forms: time study. Samples consisting of 2 pg of form B were subjected to isoelectric focusing on pH 5-7 gels at various times after purification. Gels were stained for activity and scanned as described under Materials and Methods. Storage conditions were at 4°C. Fresh form B (-1, I-day-old form B (- - -), and 11-day-old form B (- - -1.
OXIDOREDUCTASE
597
0.6
t
03
,o 2 0 cm of gel
FIG. 3. Electrofocusing of reductase after per-formic acid oxidation. Ferredoxin-NADP reductase was oxidized with performic acid and treated as described under Materials and Methods. A sample consisting of 10 pg of performic acid-oxidized reductase was electrofocused on pH 3.5-10 gels and stained for protein as described under Materials and Methods.
its presence does not alter the observed pattern on gels or columns. DISCUSSION
The results obtained in our laboratory agree well with those observed by Gozzer et al. (1) that, within the limits of the physical methods employed, i.e., N-terminal analysis, molecular weight analysis by SDS-gel electrophoresis and gel filtration, amino acid composition, and immunodiffusion, the multiple forms of reductase are indistinguishable except by isoelectric focusing. However, a comparison of the specific activities of each of the forms in four reductase dependent activities does reveal some differences. Although all forms catalyze a similar rate of NADP photoreduction, form E catalyzed a significantly lower rate of diaphorase, transhydrogenase, and ferredoxin-dependent cytochrome c reductase activity. This lower activity found for form E in the diaphorase, transhydrogenase, and the cytochrome c reductase assays is the result of a higher K,,,,, of this form. Initial velocity experiments conducted by several investigators all suggest an identical reaction mechanism for these three activitiesthe first steps of which are the binding of NADPH by the enzyme, reduction of the enzyme-bound FAD, and release of NADP prior to binding of the reducible substrate
598
ELLEFSONANDKROGMANN
(5, 19, 20). Therefore, the correlation between diaphorase specific activity and K NADPHfound for form E should affect its performance in these other reactions, which appears to be the case. The cause of this lowered affinity for NADPH by form E is not known at this time. Davis and San Pietro studied the interconversion between high and low activity forms of ferredoxin-NADP oxidoreductase and attributed these interconversions to a modification of a sulfhydryl group within the enzyme (21). This is consistent with results presented here which suggest that modifications of sulfhydryl groups play a role in the interconversion of multiple forms of this enzyme. The decline in ferredoxin-dependent cytochrome c reductase activity with decreasing isoelectric point of the reductase form provides another kinetic distinction among the forms. Preliminary experiments reveal that the KNADpH determined for forms D and A (50% difference in specific activity) in the cytochrome c reductase activity are identical within error. Thus it would appear that the differences in specific activity observed are due to differences in the affinity for the ferredoxin-cytochrome c acceptor. Although Gozzer et al. (1) have reported no difference in affinity for ferredoxin by the various reductase forms, the true acceptor for this activity is a cytochrome c-ferredoxin complex (22) which may have a different attraction for the reductase forms. Refocusing experiments with each of the reductase forms gave multiple peaks, representative of those found for the unresolved reductase, after storage at 4°C. The time interval required for maximum interconversion was accelerated fivefold by the removal of ampholytes from the sample. Although the original form still predominated, other more basic and acidic forms were generated. Several attempts to prevent this interconversion were unsuccessful. From the data and experiments described above, several conclusions can be made concerning the process of reductase interconversion. First deamidation can be ruled out as the sole cause of these forms because only more acidic species would be formed and this is not the case. Likewise, simple
conformational equilibrium, which would be eliminated by prior treatment of reductase with 6 M urea and 5 mM dithioerythritol followed by electrofocusing on 6 M ureacontaining gels, did not give a simplified gel pattern. The possibility of oxidation-reduction differences among the forms has the best support at the present time. Treatment of reductase with performic acid would be expected to oxidize all sulfur-containing amino acids and other readily oxidizable groups to a uniform oxidation state. Isoelectric focusing after this treatment reduced the gel pattern from five to six bands to two bands. The remaining two forms found after treatment with performic acid may represent two different oxidized products or the forms might reflect accessibility differences of a labile group to oxidation. Further experimentation will be required to answer these questions. The meaning of the multiple forms of ferredoxin-NADP reductase is obscure. All reasonable efforts to suppress the appearance of multiple forms of the enzyme from appearing on refocusing of a single form have failed. For example, inclusion of excess P-mercaptoethanol does not prevent the conversion of one form into several forms. Our failure to gain chemical control over formation of these isozymes suggests that the process is not physiologically significant. However, Ortega et al. (23) have found that activity of ferredoxin-NADP reductase ofNostoc muscorum can be lowered by exposure to NADPH and they suggest that this is a physiological regulation achieved by reduction of the enzyme. It will be of interest to learn if this kind of regulation occurs with the spinach enzyme and if it can be related to the multiple forms of the enzyme described in this paper. REFERENCES 1. GOZZER, C., ZANETTI, G. A., MINCHIOTTI, Biochim. Biophys.
G., GALLIANO, M., SACCHI, L., AND CURTI, B. (1977) Acta 485, 278-290.
2. JAGENDORF, A. T., AND AVRON, M. (1958) J. Biol. Chem. 231,2’7-290. 3. ARNON, D. I. (1949) Plant Physiol. 24, l-15. 4. WARBURG, them.
O., AND CHRISTIAN, Z. 310,384~389.
W. (1941)
Bio-
MULTIPLE
FORMS
OF FERREDOXIN-NADP
5. ZANETTI, G., AND FORTI, G. (1966)J. Biol. Chem. 241, 279-285. 6. ARNON, D. I., AND BUCHANAN, B. (1971)in Methods in Enzymology (San Pietro, A., ed.), Vol. 23, pp. 413-440, Academic Press, New York. 7. BORCHERT, M. T., AND WESSELS, J. S. C. (1970) Biochim. Biophys. Acta 197, 78-83. 8. SHIN, M. (1971) in Methods in Enzymology (San Pietro, A., ed.), Vol. 23, pp. 440-447, Academic Press, New York. 9. RIGHETTI, P., AND DRYSDALE, J. W. (1971) Biochim. Biophys. Acta 236, 17-28. 10. FAIRBANKS, G., STECK, T. L., AND WALLACH, D. F. H. (1971) Biochem,istry 10, 2606-2617. 11. HIRS, C. H. W. (1967) in Methods in Enzymology (Hirs, C. H. W., ed.), Vol. 11, pp. 59-62, Academic Press, New York. 12. NELSON, N., AND NEUMANN, J. (1969) J. Biol. Chem. 244, 1926-1931. 13. AVRON, M., AND JAGENDORF, A. T. (1956) Arch. Biochem. Biophys. 65, 475-490. 14. GROS, C., AND LABOUESSE, B. (1969) Eur. J. Biothem. 7, 463-470. 15. GRAY, W. (1972) in Methods in Enzymology (Hirs, C. H. W., and Timashoff, S. N., eds.), Vol. 25,
16.
17.
18.
19. 20.
21. 22. 23.
OXIDOEEDUCTASE
599
Pt. B, pp. 121-138, Academic Press, New York. MOORE, S., AND STEIN, W. (1963) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 6, pp. 819-830, Academic Press, New York. KEISTER, D. L., SAN PIETRO, A., AND STOLZENBACH, F. E. (1960) Arch. Biochem. Biophys. 98, 235-241. MEANS, G. E., AND FEENEY, R. E. (1971) Chemical Modification of Proteins, pp. 160-162, Holden-Day, San Francisco. NAKAMURA, S., ,~ND KIMURA, T. (1971) J. Biol. Chem. 246, 6235-6241. SHIN, M. (1968) in Flavins and Flavoproteins, Proc. of 2nd Conference on Flavins and Flavoproteins (Yagi, K., ed), p. 1, Univ. Park Press, Baltimore. DAVIS, D. J., AND SAN PIETRO, A. (1977) Adz. Biochem. Biophys. 184, 572-577. DAVIS, D. J., AND SAN PIETRO, A. (1977) Arch. Biochem. Biophys. 182, 266-272. ORTEGA, T., RIVAS, J., CARDENAS, J., AND LoSADA, M. (1971) Biochem. Biophys. Res. Commun. 78, 185-193.