Planta
Planta 130, 1 5 - 2 1 (1976)
9 by Springer-Verlag 1976
Separation and Localization of Two Classes of Auxin Binding Sites in Corn Coleoptile Membranes* Susan Batt** Department of Botany, University of Glasgow, Glasgow, G12 8QQ, U.K.
Michael A. Venis Woodstock Laboratory, Sittingbourne Research Centre, Sittingbourne, Kent, ME9 SAG, U.K.
Summary. Further evidence is presented for the discrete nature of the two classes of high affinity auxin binding sites in corn (Zea mays L.) coleoptile membranes, site l and site 2. Fractions can be obtained by differential centrifugation that exhibit binding kinetics characteristic of site 2, but not site 1. Membrane preparations containing both binding sites may be resolved on sucrose gradients into a light and a heavy band, whose binding kinetics and analogue binding specificities correspond to those deduced for site I and site 2 respectively in unfractionated membranes. Evidence from enzymic and chemical assays and from electron microscopy suggests that site 2, the auxin-specific binding site, is located in fractions enriched in plasma membrane, whereas site 1 is associated with Golgi membranes and/or endoplasmic reticulnm.
Introduction In the preceding paper (Batt et al., 1976) we presented kinetic evidence for the existence of two classes of high affinity auxin binding sites in crude membrane preparations from corn coleoptiles. The dissociation constants for 1-naphthylacetic acid (NAA) were estimated at 1 . 8 • (site 1) and 1 4 . 5 • (site 2). Both physiologically active and inactive auxin analogues were found to bind to site 1, whereas the binding properties of site 2 were more compatible with those expected of an auxin receptor, since the only compounds that competed with N A A for binding were either active auxins or analogues that might * Abbreviations: NAA = 1-naphthylacetic acid, I A A - 3-indolylacetic acid, TIBA=2,3,5-triiodobenzoic acid, SDH=succinic dehydrogenase, IDPase-inosine diphosphatase ** Present address: Department of Biology, University of Utah, Salt Lake City, Utah 841 I2, U.S.A.
reasonably be expected to bind at an auxin receptor, namely anti-auxins and polar transport inhibitors. We have been able to achieve substantial resolution of the binding sites and we report here on the localization of site 1 and site 2 in different membrane populations. Materials and Methods Sources of chemicals and radiochemicals, methods of assaying binding of [I~CINAA, and analyses of binding data were as described by B a t t e t al. (1976). Discontinuous Sucrose Gradients. Crude membrane fractions were prepared from coleoptiles of 45 day old Zea mays, cv. Kelvedon 33 as described previously (Batt et aI., 1976). Membrane pellets (4,000-38,000xg or 10,00038,000 • flactions) were resuspended in 18% w/w (0.56 M) sucrose in 50 mM Tris-acetate pH 8.04).1 mM MgCI2 and layered (5 ml containing l0 20 mg protein per gradient) on gradients of the type described by Hodges et al. (1972). "Complete" gradients consisted of 2 ml of 45% w/w (1.58 M) and 3 ml each of 38% (1.29 M), 34% (1.14 M), 30% (0.99 M) and 25% (0.81 M) sucrose, all in 50mM Tris-acetate pH 8.0-0.1 mM MgC1 a. "Simple" gradients consisted of 6 ml of 45% w/w and 8 ml of 30% w/w sucrose. Gradients were centrifuged for 1.5 h at 100,000xg (29,000 rpm in MSE 3 x 20 ml swing-out rotor). The visible bands at the sucrose interfaces were collected with a Pasteur pipette, diluted with grinding buffer (0.25 M sucrose, 50 mM Tris-acetate pH 8.0, 1 mM EDTA, 0.1 mM MgC12) and repelleted at 80,000 x g for 30 min. These pellets were assayed for [14C]NA A binding, for enzyme activity, or used for chemical analyses.
Enzyme Assays
l. ATPase was assayed at pH 6.0 using a modified Lowry and Lopez (1946) procedure for measurement of inorganic phosphate release. Assays consisted of 20 mM Tris-2(N-morpholino) ethane sulphonic acid, pH 6.0, 5mM ATP (Sigma, disodium salt titrated to pH 6.0 with NaOH), 17raM Na + (from the ATP), 33 mM KCI, 5 mM MgC12 and membrane sample, in a total volume of 1.0 ml. After incubation at 30 ~ C for 15 min, reactions were stopped with 0.5 ml of 10% w/v trichloroacetic acid (TCA), followed by the successive additon of 4ml acetate buffer (0. i M acetic acid, 25 mM sodium acetate, pH 4.0), I ml ammonium molybdate (BDH, 1% solution in 2N H2SO4) and 0.5 ml of freshly prepared
I6 10% w/v FeSO~ containing one drop of concentrated HzSO 4. Tube contents were mixed thoroughly, centrifuged at full speed in a bench centrifuge for 7 4 rain and absorbance of the supernatant was determined at 660 nm. 2. Succinic dehydrogenase (SDH) was assayed by a procedure similar to that described by Pennington (1961). Reaction mixtures consisting of 0.2 ml 0.2 M phosphate buffer pH 7.4 containing 5 mg/ml bovine serum albumin, 0.4 ml 0.125 M sodium succinate pH 7.4, and 0.2 ml of membrane suspension were pre-incubated at 37~ for 5 rain. The reaction was started by the addition of 0.2 ml of 0.5% w/v INT in 1 mM EDTA (INT =2-(p-indophenyl)3-(p-nitrophenyl)-5-phenyl tetrazolium) and stopped (after 10 rain) by the addition of 1 ml 10% w/v TCA. The formazan colour was extracted into 4 ml of ethyl acetate, the phases were separated by briefcentrifugation, and absorbance at 490 nm was determined in the organic layer. Enzyme activity was calculated from the relationship 1 nmol formazan/ml=20.1 absorbance units. 3. Glucan synthetase was determined as described by Van der Woude et al. (1974). Activities quoted are nmoles of glucose incorporated into the hot water insoluble, lipid-insoluble fraction at high (1 raM) UDP-glucose concentration. 4. Latent inosine diphosphatase (IDPase) was determined by the method of Ray et al. (1969), except that the enzyme was activated by including sodium deoxycholate in the assay at a final concentration of 0.1% (Powell and Brew, 1974). Release of inorganic phosphate was estimated as described for ATPase. 5. NADPH-cytochrome c reductase was assayed as described by Leonard et al. (1973) except that the reaction volume was 1.5 ml and the reaction temperature was 30~ C. Protein content was measured by the method of Lowry et al. (1951). Estimation of Phospholipid and Sterol Content. Lipids were extracted by the general method of Folch et al. (1957). Membrane fractions resuspended in water were mixed with 20 volumes of chloroform:methanol (2:1 v/v) and left at room temperature overnight. After removal of insoluble matter by brief centrifugation, the supernatant was extracted with 0.2 volumes of 0.04% MgCI2 and the aqueous phase discarded. The lower (organic) phase was washed twice with small volumes of upper phase medium and the washes discarded. Sufficient methanol was added to give a single phase and portions of the extract taken for phosphorus analysis. The samples were evaporated down in Kjeldahl flasks and digested and assayed for phosphate as described by Rouser et al. (1966), except that it was found necessary to develop the colour by heating the final mixture at 60 ~ C for 5 min. Sterols were determined directly on resuspended membrane fractions by a modification of the method of Stadtman (1957). 5 ml of a reagent consisting of 640 ml acetic anhydride, 36 ml glacial acetic acid and 6 g p-toluene sulphonic-acid were added, with mixing, to 0.2 ml of membrane suspension. After standing for 5min the samples were clear and 0.5ml of concentrated H2SO4 was added dropwise, with cooling. Samples were maintained at 25 ~ C for 15 min before measurement of absorbance at 625 nm. Sterol content was calculated by reference to a standard curve constructed with a sitosterol sample (Sigma, assayed by g.l.c, as 60% sitosterol, 40% campesterol and using an average sterol molecular weight of 400.
Results
pH Optimum of the Binding Sites All the data reported in the preceding paper (Batt et al., 1976) were obtained using a binding buffer of pH 5.5 since it was found early on in the investigation that greatest total binding of [14C]NAA was ob-
S. Batt and M.A. Venis: Auxin Binding Sites
300
z '~
,"
\82 '\
200
100
0
-4.0
4.5
I 5.0
i 5.5
I 6.0
4 6.5
7.0
pH
Fig. l. Effect of pH on binding of [I~C]NAA to site 1 and site 2 in a 4,000-38,000 • g membrane fraction. Site 1 and site 2 binding were estimated from the differences in radioactivity bound between 2 x 1 0 - T M - 4 x 1 0 7 M NAA and 4 x l 0 - T M - 1 0 x l 0 - T M NAA respectively
served at this pH. Having obtained kinetic evidence for the presence of two sets of binding sites in the membrane, it was important to discover whether there was an appreciable difference between the sites in terms of their pH requirements for binding. From Figure 1 it can be seen that site 1 binding (estimated as described in the figure legend) shows an optimum between pH 5.0 and pH 5.5, perhaps slightly lower than the pH optimum for site 2. The difference is very slight however, and it was therefore decided to continue using a pH 5.5 binding buffer in subsequent investigations.
Binding Kinetics of 4,000-10,O00 x g and 10,000-38,000 • g Fractions The binding studies reported previously (Batt et al., 1976) employed either a 4,000-38,000 x g or a 4,00080,000xg membrane preparation. To determine whether any separation of the binding sites could be achieved by differential centrifugation, the kinetics of [14C]NAA binding were examined in 4,00010,000 x g and in 1'0,000 38,000 x g fractions (Fig. 2). The 4,000-10,000xg fraction appears to contain a homogeneous population of binding sites with a dissociation constant for NAA of 15.7 x 10- 7 M, characteristic of site 2, the auxin-specific site. The binding kinetics of the 10,000-38,000 • g fraction suggests the presence of both site 1 and site 2 binding (Fig. 2b). Binding of [14C]NAA by 1,0004,000 x g and by 1,000-10,000 x g fractions was found to be kinetically similar to that illustrated for the 4,000-10,000xg
S. Batt and M.A. Venis: Auxin Binding Sites
t7
(a) 120Q
(b)
0
8
( ~ K I = 2.9x 10-7M
0 38,000x g t~
= 10.2x 1 0 - 7 M
2 800
_~
i
~
~
9
4
O
K= 15.7x 10-7M
-~e,~
4
I 0.2
400 0.1
NAA conch,
I 0.5
I
1.0
0
I
I 20
10
pM (logscale)
p mo[
I 30
bound/g fresh
wt
Fig. 2a and b. Kinetics of [14C]NAA binding by 4,000-10,000xg and 10,000-38,000• as a function of NAA concentration_ (b) Scatchard analysis of the binding data
membrane fractions. (a) pellet radioactivity
Table 1. Auxin binding and SDH activity in 1,000 10,000 • g fractions resolved on discontinuous sucrose gradients. Binding of [14C]NAA
is the difference in radioactivity bound at 2 x 10-7M and 10 -6 M NAA Sucrose interface Molarity (% w/w)
0.56 0.90 0.90-1.20 1.20-1.35 1.35-1.50 1.50-1.95
(18.0-27.6) (27.6-35.6) (35.6 39.3) (39.3M-3.0) (43.0-53.4)
Total activities [14C]NAA binding Adpm/assay
SDH n moles formazan/min/assay
39 181 128 26 0
0.06 0.52 13.13 2.27 0.26
fraction, i.e. characteristic of site 2. Since all these fractions can be expected to contain mitochondria it might be suggested that site 2 binding is a property ofmitochondria. To examine this possibility, a 1,00010,000 x g preparation was fractionated on the discontinuous sucrose gradient of Table 1 which is designed to band mitochondria predominantly at the 1.20-1.35 M sucrose interface (Dr. C.J. Leaver, Botany Dept, Edinburgh University, personal communication). Binding of [14C]NAA and activity of the mitochondrial marker enzyme SDH were examined in each of the recovered fractions. Most of the SDH activity resides in the 1.20-1.35 M band and this fraction is also able to bind NAA (Table 1). However, it is clear from the Table that there is no correlation between NAA binding and SDH activity in the various fractions, suggesting that the presence of mitochondria in fractions containing site 2 is no more than coincidental.
[14ClNAA binding Adpm/mg protein
SDH n moles formazan/min/mg protein
743 473 103 125 0
5.6 6.6 51.1 47.1 13.9
Enzymic and Chemical Characterization of Gradient-Fractionated Membranes To obtain information on the types of membrane in preparations containing both site 1 and site 2, 10,000-38,000 x g fractions were resolved on discontinuous sucrose gradients similar to those described by Hodges et al. (1972) and the isolated membrane bands analysed for activity of various marker enzymes and for sterol and phospholipid content. High steroI: phospholipid ratios have been reported as characteristic of plasma membrane enrichment, as have glucan synthetase (assayed at 1 mM UDP-glucose) and acidic ATPase activities. Latent IDPase is a marker for Golgi membranes, while NADPH-cytochrome c reductase may be associated with endoplasmic reticulure (see Hodges and Leonard, 1974 and references therein). IDPase activity peaks clearly in band 2 of the
18
S. Batt and M.A. Venis: Auxin Binding Sites
Table2. Enzymic and chemical analyses of 10,000-38,000xg membrane preparations fractionated on discontinuous sucrose gradients, a) total activities, n moles/min]g fresh wt., and sterol: phospholipid ratios b) specific activities, n moles/rain/rag protein Band % w/w sucrose
ATPase
Glucan synthetase
IDPase
NADPHcytochrome c reductase
Sterol: phospholipid molar ratio
a) total activities 1. 18-25 2. 25-30 3. 30-34 4. 34-38 5. 3 8 4 5
2.4 8.8 11.1 12.4 12.5
0,20 2.02 1.95 1.95 1.55
6.8 30.7 9.0 5.9 2.6
4.89 6.58 4.49 1,87 0.59
0.36 0.35 0.47 0.64 0.79
b) specific activities 1. 18-25 2. 25-30 3. 30-34 4. 34-38 5. 3 8 4 5
52 88 174 167 183
5.1 19.5 43.3 36.1 41.3
148 307 141 80 38
105.8 60.4 43.2 27.0 7.1
gradient, while ATPase is highest in bands 3-5 (Table 2). The specific activity of glucan synthetase is also greatest in bands 3-5, although the total distribution shows that high activity is also associated with band 2 (Table 2 a), no doubt because activity of this enzyme is apparently related to both plasma membrane and dictyosome content (Van der Woude et al., 1974). NADPH-cytochrome c reductase displays the highest specific activity in band 1, while total activity is greatest in band 2. The sterol-phospholipid ratios increase down the gradient, with the densest regions, band 4 and 5, having the highest ratios. Electron microscopy of sections stained with the plasma membrane stain of Roland et al, (1972) also indicates that plasma membrane is located very largely in bands 4 and 5, with a few profiles observable in band 3. Any mitochondria present are found, in lead-stained sections, to reside exclusively in band 4.
Auxin-Binding Characteristics of Gradient Fractionated Membranes The distribution of [14C]NAA binding in bands recovered from gradients of the type described above is shown in Table 3. All fractions are able to bind [14C]NAA,the greatest binding activity on the basis of Adpm over the selected concentration range being in the lightest regions, bands 1 and 2. It is difficult to recover sufficient material from each band to permit a full kinetic analysis on each fraction and as a compromise, the "simple" gradient was used for this purpose (See Materials and Methods). This gradient yields two fractions, the light fraction representing the combination of bands 1 and 2 and the heavy fraction representing bands 3-5.
Table3. Binding of [a4C]NAA by 4,000-38,000xg membrane preparations fractionated on discontinuous sucrose gradients. Binding is expressed as the difference in radioactivity bound at2xl0 VMand7xl0 7MNAA Band % w/w sucrose
Total activity Adpm/assay
Specific activity Adpm/mg protein
1. 18-25 2. 25-30 3. 30-34 4. 34-38 5. 3845
306 455 218 115 107
2,037 1,515 727 218 173
The kinetics of [14C]NA A binding to each of these bands (Fig. 3) suggest that each band contains one distinct set of binding sites. The dissociation constants, 3.9x 10 7 M for the light band, and l l . 6 x 10-7 M for the heavy band, compare favourably with those determined previously for site 1 and site 2 in unfractionated membrane preparations, namely 1.8 X 10 - 7 M and 14.5 x 10 7 M respectively (Batt et al., 1976). This similarity suggests that substantial resolution of the binding sites has been achieved. If the light and heavy bands do indeed contain site 1 and site 2 respectively, then the binding specificities of each site as determined in unfractionated preparations should be retained. Specificity was examined using three compounds, the auxin 3-indolylacetic acid (IAA), the anti-auxin and auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) and the inactive compound benzoic acid (BA). The ability of these compounds to interact with the NAA binding sites in each band was determined by constructing [J4C]NAA binding curves in the presence and absence of a fixed concentration of the analogue and analysing
S. Batt and M.A. Venis: Auxin Binding Sites
19
(a)
~
(b)
4
1000
K = 3.9 x 1 0 - 7 M
~
n=24pmol/g
~
X
~o0
K=11,6x10_7M
_-#.
n = 32 p mol/g
Heavy band
300
I
I
I
I
I
0.2
0.5
1.0
10
20
NAA
concn.,/IM(logscale)
pmolbound/gfreshwt
Fig. 3a and b. Kinetics of [14C]NAA binding to light and heavy bands obtained by fractionation of a 4 , 0 0 0 . 3 8 , 0 0 0 x g m e m b r a n e preparation on a " s i m p l e " sucrose gradient. (a) pellet radioactivity as a function of N A A concentration (b) Scatchard analysis of the binding data
TaMe 4. Comparison of Ki values calculated for site 1 and site 2 in unfractionated 4,000-38,000 x g m e m b r a n e preparations (data from Batt et al., 1976) with those calculated for the two bands from a ' s i m p l e ' sucrose gradient (data from Fig. 4)
Compound
IAA Benzoic acid TIBA
Unfractionated K i (M)
Fractionated K~ (M)
Site 1
Site 2
Site 1
Site 2
2 . 5 x 10 -6 3.6 x 10- 6 1.9x 10 6
7.3x I0 -6 No competition 2.4 x 10-6
7 . 9 x 10 -6 12 x 10- 6 1.3 x 10-6
9.1 x 10 -6 No competition 1.9 x 10-6
the data on double reciprocal plots as described previously (Batt et al., 1976). From these plots, (Fig. 4) it may be concluded that IAA and TIBA are able to compete with NAA for the binding sites present in both membrane bands. Benzoic, on the other hand, is competitive with NAA for the binding sites in the light band, but does not compete for binding sites in the heavy band. In Table 4, the K i values calculated from Figure 4 are compared with those estimated from earlier experiments with unfractionated membrane preparations (Batt et al., 1976). It would seem that the binding specificities of the light and heavy bands do indeed correspond to those deduced previously for site 1 and site 2 respectively (Batt et al., 1976), since only physiologically active auxin analogues bind to the heavy fraction, while both active and inactive compounds can bind to the light fraction. Discussion
From analyses of the kinetics of [14C]NAAbinding and of the interactions of a range of auxin analogues, it was concluded that crude membrane preparations from corn coleoptiles contain two classes of high
affinity auxin binding sites, termed site 1 and site 2 (Batt et al., 1976). The results presented here reinforce the earlier evidence that these sites are indeed distinct and present evidence that the auxin-specific site 2 binding is associated with plasma membraneenriched fractions. By differential centrifugation, a fraction can be obtained that contains site 2 but not site 1 binding activity (Fig. 2). This fraction contains mitochondria, but when a low speed pellet is resolved on a sucrose gradient, [14C]NAA binding and SDH activities in the different fractions are not correlated (Table 1). Site 2 binding is still detected in 10,000 38,000 x g fractions (Fig. 2) which are considerably depleted in mitochondria on the basis of SDH activity (unpublished observations). Furthermore, Hertel et al. (1972) detected binding which was predominantly site 2 binding (see Batt et al., 1976) in fractions which were substantially free of cytochrome c oxidase activity. We conclude, therefore, that site 2 binding is not to mitochondria, and that the binding detected in low force pellets probably represents binding to heavier members of membrane population present in higher speed fractions (perhaps attached to other cellular constituents).
20
S. Batt and M.A. Venis: Auxin Binding Sites (a)
1.4
Light Band
1.4
+ I A A 5 % ~ / ~
0.7
I
//
-3
I
1
-1
(b)
0.7
I
I
3
5
I
--2
S
,/~
I
1
1.0
I
I
3
5
1.0
+ BA5pM E3
0.5
0.5
=> / _
_x
I
-1
1
[
I
3
5
1
--2
I
J
I
1
3
1 1
[ 3
I
5
1.8
(c)
Q
0.9
0.9
-3
-1
1
i
3
[
5
/r
-2
I 5
1 Free,/zM
Fig. 4a-e. Competition for [14C]NAA binding sites in the light and heavy bands obtained by fractionation of 4,000-38,000x g membrane preparation on "simple" sucrose gradients. Double reciprocal plots of NAA binding in the presence and absence of (a) IAA (b) benzoic acid (BA) (c) TIBA
Crude membrane pellets can be fractionated on discontinuous sucrose gradients into a light and a heavy band whose properties, in terms of [14C]NAA binding kinetics (Fig. 3) and analogue binding specificity (Fig. 4, Table 4) are in accord with those found for site 1 and site 2 respectively in unfractionated preparations. The somewhat higher N A A dissociation constant in the light band (Fig. 3) and the higher K~ values for IAA and benzoic acid in this band
(Table 4), relative to those observed for site 1 in crude fractions, suggest that resolution is not complete and that the light band is partly contaminated with site 2. F r o m the IDPase and N A D P H - c y t o c h r o m e c reductase activities (Table 2) the light band containing site 1 would appear to be enriched in Golgi membranes and endoplasmic reticulum. Leonard et al. (1973) used an additional 20% w/w sucrose step in their gradients and found (in oat roots) that the speci-
S. Batt and M.A. Venis: Auxin Binding Sites
fic activity peak of NADPH-cytochrome c reductase was at the 18-20% sucrose interface whereas NADHcytochrome c reductase (used as a tonoplast and/or endoplasmic reticulum marker) peaked in the 20-25% sucrose band. In our experience with corn coleoptiles, we very seldom observed any visible band at an 18 20% sucrose interface and the 20% sucrose step was therefore routinely omitted. On the basis of sterol:phospholipid ratios (Table 2) and of electron microscopy of specificallystained sections, the gradient bands containing site 2 seem to be enriched in plasma membrane, particularly bands 4 and 5. The use of enzymic markers for plasma membrane is not altogether satisfactory. Glucan synthetase activity appears to be related to both plasma membrane and dictyosome content (Table 2 and Van der Woude et al., 1974), while Leigh et al. (1975) report that acidic ATPase of maize roots is associated with both plasmalemma and with another membrane fraction resolvable on Ficoll gradients. The specific staining method of Roland et al. (1972) is probably a fairly reliable indicator of plasma membrane content, but is a time-consuming procedure. High sterol:phospholipid ratios correlate quite well with specifically-stained membrane content, and their determination is somewhat less laborious, but an even more rapid procedure for plasma membrane evaluation would be desirable. Site 2 is thus present in membrane fractions that are enriched in plasma membrane. The analogue binding specificity of site 2 suggests that it may represent an auxin receptor site, though its precise role in terms of a physiological response cannot be evaluated at present. Site 1 appears to be associated with dictyosomes and/or endoplasmic reticulum, and the observations of Gawlik and Shen-Miller (1974), that IAA produces rapid changes in dictyosome content of oat coleoptile cells, are of interest in this respect. However, since site 1 binds active and inactive auxin analogues, its possible function remains mysterious, yet it is improbable that a binding site of such high affinity would be evolved without some physiological value. We are indebted to Shirley Potter, Shell Research Ltd, for the electron microscopic studies. We are also grateful to several members of the Biochemistry and Physiology Division, Shell Research Ltd., in particular Winifred Tyers and Richard Bumpus, for assistance with coleoptile harvesting. S. Batt wishes to thank the Science Research Council for financial support.
21
References Batt, S., Wilkins, M.B., Venis, M.A.: Auxin binding to corn coleoptile membranes: kinetics and specificity. Planta (Bed.) 130, 7-13 (1976) Folch, J., Lees, M., Sloane-Stanley, G.H.: A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 497-509 (1957) Gawlik, S.R., Shen-Miller, J. : Effects of indoleacetic acid on dicytosomes of apical and expanding cells of oat coleoptiles. Plant Physiol. 54, 217-221 (1974) Hertel, R., Thomson, K., Russo, V.E.A.: In vitro auxin binding to particulate cell fractions from corn coleoptiles. Planta (Bed.) 107, 325 340 (1972) Hodges, T.K., Leonard, R.T. : Purification of a plasma-membranebound adenosine triphosphatase from plant roots. In: Methods in Enzymology XXXII, pp. 392-406. Fleischer, S., Packer, L. (eds) NewYork-SanFrancisco-London: Academic Press 1974 Hodges, T.K., Leonard, R.T., Bracker, C.E., Keenan, T.W.: Purification of an ionstimulated adenosine trophosphatase from plant roots:association with plasma membranes. Proc. Nat. Acad. Sci. U.S.A. 69, 3307-3311 (1972) Leigh, R.A., Williamson, F.A., Wyn-Jones, G.: Presence of two different membrane bound, KCl-stinmlated adenosine triphosphatase activities in maize roots. Plant Physiol. 55, 678-685 (1975) Leonard, R.T., Hansen, D., Hodges, T.K.: Membrane-bound adenosine triphosphatase activities of oat roots. Plant Physiol. 51, 749-754 (1973) Lowry, O.N., Lopez, J.H.: The determination of inorganic phosphate in the presence of labile phosphate esters. J. Biol. Chem. 162, 421-428 (1946) Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, K.J. : Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 262-275 (1951) Pennington, R.J. : Biochemistry of dystrophic muscle. Mitochondrial succinate-tetrazolium reductase and adenosine triphosphatase. Biochem. J. 80, 649454 (1961) Powell, J.T., Brew, K.: Glycosyltransferases in the Golgi membranes of onion stem. Biochem. J. 142, 203 209 (1974) Ray, P.M., Shininger, T.L., Ray, M.M.: Isolation of fl-glucan synthetase particles from plant cells and identification with Golgi membranes. Proc. Nat. Acad. Sci. USA 64, 605-612 (1969) Roland, J.C., Lembi, C.A., Morr'e, D.J.: Phosphotungstic acid-chromic acid as a selective electron-dense stain for plasma membranes of plant cells. Stain Technology 47, 195 200 (1972) Rouser, G., Siakotoj, A.N., Fleischer, S.: Quantitative analysis of phospholipids by thinlayer chromatography and phosphorus analysis of spots. Lipids 1, 85 86 (1966) Stadtman, T.C.: Preparation and assay of cholesterol and ergosterol. In: Methods in Enzymology III, pp 392-394, Colowick, S.P., Kaplan, N.O. (eds) New York: Academic Press 1957 Van der Woude, W.J., Lembi, C,A., Mor/e, D.J., Kindinger, J.I., Ordin, L. : fl-Glucansynthetases of plasma membrane and Golgi apparatus from onion stem. Plant Physiol. 54, 333-340 (1974) Wilkinson, G. : Statistical estimations in enzyme kinetics. Biochem. J. 80, 324 332 (1961) Received 7 November; accepted 8 December 1975
Planta 130, 1 5 - 2 1 (1976)
9 by Springer-Verlag 1976
Separation and Localization of Two Classes of Auxin Binding Sites in Corn Coleoptile Membranes* Susan Batt** Department of Botany, University of Glasgow, Glasgow, G12 8QQ, U.K.
Michael A. Venis Woodstock Laboratory, Sittingbourne Research Centre, Sittingbourne, Kent, ME9 SAG, U.K.
Summary. Further evidence is presented for the discrete nature of the two classes of high affinity auxin binding sites in corn (Zea mays L.) coleoptile membranes, site l and site 2. Fractions can be obtained by differential centrifugation that exhibit binding kinetics characteristic of site 2, but not site 1. Membrane preparations containing both binding sites may be resolved on sucrose gradients into a light and a heavy band, whose binding kinetics and analogue binding specificities correspond to those deduced for site I and site 2 respectively in unfractionated membranes. Evidence from enzymic and chemical assays and from electron microscopy suggests that site 2, the auxin-specific binding site, is located in fractions enriched in plasma membrane, whereas site 1 is associated with Golgi membranes and/or endoplasmic reticulnm.
Introduction In the preceding paper (Batt et al., 1976) we presented kinetic evidence for the existence of two classes of high affinity auxin binding sites in crude membrane preparations from corn coleoptiles. The dissociation constants for 1-naphthylacetic acid (NAA) were estimated at 1 . 8 • (site 1) and 1 4 . 5 • (site 2). Both physiologically active and inactive auxin analogues were found to bind to site 1, whereas the binding properties of site 2 were more compatible with those expected of an auxin receptor, since the only compounds that competed with N A A for binding were either active auxins or analogues that might * Abbreviations: NAA = 1-naphthylacetic acid, I A A - 3-indolylacetic acid, TIBA=2,3,5-triiodobenzoic acid, SDH=succinic dehydrogenase, IDPase-inosine diphosphatase ** Present address: Department of Biology, University of Utah, Salt Lake City, Utah 841 I2, U.S.A.
reasonably be expected to bind at an auxin receptor, namely anti-auxins and polar transport inhibitors. We have been able to achieve substantial resolution of the binding sites and we report here on the localization of site 1 and site 2 in different membrane populations. Materials and Methods Sources of chemicals and radiochemicals, methods of assaying binding of [I~CINAA, and analyses of binding data were as described by B a t t e t al. (1976). Discontinuous Sucrose Gradients. Crude membrane fractions were prepared from coleoptiles of 45 day old Zea mays, cv. Kelvedon 33 as described previously (Batt et aI., 1976). Membrane pellets (4,000-38,000xg or 10,00038,000 • flactions) were resuspended in 18% w/w (0.56 M) sucrose in 50 mM Tris-acetate pH 8.04).1 mM MgCI2 and layered (5 ml containing l0 20 mg protein per gradient) on gradients of the type described by Hodges et al. (1972). "Complete" gradients consisted of 2 ml of 45% w/w (1.58 M) and 3 ml each of 38% (1.29 M), 34% (1.14 M), 30% (0.99 M) and 25% (0.81 M) sucrose, all in 50mM Tris-acetate pH 8.0-0.1 mM MgC1 a. "Simple" gradients consisted of 6 ml of 45% w/w and 8 ml of 30% w/w sucrose. Gradients were centrifuged for 1.5 h at 100,000xg (29,000 rpm in MSE 3 x 20 ml swing-out rotor). The visible bands at the sucrose interfaces were collected with a Pasteur pipette, diluted with grinding buffer (0.25 M sucrose, 50 mM Tris-acetate pH 8.0, 1 mM EDTA, 0.1 mM MgC12) and repelleted at 80,000 x g for 30 min. These pellets were assayed for [14C]NA A binding, for enzyme activity, or used for chemical analyses.
Enzyme Assays
l. ATPase was assayed at pH 6.0 using a modified Lowry and Lopez (1946) procedure for measurement of inorganic phosphate release. Assays consisted of 20 mM Tris-2(N-morpholino) ethane sulphonic acid, pH 6.0, 5mM ATP (Sigma, disodium salt titrated to pH 6.0 with NaOH), 17raM Na + (from the ATP), 33 mM KCI, 5 mM MgC12 and membrane sample, in a total volume of 1.0 ml. After incubation at 30 ~ C for 15 min, reactions were stopped with 0.5 ml of 10% w/v trichloroacetic acid (TCA), followed by the successive additon of 4ml acetate buffer (0. i M acetic acid, 25 mM sodium acetate, pH 4.0), I ml ammonium molybdate (BDH, 1% solution in 2N H2SO4) and 0.5 ml of freshly prepared
I6 10% w/v FeSO~ containing one drop of concentrated HzSO 4. Tube contents were mixed thoroughly, centrifuged at full speed in a bench centrifuge for 7 4 rain and absorbance of the supernatant was determined at 660 nm. 2. Succinic dehydrogenase (SDH) was assayed by a procedure similar to that described by Pennington (1961). Reaction mixtures consisting of 0.2 ml 0.2 M phosphate buffer pH 7.4 containing 5 mg/ml bovine serum albumin, 0.4 ml 0.125 M sodium succinate pH 7.4, and 0.2 ml of membrane suspension were pre-incubated at 37~ for 5 rain. The reaction was started by the addition of 0.2 ml of 0.5% w/v INT in 1 mM EDTA (INT =2-(p-indophenyl)3-(p-nitrophenyl)-5-phenyl tetrazolium) and stopped (after 10 rain) by the addition of 1 ml 10% w/v TCA. The formazan colour was extracted into 4 ml of ethyl acetate, the phases were separated by briefcentrifugation, and absorbance at 490 nm was determined in the organic layer. Enzyme activity was calculated from the relationship 1 nmol formazan/ml=20.1 absorbance units. 3. Glucan synthetase was determined as described by Van der Woude et al. (1974). Activities quoted are nmoles of glucose incorporated into the hot water insoluble, lipid-insoluble fraction at high (1 raM) UDP-glucose concentration. 4. Latent inosine diphosphatase (IDPase) was determined by the method of Ray et al. (1969), except that the enzyme was activated by including sodium deoxycholate in the assay at a final concentration of 0.1% (Powell and Brew, 1974). Release of inorganic phosphate was estimated as described for ATPase. 5. NADPH-cytochrome c reductase was assayed as described by Leonard et al. (1973) except that the reaction volume was 1.5 ml and the reaction temperature was 30~ C. Protein content was measured by the method of Lowry et al. (1951). Estimation of Phospholipid and Sterol Content. Lipids were extracted by the general method of Folch et al. (1957). Membrane fractions resuspended in water were mixed with 20 volumes of chloroform:methanol (2:1 v/v) and left at room temperature overnight. After removal of insoluble matter by brief centrifugation, the supernatant was extracted with 0.2 volumes of 0.04% MgCI2 and the aqueous phase discarded. The lower (organic) phase was washed twice with small volumes of upper phase medium and the washes discarded. Sufficient methanol was added to give a single phase and portions of the extract taken for phosphorus analysis. The samples were evaporated down in Kjeldahl flasks and digested and assayed for phosphate as described by Rouser et al. (1966), except that it was found necessary to develop the colour by heating the final mixture at 60 ~ C for 5 min. Sterols were determined directly on resuspended membrane fractions by a modification of the method of Stadtman (1957). 5 ml of a reagent consisting of 640 ml acetic anhydride, 36 ml glacial acetic acid and 6 g p-toluene sulphonic-acid were added, with mixing, to 0.2 ml of membrane suspension. After standing for 5min the samples were clear and 0.5ml of concentrated H2SO4 was added dropwise, with cooling. Samples were maintained at 25 ~ C for 15 min before measurement of absorbance at 625 nm. Sterol content was calculated by reference to a standard curve constructed with a sitosterol sample (Sigma, assayed by g.l.c, as 60% sitosterol, 40% campesterol and using an average sterol molecular weight of 400.
Results
pH Optimum of the Binding Sites All the data reported in the preceding paper (Batt et al., 1976) were obtained using a binding buffer of pH 5.5 since it was found early on in the investigation that greatest total binding of [14C]NAA was ob-
S. Batt and M.A. Venis: Auxin Binding Sites
300
z '~
,"
\82 '\
200
100
0
-4.0
4.5
I 5.0
i 5.5
I 6.0
4 6.5
7.0
pH
Fig. l. Effect of pH on binding of [I~C]NAA to site 1 and site 2 in a 4,000-38,000 • g membrane fraction. Site 1 and site 2 binding were estimated from the differences in radioactivity bound between 2 x 1 0 - T M - 4 x 1 0 7 M NAA and 4 x l 0 - T M - 1 0 x l 0 - T M NAA respectively
served at this pH. Having obtained kinetic evidence for the presence of two sets of binding sites in the membrane, it was important to discover whether there was an appreciable difference between the sites in terms of their pH requirements for binding. From Figure 1 it can be seen that site 1 binding (estimated as described in the figure legend) shows an optimum between pH 5.0 and pH 5.5, perhaps slightly lower than the pH optimum for site 2. The difference is very slight however, and it was therefore decided to continue using a pH 5.5 binding buffer in subsequent investigations.
Binding Kinetics of 4,000-10,O00 x g and 10,000-38,000 • g Fractions The binding studies reported previously (Batt et al., 1976) employed either a 4,000-38,000 x g or a 4,00080,000xg membrane preparation. To determine whether any separation of the binding sites could be achieved by differential centrifugation, the kinetics of [14C]NAA binding were examined in 4,00010,000 x g and in 1'0,000 38,000 x g fractions (Fig. 2). The 4,000-10,000xg fraction appears to contain a homogeneous population of binding sites with a dissociation constant for NAA of 15.7 x 10- 7 M, characteristic of site 2, the auxin-specific site. The binding kinetics of the 10,000-38,000 • g fraction suggests the presence of both site 1 and site 2 binding (Fig. 2b). Binding of [14C]NAA by 1,0004,000 x g and by 1,000-10,000 x g fractions was found to be kinetically similar to that illustrated for the 4,000-10,000xg
S. Batt and M.A. Venis: Auxin Binding Sites
t7
(a) 120Q
(b)
0
8
( ~ K I = 2.9x 10-7M
0 38,000x g t~
= 10.2x 1 0 - 7 M
2 800
_~
i
~
~
9
4
O
K= 15.7x 10-7M
-~e,~
4
I 0.2
400 0.1
NAA conch,
I 0.5
I
1.0
0
I
I 20
10
pM (logscale)
p mo[
I 30
bound/g fresh
wt
Fig. 2a and b. Kinetics of [14C]NAA binding by 4,000-10,000xg and 10,000-38,000• as a function of NAA concentration_ (b) Scatchard analysis of the binding data
membrane fractions. (a) pellet radioactivity
Table 1. Auxin binding and SDH activity in 1,000 10,000 • g fractions resolved on discontinuous sucrose gradients. Binding of [14C]NAA
is the difference in radioactivity bound at 2 x 10-7M and 10 -6 M NAA Sucrose interface Molarity (% w/w)
0.56 0.90 0.90-1.20 1.20-1.35 1.35-1.50 1.50-1.95
(18.0-27.6) (27.6-35.6) (35.6 39.3) (39.3M-3.0) (43.0-53.4)
Total activities [14C]NAA binding Adpm/assay
SDH n moles formazan/min/assay
39 181 128 26 0
0.06 0.52 13.13 2.27 0.26
fraction, i.e. characteristic of site 2. Since all these fractions can be expected to contain mitochondria it might be suggested that site 2 binding is a property ofmitochondria. To examine this possibility, a 1,00010,000 x g preparation was fractionated on the discontinuous sucrose gradient of Table 1 which is designed to band mitochondria predominantly at the 1.20-1.35 M sucrose interface (Dr. C.J. Leaver, Botany Dept, Edinburgh University, personal communication). Binding of [14C]NAA and activity of the mitochondrial marker enzyme SDH were examined in each of the recovered fractions. Most of the SDH activity resides in the 1.20-1.35 M band and this fraction is also able to bind NAA (Table 1). However, it is clear from the Table that there is no correlation between NAA binding and SDH activity in the various fractions, suggesting that the presence of mitochondria in fractions containing site 2 is no more than coincidental.
[14ClNAA binding Adpm/mg protein
SDH n moles formazan/min/mg protein
743 473 103 125 0
5.6 6.6 51.1 47.1 13.9
Enzymic and Chemical Characterization of Gradient-Fractionated Membranes To obtain information on the types of membrane in preparations containing both site 1 and site 2, 10,000-38,000 x g fractions were resolved on discontinuous sucrose gradients similar to those described by Hodges et al. (1972) and the isolated membrane bands analysed for activity of various marker enzymes and for sterol and phospholipid content. High steroI: phospholipid ratios have been reported as characteristic of plasma membrane enrichment, as have glucan synthetase (assayed at 1 mM UDP-glucose) and acidic ATPase activities. Latent IDPase is a marker for Golgi membranes, while NADPH-cytochrome c reductase may be associated with endoplasmic reticulure (see Hodges and Leonard, 1974 and references therein). IDPase activity peaks clearly in band 2 of the
18
S. Batt and M.A. Venis: Auxin Binding Sites
Table2. Enzymic and chemical analyses of 10,000-38,000xg membrane preparations fractionated on discontinuous sucrose gradients, a) total activities, n moles/min]g fresh wt., and sterol: phospholipid ratios b) specific activities, n moles/rain/rag protein Band % w/w sucrose
ATPase
Glucan synthetase
IDPase
NADPHcytochrome c reductase
Sterol: phospholipid molar ratio
a) total activities 1. 18-25 2. 25-30 3. 30-34 4. 34-38 5. 3 8 4 5
2.4 8.8 11.1 12.4 12.5
0,20 2.02 1.95 1.95 1.55
6.8 30.7 9.0 5.9 2.6
4.89 6.58 4.49 1,87 0.59
0.36 0.35 0.47 0.64 0.79
b) specific activities 1. 18-25 2. 25-30 3. 30-34 4. 34-38 5. 3 8 4 5
52 88 174 167 183
5.1 19.5 43.3 36.1 41.3
148 307 141 80 38
105.8 60.4 43.2 27.0 7.1
gradient, while ATPase is highest in bands 3-5 (Table 2). The specific activity of glucan synthetase is also greatest in bands 3-5, although the total distribution shows that high activity is also associated with band 2 (Table 2 a), no doubt because activity of this enzyme is apparently related to both plasma membrane and dictyosome content (Van der Woude et al., 1974). NADPH-cytochrome c reductase displays the highest specific activity in band 1, while total activity is greatest in band 2. The sterol-phospholipid ratios increase down the gradient, with the densest regions, band 4 and 5, having the highest ratios. Electron microscopy of sections stained with the plasma membrane stain of Roland et al, (1972) also indicates that plasma membrane is located very largely in bands 4 and 5, with a few profiles observable in band 3. Any mitochondria present are found, in lead-stained sections, to reside exclusively in band 4.
Auxin-Binding Characteristics of Gradient Fractionated Membranes The distribution of [14C]NAA binding in bands recovered from gradients of the type described above is shown in Table 3. All fractions are able to bind [14C]NAA,the greatest binding activity on the basis of Adpm over the selected concentration range being in the lightest regions, bands 1 and 2. It is difficult to recover sufficient material from each band to permit a full kinetic analysis on each fraction and as a compromise, the "simple" gradient was used for this purpose (See Materials and Methods). This gradient yields two fractions, the light fraction representing the combination of bands 1 and 2 and the heavy fraction representing bands 3-5.
Table3. Binding of [a4C]NAA by 4,000-38,000xg membrane preparations fractionated on discontinuous sucrose gradients. Binding is expressed as the difference in radioactivity bound at2xl0 VMand7xl0 7MNAA Band % w/w sucrose
Total activity Adpm/assay
Specific activity Adpm/mg protein
1. 18-25 2. 25-30 3. 30-34 4. 34-38 5. 3845
306 455 218 115 107
2,037 1,515 727 218 173
The kinetics of [14C]NA A binding to each of these bands (Fig. 3) suggest that each band contains one distinct set of binding sites. The dissociation constants, 3.9x 10 7 M for the light band, and l l . 6 x 10-7 M for the heavy band, compare favourably with those determined previously for site 1 and site 2 in unfractionated membrane preparations, namely 1.8 X 10 - 7 M and 14.5 x 10 7 M respectively (Batt et al., 1976). This similarity suggests that substantial resolution of the binding sites has been achieved. If the light and heavy bands do indeed contain site 1 and site 2 respectively, then the binding specificities of each site as determined in unfractionated preparations should be retained. Specificity was examined using three compounds, the auxin 3-indolylacetic acid (IAA), the anti-auxin and auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) and the inactive compound benzoic acid (BA). The ability of these compounds to interact with the NAA binding sites in each band was determined by constructing [J4C]NAA binding curves in the presence and absence of a fixed concentration of the analogue and analysing
S. Batt and M.A. Venis: Auxin Binding Sites
19
(a)
~
(b)
4
1000
K = 3.9 x 1 0 - 7 M
~
n=24pmol/g
~
X
~o0
K=11,6x10_7M
_-#.
n = 32 p mol/g
Heavy band
300
I
I
I
I
I
0.2
0.5
1.0
10
20
NAA
concn.,/IM(logscale)
pmolbound/gfreshwt
Fig. 3a and b. Kinetics of [14C]NAA binding to light and heavy bands obtained by fractionation of a 4 , 0 0 0 . 3 8 , 0 0 0 x g m e m b r a n e preparation on a " s i m p l e " sucrose gradient. (a) pellet radioactivity as a function of N A A concentration (b) Scatchard analysis of the binding data
TaMe 4. Comparison of Ki values calculated for site 1 and site 2 in unfractionated 4,000-38,000 x g m e m b r a n e preparations (data from Batt et al., 1976) with those calculated for the two bands from a ' s i m p l e ' sucrose gradient (data from Fig. 4)
Compound
IAA Benzoic acid TIBA
Unfractionated K i (M)
Fractionated K~ (M)
Site 1
Site 2
Site 1
Site 2
2 . 5 x 10 -6 3.6 x 10- 6 1.9x 10 6
7.3x I0 -6 No competition 2.4 x 10-6
7 . 9 x 10 -6 12 x 10- 6 1.3 x 10-6
9.1 x 10 -6 No competition 1.9 x 10-6
the data on double reciprocal plots as described previously (Batt et al., 1976). From these plots, (Fig. 4) it may be concluded that IAA and TIBA are able to compete with NAA for the binding sites present in both membrane bands. Benzoic, on the other hand, is competitive with NAA for the binding sites in the light band, but does not compete for binding sites in the heavy band. In Table 4, the K i values calculated from Figure 4 are compared with those estimated from earlier experiments with unfractionated membrane preparations (Batt et al., 1976). It would seem that the binding specificities of the light and heavy bands do indeed correspond to those deduced previously for site 1 and site 2 respectively (Batt et al., 1976), since only physiologically active auxin analogues bind to the heavy fraction, while both active and inactive compounds can bind to the light fraction. Discussion
From analyses of the kinetics of [14C]NAAbinding and of the interactions of a range of auxin analogues, it was concluded that crude membrane preparations from corn coleoptiles contain two classes of high
affinity auxin binding sites, termed site 1 and site 2 (Batt et al., 1976). The results presented here reinforce the earlier evidence that these sites are indeed distinct and present evidence that the auxin-specific site 2 binding is associated with plasma membraneenriched fractions. By differential centrifugation, a fraction can be obtained that contains site 2 but not site 1 binding activity (Fig. 2). This fraction contains mitochondria, but when a low speed pellet is resolved on a sucrose gradient, [14C]NAA binding and SDH activities in the different fractions are not correlated (Table 1). Site 2 binding is still detected in 10,000 38,000 x g fractions (Fig. 2) which are considerably depleted in mitochondria on the basis of SDH activity (unpublished observations). Furthermore, Hertel et al. (1972) detected binding which was predominantly site 2 binding (see Batt et al., 1976) in fractions which were substantially free of cytochrome c oxidase activity. We conclude, therefore, that site 2 binding is not to mitochondria, and that the binding detected in low force pellets probably represents binding to heavier members of membrane population present in higher speed fractions (perhaps attached to other cellular constituents).
20
S. Batt and M.A. Venis: Auxin Binding Sites (a)
1.4
Light Band
1.4
+ I A A 5 % ~ / ~
0.7
I
//
-3
I
1
-1
(b)
0.7
I
I
3
5
I
--2
S
,/~
I
1
1.0
I
I
3
5
1.0
+ BA5pM E3
0.5
0.5
=> / _
_x
I
-1
1
[
I
3
5
1
--2
I
J
I
1
3
1 1
[ 3
I
5
1.8
(c)
Q
0.9
0.9
-3
-1
1
i
3
[
5
/r
-2
I 5
1 Free,/zM
Fig. 4a-e. Competition for [14C]NAA binding sites in the light and heavy bands obtained by fractionation of 4,000-38,000x g membrane preparation on "simple" sucrose gradients. Double reciprocal plots of NAA binding in the presence and absence of (a) IAA (b) benzoic acid (BA) (c) TIBA
Crude membrane pellets can be fractionated on discontinuous sucrose gradients into a light and a heavy band whose properties, in terms of [14C]NAA binding kinetics (Fig. 3) and analogue binding specificity (Fig. 4, Table 4) are in accord with those found for site 1 and site 2 respectively in unfractionated preparations. The somewhat higher N A A dissociation constant in the light band (Fig. 3) and the higher K~ values for IAA and benzoic acid in this band
(Table 4), relative to those observed for site 1 in crude fractions, suggest that resolution is not complete and that the light band is partly contaminated with site 2. F r o m the IDPase and N A D P H - c y t o c h r o m e c reductase activities (Table 2) the light band containing site 1 would appear to be enriched in Golgi membranes and endoplasmic reticulum. Leonard et al. (1973) used an additional 20% w/w sucrose step in their gradients and found (in oat roots) that the speci-
S. Batt and M.A. Venis: Auxin Binding Sites
fic activity peak of NADPH-cytochrome c reductase was at the 18-20% sucrose interface whereas NADHcytochrome c reductase (used as a tonoplast and/or endoplasmic reticulum marker) peaked in the 20-25% sucrose band. In our experience with corn coleoptiles, we very seldom observed any visible band at an 18 20% sucrose interface and the 20% sucrose step was therefore routinely omitted. On the basis of sterol:phospholipid ratios (Table 2) and of electron microscopy of specificallystained sections, the gradient bands containing site 2 seem to be enriched in plasma membrane, particularly bands 4 and 5. The use of enzymic markers for plasma membrane is not altogether satisfactory. Glucan synthetase activity appears to be related to both plasma membrane and dictyosome content (Table 2 and Van der Woude et al., 1974), while Leigh et al. (1975) report that acidic ATPase of maize roots is associated with both plasmalemma and with another membrane fraction resolvable on Ficoll gradients. The specific staining method of Roland et al. (1972) is probably a fairly reliable indicator of plasma membrane content, but is a time-consuming procedure. High sterol:phospholipid ratios correlate quite well with specifically-stained membrane content, and their determination is somewhat less laborious, but an even more rapid procedure for plasma membrane evaluation would be desirable. Site 2 is thus present in membrane fractions that are enriched in plasma membrane. The analogue binding specificity of site 2 suggests that it may represent an auxin receptor site, though its precise role in terms of a physiological response cannot be evaluated at present. Site 1 appears to be associated with dictyosomes and/or endoplasmic reticulum, and the observations of Gawlik and Shen-Miller (1974), that IAA produces rapid changes in dictyosome content of oat coleoptile cells, are of interest in this respect. However, since site 1 binds active and inactive auxin analogues, its possible function remains mysterious, yet it is improbable that a binding site of such high affinity would be evolved without some physiological value. We are indebted to Shirley Potter, Shell Research Ltd, for the electron microscopic studies. We are also grateful to several members of the Biochemistry and Physiology Division, Shell Research Ltd., in particular Winifred Tyers and Richard Bumpus, for assistance with coleoptile harvesting. S. Batt wishes to thank the Science Research Council for financial support.
21
References Batt, S., Wilkins, M.B., Venis, M.A.: Auxin binding to corn coleoptile membranes: kinetics and specificity. Planta (Bed.) 130, 7-13 (1976) Folch, J., Lees, M., Sloane-Stanley, G.H.: A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 497-509 (1957) Gawlik, S.R., Shen-Miller, J. : Effects of indoleacetic acid on dicytosomes of apical and expanding cells of oat coleoptiles. Plant Physiol. 54, 217-221 (1974) Hertel, R., Thomson, K., Russo, V.E.A.: In vitro auxin binding to particulate cell fractions from corn coleoptiles. Planta (Bed.) 107, 325 340 (1972) Hodges, T.K., Leonard, R.T. : Purification of a plasma-membranebound adenosine triphosphatase from plant roots. In: Methods in Enzymology XXXII, pp. 392-406. Fleischer, S., Packer, L. (eds) NewYork-SanFrancisco-London: Academic Press 1974 Hodges, T.K., Leonard, R.T., Bracker, C.E., Keenan, T.W.: Purification of an ionstimulated adenosine trophosphatase from plant roots:association with plasma membranes. Proc. Nat. Acad. Sci. U.S.A. 69, 3307-3311 (1972) Leigh, R.A., Williamson, F.A., Wyn-Jones, G.: Presence of two different membrane bound, KCl-stinmlated adenosine triphosphatase activities in maize roots. Plant Physiol. 55, 678-685 (1975) Leonard, R.T., Hansen, D., Hodges, T.K.: Membrane-bound adenosine triphosphatase activities of oat roots. Plant Physiol. 51, 749-754 (1973) Lowry, O.N., Lopez, J.H.: The determination of inorganic phosphate in the presence of labile phosphate esters. J. Biol. Chem. 162, 421-428 (1946) Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, K.J. : Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 262-275 (1951) Pennington, R.J. : Biochemistry of dystrophic muscle. Mitochondrial succinate-tetrazolium reductase and adenosine triphosphatase. Biochem. J. 80, 649454 (1961) Powell, J.T., Brew, K.: Glycosyltransferases in the Golgi membranes of onion stem. Biochem. J. 142, 203 209 (1974) Ray, P.M., Shininger, T.L., Ray, M.M.: Isolation of fl-glucan synthetase particles from plant cells and identification with Golgi membranes. Proc. Nat. Acad. Sci. USA 64, 605-612 (1969) Roland, J.C., Lembi, C.A., Morr'e, D.J.: Phosphotungstic acid-chromic acid as a selective electron-dense stain for plasma membranes of plant cells. Stain Technology 47, 195 200 (1972) Rouser, G., Siakotoj, A.N., Fleischer, S.: Quantitative analysis of phospholipids by thinlayer chromatography and phosphorus analysis of spots. Lipids 1, 85 86 (1966) Stadtman, T.C.: Preparation and assay of cholesterol and ergosterol. In: Methods in Enzymology III, pp 392-394, Colowick, S.P., Kaplan, N.O. (eds) New York: Academic Press 1957 Van der Woude, W.J., Lembi, C,A., Mor/e, D.J., Kindinger, J.I., Ordin, L. : fl-Glucansynthetases of plasma membrane and Golgi apparatus from onion stem. Plant Physiol. 54, 333-340 (1974) Wilkinson, G. : Statistical estimations in enzyme kinetics. Biochem. J. 80, 324 332 (1961) Received 7 November; accepted 8 December 1975
