- Email: [email protected]
[222a] Leucine binding proteins from Escherichia coli
[222a]
LEUCINE BINDING PROTEINS (E.
coli)
639
[222a] Leucine Binding Proteins from Escherichia coli t By
CLEMENT E. FURLONG and LEON A. HEPPEL
Assay Methods
Principle. Binding proteins have been described for a number of L-amino acids, ~-5 as well as for o-galactose, 4 inorganic sulfate,6'7 Larabinose, s and calcium. 9 These proteins reversibly bind substrate and so far have not been found to have enzymatic activity. It is believed that they are implicated in active transport. For assay purposes, binding is most commonly measured by equilibrium dialysis. A more rapid, but in some cases less accurate, assay depends on filtration of a mixture of labeled substrate and binding protein through a nitrocellulose membrane filter, followed by cautious washing. The protein is retained on the filter, together with bound substrate. 1° Equilibrium Dialysis Assay. In testing column fractions a subsaturating level of leucine is used, but for kinetic and other quantitative studies a saturating concentration is required. The assays are carried out in Plexiglas cells containing two wells (0.2 ml each), separated by a disc of dialysis tubing, l°a In the assay using a subsaturating level of leucine (assay 1), side A contains protein, 0.05 M NaC1, and 0.01 M potassium phosphate buffer, pH 7.0, in a total volume of 0.10 or 0.05 ml. Side B contains 2 × 10 -6 M ~4C-leucine, 0.05 M NaCI, and 0.01 M phosphate
1The purification of the leucine-specific binding protein is according to the procedure of Furlong and Weiner. 2 The method described here for purification of the leucineisoleucine-valine binding protein was developed by C. E. Furlong (unpublished). zC. E. Furlong andJ. H. Weiner, Biochem. Biophys. Res. Commun., 38, 1(176 (1970). aj. R. Piperno and D. L. Oxender,J. Biol. Chem., 241, 5732 (1966). 4y. Anraku,J. Biol. Chem., 243, 3116 (1968). 50. H. Wilson and J. T. Holden,J. Biol. Chem., 244, 2743 (1969). CA. B. Pardee,J. Biol. Chem., 241, 5886 (1966). rA. B. Pardee, Science 156, 1627 (1967). 8R. W. Hogg and E. Englesberg,J. Bacteriol. 100, 423 (1969). 9R. H. Wasserman, R. A. Corradino, and A. N. Taylor, J. Biol. Chem. 243, 3978 (1968). I°A. D. Riggs and S. Bourgeois,J. Mol. Biol. 34, 361 (1968). l°aThese cells are available commercially. A detailed description of the dialysis chamber is given in P. T. Englund, J. A. Huberman, T. M. Jovin, and A. Kornberg, J. Biol. Chem. 244, 3038 (1969).
640
AMINO ACID BINDING PROTEINS
[222al
buffer in a volume equal to that in side A. When a saturating level of leucine is used (assay 2), side B contains 2 × 10-5 M leucine. The solutions are saturated with chloroform to prevent bacterial growth. After overnight equilibration at 2° on a rotating apparatus, aliquots are removed from each side and counted in a Triton-toluene counting solution. 11 Membrane Filter Assay (assay 3). In this assay, 0.05 ml of 2 X 10 -5 M leucine is added to 0.1 ml of column fraction, followed by 0.05 ml of 1M MgC12. The mixture is filtered on a 25 mm Schleicher and Schuell B-6 filter and washed with 0.5 ml of 0.2 M MgC12. The filters are dried and counted in a solution of 15 g PPO and 0.2 g POPOP (Packard Instrument Co.) in 3.8 liters of toluene.
Purification Procedure The first leucine binding protein was discovered by Piperno and Oxender; 3 it binds leucine, isoleucine, and valine. A simple purification leading to crystallization has been described. TM The protein was independently purified by Anraku. 4 A second binding protein, specific for leucine, was discovered later. 2 We describe here conditions for growth of cells and a purification procedure designed to make both leucine binding proteins available in a homogeneous state and in good yield. All steps of purification are carried out at 0-3 °. Growth of Cells. Escherichia coli Strain 7 (derived from K10, from Dr. E. C. C. Lin) are grown in minimal medium 13 supplemented with 1% succinic acid. (We used Baker and Adamson, Analytical Grade succinic acid; certain other preparations contained a growth inhibitor). Strain K 12 gave lower yields of the leucine specific protein. This was also true when succinic acid was replaced by glycerol or glucose. The cells are harvested in early stationary phase with a Sharpies continuous flow centrifuge. A large-scale osmotic shock procedure is carried out with freshly harvested cells as previously described 4 except that the concentration of EDTA is 2 mM, and MgCIz is added to a final concentration of 1 mM about 1 minute after the shock treatment with cold deionized water. Following a suggestion of Dr. D. L. Oxender, the cell paste is rapidly dispersed in liquid medium by means of a large stainless steel Waring blendor, operated twice for 5 seconds at a low speed. DEAE-Cellulose Chromatography. Shock fluids are concentrated to about 10 mg protein/ml in an Amicon model 401 ultrafiltration cell 11M. S. Patterson and R. C. Green, Anal. Chem. 37,854 (1965). 12W. R. Penrose, G. E. Nichoalds, J. R. Piperno, and D. L. Oxender, J. Biol. Chem. 243, 5921 (1968). 13S. Tanaka, S. A. Lerner, and E. C. C. Lin,J. Bacteriol. 93, 642 (1967).
[222a]
LEUCINE BINDING PROTEINS (E.
coli)
641
fitted with a UM-10 filter. Concentrated shock fluid (1.3 g protein from 369 g wet cell paste) is passed through a Biogel-Pl0 column equilibrated with deionized water. The sample volume may be up to 20% of the resin bed volume. This removes small molecules as well as considerable 260 m/x absorbing material. The protein peak (1.3 g protein in 320 ml) is applied to a 2.5 cm x 54 cm column of DEAE-cellulose (Brown and Co.) prepared by the usual washing procedure, 14 followed by equilibration with 5 mM Tris-HCl buffer, pH 7.6. This corresponds to about 5 mg protein/ml resin bed volume. The column is eluted with a linear gradient consisting of 3500 ml (10 resin bed volumes) of 5 mM Tris-HC1 buffer, pH 7.6, in the mixing chamber, and 3500 ml of the same buffer containing 0.15 M NaCI in the reservoir chamber. Two leucine-binding peaks are found to elute from the DEAE-cellulose column. Fractions from the first peak also bind isoleucine and valine, while fractions from the second peak bind only leucine. The procedure is easily scaled down to smaller amounts.
Further Purificatio~l of the Leucine-Specific Binding Protein. Preparative Polyaco,lamide (;el Electrophoresis. The fractions from the DEAE-cellulose peak which bind only leucine are pooled, concentrated by ultrafihration, and dialyzed against a 1:4 dilution of stacking gel buffer (see below). Electrophoresis is carried out in a Canalco preparative electrophoresis apparatus (in two runs) using column number PD-2/320. Any other apparatus suitable for preparative gel electrophoresis could be used. A published procedure 1~ is modified in that a buffer system developed by Jovin ~6 is used. The 3 cm high resolving gel contains 0.083 M Tris-HCl, pH 7.4; the 4 ml stacking gel contains 0.069 M Tris b a s e 0.052 M HaPO4, pH 6.7; the upper buffer contains 0.05 M Tris b a s e 0.06 M Tricine, pH 7.9; and the lower and elution buffers contain 0.06 M Tris-HC1, pH 7.3. Electrophoresis is monitored with a recorder and flow-cell spectrophotometer at 280 m/x, and requires up to 5 hours. The current is 20 milliamperes, the flow rate about one drop per 8 seconds, and the fraction size is 5 ml. After running analytical gels on all of the fractions which have leucine-binding activity, the ones which show a single band are pooled, concentrated by ultrafiltration and dialyzed against deionized water. A purification of 59-fold over the crude, desahed shock fluid is obtained with an overall yield of 58%. Custallization of the Leucine-Specific Binding Protein. Crystallization is achieved by slowly dialyzing ammonium sulfate into a solution of binding protein using a large binding chamber (1-mi capacity). Crystal14E. A. Peterson and H. A. Sober, Vol V. ~C. E. Furlong, and J. Preiss,J. Biol. Chem., 244, 2539 (1969). l~D. Wilson, personal communication.
642
AMINO ACID BINDING PROTEINS
[222a]
lization does not alter the specific activity of material obtained from the preparative gel electrophoresis step. Further Purification of the Leucine-Isoleucine-Valine Binding Protein. The leucine-isoleucine-valine binding protein from the DEAE-cellulose column may be purified to homogeneity in two additional steps. Fractions from the column are pooled, concentrated by ultrafiltration as before, and dialyzed against cold 5 mM potassium phosphate buffer, pH 7.3. Hydroxylapatite Chromatography. The concentrated protein solution is adsorbed on to a hydroxylapatite column (Bio-Gel HT) which has been equilibrated with 5 mM potassium phosphate buffer, pH 7.3. One milliliter resin bed volume of hydroxylapatite is used per 4 mg of protein. The column is washed with 2 resin bed volumes of equilibration buffer before starting a linear gradient consisting of 10 resin bed volumes of 5 mM phosphate buffer, pH 7.3, in the mixing vessel and 10 resin bed volumes of 120 mM potassium phosphate buffer in the reservoir vessel. The binding protein usually elutes as a symmetrical peak about midway through the gradient (assay 1 or 3). The leucine-isoleucine-valine binding fractions are pooled, concentrated as before, and dialyzed against cold deionized water. The yield from the hydroxylapatite step should be about 90% with a specific activity of 10 m/zmoles of leucine bound per milligram of protein. Isoelectrofocusing. An electrofocusing column is prepared using carrier ampholytes with a pH range of 3-6. (These are obtained from LKB Instruments, Inc., Rockville, Maryland.) The pH gradient is formed for about 24 hours before applying the sample. To introduce the sample, the power is turned off and a volume of the carrier ampholytes slightly in excess of the volume of sample to be applied is removed from the column just below the pH 6 region. The volume is about 3-5 ml, and the operation is carried out using a syringe fitted with a long, flexible tube. (The cathode is at the top of the column.) The carrier ampholyte solution is evaporated to dryness by vacuum distillation at 37 °. The sample of protein solution is used to dissolve the ampholytesucrose pellet. This gives the protein solution the proper density to return it with the syringe to the same region of the gradient from which the ampholytes were removed. The power is turned on for an additional 24 hours or more. The column is then emptied according to the manufacturer's instructions. The activity is found at pH 4.8. We have routinely carried out analytical disc gel electrophoresis on all of the fractions containing binding activity as judged by assay 1 or assay 3. Tubes containing a single band of protein are pooled. Iso-
[222a]
LEUClYE ~ I N D X N G PROTEIns (E. coil)
643
electric focusing should result in pure protein (specific activity 15 mt~moles leucine bound per milligram of protein) with a yield of 60%. The recovery of total leucine binding activity in the DEAE-cellulose step is quantitative. Overall recovery of the leucine-isoleucine-valine binding protein is 50-60%, with a purification of 25-fold from the crude, desahed shock fluid. Tile protein is crystallized by dialysis against 2-methyl-2, 4-pentanediol, as described by Penrose et al. v' Properties °/ the Leucine Specific Binding Protein. This material appeared to be homogeneous as judged by disc gel electrophoresis at two pH's and with different percent gels. Its molecular weight was estimated al 36,000 by the disc gel method of Hedrick and Smith, ~7 37,000 by the Sephadex gel filtration method of Andrews, TM and 39,400 by equilibrium ultracentrifugation (assuming v equals 0.73). Of the many k-amino acids tested, only leucine was hound, and no other nonradioactive amino acid inhibited the binding of 14C-leucine. The binding of leucine was, however, quite sensitive to inhibition by the analog trifluoroleucine. The Ka tot leucine binding was 0.7 /xM. Properties ~?/ the Leucine-lsoleuci~le-Valine Binding Protein. This protein also appeared to be homogeneous as judged by disc gel electrophoresis and sedimentation pattern. It bound leucine, isoleucine, and valine. Other amino acids did not affect the binding except fi)r weak inhibition by threonine. Trifluoroleucine did not inhibit the binding of leucine by this particular binding protein. The K,l t:or leucine binding was 1
/~M.
hmmmolog~ieal Properties ~!/the Two 1,eueine Binding Protein, s. Antiserum prepared against either of the leucine binding proteins reacts with the other binding protein. Neither antiserum reacts with binding proteins for galactose, arginine (3 proteins), cystine, or glutamine) '~
17.1.L. Hedrick and A.J. Smith, Arch. Biochem. Biophys. 126, 155 (1968). l~p. Andrews, Biochem..]. 91,222 (1964). ~9C. E. Furlong and J. H. Weiner (unpublished).
LEUCINE BINDING PROTEINS (E.
coli)
639
[222a] Leucine Binding Proteins from Escherichia coli t By
CLEMENT E. FURLONG and LEON A. HEPPEL
Assay Methods
Principle. Binding proteins have been described for a number of L-amino acids, ~-5 as well as for o-galactose, 4 inorganic sulfate,6'7 Larabinose, s and calcium. 9 These proteins reversibly bind substrate and so far have not been found to have enzymatic activity. It is believed that they are implicated in active transport. For assay purposes, binding is most commonly measured by equilibrium dialysis. A more rapid, but in some cases less accurate, assay depends on filtration of a mixture of labeled substrate and binding protein through a nitrocellulose membrane filter, followed by cautious washing. The protein is retained on the filter, together with bound substrate. 1° Equilibrium Dialysis Assay. In testing column fractions a subsaturating level of leucine is used, but for kinetic and other quantitative studies a saturating concentration is required. The assays are carried out in Plexiglas cells containing two wells (0.2 ml each), separated by a disc of dialysis tubing, l°a In the assay using a subsaturating level of leucine (assay 1), side A contains protein, 0.05 M NaC1, and 0.01 M potassium phosphate buffer, pH 7.0, in a total volume of 0.10 or 0.05 ml. Side B contains 2 × 10 -6 M ~4C-leucine, 0.05 M NaCI, and 0.01 M phosphate
1The purification of the leucine-specific binding protein is according to the procedure of Furlong and Weiner. 2 The method described here for purification of the leucineisoleucine-valine binding protein was developed by C. E. Furlong (unpublished). zC. E. Furlong andJ. H. Weiner, Biochem. Biophys. Res. Commun., 38, 1(176 (1970). aj. R. Piperno and D. L. Oxender,J. Biol. Chem., 241, 5732 (1966). 4y. Anraku,J. Biol. Chem., 243, 3116 (1968). 50. H. Wilson and J. T. Holden,J. Biol. Chem., 244, 2743 (1969). CA. B. Pardee,J. Biol. Chem., 241, 5886 (1966). rA. B. Pardee, Science 156, 1627 (1967). 8R. W. Hogg and E. Englesberg,J. Bacteriol. 100, 423 (1969). 9R. H. Wasserman, R. A. Corradino, and A. N. Taylor, J. Biol. Chem. 243, 3978 (1968). I°A. D. Riggs and S. Bourgeois,J. Mol. Biol. 34, 361 (1968). l°aThese cells are available commercially. A detailed description of the dialysis chamber is given in P. T. Englund, J. A. Huberman, T. M. Jovin, and A. Kornberg, J. Biol. Chem. 244, 3038 (1969).
640
AMINO ACID BINDING PROTEINS
[222al
buffer in a volume equal to that in side A. When a saturating level of leucine is used (assay 2), side B contains 2 × 10-5 M leucine. The solutions are saturated with chloroform to prevent bacterial growth. After overnight equilibration at 2° on a rotating apparatus, aliquots are removed from each side and counted in a Triton-toluene counting solution. 11 Membrane Filter Assay (assay 3). In this assay, 0.05 ml of 2 X 10 -5 M leucine is added to 0.1 ml of column fraction, followed by 0.05 ml of 1M MgC12. The mixture is filtered on a 25 mm Schleicher and Schuell B-6 filter and washed with 0.5 ml of 0.2 M MgC12. The filters are dried and counted in a solution of 15 g PPO and 0.2 g POPOP (Packard Instrument Co.) in 3.8 liters of toluene.
Purification Procedure The first leucine binding protein was discovered by Piperno and Oxender; 3 it binds leucine, isoleucine, and valine. A simple purification leading to crystallization has been described. TM The protein was independently purified by Anraku. 4 A second binding protein, specific for leucine, was discovered later. 2 We describe here conditions for growth of cells and a purification procedure designed to make both leucine binding proteins available in a homogeneous state and in good yield. All steps of purification are carried out at 0-3 °. Growth of Cells. Escherichia coli Strain 7 (derived from K10, from Dr. E. C. C. Lin) are grown in minimal medium 13 supplemented with 1% succinic acid. (We used Baker and Adamson, Analytical Grade succinic acid; certain other preparations contained a growth inhibitor). Strain K 12 gave lower yields of the leucine specific protein. This was also true when succinic acid was replaced by glycerol or glucose. The cells are harvested in early stationary phase with a Sharpies continuous flow centrifuge. A large-scale osmotic shock procedure is carried out with freshly harvested cells as previously described 4 except that the concentration of EDTA is 2 mM, and MgCIz is added to a final concentration of 1 mM about 1 minute after the shock treatment with cold deionized water. Following a suggestion of Dr. D. L. Oxender, the cell paste is rapidly dispersed in liquid medium by means of a large stainless steel Waring blendor, operated twice for 5 seconds at a low speed. DEAE-Cellulose Chromatography. Shock fluids are concentrated to about 10 mg protein/ml in an Amicon model 401 ultrafiltration cell 11M. S. Patterson and R. C. Green, Anal. Chem. 37,854 (1965). 12W. R. Penrose, G. E. Nichoalds, J. R. Piperno, and D. L. Oxender, J. Biol. Chem. 243, 5921 (1968). 13S. Tanaka, S. A. Lerner, and E. C. C. Lin,J. Bacteriol. 93, 642 (1967).
[222a]
LEUCINE BINDING PROTEINS (E.
coli)
641
fitted with a UM-10 filter. Concentrated shock fluid (1.3 g protein from 369 g wet cell paste) is passed through a Biogel-Pl0 column equilibrated with deionized water. The sample volume may be up to 20% of the resin bed volume. This removes small molecules as well as considerable 260 m/x absorbing material. The protein peak (1.3 g protein in 320 ml) is applied to a 2.5 cm x 54 cm column of DEAE-cellulose (Brown and Co.) prepared by the usual washing procedure, 14 followed by equilibration with 5 mM Tris-HCl buffer, pH 7.6. This corresponds to about 5 mg protein/ml resin bed volume. The column is eluted with a linear gradient consisting of 3500 ml (10 resin bed volumes) of 5 mM Tris-HC1 buffer, pH 7.6, in the mixing chamber, and 3500 ml of the same buffer containing 0.15 M NaCI in the reservoir chamber. Two leucine-binding peaks are found to elute from the DEAE-cellulose column. Fractions from the first peak also bind isoleucine and valine, while fractions from the second peak bind only leucine. The procedure is easily scaled down to smaller amounts.
Further Purificatio~l of the Leucine-Specific Binding Protein. Preparative Polyaco,lamide (;el Electrophoresis. The fractions from the DEAE-cellulose peak which bind only leucine are pooled, concentrated by ultrafihration, and dialyzed against a 1:4 dilution of stacking gel buffer (see below). Electrophoresis is carried out in a Canalco preparative electrophoresis apparatus (in two runs) using column number PD-2/320. Any other apparatus suitable for preparative gel electrophoresis could be used. A published procedure 1~ is modified in that a buffer system developed by Jovin ~6 is used. The 3 cm high resolving gel contains 0.083 M Tris-HCl, pH 7.4; the 4 ml stacking gel contains 0.069 M Tris b a s e 0.052 M HaPO4, pH 6.7; the upper buffer contains 0.05 M Tris b a s e 0.06 M Tricine, pH 7.9; and the lower and elution buffers contain 0.06 M Tris-HC1, pH 7.3. Electrophoresis is monitored with a recorder and flow-cell spectrophotometer at 280 m/x, and requires up to 5 hours. The current is 20 milliamperes, the flow rate about one drop per 8 seconds, and the fraction size is 5 ml. After running analytical gels on all of the fractions which have leucine-binding activity, the ones which show a single band are pooled, concentrated by ultrafiltration and dialyzed against deionized water. A purification of 59-fold over the crude, desahed shock fluid is obtained with an overall yield of 58%. Custallization of the Leucine-Specific Binding Protein. Crystallization is achieved by slowly dialyzing ammonium sulfate into a solution of binding protein using a large binding chamber (1-mi capacity). Crystal14E. A. Peterson and H. A. Sober, Vol V. ~C. E. Furlong, and J. Preiss,J. Biol. Chem., 244, 2539 (1969). l~D. Wilson, personal communication.
642
AMINO ACID BINDING PROTEINS
[222a]
lization does not alter the specific activity of material obtained from the preparative gel electrophoresis step. Further Purification of the Leucine-Isoleucine-Valine Binding Protein. The leucine-isoleucine-valine binding protein from the DEAE-cellulose column may be purified to homogeneity in two additional steps. Fractions from the column are pooled, concentrated by ultrafiltration as before, and dialyzed against cold 5 mM potassium phosphate buffer, pH 7.3. Hydroxylapatite Chromatography. The concentrated protein solution is adsorbed on to a hydroxylapatite column (Bio-Gel HT) which has been equilibrated with 5 mM potassium phosphate buffer, pH 7.3. One milliliter resin bed volume of hydroxylapatite is used per 4 mg of protein. The column is washed with 2 resin bed volumes of equilibration buffer before starting a linear gradient consisting of 10 resin bed volumes of 5 mM phosphate buffer, pH 7.3, in the mixing vessel and 10 resin bed volumes of 120 mM potassium phosphate buffer in the reservoir vessel. The binding protein usually elutes as a symmetrical peak about midway through the gradient (assay 1 or 3). The leucine-isoleucine-valine binding fractions are pooled, concentrated as before, and dialyzed against cold deionized water. The yield from the hydroxylapatite step should be about 90% with a specific activity of 10 m/zmoles of leucine bound per milligram of protein. Isoelectrofocusing. An electrofocusing column is prepared using carrier ampholytes with a pH range of 3-6. (These are obtained from LKB Instruments, Inc., Rockville, Maryland.) The pH gradient is formed for about 24 hours before applying the sample. To introduce the sample, the power is turned off and a volume of the carrier ampholytes slightly in excess of the volume of sample to be applied is removed from the column just below the pH 6 region. The volume is about 3-5 ml, and the operation is carried out using a syringe fitted with a long, flexible tube. (The cathode is at the top of the column.) The carrier ampholyte solution is evaporated to dryness by vacuum distillation at 37 °. The sample of protein solution is used to dissolve the ampholytesucrose pellet. This gives the protein solution the proper density to return it with the syringe to the same region of the gradient from which the ampholytes were removed. The power is turned on for an additional 24 hours or more. The column is then emptied according to the manufacturer's instructions. The activity is found at pH 4.8. We have routinely carried out analytical disc gel electrophoresis on all of the fractions containing binding activity as judged by assay 1 or assay 3. Tubes containing a single band of protein are pooled. Iso-
[222a]
LEUClYE ~ I N D X N G PROTEIns (E. coil)
643
electric focusing should result in pure protein (specific activity 15 mt~moles leucine bound per milligram of protein) with a yield of 60%. The recovery of total leucine binding activity in the DEAE-cellulose step is quantitative. Overall recovery of the leucine-isoleucine-valine binding protein is 50-60%, with a purification of 25-fold from the crude, desahed shock fluid. Tile protein is crystallized by dialysis against 2-methyl-2, 4-pentanediol, as described by Penrose et al. v' Properties °/ the Leucine Specific Binding Protein. This material appeared to be homogeneous as judged by disc gel electrophoresis at two pH's and with different percent gels. Its molecular weight was estimated al 36,000 by the disc gel method of Hedrick and Smith, ~7 37,000 by the Sephadex gel filtration method of Andrews, TM and 39,400 by equilibrium ultracentrifugation (assuming v equals 0.73). Of the many k-amino acids tested, only leucine was hound, and no other nonradioactive amino acid inhibited the binding of 14C-leucine. The binding of leucine was, however, quite sensitive to inhibition by the analog trifluoroleucine. The Ka tot leucine binding was 0.7 /xM. Properties ~?/ the Leucine-lsoleuci~le-Valine Binding Protein. This protein also appeared to be homogeneous as judged by disc gel electrophoresis and sedimentation pattern. It bound leucine, isoleucine, and valine. Other amino acids did not affect the binding except fi)r weak inhibition by threonine. Trifluoroleucine did not inhibit the binding of leucine by this particular binding protein. The K,l t:or leucine binding was 1
/~M.
hmmmolog~ieal Properties ~!/the Two 1,eueine Binding Protein, s. Antiserum prepared against either of the leucine binding proteins reacts with the other binding protein. Neither antiserum reacts with binding proteins for galactose, arginine (3 proteins), cystine, or glutamine) '~
17.1.L. Hedrick and A.J. Smith, Arch. Biochem. Biophys. 126, 155 (1968). l~p. Andrews, Biochem..]. 91,222 (1964). ~9C. E. Furlong and J. H. Weiner (unpublished).