[71]
AMINO ACID TRANSPORT PROTEINS
607
[71] A m i n o A c i d T r a n s p o r t P r o t e i n s B y G. I. GLOVER
Rationale for the Approach Historically, it has been useful to approach the study of a complex system by breaking it down into its components to simplify the investigation and then reconstructing it. The study of active transport by this general methodology has taken a variety of directions. Membrane vesicles have allowed the study of active transport apart from possible interactions with cytoplasmic and genetic material and have given insight into the coupling of energy to transport. 1 Other techniques have been applied to the identification and purification of the molecular components (proteins) of transport systems. Osmotic shock has led to the release of proteins capable of binding a variety of transport substrates. 2 In the event the transport system is inducible, differential labeling techniques appear to be useful in introducing radioactive markers into transport proteins to guide their isolation. ~,4 Utilizing equilibrium dialysis to assay for binding, proline binding proteins have been solubilized by means of detergents from the membranes of Eschericia coli. ~ These approaches have all depended on one or more characteristics of individual transport systems that are not common to all systems: the transport proteins must be removable from the membrane by osmotic shock, or be inducible, or retain binding activity upon solubilization from the membrane or removal by osmotic shock. An alternative that depends on a transport system having specificity for a particular substrate, or class of substrate, and high affinity for the substrate(s) is affinity labeling. A radioactive affinity label could, in principle, be used to specifically label the substrate binding proteins of a transport system and the label could be used as a marker to follow the purification of the protein. The experience gained could then be applied to isolation of the potentially active, unlabeled protein. Few reports of affinity labeling of transport sites have appeared.
1 H. R. Kaback, CRC Critical Reviews in Microbiology, p. 333 (1973). 2 D. L. Oxender, Annu. Rev. Biochem. 41, 777 (1972). 3 C. F. Fox and E. P. Kennedy, Proc. Natl. Acad. Sci. U,S.A. 54, 891 (1965). 4 A. R. Kolber and W. D. Stein, Curt. Mol. Biol. 1, 244 (1967). A. S. Gordon, F. J. Lombardi, and H. R. Kaback, Proc. Natl. Acad. Sci. U.S.A. 69, 358 (1972).
608
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[71]
Becker et al2 specifically inactivated the biotin transport system of E . coli using biotin p-nitrophenyl ester. The lactose transport protein of E . coli was labeled by N-bromoacetyl-fl-D-galactopyranosylamine.7 In the only reported attempt to isolate an affinity labeled protein, s glucose 6-isothiocyanate, which is an affinity label for the glucose transport system in human erythrocytes, gave enough nonspecific labeling of other membrane proteins to render identification of the transport protein difficult. It is not unexpected that electrophilic affinity labeling reagents, by virtue of their inherent chemical reactivity, might react randomly with nucleophilic groups found on the surfaces of proteins in addition to their site-specific reactions. This criticism9 is valid, but affinity labeling reagents with reactive alkylating groups on them have shown great selectivity and given stoichiometric alkylation of purified proteins. 1°
Design of an Affinity Labeling Reagent The approach to selecting a suitable site for introducing a reactive grouping into the substrate is the same whether one is seeking to label a purified enzyme or one that is membrane bound. We investigated the substrate specificity of the general amino acid transport system in N e u r o s p o r a crassa 11 and the tyrosine/phenylalanine transport system in Bacillus subtilis 12 by testing amino acids and amino acid derivatives and analogs as competitive inhibitors. In N e u r o s p o r a we found that all common amino acids, with exception to the acidic amino acids and their amides, had high affinities for the general system. N-Acyl amino acids were poor inhibitors while those amino acids modified in the carboxylate group (esters or amides) were reasonable inhibitors with Kl values in the millimolar range. The tyrosine/phenylalanine transport system is specific for these amino acids, and it was found that carboxyl modification led to better inhibitors than did alteration of the amino groups. sj. M. Becker, M. Wilcheck, and E. Katchalski, Proc. Natl. Acad. Sci. U.S.A. 68, 2604 (1971). ~J. Yariv, A. J. Kolb, and M. Yariv, FEBS Lett. 27, 27 (1972). s R. D. Taverna and R. G. Langdon, Biochem. Biophys. Res. Commun. 54, 593 (1973). See also this volume [13]. 9S. I. Chavin, FEBS Left. 14, 269 (1971). 10E. Shaw, Physiol. Rev. 50, 244 (1970). 11C. W. Magill, S. O. Nelson, S. M. D'Ambrosio, and G. I. Glover, J. Bacteriol. 113, 1320 (1973). 12S. M. D'Ambrosio, G. I. Glover, S. O. Nelson, and R. A. Jensen, J. Bacteriol. 115, 673 (1973).
[71]
AMINO ACID TRANSPORT PROTEINS
609
Accordingly, the logical site for modification of the substrate molecule in either case was the carboxyl group. We chose to utilize the chloromethyl ketones of leucine (LUCK) and lysine (LCK) for Neurospora, and those of phenylalanine (PCK) and tyrosine (TCK) for Bacillus. +
NH3
/
R-- CH2--CH
\
C II--CHi---CI O
Synthesis of Amino Acid Chloromethyl Ketones Syntheses of TCK, TM PCK, '3 LUCK, TM and LCK 15 have been reported. With the exception of TCK, the procedures are relatively successful. TCK is obtained in low yield and is sufficiently unstable and difficult to purify that we did not use it in our extensive studies. We have developed a general synthetic procedure involving a minimum of handling and purification. Although the published procedures use crystallization for purification, we found it to be inadequate, particularly with regard to the radioactive reagents. 1~ TCK, 12 PCK, and LUCK can be purified by ionexchange chromatography on SE-Sephadex, but impurities coehromatographed with tritiated PCK. A new method for adsorption chromatography of water-soluble organic compounds on Sephadex G-10 'mS has allowed the purification of tritiated LUCK and PCK to homogeneity. The detailed procedure presented below is applicable to any amino acid with an unfunctionalized side chain and to lysine in which the amino groups can be protected with the t-butyloxycarbonyl group. The procedure generally follows that reported for synthesis of LUCK 14 except that intermediates are not isolated at any point and the products are purified by adsorption chromatography on Sephadex G-10. We believe that this is a superior method, particularly for radioactive syntheses. It should be noted that taking melting points of these compounds is not a reliable method of identification and that nuclear magnetic resonance (NMR) spectroscopy is the only sure method of confirming the 1~E. Shaw and J. Ruscica, Arch. Biochem. Biophys. 145, 484 (1971). 14p. L. Birch, H. A. El-Obeid, and M. Akhtar, Arch. Biochem. Biophys. 148, 447 (1972). 15E. Shaw and G. Glover, Arch. Biochem. Biophys. 139, 298 (1970). 1~S. M. D'Ambrosio, G. I. Glover, and R. A. Jensen, Arch. Biochera. Biophys. 1{}7, 754 (1975). 17G. I. Glover, P. S. Mariano, and T. J. Wilkinson,Sep. Sci. 10, 795 (1975). L~G. I. Glover, P. S. Mariano, and S. Cheowtirakul,Sep. Sci. 11, 147 (1976).
610
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[71]
structures of the products. In this regard, the chloromethyl ketone methylene proton singlet (--CO---CH~--C1) appears reliably at 4.1 ppm relative to internal tetramethylsilane in trifluoroacetic acid solvent. 1~ General Synthetic Procedure 19
The N-t-butyloxycarbonyl amino acids are prepared according to the published procedure :° and used without crystallization after drying the crude product at reduced pressure for several days in the presence of powdered phosphorus pentoxide. The BOC-amino acid (10 mmoles) is stirred at --15 ° in 100 ml of anhydrous ether 21 and 1.4 ml (1.0 g, 10 mmoles) of triethylamine,22 and 0.95 ml (1.08 g, 10 mmoles) of ethyl chloroformate2s is added. The suspension, which contains a white precipitate of triethylamine hydrochloride, is stirred at 0 ° for 10 min and added during a few minutes to a solution of about 30 mmoles of diazomethane in ether. :4 A vigorous evolution of nitrogen occurs, and the intensity of the yellow color of the diazomethane should decrease, but not disappear. The suspension is stirred for 30 min at 25 °, then is freed of excess diazomethane by a vigorous stream of nitrogen; the yellow color fades. The solution is extracted sequentially with four 50-ml portions of deionized water to remove triethylamine hydrochloride and with 50 ml of a 5% sodium bicarbonate solution to remove unreacted BOC-amino acid. The solution is dried by stirring over several grams of powdered anhydrous sodium sulfate and filtered. The diazoketone is converted to the chloromethyl ketone by cooling the solution to 0 ° and bubbling anhydrous hydrogen chloride gas through it. If a precipitate forms within 5 min, interrupt the reaction, remove the triethylamine hydrochloride by filtration, and continue the hydrogen chloride treatment for 30 rain. Store the hydrogen chloride-saturated solution at 0 ° for at least 2 hr, during which time crystals of the amino acid chloromethyl ketone hydrochloride form. The yield of crystals obtained by filtration is about 50%. If no crystals appear, evaporate the solvent in v a c u o and proceed with purification of the residue. 19NOTE : All procedures must be carried out in a fume hood. A. Ali, F. Fahrenholz, and B. Weinstein, Angew. Chem. Int. Ed. Engl. 11, 289 (1972). ,1 Anhydrousether can be purchased in 1-pint cans and used freshly opened. 2~Dried by storage of reagent grade over potassium hydroxidepellets. ~aUsed as purchased from Aldrich Chemical. Diazomethane is prepared from Diazald using the diazomethane kit and instructions from Aldrich. Request literature from Aldrich prior to carrying out the reaction. The standard procedure yields a yellow solution of about 3 g (70 mmoles) of diazomethanein about 250 ml of ether.
[71]
AMINO ACID TRANSPORT PROTEINS
611
Purification
The crude product is first chromatographed on SE-Sephadex as described for TCK, 12 removing impurities not removed by absorption chromatography on Sephadex G-10. A column, 2.5 X 200 cm, of Sephadex G-10 is poured and equilibrated with 1 mM hydrochloric acid. Do not substitute another brand or crosslinkage type, since this is not gel filtration, but adsorption chromatography, lm8 The crystalline amino acid chloromethyl ketone obtained above is dissolved in 2-5 ml of 1 mM HC1 and applied and eluted from the column with the same solvent. The purified amino acid chloromethyl ketone elutes as a broad peak at an elution volume of several hundred milliliters. The peaks can be detected by the UV absorption of the ketone grouping at 280 nm or by spotting samples on filter paper and using n[nhydrin to detect the amino group. The tubes containing the product are combined and lyophilized to yield the slightly off-white crystals of the chloromethyl ketone hydrochlorides.
Affinity Labeling Studies
We found that LUCK irreversibly inhibited the neutral and general transport systems of Neurospora whereas LCK had little effect on these systems. ~5 Leucine is a substrate for both systems inactivated, whereas lysine is a substrate for only the general system. Both TCK and PCK inactivated the specific tyrosine/phenylalanine transport system of Bacillus as well the transport system(s) for neutral, aliphatic amino acids. The advantage of using affinity labeling reagents to "tag" proteins in complex mixtures is that inactivation via an enzyme-inhibitor complex can be distinguished kinetically from simple bimolecular alkylation. There are two criteria that should be met by a site-specific reagent: (1) The rate of inactivation should be retarded by substrate; (2) an appropriate kinetic analysis should reveal the intermediacy of the enzymeinhibitor complex in the inactivation process. In whole-cell systems additional controls are needed: (1) Incubation of the cells with the reagent must have no effect on cell viability as determined by dilution and replicate plating. (2) The inactivation could be due to a general effect on transport, such as inactivation of the system coupling energy to active transport. This must be tested by assaying the effect of the affinity 2~S. O. Nelson, G. I. Glover, and C. W. Magill, Arch. Biochem. Biophys. 168, 483 (1975).
612
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[71]
labeling on related and unrelated transport systems found in the same cells. N e u r o s p o r a 2s
The rate of inactivation of the neutral and general transport systems by LUCK could not be retarded by three substrates (phenylalanine, leucine, or histidine), a finding indicative that this reagent was not functioning as an affinity label. This is interesting, since LUCK is a competitive inhibitor of the general transport system and, therefore, binds to the site of interest. We have shown that the mode of inactivation is the alkylation of sulfhydryl groups that are essential for transport. This attempted affinity labeling study is an example of what one can learn from a chemical approach even though original expectations are not realized. For example, LCK, which should be as good an alkylating agent as LUCK, does not inactivate the transport systems for amino acids or glucose. Furthermore, N-ethylmaleimide and iodoacetamide irreversibly inhibit amino acid and glucose transport. Thus, it is clear that LUCK is remarkably specific for sulfhydryl groups essential to the neutral and general transport systems even though there are sulfhydryl groups in the membrane that are essential to other transport systems and are accessible to other alkylating agents. The selectivity observed suggests that the use of radioactive LUCK to inactivate the neutral and general transport systems could result in the alkylation of no more than two sulfhydryl group-containing proteins that are involved in transport. Although these proteins may not be the substrate binding proteins, they may have some activity or characteristic that would suggest their role in the transport process. We are presently working with a mutant that lacks the neutral transport system in an effort to selectively alkylate the protein involved in the general amino acid transport system. BaciIlus
TCK and PCK appeared to give the same results, and we elected to continue our work with FCK since TCK was more difficult to prepare and purify. 12 PCK is a competitive inhibitor of the transport of tyrosine and phenylalanine, and the rate of inactivation of the tyrosine/phenylalanine transport system can be effectively retarded by either natural ligand. On the other hand, the rate of inactivation of the transport system (s) for the neutral, aliphatic amino acids is unaffected by any of the substrates or by phenylalanine and tyrosine. Clearly, PCK is an affinity
[72]
BIOTIN TRANSPORT SYSTEM
613
label for the tyrosine/phenylalanine transport system and inactivates and the transport of neutral, alphatic amino acids by simple bimolecular alkylation. We determined the apparent first-order rate constant for the inactivation of the tyrosine/phenylalanine transport system by several concentrations of PCK. By plotting these data in double-reciprocal form we were able to demonstrate the intermediacy of an enzyme-PCK complex in the inactivation reaction and determine Ki values of 194 and 177 tLM for PCK inhibition of tyrosine and phenylalanine transport, respectively. In addition, this analysis gives the actual first-order rate constants for the inactivation of tyrosine and phenylalanine transport by the enzymePCK complex of 0.016 and 0.012 M -1, respectively. Given the level of imprecision in the assays using whole-cell systems, these numbers are in agreement. The rate of loss of leucine transport activity is comparable to that for tyrosine and phenylalanine transport activity. The transport systems for adenine and basic and acidic amino acids are unaffected by PCK. Cells treated with PCK and then diluted and plated showed no decrease in viability compared to untreated controls. The effects of PCK are, therefore, limited to those observed.
[72] T h e B i o t i n T r a n s p o r t S y s t e m B y EDWARD A. BAYER and .~,~EIR WILCHEK
In the following account we used a well characterized active transport system as a model for affinity labeling studies on intact cells. The biotin transport system in yeasts has been characterized as a high-affinity, carrier-mediated, energy-requiring process. 1,2 Since the carrier recognizes the ureido ring of the biotin molecule, a broad range of modifications of the valeric acid side chain are possible without affecting the inherent affinity. Thus, several potential candidates for affinity labeling studies were synthesized, and biotinyl-p-nitrophenyl ester (pBNP) was found to be a potent inhibitor of biotin transport. 3 Evidence, which supports the contention that this compound acts as an affinity label, includes (a) time and concentration dependence of I)BNP inactivation at relatively low concentrations; (b) protection of the transport system from pBNP inactivation by high concentrations of free biotin; (c) inability of 1T. O. :Rogers and H. C. Lichstein, J. Bacteriol. 100, 557 (1969). : T. O. Rogers and H. C. Lichstein, J. Bacteriol. 100, 564 (1969). ~J. M. Becket, M. Wilchek, and E. Katchalski, Prec, Natl. Acad. Sci. U.S.A. 68, 2604 (1971).
AMINO ACID TRANSPORT PROTEINS
607
[71] A m i n o A c i d T r a n s p o r t P r o t e i n s B y G. I. GLOVER
Rationale for the Approach Historically, it has been useful to approach the study of a complex system by breaking it down into its components to simplify the investigation and then reconstructing it. The study of active transport by this general methodology has taken a variety of directions. Membrane vesicles have allowed the study of active transport apart from possible interactions with cytoplasmic and genetic material and have given insight into the coupling of energy to transport. 1 Other techniques have been applied to the identification and purification of the molecular components (proteins) of transport systems. Osmotic shock has led to the release of proteins capable of binding a variety of transport substrates. 2 In the event the transport system is inducible, differential labeling techniques appear to be useful in introducing radioactive markers into transport proteins to guide their isolation. ~,4 Utilizing equilibrium dialysis to assay for binding, proline binding proteins have been solubilized by means of detergents from the membranes of Eschericia coli. ~ These approaches have all depended on one or more characteristics of individual transport systems that are not common to all systems: the transport proteins must be removable from the membrane by osmotic shock, or be inducible, or retain binding activity upon solubilization from the membrane or removal by osmotic shock. An alternative that depends on a transport system having specificity for a particular substrate, or class of substrate, and high affinity for the substrate(s) is affinity labeling. A radioactive affinity label could, in principle, be used to specifically label the substrate binding proteins of a transport system and the label could be used as a marker to follow the purification of the protein. The experience gained could then be applied to isolation of the potentially active, unlabeled protein. Few reports of affinity labeling of transport sites have appeared.
1 H. R. Kaback, CRC Critical Reviews in Microbiology, p. 333 (1973). 2 D. L. Oxender, Annu. Rev. Biochem. 41, 777 (1972). 3 C. F. Fox and E. P. Kennedy, Proc. Natl. Acad. Sci. U,S.A. 54, 891 (1965). 4 A. R. Kolber and W. D. Stein, Curt. Mol. Biol. 1, 244 (1967). A. S. Gordon, F. J. Lombardi, and H. R. Kaback, Proc. Natl. Acad. Sci. U.S.A. 69, 358 (1972).
608
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[71]
Becker et al2 specifically inactivated the biotin transport system of E . coli using biotin p-nitrophenyl ester. The lactose transport protein of E . coli was labeled by N-bromoacetyl-fl-D-galactopyranosylamine.7 In the only reported attempt to isolate an affinity labeled protein, s glucose 6-isothiocyanate, which is an affinity label for the glucose transport system in human erythrocytes, gave enough nonspecific labeling of other membrane proteins to render identification of the transport protein difficult. It is not unexpected that electrophilic affinity labeling reagents, by virtue of their inherent chemical reactivity, might react randomly with nucleophilic groups found on the surfaces of proteins in addition to their site-specific reactions. This criticism9 is valid, but affinity labeling reagents with reactive alkylating groups on them have shown great selectivity and given stoichiometric alkylation of purified proteins. 1°
Design of an Affinity Labeling Reagent The approach to selecting a suitable site for introducing a reactive grouping into the substrate is the same whether one is seeking to label a purified enzyme or one that is membrane bound. We investigated the substrate specificity of the general amino acid transport system in N e u r o s p o r a crassa 11 and the tyrosine/phenylalanine transport system in Bacillus subtilis 12 by testing amino acids and amino acid derivatives and analogs as competitive inhibitors. In N e u r o s p o r a we found that all common amino acids, with exception to the acidic amino acids and their amides, had high affinities for the general system. N-Acyl amino acids were poor inhibitors while those amino acids modified in the carboxylate group (esters or amides) were reasonable inhibitors with Kl values in the millimolar range. The tyrosine/phenylalanine transport system is specific for these amino acids, and it was found that carboxyl modification led to better inhibitors than did alteration of the amino groups. sj. M. Becker, M. Wilcheck, and E. Katchalski, Proc. Natl. Acad. Sci. U.S.A. 68, 2604 (1971). ~J. Yariv, A. J. Kolb, and M. Yariv, FEBS Lett. 27, 27 (1972). s R. D. Taverna and R. G. Langdon, Biochem. Biophys. Res. Commun. 54, 593 (1973). See also this volume [13]. 9S. I. Chavin, FEBS Left. 14, 269 (1971). 10E. Shaw, Physiol. Rev. 50, 244 (1970). 11C. W. Magill, S. O. Nelson, S. M. D'Ambrosio, and G. I. Glover, J. Bacteriol. 113, 1320 (1973). 12S. M. D'Ambrosio, G. I. Glover, S. O. Nelson, and R. A. Jensen, J. Bacteriol. 115, 673 (1973).
[71]
AMINO ACID TRANSPORT PROTEINS
609
Accordingly, the logical site for modification of the substrate molecule in either case was the carboxyl group. We chose to utilize the chloromethyl ketones of leucine (LUCK) and lysine (LCK) for Neurospora, and those of phenylalanine (PCK) and tyrosine (TCK) for Bacillus. +
NH3
/
R-- CH2--CH
\
C II--CHi---CI O
Synthesis of Amino Acid Chloromethyl Ketones Syntheses of TCK, TM PCK, '3 LUCK, TM and LCK 15 have been reported. With the exception of TCK, the procedures are relatively successful. TCK is obtained in low yield and is sufficiently unstable and difficult to purify that we did not use it in our extensive studies. We have developed a general synthetic procedure involving a minimum of handling and purification. Although the published procedures use crystallization for purification, we found it to be inadequate, particularly with regard to the radioactive reagents. 1~ TCK, 12 PCK, and LUCK can be purified by ionexchange chromatography on SE-Sephadex, but impurities coehromatographed with tritiated PCK. A new method for adsorption chromatography of water-soluble organic compounds on Sephadex G-10 'mS has allowed the purification of tritiated LUCK and PCK to homogeneity. The detailed procedure presented below is applicable to any amino acid with an unfunctionalized side chain and to lysine in which the amino groups can be protected with the t-butyloxycarbonyl group. The procedure generally follows that reported for synthesis of LUCK 14 except that intermediates are not isolated at any point and the products are purified by adsorption chromatography on Sephadex G-10. We believe that this is a superior method, particularly for radioactive syntheses. It should be noted that taking melting points of these compounds is not a reliable method of identification and that nuclear magnetic resonance (NMR) spectroscopy is the only sure method of confirming the 1~E. Shaw and J. Ruscica, Arch. Biochem. Biophys. 145, 484 (1971). 14p. L. Birch, H. A. El-Obeid, and M. Akhtar, Arch. Biochem. Biophys. 148, 447 (1972). 15E. Shaw and G. Glover, Arch. Biochem. Biophys. 139, 298 (1970). 1~S. M. D'Ambrosio, G. I. Glover, and R. A. Jensen, Arch. Biochera. Biophys. 1{}7, 754 (1975). 17G. I. Glover, P. S. Mariano, and T. J. Wilkinson,Sep. Sci. 10, 795 (1975). L~G. I. Glover, P. S. Mariano, and S. Cheowtirakul,Sep. Sci. 11, 147 (1976).
610
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[71]
structures of the products. In this regard, the chloromethyl ketone methylene proton singlet (--CO---CH~--C1) appears reliably at 4.1 ppm relative to internal tetramethylsilane in trifluoroacetic acid solvent. 1~ General Synthetic Procedure 19
The N-t-butyloxycarbonyl amino acids are prepared according to the published procedure :° and used without crystallization after drying the crude product at reduced pressure for several days in the presence of powdered phosphorus pentoxide. The BOC-amino acid (10 mmoles) is stirred at --15 ° in 100 ml of anhydrous ether 21 and 1.4 ml (1.0 g, 10 mmoles) of triethylamine,22 and 0.95 ml (1.08 g, 10 mmoles) of ethyl chloroformate2s is added. The suspension, which contains a white precipitate of triethylamine hydrochloride, is stirred at 0 ° for 10 min and added during a few minutes to a solution of about 30 mmoles of diazomethane in ether. :4 A vigorous evolution of nitrogen occurs, and the intensity of the yellow color of the diazomethane should decrease, but not disappear. The suspension is stirred for 30 min at 25 °, then is freed of excess diazomethane by a vigorous stream of nitrogen; the yellow color fades. The solution is extracted sequentially with four 50-ml portions of deionized water to remove triethylamine hydrochloride and with 50 ml of a 5% sodium bicarbonate solution to remove unreacted BOC-amino acid. The solution is dried by stirring over several grams of powdered anhydrous sodium sulfate and filtered. The diazoketone is converted to the chloromethyl ketone by cooling the solution to 0 ° and bubbling anhydrous hydrogen chloride gas through it. If a precipitate forms within 5 min, interrupt the reaction, remove the triethylamine hydrochloride by filtration, and continue the hydrogen chloride treatment for 30 rain. Store the hydrogen chloride-saturated solution at 0 ° for at least 2 hr, during which time crystals of the amino acid chloromethyl ketone hydrochloride form. The yield of crystals obtained by filtration is about 50%. If no crystals appear, evaporate the solvent in v a c u o and proceed with purification of the residue. 19NOTE : All procedures must be carried out in a fume hood. A. Ali, F. Fahrenholz, and B. Weinstein, Angew. Chem. Int. Ed. Engl. 11, 289 (1972). ,1 Anhydrousether can be purchased in 1-pint cans and used freshly opened. 2~Dried by storage of reagent grade over potassium hydroxidepellets. ~aUsed as purchased from Aldrich Chemical. Diazomethane is prepared from Diazald using the diazomethane kit and instructions from Aldrich. Request literature from Aldrich prior to carrying out the reaction. The standard procedure yields a yellow solution of about 3 g (70 mmoles) of diazomethanein about 250 ml of ether.
[71]
AMINO ACID TRANSPORT PROTEINS
611
Purification
The crude product is first chromatographed on SE-Sephadex as described for TCK, 12 removing impurities not removed by absorption chromatography on Sephadex G-10. A column, 2.5 X 200 cm, of Sephadex G-10 is poured and equilibrated with 1 mM hydrochloric acid. Do not substitute another brand or crosslinkage type, since this is not gel filtration, but adsorption chromatography, lm8 The crystalline amino acid chloromethyl ketone obtained above is dissolved in 2-5 ml of 1 mM HC1 and applied and eluted from the column with the same solvent. The purified amino acid chloromethyl ketone elutes as a broad peak at an elution volume of several hundred milliliters. The peaks can be detected by the UV absorption of the ketone grouping at 280 nm or by spotting samples on filter paper and using n[nhydrin to detect the amino group. The tubes containing the product are combined and lyophilized to yield the slightly off-white crystals of the chloromethyl ketone hydrochlorides.
Affinity Labeling Studies
We found that LUCK irreversibly inhibited the neutral and general transport systems of Neurospora whereas LCK had little effect on these systems. ~5 Leucine is a substrate for both systems inactivated, whereas lysine is a substrate for only the general system. Both TCK and PCK inactivated the specific tyrosine/phenylalanine transport system of Bacillus as well the transport system(s) for neutral, aliphatic amino acids. The advantage of using affinity labeling reagents to "tag" proteins in complex mixtures is that inactivation via an enzyme-inhibitor complex can be distinguished kinetically from simple bimolecular alkylation. There are two criteria that should be met by a site-specific reagent: (1) The rate of inactivation should be retarded by substrate; (2) an appropriate kinetic analysis should reveal the intermediacy of the enzymeinhibitor complex in the inactivation process. In whole-cell systems additional controls are needed: (1) Incubation of the cells with the reagent must have no effect on cell viability as determined by dilution and replicate plating. (2) The inactivation could be due to a general effect on transport, such as inactivation of the system coupling energy to active transport. This must be tested by assaying the effect of the affinity 2~S. O. Nelson, G. I. Glover, and C. W. Magill, Arch. Biochem. Biophys. 168, 483 (1975).
612
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[71]
labeling on related and unrelated transport systems found in the same cells. N e u r o s p o r a 2s
The rate of inactivation of the neutral and general transport systems by LUCK could not be retarded by three substrates (phenylalanine, leucine, or histidine), a finding indicative that this reagent was not functioning as an affinity label. This is interesting, since LUCK is a competitive inhibitor of the general transport system and, therefore, binds to the site of interest. We have shown that the mode of inactivation is the alkylation of sulfhydryl groups that are essential for transport. This attempted affinity labeling study is an example of what one can learn from a chemical approach even though original expectations are not realized. For example, LCK, which should be as good an alkylating agent as LUCK, does not inactivate the transport systems for amino acids or glucose. Furthermore, N-ethylmaleimide and iodoacetamide irreversibly inhibit amino acid and glucose transport. Thus, it is clear that LUCK is remarkably specific for sulfhydryl groups essential to the neutral and general transport systems even though there are sulfhydryl groups in the membrane that are essential to other transport systems and are accessible to other alkylating agents. The selectivity observed suggests that the use of radioactive LUCK to inactivate the neutral and general transport systems could result in the alkylation of no more than two sulfhydryl group-containing proteins that are involved in transport. Although these proteins may not be the substrate binding proteins, they may have some activity or characteristic that would suggest their role in the transport process. We are presently working with a mutant that lacks the neutral transport system in an effort to selectively alkylate the protein involved in the general amino acid transport system. BaciIlus
TCK and PCK appeared to give the same results, and we elected to continue our work with FCK since TCK was more difficult to prepare and purify. 12 PCK is a competitive inhibitor of the transport of tyrosine and phenylalanine, and the rate of inactivation of the tyrosine/phenylalanine transport system can be effectively retarded by either natural ligand. On the other hand, the rate of inactivation of the transport system (s) for the neutral, aliphatic amino acids is unaffected by any of the substrates or by phenylalanine and tyrosine. Clearly, PCK is an affinity
[72]
BIOTIN TRANSPORT SYSTEM
613
label for the tyrosine/phenylalanine transport system and inactivates and the transport of neutral, alphatic amino acids by simple bimolecular alkylation. We determined the apparent first-order rate constant for the inactivation of the tyrosine/phenylalanine transport system by several concentrations of PCK. By plotting these data in double-reciprocal form we were able to demonstrate the intermediacy of an enzyme-PCK complex in the inactivation reaction and determine Ki values of 194 and 177 tLM for PCK inhibition of tyrosine and phenylalanine transport, respectively. In addition, this analysis gives the actual first-order rate constants for the inactivation of tyrosine and phenylalanine transport by the enzymePCK complex of 0.016 and 0.012 M -1, respectively. Given the level of imprecision in the assays using whole-cell systems, these numbers are in agreement. The rate of loss of leucine transport activity is comparable to that for tyrosine and phenylalanine transport activity. The transport systems for adenine and basic and acidic amino acids are unaffected by PCK. Cells treated with PCK and then diluted and plated showed no decrease in viability compared to untreated controls. The effects of PCK are, therefore, limited to those observed.
[72] T h e B i o t i n T r a n s p o r t S y s t e m B y EDWARD A. BAYER and .~,~EIR WILCHEK
In the following account we used a well characterized active transport system as a model for affinity labeling studies on intact cells. The biotin transport system in yeasts has been characterized as a high-affinity, carrier-mediated, energy-requiring process. 1,2 Since the carrier recognizes the ureido ring of the biotin molecule, a broad range of modifications of the valeric acid side chain are possible without affecting the inherent affinity. Thus, several potential candidates for affinity labeling studies were synthesized, and biotinyl-p-nitrophenyl ester (pBNP) was found to be a potent inhibitor of biotin transport. 3 Evidence, which supports the contention that this compound acts as an affinity label, includes (a) time and concentration dependence of I)BNP inactivation at relatively low concentrations; (b) protection of the transport system from pBNP inactivation by high concentrations of free biotin; (c) inability of 1T. O. :Rogers and H. C. Lichstein, J. Bacteriol. 100, 557 (1969). : T. O. Rogers and H. C. Lichstein, J. Bacteriol. 100, 564 (1969). ~J. M. Becket, M. Wilchek, and E. Katchalski, Prec, Natl. Acad. Sci. U.S.A. 68, 2604 (1971).
