[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).
614
~NZYMES, ANTIBODIES, AND OTHER PROTEINS
[72]
model compounds, lacking the biotin moiety or chemically reactive group, to inhibit biotin uptake; and (d) the neutrality of pBNP with respect to other transport systems. The major disadvantage in the use of affinity labels is the inherent loss of activity. Subsequent isolation of such affinity-labeled components from a heterogeneous mixture is valueless, since there is no direct method of proving their association with the original system; no additional mechanistic information can be obtained from an inactivated component, the identity of which is in question. Therefore, a salient feature of this system is the discovery that the pBNP-affinity-labeled biotin transport components can be chemically reactivated with thiols. 4 This discovery, the first of its kind in whole cells, is of multifold significance, since it provides (a) chemical information as to the nature of the bond between the affinity label and the transport protein; (b) a rapid method for the localization, quantification, and identification of transport components during various stages of purification; (c) a potential method for the ultimate regaining of binding activity in the purified transport components (s) ; and (d) a method for switching on and off the biological transport system of whole cells. In an expansion of the above approach, a homologous series of affinity labeling reagents was synthesized in which the chemically reactive p-nitrophenyl ester group is located at increasing distances from the biologically active ureido moiety2 The affinity labels were .selected in order to encompass chain lengths corresponding to those of biotin and biocytin, both of which occurs in nature. The inhibitory capacity of each derivative was examined and compared in order to map the topography of the biotin receptor. The.results suggest the existence of at least two nucleophilic amino acid residues adjacent to the binding site of the biotin transport system in yeast. From studies of this type, structural information may be obtained for heterogeneous systems in which X-ray crystallographic analysis is inconceivable. Preparation of Biotinyl-p-nitrophenyl Ester (BNP) p-Nitrophenol (175 mg, 1.3 mmoles) and dicyclohexylcarbodiimide (206 mg, 1 mmole) are added to a suspension of biotin (244 mg, 1.0 mmole) in 3 ml of methylene chloride. The above mixture is stirred for 24 hr at 25 °, then the reaction mixture is taken to dryness under reduced pressure, and the yellow, gummy residue is washed several times with 4T. Viswanatha, E. Bayer, and M. Wilchek, Biochim. Biophys. Acta 4{)1, 152 (1974). E. A. Bayer, T. Viswanatha, and M. Wilchek,FEBS Lett. @O,309 (1975).
[72]
BIOTIN TRANSPORT SYSTEM
615
absolute ether. The residue is taken up in isopropanol. Following filtration, the solution is reduced to minimum volume and allowed to crystallize overnight. The crystals of pBNP are collected by filtration and washed with anhydrous ether. Yield: 120 mg (33%), m.p. 156°-158 °. The synthesis of norbiotinyl- and homobiotinyl-p-nitrophenyl esters is accomplished in a similar fashion to that of the biotinyl derivative by substituting the appropriate analog. Preparation of Model Affinity Labels Members of the homologous series of biotin-containing affinity labels (see the table) are synthesized by reacting biotinyl-N-hydroxysuceiniHOMOLOGOUS SERIES OF BIOTIN-CONTAINING AFFINITY LABELS O
II
HN
NH
0) O
II
j c ~ HN NH
O ~S//'-(CH2 )~-- C--NH--(CH2 (ll)
p-Nitrophenyl ester
Abbreviation
m
n
A Chain length a
Norbiotinyl Biotinyl Homobiotinyl Norbiotinyl-glycyl Biotinyl-glycyl Biotinyl-~-alanyl Biotinyl-~,-aminobutyryl Biotinyl-b-aminovaleryl Biotinyl-~-aminocaproyl
- 1BNP BNP + 1BNP - 1B3NP B3NP B4NP B5NP B6NP B7NP
3 4 5 3 4 4 4 4 4
---1 1 2 3 4 5
- 1 0 1 2 3 4 5 6 7
D e v i a t i o n of side a r m f r o m r e f e r e n c e affinity label ( B N P ) in n u m b e r of b a c k b o n e atoms.
616
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[72]
mide ester (341 rag, 1 mmole),6 dissolved in a minimum of dimethylformamide, with the corresponding w-amino carboxylic acid (1.2 mmoles) dissolved in 0.1 M sodium bicarbonate. The reaction is allowed to proceed for 4 hr at room temperature. The solution is acidified with 1 N HC1, and the resulting precipitate is filtered, washed well with deionized water, and dried in a desiccator over calcium chloride. The purity of the above intermediates is checked by thin-layer chromatography (TLC) (chloroform-methanol-acetic acid, 9:1:1). The biotinyl-amino carboxylic acid derivative is allowed to react further with p-nitrophenol (20% excess) as described above for the synthesis of pBNP. Purity is established by TLC (chloroform-methanol 9:1) and by the stoichiometric release (A,oo) of pnitrophenol from a weighed sample hydrolyzed in 0.2 M sodium hydroxide.
Biotin Uptake Assay
Saccharomyces cerevisiae, Fleischman strain 139 (ATCC 9896) is grown at 30 ° in a "biotin-sufficient''1 Vogel's medium, ~ with the following additions: inositol (36 ~g/ml), calcium pantothenate (2 ~g/ml), pyridoxine hydrochloride and thiamine hydrochloride (4 ~g/ml each), biotin (0.25 ng/ml), and glucose (1%) instead of sucrose. In a typical experiment, growth cultures (200 ml) are harvested, washed three times, and resuspended to l0 s cells/ml with 50 mM potassium phosphate at pH 4.0. Cell samples (9 ml) are treated for 30 min at 30 ° with an ethanolic solution (1 ml) containing one of the affinity label homologs (to a final concentration of 25 ~J/) ; ethanol (1 ml) is added to control cells. Cells treated in this manner are washed three times with distilled water and resuspended to 10 ml. A sample (0.5 ml) is added to 50 mM potassium phosphate at pH 4.0 containing glucose (1% final concentration; 5.0 ml final volume). After incubation for 20 min at 30 °, 50 ~l of [14C]biotin (5 ~g/ml) are added. Aliquots (1 ml) are taken at intervals, and the cells are collected and washed on glass-fiber filters (Tamar, Israel), after which radioactive samples are counted. 8 The remainder of the inhibited cells is centrifuged and treated with a solution (5.0 ml) of 0.2 M mercaptoethanol (pH 4.0). After 30 min at 30 °, the cells are washed three times and resuspended to the original volume (9.5 ml). A sample (0.5 ml) is assayed as above for biotin uptake. e E. Bayer and M. Wilchek, this series, Vol. 34, pp. 265-267. It. J. Vogel, Microbiol Gen. Bull. 13, 42 (1956). 8 G. A. Bray, Anal. Biochem. 1, 279 (1960).
[72]
BIOTIN TR2~NSPORT SYSTEM
617
Comments
The above affinity labeling reagents were found to inhibit specifically the uptake of biotin, but not other nutrients, into yeast cells. From the reactivation studies, one can conclude that an essential cysteine or histidine residue was modified in the transport system. The biotinylated protein can be specifically adsorbed to avidin columns, and subsequent elution with thiols should yield biotin transport component(s) in an active form.
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).
614
~NZYMES, ANTIBODIES, AND OTHER PROTEINS
[72]
model compounds, lacking the biotin moiety or chemically reactive group, to inhibit biotin uptake; and (d) the neutrality of pBNP with respect to other transport systems. The major disadvantage in the use of affinity labels is the inherent loss of activity. Subsequent isolation of such affinity-labeled components from a heterogeneous mixture is valueless, since there is no direct method of proving their association with the original system; no additional mechanistic information can be obtained from an inactivated component, the identity of which is in question. Therefore, a salient feature of this system is the discovery that the pBNP-affinity-labeled biotin transport components can be chemically reactivated with thiols. 4 This discovery, the first of its kind in whole cells, is of multifold significance, since it provides (a) chemical information as to the nature of the bond between the affinity label and the transport protein; (b) a rapid method for the localization, quantification, and identification of transport components during various stages of purification; (c) a potential method for the ultimate regaining of binding activity in the purified transport components (s) ; and (d) a method for switching on and off the biological transport system of whole cells. In an expansion of the above approach, a homologous series of affinity labeling reagents was synthesized in which the chemically reactive p-nitrophenyl ester group is located at increasing distances from the biologically active ureido moiety2 The affinity labels were .selected in order to encompass chain lengths corresponding to those of biotin and biocytin, both of which occurs in nature. The inhibitory capacity of each derivative was examined and compared in order to map the topography of the biotin receptor. The.results suggest the existence of at least two nucleophilic amino acid residues adjacent to the binding site of the biotin transport system in yeast. From studies of this type, structural information may be obtained for heterogeneous systems in which X-ray crystallographic analysis is inconceivable. Preparation of Biotinyl-p-nitrophenyl Ester (BNP) p-Nitrophenol (175 mg, 1.3 mmoles) and dicyclohexylcarbodiimide (206 mg, 1 mmole) are added to a suspension of biotin (244 mg, 1.0 mmole) in 3 ml of methylene chloride. The above mixture is stirred for 24 hr at 25 °, then the reaction mixture is taken to dryness under reduced pressure, and the yellow, gummy residue is washed several times with 4T. Viswanatha, E. Bayer, and M. Wilchek, Biochim. Biophys. Acta 4{)1, 152 (1974). E. A. Bayer, T. Viswanatha, and M. Wilchek,FEBS Lett. @O,309 (1975).
[72]
BIOTIN TRANSPORT SYSTEM
615
absolute ether. The residue is taken up in isopropanol. Following filtration, the solution is reduced to minimum volume and allowed to crystallize overnight. The crystals of pBNP are collected by filtration and washed with anhydrous ether. Yield: 120 mg (33%), m.p. 156°-158 °. The synthesis of norbiotinyl- and homobiotinyl-p-nitrophenyl esters is accomplished in a similar fashion to that of the biotinyl derivative by substituting the appropriate analog. Preparation of Model Affinity Labels Members of the homologous series of biotin-containing affinity labels (see the table) are synthesized by reacting biotinyl-N-hydroxysuceiniHOMOLOGOUS SERIES OF BIOTIN-CONTAINING AFFINITY LABELS O
II
HN
NH
0) O
II
j c ~ HN NH
O ~S//'-(CH2 )~-- C--NH--(CH2 (ll)
p-Nitrophenyl ester
Abbreviation
m
n
A Chain length a
Norbiotinyl Biotinyl Homobiotinyl Norbiotinyl-glycyl Biotinyl-glycyl Biotinyl-~-alanyl Biotinyl-~,-aminobutyryl Biotinyl-b-aminovaleryl Biotinyl-~-aminocaproyl
- 1BNP BNP + 1BNP - 1B3NP B3NP B4NP B5NP B6NP B7NP
3 4 5 3 4 4 4 4 4
---1 1 2 3 4 5
- 1 0 1 2 3 4 5 6 7
D e v i a t i o n of side a r m f r o m r e f e r e n c e affinity label ( B N P ) in n u m b e r of b a c k b o n e atoms.
616
ENZYMES, ANTIBODIES, AND OTHER PROTEINS
[72]
mide ester (341 rag, 1 mmole),6 dissolved in a minimum of dimethylformamide, with the corresponding w-amino carboxylic acid (1.2 mmoles) dissolved in 0.1 M sodium bicarbonate. The reaction is allowed to proceed for 4 hr at room temperature. The solution is acidified with 1 N HC1, and the resulting precipitate is filtered, washed well with deionized water, and dried in a desiccator over calcium chloride. The purity of the above intermediates is checked by thin-layer chromatography (TLC) (chloroform-methanol-acetic acid, 9:1:1). The biotinyl-amino carboxylic acid derivative is allowed to react further with p-nitrophenol (20% excess) as described above for the synthesis of pBNP. Purity is established by TLC (chloroform-methanol 9:1) and by the stoichiometric release (A,oo) of pnitrophenol from a weighed sample hydrolyzed in 0.2 M sodium hydroxide.
Biotin Uptake Assay
Saccharomyces cerevisiae, Fleischman strain 139 (ATCC 9896) is grown at 30 ° in a "biotin-sufficient''1 Vogel's medium, ~ with the following additions: inositol (36 ~g/ml), calcium pantothenate (2 ~g/ml), pyridoxine hydrochloride and thiamine hydrochloride (4 ~g/ml each), biotin (0.25 ng/ml), and glucose (1%) instead of sucrose. In a typical experiment, growth cultures (200 ml) are harvested, washed three times, and resuspended to l0 s cells/ml with 50 mM potassium phosphate at pH 4.0. Cell samples (9 ml) are treated for 30 min at 30 ° with an ethanolic solution (1 ml) containing one of the affinity label homologs (to a final concentration of 25 ~J/) ; ethanol (1 ml) is added to control cells. Cells treated in this manner are washed three times with distilled water and resuspended to 10 ml. A sample (0.5 ml) is added to 50 mM potassium phosphate at pH 4.0 containing glucose (1% final concentration; 5.0 ml final volume). After incubation for 20 min at 30 °, 50 ~l of [14C]biotin (5 ~g/ml) are added. Aliquots (1 ml) are taken at intervals, and the cells are collected and washed on glass-fiber filters (Tamar, Israel), after which radioactive samples are counted. 8 The remainder of the inhibited cells is centrifuged and treated with a solution (5.0 ml) of 0.2 M mercaptoethanol (pH 4.0). After 30 min at 30 °, the cells are washed three times and resuspended to the original volume (9.5 ml). A sample (0.5 ml) is assayed as above for biotin uptake. e E. Bayer and M. Wilchek, this series, Vol. 34, pp. 265-267. It. J. Vogel, Microbiol Gen. Bull. 13, 42 (1956). 8 G. A. Bray, Anal. Biochem. 1, 279 (1960).
[72]
BIOTIN TR2~NSPORT SYSTEM
617
Comments
The above affinity labeling reagents were found to inhibit specifically the uptake of biotin, but not other nutrients, into yeast cells. From the reactivation studies, one can conclude that an essential cysteine or histidine residue was modified in the transport system. The biotinylated protein can be specifically adsorbed to avidin columns, and subsequent elution with thiols should yield biotin transport component(s) in an active form.
