Planta

Planta (Berl.) I30, 7 - 1 3 (1976)

9 by Springer-Verlag 1976

Auxin Binding to Corn Coleoptile Membranes: Kinetics and Specificity* Susan Batt** and Malcolm B. Wilkins Department of Botany, University of Glasgow, Glasgow G12 8QQ, U.K.

Michael A. Venis Shell Research Ltd., Woodstock Laboratory, Sittingbourne Research Centre, Sittingbourne, Kent, U.K.

Summary. Detailed examination of binding over the range 1 0 - 7 - i 0 - 6 M suggests that membrane preparations from coleoptiles of Zea mays L., cv Kelvedon 33 contain at least two sets of high affinity binding sites for 1-naphthylacetic acid (NAA), with dissociation constants of 1.8 • 10-7 M (site 1) and 14.5 x 10 -7 M (site 2). Similar studies with 3-indolylacetic acid (IAA) also indicate two sets of binding sites, whose concentrations are closely comparable to those deduced for NAA. A substantial proportion of the total binding activity is retained in a detergent-solubilized preparation. Using [t~C]NAA the interactions of a range of analogues with each of the binding sites have been examined with the aid of double reciprocal plots. The specificity of site 2 is compatible with that expected for an auxin receptor, in that only active auxins, antiauxin transport inhibitors are able to compete with [14C]NAA for the binding sites. Site 1 on the other hand is less specific, since it appears to bind all compounds tested, including physiologically inactive analogues.

Introduction

Several lines of evidence, including rapid auxin responses such as elongation growth and proton extrusion (see Evans, 1974 for review) suggest a primary site of action for auxins at the cell surface, probably involving the plasma membrane. The studies of Lembi et al. (1971) with the auxin transport inhibitor * Abbreviations." NAA = l-naphthylacetic acid, IAA=3-indolyIacetic acid, 2,4-D=2,4-dichlorophenoxyacetic acid, 2,6-D=2,6-dichlorophenoxyacetic acid, 2,4,5-T=2,4,5-trichlorophenoxyacetic acid, 2 - C P I B = ~ - ( 2 - c h l o r o p h e n o x y ) - i s o b u t y r i c acid, 2,4-B =2,4-dichlorobenzoic acid, 2,6-B=2,6-dichlorobenzoic acid, TIBA=2,3,5-triiodobenzoic acid, NPA = 1-N-naphthylphthalamic acid ** Present address: Department of Biology, University of Utah, Salt Lake City, Utah 84112, USA

1-N-naphthylphthalamic acid (NPA) and of Hertel et al. (1972) with the auxins 1-naphthylacetic acid (NAA) and 3-indolylacetic acid (IAA) demonstrated binding of these compounds to heterogeneous membrane preparations from corn coleoptiles and afforded direct evidence for auxin-membrane interaction. In the auxin studies, binding was examined over a wide concentration range (> 1,000-fold) and the observed levels of saturable binding were somewhat low. Estimates of dissociation constants, K were 1 - 2 x 10 .6 M for NAA and 3 - 4 x 10 .6 M for IAA (Hertel et al., 1972). Subsequent changes in procedure were reported to give improved binding and KNAA was revised to 5 x 10 -7 M (Ray and Hertel, unpublished, quoted in Hertel, 1974). Using these and other modifications to the original techniques, it has proved possible to enhance greatly the levels of saturable binding and hence obtain more precise kinetics. By studying binding in detail over a restricted concentration range ( 1 0 - v - 1 0 - 6 M ) we have obtained evidence for two distinct sets of auxin binding sites which differ in their specificities.

Materials and Methods Chemicals, IAA, NAA, 2,4-dichlorophenoxyacetic acid (2,4-D) and ~-(2-chIorophenoxy)-isobutyric acid (2-CPIB) were obtained from Sigma, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), 2,4-dichlorobenzoic acid (2,4-B) and 2,6-dichlorobenzoic acid (2,6-B) from British Drug Houses, benzoic acid from Hopkin and Williams, and 2,3,5-triiodobenzoic acid (TIBA) from Koch-Light. Compounds were recrystallised from water where necessary. NPA and 2,6-dichlorophenoxyacetic acid (2,6-D) were gifts from Dr. R. Hertel, University of Freiburg, and Dr. V. Math, Botany Department, University of Glasgow respectively. Purity of all compounds was verified by mass spectrometry (Dr. V. Math). l-naphthyl [1-1~C]acetic acid, 44-61 mCi/m mole and 3-indolyl (acetic acid-l-14C), 52mCi/m mole, were obtained from the Radiochemical Centre, Amersham. No radiochemical impurities were detected by thin layer chromatography (silica) in two solvent systems.

8

S. Batt et al.: Auxin Binding to Coleoptile Membranes

Kinetic Analyses. Binding data were analysed by the method of

Preparation of Binding Fractions. Coleoptiles were harvested from seedlings of 4-5 day old Zea mays L., cv. Kelvedon 33, grown in

Scatchard (1949). For a homogeneous population of binding sites a graph of Bound/Free against moles bound yields a straight line,

the dark at 25~ C. Leaf rolls were removed and the coleoptiles kept on ice. All subsequent operations were at 0-4 ~ C. Homogenization, washing and binding media were those described in Hertel (1974). The tissue was cut into small pieces with a razor blade and homogenized, by pestle and mortar, in an equal volume of grinding buffer (0.25 M sucrose, 50 mM Tris, 1 mM disodium EDTA, 0.1 mM MgC12, adjusted with acetic acid to pH 8.0). The homogenate was squeezed through 20/~m nylon cloth and the residue reground and extracted twice more in equal volumes of the buffer. Generally, the amount of tissue used was designed to give the equivalent of ca. 0.5 g fresh weight per assay. The combined extracts were centrifuged at 4,000 x g for 20 min and the pellets discarded. The supernatant was recentrifuged for 30 min at either 38,000 x g or at 80,000 x g. In some cases (see text) the resulting pellet was resuspended and repelleted at the same speed from 4 volumes (based on original tissue wt.) of a wash medium (0.25 M sucrose, 10 mM trisodium citrate, 0.5 mM MgC12, pH 6.0 with acetic acid).

from which a dissociation constant, K = -

and the concen-

tration of sites, n ( = intercept on abscissa) may be obtained. Scatchard analysis of auxin binding to unfractionated membrane preparations yielded a biphasic plot to which two straight lines were fitted by regression analyses through the appropriate points. Binding constants (K~, K2, n~, n~ +n=) were calculated from the slopes and intercepts of each line. Competition experiments were analysed by double reciprocal plots of 1/Bound against 1/Free (Klotz, 1953), analogous to Lineweaver-Burk plots. Lines in the presence and absence of competitor were computed by weighted regression analysis (Wilkinson, 1961). For the present purposes, three types of interaction have been distinguished: (1) "No competition" (regression lines superimposable), (2) "Competitive" (regression lines have common ordinate intercept, =l/n), (3) "Not competitive" (regression lines with ordinate intercepts significantly different by 'if-test; lines may be parallel or intersecting at or near the abscissa). In the case of competitive interactions, inhibitor constants, Ki were calculated i from the relationship Ki (Dixon and Webb, 1958), where

Binding Assay. The membrane pellet was resuspended in binding medium (0.25 M sucrose, 10 mM trisodium citrate, 5 mM MgSO4, pH 5.5 with acetic acid) using a Teflon-glass homogeniser and [14C]NAA added, usually to a final concentration of 2 x 10 .7 M. The labelled suspension was divided into 5 ml portions to which equal small aliquots of either water or an appropriate concentration of unlabelled NAA were added to give final NAA concentrations ranging generally from 2x 10-TM to 10 -6 M. Triplicate 1.5ml samples from each aliquot were centrifuged in 2.5 ml tubes either at 38,000 x g for 45 min (MSE High Speed 18, using three-place adaptors custom-made by MSE for the 8 x 50 ml angle rotor) or at 150,000 x g for 15 min (MSE Superspeed 50, using 2.5 ml adaptors in the 10 x 10 mt angle rotor). The supernatants were decanted and the tubes drained for 5 - 1 0 rain. The tubes and surfaces of the pellets were rinsed with ca. 2 ml distilled water, followed by draining for a further 5-10 rain. Pellets were recovered with Pasteur pipettes using 3 x 0.4 ml aliquots of distilled water, transferred to scintillation vials and counted in 10 ml of scintillation fluid (4 g 2,5-diphenyloxazole per litre of toluene, mixed 2:1 v/v with Triton X-100). Total radioactivity per assay was determined by counting 100 ~1 samples from the original 5 ml aliquots. Experiments with [14C] IAA followed a similar protocol. Values on graphs are mean values + S.E. where these exceeded the size of the points. When competitive effects of auxins and analogues were to be studied, the membrane suspension after addition of [14C]NAA was divided into two equal portions, to one of which the competitor was added to the required concentration. Each preparation was then subdivided for addition of unlabelled NAA, and the procedure continued as already described.

Kp/K- 1

K = dissociation constant in the absence of competitor and Kp = dissociation constant in the presence of competitor at concentration i. K and Kp are equal to the negative reciprocals of the respective abscissa intercepts.

Results

Kinetics of NAA Binding Initial experiments established that over the range pH 3.5-7.5, saturable [14C]NAA binding exhibits a clear optimum at pH 5.5. When experiments were carried out over a wide concentration range, results similar to those described by Hertel et al. (1972) were obtained, with an apparent KNAA=1.3 X 10-6M for the sites of highest affinity. Above 10-a M, binding was found to saturate very slowly with increasing NAA concentration, indicating low affinity binding at a large number of sites.

2400

(bl (bl 12

2200

K 1 --=1,5 x 10-7M 9 2000

ii-

w 1800 ~160fl "o

= s 1400

g

K 2 = 16,1 x 10-7M n 2 = 96 p rnol/g

1200 1000

0.2

0.5 N A A concentration,/zM (Io9 scale)

1.0

i

I

2O

4O

p mol bound/g fresh wt.

60

Fig. 1. Kinetics of [14CJNAA binding, a: pellet radioactivity as a function of NAA concentration, b: Scatchard analysis of the binding data

S.Batt et al.: Auxin Binding to Coleoptile Membranes

9

In o r d e r to s t u d y in g r e a t e r detail the high affinity region of the b i n d i n g curve, s u b s e q u e n t e x p e r i m e n t s were d e s i g n e d to e x a m i n e m o r e fully the kinetics of b i n d i n g over the r a n g e 2 x 10 - 7 M - 10 . 6 M. A s the N A A c o n c e n t r a t i o n is increased, r a d i o a c t i v i t y in the pellet decreases (Fig. la), i n d i c a t i n g progressive s a t u r a t i o n of the N A A b i n d i n g sites. S c a t c h a r d analysis of the d a t a (Fig. 1 b) gives a b i p h a s i c plot, suggesting the presence within the m e m b r a n e p r e p a r a t i o n of at least two sets of high affinity b i n d i n g sites for N A A , with d i s s o c i a t i o n c o n s t a n t s of 1.5 x 1 0 - T M (site 1) a n d 16.1 x 10 -~ M (site 2). F r o m five e x p e r i m e n t s using a 4 , 0 0 0 - 8 0 , 0 0 0 x g fraction, the m e a n b i n d i n g p a r a m eters were: Site 1: K 1 = 1.8 x 1 0 - 7 M ; n 1 = 52 p m o l / g fresh wt. Site 2: K a = 1 4 . 5 x 10 . 7 M ; n z = 1 0 1 p m o l / g fresh wt. The use of the direct intercept-slope analysis for evaluating binding parameters in a two site system is not strictly correct, but the values so obtained are related algebraically to the true site binding constants (Hunston, 1975). Analysis of the data from the same five experiments by the Hunston method yields the following values:

competitive effects of auxin analogues and since the inter-experiment variation in dissociation constants is at least as great as the difference between the methods of analysis, the simpler intercept-slope evaluation has been used.

Effect of Pellet Washing Hertel (1974) r e p o r t e d briefly t h a t r e m o v i n g the s u p e r n a t a n t a n d w a s h i n g the m e m b r a n e pellet (see M a t e r i a l s a n d M e t h o d s ) led to i m p r o v e d b i n d i n g activity, p o s s i b l y due to r e m o v a l of s o m e i n h i b i t o r y c o m p o n e n t . W i t h the initial b a t c h e s of seed used in the p r e s e n t work, rio i m p r o v e m e n t was o b s e r v e d by i n t r o d u c i n g a w a s h i n g step, a n d u n w a s h e d pellets gave satisfactory b i n d i n g kinetics. W i t h the seed used d u r i n g 1975 however, the wash step was f o u n d to be essential, especially for clear r e s o l u t i o n of site 1 b i n d i n g (Table 1). Different b a t c h e s of seed (and p e r h a p s varieties also) w o u l d therefore a p p e a r to differ in this respect. It was also often necessary, with the later seed batch, to use higher force pellets ( 4 , 0 0 0 - 8 0 , 0 0 0 • g) in o r d e r to distinguish clearly the presence of two sets of b i n d i n g sites.

Site 1: K 1= 1.4 x 1 0 - 7 M: n I = 31 pmol/g fresh wt. Site 2: K2=16.7x 10 .7 M; n2= 122pmol/g fresh wt. This method thus results in a somewhat different distribution of binding sites between n~ and n2, but the dissociation constants are not markedly different from those obtained by the direct method. Since the main interest in the present work lies in the relative Table 1. Effect of pellet washing on binding of NAA-I*C at 3

concentrations. Site 1 binding is estimated from Adpm (2.2x 10-VM-4.2xl0-YM) and site 2 from Adpm (4.2x10-TM 10.2 x 10- 7M) Pellet dpm/g fresh wt

Adpm/g

2.2xl0-VM 4.2x10-TM 10.2x10-TM Sitel Site2 No wash Washed

1,959 3,090

1,819 2,416

1,507 1,752

140 674

312 664

Solubilization of Binding Activity In o r d e r to e x a m i n e the p o s s i b i l i t y t h a t the o b s e r v e d b i n d i n g m i g h t r e p r e s e n t t r a n s p o r t into closed vesicles, a m e m b r a n e p r e p a r a t i o n was t r e a t e d with the d e t e r g e n t T r i t o n X-100. After c e n t r i f u g a t i o n at 120,000 x g for 1 h to r e m o v e i n s o l u b l e material, the d e t e r g e n t conc e n t r a t i o n was l o w e r e d b y gel filtration a n d b i n d i n g activity in the soluble fraction d e t e r m i n e d b y equil i b r i u m dialysis. C o m p a r e d with a crude, u n t r e a t e d m e m b r a n e p r e p a r a t i o n , it is evident (Table 2) t h a t 87 ~ of the t o t a l b i n d i n g activity (A d p m / m l / g tissue) is r e t a i n e d in the T r i t o n - s o l u b l e extract, suggesting t h a t b i n d i n g is n o t d e p e n d e n t u p o n t r a n s p o r t into vesicles.

Table 2. Binding of [14C] NAA by a solubilized membrane preparation. A 4,000 - 38,000 x g pellet was suspended in 0.2 ~s w/v Triton X-100 in 50 mM Tris-acetate pH 7.6 for 2 h at 0~ C, then centrifuged at 120,000 x g for 1 h. The supernatant was passed through a short bed of Sephadex G25 (Pharmacia) equilibrated in binding buffer and the eluate monitored at 280 nm. Aliquots (1 ml) of the excluded fraction were dialysed against 10 ml of either 2.2 x 10 -7 M [1~C] NAA in binding buffer, or the same plus 5 x 10 `6 M unlabelled NAA. After equilibration on a rotary turntable (4~ C, 23 h) duplicate 250 I.tlsamples of the solutions inside and outside the dialysis bags were counted for radio-activity. Binding by an untreated membrane preparation was similarly determined by equilibrium dialysis. Binding is calculated as Adpm 1- Adpm 2 where Adpmj and Adpm2 are the differences in radioactivity between inside and outside solutions at 2.2 x 10-7 M and 5 x 10-6 M NAA respectively Sample

Untreated Triton

Fresh wt

2.2 x 10 -7 M

5 X 10 -6

g/ml

Adpmt/250 [xl

Adpm2/250 gl

Adpm 1- dpm 2

AAdpm/ml/g/tissue

0.80 1.18

462 592

144 185

318 407

1,595 1,380

M

Binding activity

l0

S. Battet al.: Auxin Binding to Coleoptile Membranes (b)

(a)

700

Fig. 2. Kinetics of [14C]IAA binding. a: pellet radioactivity as a function of IAA concentration, b: Scatchard analysis of the binding data

600 K2 = 5.8 x 10-~M E

o 2 = 100 p mol/g

-~ 500

400

1

) 0,2

I I 0.5 1.0 IAA concentration, pM (logscale)

~

I 10

I 20

30

p mol bound/g fresh wt

(a)

I

/ / eS tio 1( b )

3000

1,0

+IAA

(c) Site2 ~ / g /

+ IAA

i

20oo

0.4

m

E d. -,5

/

0.5

f 0.2

0.2

0.5

1.0

-3

-1

NAA concentratlon,/~M (log scale)

0

1

3

5

1

2

I Free,/JM

Fig. 3. Effect of IAA on [J4C]NAA binding a: experimental binding values in the presence and absence of IAA, b, c: double reciprocal plots of the data for site 1 and site 2 respectively

Binding of IAA When binding of the endogenous auxin I A A is examined (Fig. 2) by methods comparable to those described for NAA, the results again suggest the presence of two sets of high affinity binding sites, with dissociation constants for I A A of 1.7x 1 0 - 6 M and 5.8x 10 -6 M. The concentrations of binding sites (Fig. 2b) are in excellent agreement with those obtained for NAA. Because of the lower binding affinities (relative to NAA), the experimentally observed changes in radioactivity bound as a function of concentration are much less for I A A than for N A A (cf. Figs. 2a and la). Preliminary experiments with labelled 2,4-D and 2,4,5-T also produced A d p m values greatly

inferior to those obtained with NAA. It was therefore evident that for purposes of competitive analysis the use of N A A as the radioactive ligand was likely to yield the most reliable results.

Specificity of the Binding Sites Hertel et al. (1972) examined auxin specificity by adding increasing concentrations of an unlabelled analogue and observing whether [ 1 4 C ] N A A (at a fixed concentration) was displaced from the membrane pellet. However, we now have evidence for the presence of two classes of auxin binding sites and in order to decide whether a given analogue is binding to one or

S. Batt et al.: A u x i n B i n d i n g to C o l e o p t i l e M e m b r a n e s

Fig. 4. Effectof benzoicacid on [~4C] NAA binding a: experimental binding curves in the presenceand absence of benzoic acid, b: double reciprocal plots of the site l binding data

11

Ii 1.5 +Benzoic

(a)

1100 ~- 1.0 (b)Site1

+Ben;~aci ~ ) X ~ d~ ~ 80(1

I 500 0.2

I 0,5

2[ 4I

1.0

I

NAA concentration, HM (log scale)

Free,/LM

3000

(a)

(c) Site 2

(b) Site 1

0.8

0.4

0.4

0.2

=

B "~ 2000

1200

I

r

0.2

0.5

1.0

-2

NAA concentration, pM (log scale)

I

1

I

3

5

-1 I Free,

0

q 1

I 2

pM

Fig. 5. Effect of 2,4-B on [14C]NAA binding a: experimental binding curves in the presence and absence of 2,4-B, b, c: double reciprocal plots of the data for site 1 and site 2 respectively

to both sites, a different type of experiment is required. The approach used has been to construct complete binding curves for N A A (as in Fig. la) in the presence or absence of a fixed concentration (not exceeding 10 -5 M) of the analogue under investigation. The data were then analysed by double reciprocal plots, constructed for the site 1 and site 2 regions of the binding curve. Occasionally, the kinetics for each site were investigated individually in different experiments. Figures 3-5 illustrate representative examples of the different types of interaction which have been distinguished (see Materials and Methods). Thus IAA (Fig. 3) yields double reciprocal plots with common ordinate intercepts, indicating a competitive interaction at both binding sites. Benzoic acid (Fig. 4) is competitive for site 1, but fails to interact with site 2, for which superimposable double reciprocal plots (not shown) are obtained. The type of interaction termed

"not competitive" is shown in Figure 5 for 2,4-B. In other experiments with this compound different types of interaction (competitive for site 1, no competition for site 2) were observed (see Table 3). Table 3 summarises the results from a large number of experiments obtained with a range of active and inactive auxin analogues, anti-auxins and transport inhibitors. For competitive interactions the calculated mean Ki values are given9 In the case of site 1 it will be observed that all compounds, irrespective of auxin activity, are able to interact in a competitive manner. The compounds 2,4-D (active) and 2,4,-B (inactive) behave somewhat variably, the interaction appearing to be 'not competitive' in some experiments. Turning to site 2, compounds which are active as auxins (IAA, 2,4-D, 2,4,5-T), anti-auxins (2-CPIB, TIBA) or auxin transport inhibitors (TIBA, NPA, see also Fig. 6) all compete with NAA for the binding

12

S.Batt et al.: Auxin Binding to Coleoptile Membranes

Table 3. Summary of analogue interaction experiments examining the specificity of the two N A A binding sites. Interactions are classed as competitive, not competitive, or no competition (see Materials and Methods). Figures in parenthesis indicate the number of experiments in which that type of interaction was observed; if, for a given compound and site, a particular interaction is not mentioned, then it was never observed. For competitive interactions, mean K~ values have been calculated

Compound

Auxin Activity a

Site 1

Site 2

Interaction

Interaction

Mean K~(M)

2.5 x 10 -6

Competitive (6)

7.3 x 10-6

5.4 x 10 .6

Competitive (5)

1.1 x 10 -s

IAA

+ + +

2,4-D

+ + +

2,6-D

Inactive b

Competitive (4)

1.4 x 10 -s

2,4,5-T 2-CPIB Benzoic acid

+ + + Strong anti-auxin Inactive

Competitive (4) Competitive (4) Competitive (2)

4.4 • 10 -6 2.7 x 10-6 3.6 x 10-6

2,4-B

Inactive

2,6-B

+

Competitive (3)

2.9 x 10-6

Competitive (3) No competition (2)

4.9 x 10 -6

TIBA NPA

+ ; strong anti-auxin; transport inhibitor c Transport inhibitor c

Competitive (4)

1.9 x 10-6

Competitive (4)

2.4 x 10- 6

Competitive (3)

6.6 x 10-6

Competitive (3)

1.6 x 10 - s

" b c

Competitive (4) ~Competitive (2) ( N o t competitive (3)

Mean Ki(M )

~Competitive (2) [ N o t competitive (2)

No competition (3) Not competitive (3) Competitive (3) Competitive (4) No competition (3) No competition (2) Not competitive (2)

4.7 x 10-5

8.5 x 10 -6 4.2 x 10- 6

J6nsson (1961). + + + =highly active; + =slightly active. Slight activity reported after 6 h, but not 24 h (Osborne et al., 1954) Thomson et al. (1973)

(a)

3000

:400

(b)

,P. 1600

~L220r E

/" f

9

8.3/~M

~

~O

Fig. 6a and b. Binding of [14C]NAA as a function of N A A concentration in the presence and absence of (a) TIBA or (b) N P A

2~M

1400

I 0.2

I 0.5

I 1.0

800

NAA concentration, ~

I 0.2

0.5

1.0

(log scale)

sites. The weak auxin 2,6-B was also competitive in three out of five experiments. On the other hand, compounds generally regarded as inactive (benzoic acid, 2,4-B, 2,6-D) are either without effect on site 2 binding ("no competition") or sometimes, in the cases of 2,4-B and 2,6-D, may behave in a 'not competitive' manner.

Discussion

In the original auxin binding studies of Hertel et al. (1972) only two or three points in the range 10 . 7 - 1 0 .6 M were selected and a single population

of high affinity binding sites (KNAA = 1 - 2 x 10 . 6 M ) was reported. When this concentration range is examined in detail, Scatchard analysis of the binding data (Fig. 1) suggests that there are at least two classes of high affinity sites, with dissociation constants for NAA of 1.8 x 10-7 M (site 1)and 14.5 x 10-7 M (site 2). IAA also appears to bind to two sets of sites (Fig. 2) whose concentrations agree well with those deduced for NAA. It would seem that the binding reported by Hertel et al, (1972) represented predominantly site 2 binding. Our results on the interactions of a range of analogues with each set of sites would suggest that

S.Batt et al.: Auxin Binding to Coleoptile Membranes

site 2 shows the binding specificity compatible with the expected properties of an auxin receptor as defined previously by Hertel et al. (1972), in that only active auxins, anti-auxins or transport inhibitors are able to compete with NAA for these binding sites (Table 3, Figs. 3-6). Site 1, on the other hand, seems to be less specific since the inactive compounds benzoic acid, 2,4-B and 2,6-D are able to interact competitively with NAA. It should be noted that the K i values for IAA (2.5 x 10 .6 M and 7.3 x 10 -6 M, Table 3) compare favourably with the IAA dissociation constants for site 1 and site 2 obtained from direct binding studies (1.7 x 10 -6 M and 5.8 x 10 .6 M respectively, Fig. 2), thereby reinforcing the suggestion that the competitive interactions studied do indeed represent competition for common classes of binding sites. There are several respects in which our findings differ from those previously reported:

1. Binding by inactive analogues. We find that all compounds tested, including physiologically inactive analogues are able to compete with NAA for site 1 binding. The fact that displacement of [I~C]NAA by compounds such as benzoic acid was not observed previously (Hertel et al., 1972) is perhaps not too surprising since, as already discussed, site 1 was apparently not detected in the earlier studies. 2. Binding by TIBA. Displacement of [14C]NAA by TIBA was observed by Hertel et al. (1972), but only at TIBA concentrations of 1 0 - 4 M or higher, and subsequently the discrepancy between these concentrations and those required to inhibit polar transport of IAA (50~ inhibition at ca. 2 x 1 0 - 6 M TIBA) was discussed (Thomson et al., 1973). However, we observe clear competition for NAA binding at TIBA concentrations as low as 2 x 10 -6 M (Fig. 6) and the calculated Ki values (ca. 2 x 10 .6 M for both sites, Table 3) are well reconciled with the data of Thomson et al. (1973) for inhibition of auxin transport. 3. Binding by NPA. We obtain competition by NPA for both binding sites (Fig. 6, Table 3), contrary to the findings of Hertel et al. (1972) where no displacement of auxin by NPA was observed. It will be noted, however, that the NPA binding interactions are relatively weak ( K i = 6 . 6 x 10 -6 M, site 1, and 1.6x 10 -s M, site 2) whereas direct binding studies with [ 3 H ] N P A (Lembi et al., 1971; Thomson, 1972; Thomson et al., 1973) have shown the presence of NPA binding sites of much higher affinity ( K = 1 0 -8 - 1 0 -7 M). Furthermore, [ 3 H ] N P A could not be displaced from the binding sites by auxins. Thus although NPA seem to bind to the auxin sites, it would seems that there are in addition distinct NPA binding sites of higher affinity. [14C] NAA is a favourable ligand for auxin binding studies because its high site affinities make binding

13

readily detectable. Furthermore, the eightfold difference between the NAA dissociation constants for site 1 and site 2 means that the sites can be clearly distinguished kinetically using crude unfractionated membrane preparations. It may seem curious that NAA should bind more tightly than IAA, since in general NAA does not appear to be more active than IAA in physiological tests. However, activity in any growth test is a complex function of several properties of the molecule, only one of which is the affinity of the molecule for the receptor. It may be that the site 2 binding affinities measured here represent the intrinsic activities of the compounds, which will be modified in a growth test by factors such as penetration, transport and inactivation. We are indebted to Dr. R. Hertel for communicating details of buffer compositions in advance of publication. S. Batt wishes to thank the Science Research Council for financial support.

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