ANALYTICAL
BIOCHEMISTRY
72,255-260
(1976)
Preparation of Cytochrome c2 from Rhodospirillum rubrum D. K. SPONHOLTZ, D. L. BRAUTIGAN, P.A. AND E. MARGOLIASH Department
of Biochemistry
and Molecular Biology, Evanston, Illinois 60201
LOACH,
Northwestern
University,
Received October 6, 1975; accepted December 2, 1975 A simplified method is described for the preparation and purification of cytochrome cZ from Rhodspirillum rubrum growth photosynthetically under anaerobic conditions. The cytochrome c1 appeared as a single electrophoretic and chromatographic form at all stages of the preparation.
Several problems are encountered in the preparation of cytochrome c2 from cells of R hodospirillum rubrum by existing procedures (l-3). These arise from the repeated use ofAmberlite IRC-50 (2) at pH values at which proteins adsorb by nonionic forces and tend to denature (4) or from adsorptions on DEAE-cellulose which are difficult to reproduce because of operation at limiting conditions (3). Cumbersome repeated desaltings of large volumes by column gel filtration are also employed (3). The requirement of substantial quantities of cytochrome c2 for studies of the reconstitution of photophosphorylating systems and of the EPR spectra and electron transfer properties of the protein, led us to attempt to simplify and improve the reproducibility of the preparation. The procedure that was developed is described below. MATERIALS
AND METHODS
Culture of R. rubrum -Cells (Rhodospirilfum rubrum No. 1.1.1) were grown in Hunter’s modified medium (5) prepared with a variation of the metals “44” solution. Instead of E?DTA2 and ferrous sulphate, a solution of the ferric complex of N-hydroxyethylethylenediaminetriacetic acid was prepared as follows: 2.49 g FeSO,*7H,O in 50 ml water added 1 This work was supported by Grants GM-19121 and GM-11741 from the National Institutes of Health. D. K. S. was supported by a Predoctoral Fellowship from the National Science Foundation, and D. L. B. was a trainee under Grant 5TI-GM-626 from the National Institutes of Health. * Abbreviations. used: EDTA, ethylenediaminetetracetic acid; CM-cellulose, carboxymethyl-cellulose; A &4 ZBO,ratio of the absorbance at 550 nm of the reduced form to the absorbance at 280 nm of the oxidized form of cytochrome c,; DEAE-cellulose, diethylaminoethyl cellulose; Tris, tris (hydroxymethylaminomethane); mS, millisiemens. 255 Copyright 0 1976 by Academic Press. Inc. All rights of reproduction in any form reserved.
256
SPONHOLTZ
ET AL.
dropwise with stirring to 4.2 g N-hydroxyethylethylenediaminetriacetic acid in 50 ml water was aerated overnight and stored at 4°C in the dark; 20 ml of this solution was added to the other components to make 100 ml of the metals “44” solution. Employing a 5 to 10% inoculum, the cells were grown in filled, sterile, stoppered 10 liter cylindrical glass bottles (20 cm diameter x 48 cm high; Corning 2M gallon Pyrex bottles). Six bottles are placed between two banks of six 40 W fluorescent lamps each, and for the first 12 to 18 hr only two lamps on each side are turned on. This gives time for the inoculated cells to metabolize dissolved oxygen, and sufficient light for early photosynthetic growth in suspensions of low optical density. After this initial period all 12 lights are turned on to yield a final intensity of about IO4 ergs -cm-2. see-‘. The temperature in the light box rises to 33 to 35°C and it is kept at that level by forced air circulation with a fan. Three to 4 days after inoculation, when the growth curve is in its late logarithmic to early stationary phase and some of the cells have dropped to the bottom of the flask, harvesting is started. The cells are centrifuged at 2700g for 50 min in batches of 5 liters in a swinging bucket centrifuge. They are washed twice on the centrifuge with about 6 vol of distilled water and stored at -20”. The cells are thawed, suspended in about 2 vol of distilled water by blending for 30 set in a 1 liter Waring Blender, and lyophilized. The lyophilized material can be stored indefinitely at -20°C. The yield was 1 to 1.2 g dry weight per liter of growth medium, as compared to the 0.2 to 0.5 g previously reported (2). Preparurion ofcytochrome c2, A typical run with cells from 57 liters of growth medium was as follows: at 4°C) lyophilized cells (65 g dry weight) were mixed with 800 ml of 0.1 M potassium acetate pH 5.0 and homogenized at high speed in a CB-6 Model 91-215 Waring Blender for 2 min in 30 set pulses. The suspension is stirred 2-3 hr, keeping the pH at 5.0 with glacial acetic acid, and centrifuged at 9000g for 30 min in a Sorvall GSA-2 rotor. The supematant was adjusted to pH 5.0, and the pellet was resuspended in an additional 500 ml of 0.1 M acetate buffer and homogenized in the blender for 3 min as before. After stirring for 1 hr, the resuspended pellets were centrifuged as described above and the supematant fractions combined (total volume of about 1100 ml). Solid ammonium sulfate was added to a concentration of 250 g/liter and after 3 hr the purple-colored precipitate was removed by filtration through a fluted Whatman No. 4 filter paper. The pale-orange filtrate was mixed with ammonium sulfate to a final concentration of 450 g/liter and, after 2-3 hr settling, was filtered through a cone of Whatman No. 50 filter paper. The filtrate was dialyzed (Spectrapor tubing, 32 mm diam) for 25 hr against four changes of20 liters of distilled water, initially adding 200 mg of Na2HP04.7H,0 in each dialysis tube and 5 ml of concentrated ammonium hydroxide to the first 20 liters of water. The dialyzed solution was adjusted to pH 4.5 with
R.RUBRUM
CYTOCHROMEC~
257
glacial acetic acid, diluted with cold distilled water (1:3), and passed through a column of CM-cellulose (Whatman CM-32; 2.8 x 15 cm) equilibrated at pH 4.5 (1 mM acetate buffer) to collect the cytochrome c2. The protein was eluted with 0.5 M sodium chloride in 10 mM sodium phosphate pH 7.5, and then desalted on a column of Sephadex G-10 (5.0 x 55 cm) equilibrated in 25 mM sodium acetate pH 5.0 (Stage I preparation). The sample was then adsorbed onto a column of CM-cellulose (1.5 x 65 cm, pH 5.0) and eluted by a linear gradient established between 750 ml of 50 mM acetate pH 5.0 and 750 ml of 250 mM acetate pH 5.0. The cytochrome c2 fractions were pooled, lyophilized, and then desalted on a column of Sephadex G-10 (5.0 x 55 cm) in 0.1 M NH,HCO, before final lyophilization and storage. Yield: 75 mg. Absorbance ratio: A so/A 280 = 1.28. The fractions containing cytochrome c’ were pooled and stored at -20”. Gel electrofocusing. To test the purity of the cytochrome c2 at various stages in the preparation, polyacrylamide gel electrofocusing (pH 3 to 10) was performed according to Tonn (6), by a procedure derived from the discussion of Wrigley (7). After fixation in trichloracetic acid (5%), the gels were stained with a bromphenol blue solution (0.1% in 10% mercuric chloride-50% ethanol). All gels were scanned with a Helena Laboratories scanner both before and after staining, employing a wide band 570 nm filter. Duplicate gels were also cut into 2 mm slices, each slice soaked for 2 days in 1 ml water and the pH determined with a Beckman digital pH meter model 76 and a Markson Model 1808 electrode. RESULTS AND DISCUSSION The procedure described above for the preparation of cytochrome c2 from lyophilized cells of R. rubrum is basically a two-step method: ammonium sulfate fractionation of the extract and column chromatography of the protein collected from the ammonium sulfate supematant. This simplified procedure makes it possible to obtain the lyophilized pure protein in less than 2 weeks from the time of the original inoculum, or in 1 week from the lyophilized cells. The yields of cytochrome cZ were from 1 .O to 1.3 mg/liter of growth medium, or 1.0 to 1.15 mg/g dry cells in three separate preparations. It should be noted that these yields are only slightly better than those reported by Kamen er al. (2) on the basis of the dry weight of cells, but are considerably higher on the basis of the volume of the growth medium. This is because of the particularly large yield of cells obtained under the growth conditions described above. Figure 1 presents the results of electrofocusing of the products at the two stages of the preparation, while Fig. 2 shows the elution profile of the CM-cellulose column chromatography which constitutes the second stage of the preparation. As can be seen in Figs. lA and B, there are a number of
258
SPONHOLTZ
ET AL.
CYTCCHROYE
C’
FIG. 1. Scans of polyacrylamide gel electrophoretograms. Electrofocusing employing pH 3-10 carrier Ampholine (L.K.B.) in 7.5% gels. Gels stained and the pH gradient determined as given under Materials and Methods. In all gels, peak 2 is the cytochrome cz while peak 3 corresponds to the cytochrome c’. Peaks 1, 4, and 5 contain colorless proteins. Scan A: unstained gel of Stage I preparation; Scan B: stained gel of Stage I preparation. Scan C: stained gel of Fraction I of CM-cellulose column chromatogram (Fig. 2); Scan D: stained gel of Fraction II of CM-cellulose column chromatogram (Fig. 2).
different proteins present in the material collected on CM-cellulose from the ammonium sulfate supernatant, only two of which are colored, cytochrome c2 and cytochrome c’. At this stage, the absorbance ratio A ss0/’ 280was 0.97 and the cytochrome c2 was confined to a single well defined band, showing no trace of the multiple forms observed in the early stages of the preparation by Bartsch et al. (3). CM-cellulose chromatography of the Stage I preparation (Fig. 2) cleanly separates the cytochrome c2 (Fraction 1). This fraction includes a small proportion of the reduced form of the protein which migrates in front of the major ferric form. At this stage, the absorbance rationA,,,/A,, was 1.28 indicating that the cytochrome c2 was entirely pure (2). This was further confirmed by gel electrofocusing (Fig. 1C). Chromatographic Fraction II (Fig. 2) consisted mostly of the cytochrome c’. It was, however, still con-
R. RUBRUM
CYTOCHROME
259
cz
1.6 1.4
0.6
6c > F
0.4
42 f PQ
0.2
EFFLUENT
VOLUME
(ml)
FIG. 2. CM-cellulose column chromatography of Stage I preparation. Conditions given under Materials and Methods. Fractions of 5.0 ml were collected at a flow rate of 25 mhhr and read at 280 nm (solid line) and 434 nm (dashed line) with a Zeiss PMQ II spectrophotometer. The conductivity of the effluent in milhsiemens (dotted line) was determined with a Radiometer CDM3 conductivity meter. Fractions were pooled as indicated by the two solid bars. Fraction I consisted of cytochrome cI, the front running shoulder being the ferrous protein, while Fraction II contained the cytochrome c’.
taminated with a small amount of what appeared to be cytochrome cZ as seen in Fig. 1D. This is surprising, since the absorption at both 280 and 434 nm came down to baseline level for some 125 ml of eluate between the two corresponding chromatographic peaks (Fig. 2). The isoelectric point for cytochrome c2 measured by gel electrofocusing was 6.0 and that for cytochrome c’ was 6.5. As with the cytochrome cz, no multiple forms of cytochrome c’ were observed. Indications of binding between cytochromes c2 and c’ were encountered in the course of the development of this method. The presence of cytochrome c2 in the cytochrome c’ fraction after ionic exchange chromatography, mentioned above, suggests that there may be an interaction between these proteins. In addition, in an early attempt to duplicate an existing procedure, the dialyzed ammonium sulfate supernatant containing both cytochromes c2 and c’ was passed through a bed of DEAEcellulose at pH 8.0 (1 mM Tris-HCl) and all of the colored protein collected on the resin. However, after the two cytochromes were partially spearated by gel filtration, the resultant cytochrome c2 fraction did not bind to DEAE-cellulose under the same conditions. As the isoelectric ‘points
260
SPONHOLTZ
ETAL.
of the two proteins are not very different, this binding may be of a specific nature, even though there are, so far, no indications that the physiological functions of the two proteins are related (8). REFERENCES 1. Horio, T., and Kamen, M. D. (1961) Biuchim. Biophys. Acta 48,266-286. 2. Kamen, M. D., Bartsch, R. G., Horio, T., and De Klerk, H. (1963) In Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol VI, pp. 391-404, Academic Press, New York. 3. Bartsch, R. E., Kakuno, T., Horio, T., and Kamen, M. D. (1971)5. Biol. Chem. 246, 4489-4496. 4. Boardman, N. K., and Partridge, S. M. (1955) Biochem. J. 59, 543-552. 5. Cohen-Bazire, E., Sistrom, W. R., and Stanier, R. Y. (1957)5. Cellular Comp. Physiol. 49, 25-68. 6. Tonn, S. J. (1975) Doctoral thesis, Northwestern University, Evanston, Illinois. 7. Wrigley, C.. W. (1971) in New Techniques in Amino Acid, Peptide and Protein Analysis (Neideiweiser, A., and Pataki, E., eds.), pp. 291-339, Ann Arbor Science Publishers, Ann Arbor, Michigan. 8. Horio, T., and Kamen, M. D. (1970) Ann. Rev. Micro. 24, 399-428.
BIOCHEMISTRY
72,255-260
(1976)
Preparation of Cytochrome c2 from Rhodospirillum rubrum D. K. SPONHOLTZ, D. L. BRAUTIGAN, P.A. AND E. MARGOLIASH Department
of Biochemistry
and Molecular Biology, Evanston, Illinois 60201
LOACH,
Northwestern
University,
Received October 6, 1975; accepted December 2, 1975 A simplified method is described for the preparation and purification of cytochrome cZ from Rhodspirillum rubrum growth photosynthetically under anaerobic conditions. The cytochrome c1 appeared as a single electrophoretic and chromatographic form at all stages of the preparation.
Several problems are encountered in the preparation of cytochrome c2 from cells of R hodospirillum rubrum by existing procedures (l-3). These arise from the repeated use ofAmberlite IRC-50 (2) at pH values at which proteins adsorb by nonionic forces and tend to denature (4) or from adsorptions on DEAE-cellulose which are difficult to reproduce because of operation at limiting conditions (3). Cumbersome repeated desaltings of large volumes by column gel filtration are also employed (3). The requirement of substantial quantities of cytochrome c2 for studies of the reconstitution of photophosphorylating systems and of the EPR spectra and electron transfer properties of the protein, led us to attempt to simplify and improve the reproducibility of the preparation. The procedure that was developed is described below. MATERIALS
AND METHODS
Culture of R. rubrum -Cells (Rhodospirilfum rubrum No. 1.1.1) were grown in Hunter’s modified medium (5) prepared with a variation of the metals “44” solution. Instead of E?DTA2 and ferrous sulphate, a solution of the ferric complex of N-hydroxyethylethylenediaminetriacetic acid was prepared as follows: 2.49 g FeSO,*7H,O in 50 ml water added 1 This work was supported by Grants GM-19121 and GM-11741 from the National Institutes of Health. D. K. S. was supported by a Predoctoral Fellowship from the National Science Foundation, and D. L. B. was a trainee under Grant 5TI-GM-626 from the National Institutes of Health. * Abbreviations. used: EDTA, ethylenediaminetetracetic acid; CM-cellulose, carboxymethyl-cellulose; A &4 ZBO,ratio of the absorbance at 550 nm of the reduced form to the absorbance at 280 nm of the oxidized form of cytochrome c,; DEAE-cellulose, diethylaminoethyl cellulose; Tris, tris (hydroxymethylaminomethane); mS, millisiemens. 255 Copyright 0 1976 by Academic Press. Inc. All rights of reproduction in any form reserved.
256
SPONHOLTZ
ET AL.
dropwise with stirring to 4.2 g N-hydroxyethylethylenediaminetriacetic acid in 50 ml water was aerated overnight and stored at 4°C in the dark; 20 ml of this solution was added to the other components to make 100 ml of the metals “44” solution. Employing a 5 to 10% inoculum, the cells were grown in filled, sterile, stoppered 10 liter cylindrical glass bottles (20 cm diameter x 48 cm high; Corning 2M gallon Pyrex bottles). Six bottles are placed between two banks of six 40 W fluorescent lamps each, and for the first 12 to 18 hr only two lamps on each side are turned on. This gives time for the inoculated cells to metabolize dissolved oxygen, and sufficient light for early photosynthetic growth in suspensions of low optical density. After this initial period all 12 lights are turned on to yield a final intensity of about IO4 ergs -cm-2. see-‘. The temperature in the light box rises to 33 to 35°C and it is kept at that level by forced air circulation with a fan. Three to 4 days after inoculation, when the growth curve is in its late logarithmic to early stationary phase and some of the cells have dropped to the bottom of the flask, harvesting is started. The cells are centrifuged at 2700g for 50 min in batches of 5 liters in a swinging bucket centrifuge. They are washed twice on the centrifuge with about 6 vol of distilled water and stored at -20”. The cells are thawed, suspended in about 2 vol of distilled water by blending for 30 set in a 1 liter Waring Blender, and lyophilized. The lyophilized material can be stored indefinitely at -20°C. The yield was 1 to 1.2 g dry weight per liter of growth medium, as compared to the 0.2 to 0.5 g previously reported (2). Preparurion ofcytochrome c2, A typical run with cells from 57 liters of growth medium was as follows: at 4°C) lyophilized cells (65 g dry weight) were mixed with 800 ml of 0.1 M potassium acetate pH 5.0 and homogenized at high speed in a CB-6 Model 91-215 Waring Blender for 2 min in 30 set pulses. The suspension is stirred 2-3 hr, keeping the pH at 5.0 with glacial acetic acid, and centrifuged at 9000g for 30 min in a Sorvall GSA-2 rotor. The supematant was adjusted to pH 5.0, and the pellet was resuspended in an additional 500 ml of 0.1 M acetate buffer and homogenized in the blender for 3 min as before. After stirring for 1 hr, the resuspended pellets were centrifuged as described above and the supematant fractions combined (total volume of about 1100 ml). Solid ammonium sulfate was added to a concentration of 250 g/liter and after 3 hr the purple-colored precipitate was removed by filtration through a fluted Whatman No. 4 filter paper. The pale-orange filtrate was mixed with ammonium sulfate to a final concentration of 450 g/liter and, after 2-3 hr settling, was filtered through a cone of Whatman No. 50 filter paper. The filtrate was dialyzed (Spectrapor tubing, 32 mm diam) for 25 hr against four changes of20 liters of distilled water, initially adding 200 mg of Na2HP04.7H,0 in each dialysis tube and 5 ml of concentrated ammonium hydroxide to the first 20 liters of water. The dialyzed solution was adjusted to pH 4.5 with
R.RUBRUM
CYTOCHROMEC~
257
glacial acetic acid, diluted with cold distilled water (1:3), and passed through a column of CM-cellulose (Whatman CM-32; 2.8 x 15 cm) equilibrated at pH 4.5 (1 mM acetate buffer) to collect the cytochrome c2. The protein was eluted with 0.5 M sodium chloride in 10 mM sodium phosphate pH 7.5, and then desalted on a column of Sephadex G-10 (5.0 x 55 cm) equilibrated in 25 mM sodium acetate pH 5.0 (Stage I preparation). The sample was then adsorbed onto a column of CM-cellulose (1.5 x 65 cm, pH 5.0) and eluted by a linear gradient established between 750 ml of 50 mM acetate pH 5.0 and 750 ml of 250 mM acetate pH 5.0. The cytochrome c2 fractions were pooled, lyophilized, and then desalted on a column of Sephadex G-10 (5.0 x 55 cm) in 0.1 M NH,HCO, before final lyophilization and storage. Yield: 75 mg. Absorbance ratio: A so/A 280 = 1.28. The fractions containing cytochrome c’ were pooled and stored at -20”. Gel electrofocusing. To test the purity of the cytochrome c2 at various stages in the preparation, polyacrylamide gel electrofocusing (pH 3 to 10) was performed according to Tonn (6), by a procedure derived from the discussion of Wrigley (7). After fixation in trichloracetic acid (5%), the gels were stained with a bromphenol blue solution (0.1% in 10% mercuric chloride-50% ethanol). All gels were scanned with a Helena Laboratories scanner both before and after staining, employing a wide band 570 nm filter. Duplicate gels were also cut into 2 mm slices, each slice soaked for 2 days in 1 ml water and the pH determined with a Beckman digital pH meter model 76 and a Markson Model 1808 electrode. RESULTS AND DISCUSSION The procedure described above for the preparation of cytochrome c2 from lyophilized cells of R. rubrum is basically a two-step method: ammonium sulfate fractionation of the extract and column chromatography of the protein collected from the ammonium sulfate supematant. This simplified procedure makes it possible to obtain the lyophilized pure protein in less than 2 weeks from the time of the original inoculum, or in 1 week from the lyophilized cells. The yields of cytochrome cZ were from 1 .O to 1.3 mg/liter of growth medium, or 1.0 to 1.15 mg/g dry cells in three separate preparations. It should be noted that these yields are only slightly better than those reported by Kamen er al. (2) on the basis of the dry weight of cells, but are considerably higher on the basis of the volume of the growth medium. This is because of the particularly large yield of cells obtained under the growth conditions described above. Figure 1 presents the results of electrofocusing of the products at the two stages of the preparation, while Fig. 2 shows the elution profile of the CM-cellulose column chromatography which constitutes the second stage of the preparation. As can be seen in Figs. lA and B, there are a number of
258
SPONHOLTZ
ET AL.
CYTCCHROYE
C’
FIG. 1. Scans of polyacrylamide gel electrophoretograms. Electrofocusing employing pH 3-10 carrier Ampholine (L.K.B.) in 7.5% gels. Gels stained and the pH gradient determined as given under Materials and Methods. In all gels, peak 2 is the cytochrome cz while peak 3 corresponds to the cytochrome c’. Peaks 1, 4, and 5 contain colorless proteins. Scan A: unstained gel of Stage I preparation; Scan B: stained gel of Stage I preparation. Scan C: stained gel of Fraction I of CM-cellulose column chromatogram (Fig. 2); Scan D: stained gel of Fraction II of CM-cellulose column chromatogram (Fig. 2).
different proteins present in the material collected on CM-cellulose from the ammonium sulfate supernatant, only two of which are colored, cytochrome c2 and cytochrome c’. At this stage, the absorbance ratio A ss0/’ 280was 0.97 and the cytochrome c2 was confined to a single well defined band, showing no trace of the multiple forms observed in the early stages of the preparation by Bartsch et al. (3). CM-cellulose chromatography of the Stage I preparation (Fig. 2) cleanly separates the cytochrome c2 (Fraction 1). This fraction includes a small proportion of the reduced form of the protein which migrates in front of the major ferric form. At this stage, the absorbance rationA,,,/A,, was 1.28 indicating that the cytochrome c2 was entirely pure (2). This was further confirmed by gel electrofocusing (Fig. 1C). Chromatographic Fraction II (Fig. 2) consisted mostly of the cytochrome c’. It was, however, still con-
R. RUBRUM
CYTOCHROME
259
cz
1.6 1.4
0.6
6c > F
0.4
42 f PQ
0.2
EFFLUENT
VOLUME
(ml)
FIG. 2. CM-cellulose column chromatography of Stage I preparation. Conditions given under Materials and Methods. Fractions of 5.0 ml were collected at a flow rate of 25 mhhr and read at 280 nm (solid line) and 434 nm (dashed line) with a Zeiss PMQ II spectrophotometer. The conductivity of the effluent in milhsiemens (dotted line) was determined with a Radiometer CDM3 conductivity meter. Fractions were pooled as indicated by the two solid bars. Fraction I consisted of cytochrome cI, the front running shoulder being the ferrous protein, while Fraction II contained the cytochrome c’.
taminated with a small amount of what appeared to be cytochrome cZ as seen in Fig. 1D. This is surprising, since the absorption at both 280 and 434 nm came down to baseline level for some 125 ml of eluate between the two corresponding chromatographic peaks (Fig. 2). The isoelectric point for cytochrome c2 measured by gel electrofocusing was 6.0 and that for cytochrome c’ was 6.5. As with the cytochrome cz, no multiple forms of cytochrome c’ were observed. Indications of binding between cytochromes c2 and c’ were encountered in the course of the development of this method. The presence of cytochrome c2 in the cytochrome c’ fraction after ionic exchange chromatography, mentioned above, suggests that there may be an interaction between these proteins. In addition, in an early attempt to duplicate an existing procedure, the dialyzed ammonium sulfate supernatant containing both cytochromes c2 and c’ was passed through a bed of DEAEcellulose at pH 8.0 (1 mM Tris-HCl) and all of the colored protein collected on the resin. However, after the two cytochromes were partially spearated by gel filtration, the resultant cytochrome c2 fraction did not bind to DEAE-cellulose under the same conditions. As the isoelectric ‘points
260
SPONHOLTZ
ETAL.
of the two proteins are not very different, this binding may be of a specific nature, even though there are, so far, no indications that the physiological functions of the two proteins are related (8). REFERENCES 1. Horio, T., and Kamen, M. D. (1961) Biuchim. Biophys. Acta 48,266-286. 2. Kamen, M. D., Bartsch, R. G., Horio, T., and De Klerk, H. (1963) In Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol VI, pp. 391-404, Academic Press, New York. 3. Bartsch, R. E., Kakuno, T., Horio, T., and Kamen, M. D. (1971)5. Biol. Chem. 246, 4489-4496. 4. Boardman, N. K., and Partridge, S. M. (1955) Biochem. J. 59, 543-552. 5. Cohen-Bazire, E., Sistrom, W. R., and Stanier, R. Y. (1957)5. Cellular Comp. Physiol. 49, 25-68. 6. Tonn, S. J. (1975) Doctoral thesis, Northwestern University, Evanston, Illinois. 7. Wrigley, C.. W. (1971) in New Techniques in Amino Acid, Peptide and Protein Analysis (Neideiweiser, A., and Pataki, E., eds.), pp. 291-339, Ann Arbor Science Publishers, Ann Arbor, Michigan. 8. Horio, T., and Kamen, M. D. (1970) Ann. Rev. Micro. 24, 399-428.
