Planta
Planta 134, 145-149 (1977)
9 by Springer-Verlag 1977
Affinity Labels for Auxin Binding Sites in Corn Coleoptile Membranes M.A. Venis Shell Biosciences Laboratory, Sittingbourne Research Centre, Sittingbourne, Kent ME9 8AG, U.K.
Abstract. Two auxin analogues have been tested as
affinity labels for auxin binding sites in coleoptile membranes of Zea mays L. Reacting the membranes at pH 8 9 with the diazonium salt of CAPA (2chloro-4-aminophenoxyacetic acid) reduces their subsequent ability to bind NAA(1-naphthylacetic acid). Diazo-Chloramben (2,5-dichloro-3-aminobenzoic acid) is also effective in inhibiting NAA binding capacity and this inhibition is largely independent of reaction pH over the range pH 6-9. Similar experiments with sulphydryl reagents have shown that reaction of the membranes with p-mercuribenzoate (PMB) strongly inhibits subsequent auxin binding activity. Prior addition of NAA protects the binding sites against the action of diazo-Chloramben or PMB when the reactions are carried out at pH 6. From these results and from other considerations, several of the amino acid residues in the binding site environment have been tentatively assigned. Key words: Affinity labels - Auxin - Cell membranes - Hormone Receptors - Receptors - Zea mays.
Introduction
We have previously described evidence for the presence of two distinct classes of high-affinity auxin binding sites in corn coleoptile membranes, termed site 1 and site 2 (Batt et al., 1976; Batt and Venis, 1976). It has recently proved possible to solubilise and partially purify the binding sites from the membranes and the binding species appear to be proteins (Venis, submitted for publication). CAPA = 2-chloro-4-aminophenoxyacetic acid ; DTNB = 5,5'-dithiobis (2-nitrobenzoic acid) ; DTT = dithiothreitol; GSH = reduced form of glutathione; N A A = 1-naphthylacetic acid; PMB =p-mercuribenzoate
Abbreviations :
As one approach towards further characterisation of the binding sites in the membranes, the effects of group-modifying reagents on binding activity are being studied. This report describes results with two diazonium compounds as potential affinity labels for auxin receptors, i.e. compounds with sufficient structural resemblance to an auxin to have preferential affinity for the receptor sites over other possible binding sites, and which contain, in addition, a chemically reactive function capable of attaching the molecule covalently to a suitable amino acid residue in the active site region. From these results, and from other findings with sulphydryl reagents, inferences are drawn concerning the amino-acid environment of the auxin binding sites.
Materials and Methods Chemicals
1-Naphthyl[1-14C]acetic acid, 61 mCi/mmol, was obtained from the Radiochemical Centre, Amersham, U.K. Chloramben (2,5dichloro-3-aminobenzoic acid) was a gift from Amchem Products Inc. Ambler, U.S.A., and was recrystallised from water. For synthesis of CAPA (2-chloro-4-aminophenoxyacetic acid), ethyl bromoacetate was condensed with 2-chloro-4-nitrophenol in aqueous ethanolic solution using sodium bicarbonate. The resulting chloronitro-ethyl phenoxyacetate was reduced catalytically (Adams catalyst) and the hydrochloride of CAPA was obtained by hydrolysis of the ester with 1 M HCI under reflux.
Diazotisation
To 5 gmoles of CAPA or Chloramben in 250 gl o f 0.5 N HC1 at 0-5~ 50 gl of freshly-prepared sodimn nitrite solution (8 mg/ ml) was added. After 10-15 min the diazonium salt was diluted with 1.2 ml of 50 m M borate pH 8.5 and aliquots of the mixture were used immediately for coupling to the membranes. In all experiments a control mixture without any amino compound was prepared.
146
M.A. Venis: Affinity Labels for Auxin Binding Sites
Membrane Preparation and Modification Membranes were prepared from 5 day, dark-grown coleoptiles of Zea mays L., cv. Kelvedon 33 (Holliwell Seed and Grain Co, Wye, UK) by differential centrifugation between 4000 x g and 80,000• as described previously ( B a t t e t al., 1976) except that the citrate wash medium was used at its unadjusted pH (8.0-8.1) instead of pH 6.0. The membranes were resuspended at the required pH at a concentration equivalent to 0.5 g tissue fresh weight/ml. Resuspension buffers were of the same composition as the grinding medium (Batt et al., 1976) except that Tris-acetate was replaced either by potassium phosphate (pH 6 8) or by sodium borate (pH 8-9). Suspensions were incubated at 25~ for 15 min with diazoninm salts or sulphydryl reagents, or with the appropriate blank solutions as controls. The reaction mixtures were then diluted into 3-4 volumes of cold p H 6 wash medium and centrifuged at 80,000 x g, 30 rain. The membrane pellets were suspended in binding buffer pH 5.5, 0.2 gM [14C] N A A was added _+0.8 gM unlabelled NAA, and saturable binding of N A A was determined by a pelleting technique (Batt et al., 1976). Total binding (site 1 and site 2) was measured as the difference in pellet radioactivity (A cpm) between the 0.2 gM and 1.0 gM N A A samples (counting efficiency = 88%). Values quoted are means of triplicate determinations. Standard errors did not exceed 3% of the mean values.
70
Chloramben
0
~
'-
o
50
4 30
10
& i 6
I 7
I 8
I 9
pH
Fig. 1. Effect of reaction pH on inhibition, by diazonium salts of CAPA or Chloramben (150 gM) of [14C] N A A binding by corn membranes. Membranes were reacted at 25~ 15 min
80
--1""~
Results
Membrane preparations were reacted with diazoCAPA or diazo-Chloramben at different pH values from pH 6-9 and the ability of the re-isolated membranes to bind [14C] NAA was compared with that of control membranes treated similarly but with the omission of the diazo compound. Treatment with diazo-CAPA does reduce subsequent binding of [~4C]NAA to the membranes, but only if the coupling reaction is carried out at pH 8-9 (Fig. 1). Inhibition by diazo-Chloramben, on the other hand, is largely independent of reaction pH over the range pH 6-9. The effectiveness of different concentrations of diazoChloramben is illustrated in Figure 2; reaction at 0~ rather than 25~ reduces inhibition about four-fold. If the compounds are acting as true affinity labels, i.e. coupling covalently with a reactive residue at or near the auxin-binding site, then coupling of the diazo-compound should be impeded by prior addition of an auxin. After separation from the reaction mixture by centrifugation and resuspension in buffer the "auxin-protected" membranes should retain greater auxin-binding activity than unprotected membranes. Unfortunately, the conditions required for coupling of diazo-CAPA (pH 8 or above, Fig. 1) are exceedingly unfavourable for NAA binding (optimum pH 5.5, see Fig. 3). It was therefore not surprising to find that NAA pretreatment at pH 8 9 affords no significant protection with this compound. The compound may indeed be acting as an affinity label, but it is not possible to obtain supporting evidence by ligand protection. Experiments of this type a r e
r
60
O
o~ 40
2c
I 30
I 75
I 150
I 300
lDiazo-Chlorarnbenl/~ M
Fig. 2 . Inhibition of [~4Cj N A A binding after treatment of membranes with different concentrations of diazo-Chloramben at pH 7, 15 min. Reaction temperature was 25~ except as noted. Control binding after 25~ incubation= 1027 cpm
difficult to perform below pH 6 because incubation of the membranes at 25~ under these conditions results in an unacceptable reduction in control binding activity. Nevertheless, pH 6 is sufficiently close to the pH optimum for NAA binding to permit investigation of auxin protection against diazo-Chloramben reaction. Addition of 25 ~tM NAA immediately prior to the diazonium salt (150 gM) does reduce substantially the extent of coupling at the receptor site, as judged by subsequent ability to bind [~4C] NAA (Table 1). The use of more general group-modifying reagents is also being examined, initially with sulphydryl r e a -
M.A. Venis: Affinity Labels for Auxin Binding Sites
147
Table 1. Affinity labelling with diazo-Chloramben in the presence or absence of N A A . M e m b r a n e s were resuspended at p H 6 and reacted ( + 2 5 IxM N A A ) with 1 5 0 g M diazo-Chloramben (25 ~ C, 15min). Control membranes received diazotisation mixture without Chloramben + NAA. Binding of [1~C] N A A was determined on the reisolated m e m b r a n e s and inhibition values were calculated relative to the appropriate controls (_+ N A A during the 25~ preincubation) Expt.
1 2 3
Inhibition of subsequent auxin binding (%) - NAA
+ NAA
47.0 52.4 63.0
20.6 7.5 18.8
Table2. Effects of treating membranes with sulphydryl reagents on subsequent auxin binding. Membranes were resuspended at p H 7 and reacted at 25~ for 15 min. Additions were made at zero time unless noted otherwise. Membranes were re-isolated and binding of [1r N A A was determined Treatment
Auxin binding (Acpm)
Inhibition (%)
Control Iodoacetic acid 1 m M Iodoacetamide 1 m M N-ethyl maleimide 1 m M DTNB 1mM P M B 0.1 m M
919 947 867 886 895 382
-3.0 5.7 3.6 2.6 58.4
Control 818 P M B 0.1 m M at + 5 m i n 424 P M B 0.1 m M at 0 min + G S H I m M at + 10min 786 G S H l m M at + 1 0 m i n 793 PMB0.1mMat0min + DTTlmM at + 10 min 741 D T T I m M at + 10min 758
48.2 3.9 3.1 9.4 7.3
gents. When several -SH reagents were tested at pH 7 under conditions similar to those used for reaction of the membranes with diazo salts, only PMB effectively inhibited the ability of the re-isolated membranes to bind [14C]NAA (Table 2). Iodoacetic acid. iodoacetamide, N-ethyl maleimide and DTNB were largely ineffective, even at 10 times the PMB concentration used (Table 2). Glutathione (GSH) and dithiothreitol (DTT) added after 10 rain of the reaction are able to reverse the inhibitory effect of PMB (Table 2). The effectiveness of PMB is enhanced at lower pH; thus treatment of membranes with 0.1 mM PMB at pH 6 leads to almost complete loss of auxin-binding activity (Table 3). Prior addition of NAA affords substantial protection against the inhibitory action of PMB at pH 6 (Table 4).
Table 3. Inhibition of auxin binding after treatment of m e m b r a n e s with P M B at pH 6. M e m b r a n e s were resuspended at pH 6 and reacted at 25 ~ C, 15 rain. Binding of [1~C] N A A was determined after re-isolation of the membranes Treatment
Auxin binding (Acpm)
Inhibition (%)
Control P M B 0.02 m M P M B 0.05 m M P M B 0.1 m M
717 619 223 16
13.7 68.9 97.8
Table 4. Protection by N A A again P M B inhibition of auxin binding. M e m b r a n e s were resuspended at p H 6 and incubated at 2 5 ~ for 15 min + 50 g M PMB. Additions of N A A were made immediately prior to PMB. Binding of [t4C]NAA was determined on the reisolated m e m b r a n e s Addition
None NAA 5 gM N A A 10~tM
Auxin binding (Acpm)
P M B inhibition (%)
Control
PMB
541 503 487
133 292 401
75.4 42.0 17.7
Discussion
From the results which have been presented, from the known variation of auxin binding with pH and from consideration of structure-activity rules, several tentative conclusions can be drawn regarding the environment of the receptor sites: 1. The presence of a free anionic function, normally carboxyl, is one of the essential structural requirements for auxin activity (see Thimann, 1963), indicating binding of the carboxyl anion to a positively-charged amino acid group in the receptor site. Binding of NAA is maximal at pit 5.5 and falls to very low values at pH 4 or pH 7 (Fig. 3). The imidazolyl-NH § of histidine (pKa=6.0; Morris, 1974) is the only candidate carboxyl-binding group whose ionization properties are compatible with loss of binding activity over the range pH 6-7. Reduction of binding on the acid side of the pH optimum can be partly attributed to protonation of the carboxyl anion (pKaNAA=4.3; Charton, 1964). 2. A fractional positive charge 0.55 nm distant from the carboxyl anion is also regarded as an essential feature of active auxin molecules (Thimann, 1963). This requirement most probably implies electrostatic bonding to an anionic residue in the binding site located at the appropriate distance from the carb-
148
M.A. Venis: AffinityLabels for Auxin Binding Sites
60(]
40C
"6
Planta 134, 145-149 (1977)
9 by Springer-Verlag 1977
Affinity Labels for Auxin Binding Sites in Corn Coleoptile Membranes M.A. Venis Shell Biosciences Laboratory, Sittingbourne Research Centre, Sittingbourne, Kent ME9 8AG, U.K.
Abstract. Two auxin analogues have been tested as
affinity labels for auxin binding sites in coleoptile membranes of Zea mays L. Reacting the membranes at pH 8 9 with the diazonium salt of CAPA (2chloro-4-aminophenoxyacetic acid) reduces their subsequent ability to bind NAA(1-naphthylacetic acid). Diazo-Chloramben (2,5-dichloro-3-aminobenzoic acid) is also effective in inhibiting NAA binding capacity and this inhibition is largely independent of reaction pH over the range pH 6-9. Similar experiments with sulphydryl reagents have shown that reaction of the membranes with p-mercuribenzoate (PMB) strongly inhibits subsequent auxin binding activity. Prior addition of NAA protects the binding sites against the action of diazo-Chloramben or PMB when the reactions are carried out at pH 6. From these results and from other considerations, several of the amino acid residues in the binding site environment have been tentatively assigned. Key words: Affinity labels - Auxin - Cell membranes - Hormone Receptors - Receptors - Zea mays.
Introduction
We have previously described evidence for the presence of two distinct classes of high-affinity auxin binding sites in corn coleoptile membranes, termed site 1 and site 2 (Batt et al., 1976; Batt and Venis, 1976). It has recently proved possible to solubilise and partially purify the binding sites from the membranes and the binding species appear to be proteins (Venis, submitted for publication). CAPA = 2-chloro-4-aminophenoxyacetic acid ; DTNB = 5,5'-dithiobis (2-nitrobenzoic acid) ; DTT = dithiothreitol; GSH = reduced form of glutathione; N A A = 1-naphthylacetic acid; PMB =p-mercuribenzoate
Abbreviations :
As one approach towards further characterisation of the binding sites in the membranes, the effects of group-modifying reagents on binding activity are being studied. This report describes results with two diazonium compounds as potential affinity labels for auxin receptors, i.e. compounds with sufficient structural resemblance to an auxin to have preferential affinity for the receptor sites over other possible binding sites, and which contain, in addition, a chemically reactive function capable of attaching the molecule covalently to a suitable amino acid residue in the active site region. From these results, and from other findings with sulphydryl reagents, inferences are drawn concerning the amino-acid environment of the auxin binding sites.
Materials and Methods Chemicals
1-Naphthyl[1-14C]acetic acid, 61 mCi/mmol, was obtained from the Radiochemical Centre, Amersham, U.K. Chloramben (2,5dichloro-3-aminobenzoic acid) was a gift from Amchem Products Inc. Ambler, U.S.A., and was recrystallised from water. For synthesis of CAPA (2-chloro-4-aminophenoxyacetic acid), ethyl bromoacetate was condensed with 2-chloro-4-nitrophenol in aqueous ethanolic solution using sodium bicarbonate. The resulting chloronitro-ethyl phenoxyacetate was reduced catalytically (Adams catalyst) and the hydrochloride of CAPA was obtained by hydrolysis of the ester with 1 M HCI under reflux.
Diazotisation
To 5 gmoles of CAPA or Chloramben in 250 gl o f 0.5 N HC1 at 0-5~ 50 gl of freshly-prepared sodimn nitrite solution (8 mg/ ml) was added. After 10-15 min the diazonium salt was diluted with 1.2 ml of 50 m M borate pH 8.5 and aliquots of the mixture were used immediately for coupling to the membranes. In all experiments a control mixture without any amino compound was prepared.
146
M.A. Venis: Affinity Labels for Auxin Binding Sites
Membrane Preparation and Modification Membranes were prepared from 5 day, dark-grown coleoptiles of Zea mays L., cv. Kelvedon 33 (Holliwell Seed and Grain Co, Wye, UK) by differential centrifugation between 4000 x g and 80,000• as described previously ( B a t t e t al., 1976) except that the citrate wash medium was used at its unadjusted pH (8.0-8.1) instead of pH 6.0. The membranes were resuspended at the required pH at a concentration equivalent to 0.5 g tissue fresh weight/ml. Resuspension buffers were of the same composition as the grinding medium (Batt et al., 1976) except that Tris-acetate was replaced either by potassium phosphate (pH 6 8) or by sodium borate (pH 8-9). Suspensions were incubated at 25~ for 15 min with diazoninm salts or sulphydryl reagents, or with the appropriate blank solutions as controls. The reaction mixtures were then diluted into 3-4 volumes of cold p H 6 wash medium and centrifuged at 80,000 x g, 30 rain. The membrane pellets were suspended in binding buffer pH 5.5, 0.2 gM [14C] N A A was added _+0.8 gM unlabelled NAA, and saturable binding of N A A was determined by a pelleting technique (Batt et al., 1976). Total binding (site 1 and site 2) was measured as the difference in pellet radioactivity (A cpm) between the 0.2 gM and 1.0 gM N A A samples (counting efficiency = 88%). Values quoted are means of triplicate determinations. Standard errors did not exceed 3% of the mean values.
70
Chloramben
0
~
'-
o
50
4 30
10
& i 6
I 7
I 8
I 9
pH
Fig. 1. Effect of reaction pH on inhibition, by diazonium salts of CAPA or Chloramben (150 gM) of [14C] N A A binding by corn membranes. Membranes were reacted at 25~ 15 min
80
--1""~
Results
Membrane preparations were reacted with diazoCAPA or diazo-Chloramben at different pH values from pH 6-9 and the ability of the re-isolated membranes to bind [14C] NAA was compared with that of control membranes treated similarly but with the omission of the diazo compound. Treatment with diazo-CAPA does reduce subsequent binding of [~4C]NAA to the membranes, but only if the coupling reaction is carried out at pH 8-9 (Fig. 1). Inhibition by diazo-Chloramben, on the other hand, is largely independent of reaction pH over the range pH 6-9. The effectiveness of different concentrations of diazoChloramben is illustrated in Figure 2; reaction at 0~ rather than 25~ reduces inhibition about four-fold. If the compounds are acting as true affinity labels, i.e. coupling covalently with a reactive residue at or near the auxin-binding site, then coupling of the diazo-compound should be impeded by prior addition of an auxin. After separation from the reaction mixture by centrifugation and resuspension in buffer the "auxin-protected" membranes should retain greater auxin-binding activity than unprotected membranes. Unfortunately, the conditions required for coupling of diazo-CAPA (pH 8 or above, Fig. 1) are exceedingly unfavourable for NAA binding (optimum pH 5.5, see Fig. 3). It was therefore not surprising to find that NAA pretreatment at pH 8 9 affords no significant protection with this compound. The compound may indeed be acting as an affinity label, but it is not possible to obtain supporting evidence by ligand protection. Experiments of this type a r e
r
60
O
o~ 40
2c
I 30
I 75
I 150
I 300
lDiazo-Chlorarnbenl/~ M
Fig. 2 . Inhibition of [~4Cj N A A binding after treatment of membranes with different concentrations of diazo-Chloramben at pH 7, 15 min. Reaction temperature was 25~ except as noted. Control binding after 25~ incubation= 1027 cpm
difficult to perform below pH 6 because incubation of the membranes at 25~ under these conditions results in an unacceptable reduction in control binding activity. Nevertheless, pH 6 is sufficiently close to the pH optimum for NAA binding to permit investigation of auxin protection against diazo-Chloramben reaction. Addition of 25 ~tM NAA immediately prior to the diazonium salt (150 gM) does reduce substantially the extent of coupling at the receptor site, as judged by subsequent ability to bind [~4C] NAA (Table 1). The use of more general group-modifying reagents is also being examined, initially with sulphydryl r e a -
M.A. Venis: Affinity Labels for Auxin Binding Sites
147
Table 1. Affinity labelling with diazo-Chloramben in the presence or absence of N A A . M e m b r a n e s were resuspended at p H 6 and reacted ( + 2 5 IxM N A A ) with 1 5 0 g M diazo-Chloramben (25 ~ C, 15min). Control membranes received diazotisation mixture without Chloramben + NAA. Binding of [1~C] N A A was determined on the reisolated m e m b r a n e s and inhibition values were calculated relative to the appropriate controls (_+ N A A during the 25~ preincubation) Expt.
1 2 3
Inhibition of subsequent auxin binding (%) - NAA
+ NAA
47.0 52.4 63.0
20.6 7.5 18.8
Table2. Effects of treating membranes with sulphydryl reagents on subsequent auxin binding. Membranes were resuspended at p H 7 and reacted at 25~ for 15 min. Additions were made at zero time unless noted otherwise. Membranes were re-isolated and binding of [1r N A A was determined Treatment
Auxin binding (Acpm)
Inhibition (%)
Control Iodoacetic acid 1 m M Iodoacetamide 1 m M N-ethyl maleimide 1 m M DTNB 1mM P M B 0.1 m M
919 947 867 886 895 382
-3.0 5.7 3.6 2.6 58.4
Control 818 P M B 0.1 m M at + 5 m i n 424 P M B 0.1 m M at 0 min + G S H I m M at + 10min 786 G S H l m M at + 1 0 m i n 793 PMB0.1mMat0min + DTTlmM at + 10 min 741 D T T I m M at + 10min 758
48.2 3.9 3.1 9.4 7.3
gents. When several -SH reagents were tested at pH 7 under conditions similar to those used for reaction of the membranes with diazo salts, only PMB effectively inhibited the ability of the re-isolated membranes to bind [14C]NAA (Table 2). Iodoacetic acid. iodoacetamide, N-ethyl maleimide and DTNB were largely ineffective, even at 10 times the PMB concentration used (Table 2). Glutathione (GSH) and dithiothreitol (DTT) added after 10 rain of the reaction are able to reverse the inhibitory effect of PMB (Table 2). The effectiveness of PMB is enhanced at lower pH; thus treatment of membranes with 0.1 mM PMB at pH 6 leads to almost complete loss of auxin-binding activity (Table 3). Prior addition of NAA affords substantial protection against the inhibitory action of PMB at pH 6 (Table 4).
Table 3. Inhibition of auxin binding after treatment of m e m b r a n e s with P M B at pH 6. M e m b r a n e s were resuspended at pH 6 and reacted at 25 ~ C, 15 rain. Binding of [1~C] N A A was determined after re-isolation of the membranes Treatment
Auxin binding (Acpm)
Inhibition (%)
Control P M B 0.02 m M P M B 0.05 m M P M B 0.1 m M
717 619 223 16
13.7 68.9 97.8
Table 4. Protection by N A A again P M B inhibition of auxin binding. M e m b r a n e s were resuspended at p H 6 and incubated at 2 5 ~ for 15 min + 50 g M PMB. Additions of N A A were made immediately prior to PMB. Binding of [t4C]NAA was determined on the reisolated m e m b r a n e s Addition
None NAA 5 gM N A A 10~tM
Auxin binding (Acpm)
P M B inhibition (%)
Control
PMB
541 503 487
133 292 401
75.4 42.0 17.7
Discussion
From the results which have been presented, from the known variation of auxin binding with pH and from consideration of structure-activity rules, several tentative conclusions can be drawn regarding the environment of the receptor sites: 1. The presence of a free anionic function, normally carboxyl, is one of the essential structural requirements for auxin activity (see Thimann, 1963), indicating binding of the carboxyl anion to a positively-charged amino acid group in the receptor site. Binding of NAA is maximal at pit 5.5 and falls to very low values at pH 4 or pH 7 (Fig. 3). The imidazolyl-NH § of histidine (pKa=6.0; Morris, 1974) is the only candidate carboxyl-binding group whose ionization properties are compatible with loss of binding activity over the range pH 6-7. Reduction of binding on the acid side of the pH optimum can be partly attributed to protonation of the carboxyl anion (pKaNAA=4.3; Charton, 1964). 2. A fractional positive charge 0.55 nm distant from the carboxyl anion is also regarded as an essential feature of active auxin molecules (Thimann, 1963). This requirement most probably implies electrostatic bonding to an anionic residue in the binding site located at the appropriate distance from the carb-
148
M.A. Venis: AffinityLabels for Auxin Binding Sites
60(]
40C
"6
