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An improved scatchard analysis of fusicoccin-binding to maize coleoptile membranes
Plant Science Letters, 33 (1984) 187--193
187
Elsevier Scientific Publishers Ireland Ltd.
AN IMPROVED SCATCHARD ANALYSIS OF FUSICOCCIN-BINDING TO MAIZE COLEOPTILE
MEMBRANES
P. ADUCCI a, M. COLETTA b and M. M A R R A a
aGruppo di Chimica Biologica e Strutturistica Chimiea, c/o Institute of Biological Cheniistry, University of Rome 'La Sapienza' and bC.N.R. Centro di Biologia Molecolare, c/o Institutes of Biological Chemistry and Chemistry, Faculty of Medicine, University of Rome 'La Sapienza" Rome (Italy) (Received May 5th, 1983) (Revision received July 20th, 1983) (Accepted September 2nd, 1983)
SUMMARY
A reinvestigation of fusicoccin (FC) binding properties in plasmalemmaenriched membranes of maiz? coleoptiles has been carried out. Scatchard plots of experimental binding data have been analysed by a non-linear leastsquares fitting program and are consistent with t w o classes of independent binding sites. The Ka-values obtained are Ka = 3.3 -+ 1.6 × 10 -1° M, KA = 1.3 + 0.53 X 10 M and are independent of protein concentration. They are only in partial agreement with those previously obtained by a different m e t h o d of analysis. --8
--
°
~1
o
o
~2
Key words: Fusicoccin binding-sites --Maize coleoptile -- Scatchard analysis - - Plasmamembranes INTRODUCTION
FC, the major p h y t o t o x i c metabolite of Fusicoccum amygdali Del., interacts with specific binding-sites located at the external surface of plant plasma membranes [1]. This p h e n o m e n o n is very likely responsible for the very early events in the mechanism of action of the substance. A preliminary characterization of FC binding-sites was performed on plasmalemmaenriched fractions of maize coleoptiles [2]. Further investigations on the properties of FC-binding and on the nature of FC receptors have been subsequently reported with this and other tissues [ 3--6 ]. These studies have
Abbreviations: 3H-FC, 3H-labelled dihydrofusicoccin; FC, fusicoccin. 0304-4211/84/$03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
188 been carried out using a FC derivative, dihydrofusicoccin, which displays identical phytotoxic [ 7 ] and plant growth regulation activities [ 8,9 ] as the parent c o m p o u n d and is easily obtainable in tritiated form with a very high specific activity. The importance of the role played by these sites in the regulatory mechanism of a plasma membrane pump has been also suggested by their location at the plasmalemma [ 1]. Further interest in FC binding sites has been raised by the detection in plant tissues of substances able to prevent FC-stimulated H ÷ extrusion in vivo (maize roots) and FC-binding to membranes (maize roots, maize coleoptiles, spinach leaves) [10]. Such endogenous substances might represent the physiological modulators of the H*-extruding system triggered by FC. From the studies on the characterization of FC-binding proteins the occurrence of two classes of sites has been reported along with the estimation of their respective Kd-values from Scatchard analysis [3,4,5,11]. Recent reports on c o m m o n misinterpretations of biphasic Scatchard plots [12,13] has prompted us to reinvestigate 3H-labeled dihydrofusicoccin (3H-FC) binding properties with a more reliable fitting procedure of non-linear Scatchard plots. MATERIALS AND METHODS Chemicals FC was prepared as previously reported [14]. 3H-FC (spec. act. 62 Ci/ mmol) was prepared according to Ballio [ 5]. Plant material Coleoptiles were obtained from seeds of Zea mays L., (XL 342 Italian Dekalb, Mestre, Italy) grown on vermiculite for 5 days in the dark at 26°C. Homogenization and cell fractionation Coleoptiles, precooled in ice, were gently ground in a mortar with 2 ml of ice-cold homogenization buffer (10 mM citrate buffer (pH 5.5) containing 5 mM MgSO4, 250 mM sucrose) per g of fresh tissue and then strained through a nylon cloth. The suspension was then centrifuged as previously reported [11] to obtain the crude membrane fraction. The pellets were taken up in 10 mM citrate buffer (pH 5.5) containing 5 mM MgSO4, to provide the crude membrane suspension used in binding experiments. Binding test Each sample contained: a suitable volume of crude membrane suspension to a final concentration of 0.5 g of fresh tissue in a total volume of I ml. Experiments were run in triplicate with samples incubated at 27°C for 90 min and then analysed as previously described [ 3 ]. In the experiments performed to determine the Kd-dependence on the concentration of membrane fractions, the original suspension was used as such and after a 2-, 4-, and 5-fold dilution.
189
Fitting procedure
F i t t i n g o f all data has been carried o u t with a non-linear least-squares program using a Marquardt algorithm with first derivatives [ 15]. The data were analysecl according to the following equation: [L]b = [L]f
q ni • PT Z i= 1 Kdt + [L]f
(1)
where [L]b and [ L] f are the concentrations of b o u n d and free ligand, respectively, PT is the total protein concentration, ni is the number of sites within the single molecule which bind with the same dissociation constant Kdi, and q is the total number of site classes corresponding to different affinities. As a consequence of the above definitions, ni • PT represents the total concentrations of sites i and also a maximum estimate of the protein concentration if a single site per molecule is assumed. Data in Fig. 3 have been analysed according to the following equation:
9=
q ni [ L] f Y, i= 1 n T (Kdi + [L]f)
(2)
where n T is the total number of fusicoccin-binding sites, Y is the fraction of sites occupied b y the ligand and is expressed b y the ratio between b o u n d SH-FC dpm and the maximum dpm value obtained when all sites are saturated, and all other symbols are as in Eqn. 1.
RESULTS A N D DISCUSSION The results of 3H-FC binding to maize coleoptile membrane fractions have been analysed using the fitting procedure described in Materials and Methods. The data fit satisfactorilyinto a theoretical curve which is representative of the binding behaviour when only two classes of specific sites are present. This is obviously a m i n i m u m estimate, although no further improvement of the fitting quality has be~n obtained by considering a higher number of classes. O n the other hand, when the data have been analysed assuming only one class of sites,the calculated curve could not fit satisfactorilythe experimental data (Fig. 1). The computer analysis of data from five independent binding experiments gives the following estimate for the K d of high and low affinity sites respectively: Kdi = 3.3 -+ 1.6 × 10 -~° M, Kd~ = 1.3 + 0.53 × 10 -8 M. Such values are only hi partial agreement with those previously reported (Kd, = 0.7 × 10-9 M, Kd = 0.6X lO-SM [ 4 ] ; K d = 1 . 2 . 1 0 - 9 , K d , = ND [3]}. The difference is esl~ecially remarkable in th~ case of high affinity constant. This is reasonable in view of the higher difficulty in determining this parameter
190
accurately. Thus the use of an independent non-linear least-squares fitting program represents a major advantage in the analysis of data with respect to previous procedures [3,11] where the parameter determination was accomplished by a manual line drawing through experimental points. The confidence o f the absolute values of dissociation constants is further strengthened by the observation that a 5-fold dilution of the membrane concentration did not bring a b o u t any meaningful change of the c o m p u t e d Kd (Fig. 2). Thus, it appears that the measured Kd is a true one and no stoichiometric binding is occurring. As a matter of fact it has been pointed out [ 16 ] that, if the concentration of receptor sites is not at least one order of magnitude lower than the true dissociation constant the apparent Kdvalues result incorrect because of the stoichiometric ligand binding and they vary as a linear function of the receptor concentration. That this is not the case for our data is proved also by the protein concentration values indirectly obtained from Eqn. 1 as n i PT (see Materials and Methods), which turn o u t to be always at least 10 times smaller than the high affinity dissociation constant. An additional degree of accuracy resides on the formalism used in Eqn. 1 according to which the affinity constant to a class of sites i is defined both by the slope of the transition corresponding to the specific binding process and by the upper a s y m p t o t e when [L]f ~ Kdi. Differently from Kdl and Kd~, which are very reproducible, the estimate of n2/nl ratio is somewhat variable among different experiments, ranging between a minimum of 10 to a maximum of 100. This is principally due to the formalism of Eqn. 1, according to which the slope of each transition i
[L] B
0.2
0.0 -I0.2
o
,
, -9.4
,
,
,
-8.6
, -7.8
"~ ~--7.0
,oo[L] (M) Fig. I. Experimental points have been plotted using a logarithmic scale for the independent variable [L ]f. Dashed line is the fitting curve using Eqn. 1 with q = 1. Continuous line corresponds to the best fitting with q = 2.
191
0.4
ILl. ILl, 0.2
0.0
o
' -10.2
' -9.4
' ~
'~--'--8.6
•
•
~
-7.8
"
-
~
i
nl
-7.0
(M) Fig. 2. Experimental points characterized by the symbol (o) refer to those already reported in Fig. 1. (m) refers to the same sample diluted by a factor of 5. The two curves correspond to the same Kd and Kd which were also obtained independently from the 1 2 best fitting of each separated experimental set.
corresponding to the different classes of sites, is clearly influenced b y the ratio n i P T / [ L ] f (Eqn. 1). The slope is reduced by an increase o f n i and becomes much less sensitive even to significant variations of ni; this fact occurs mostly with low affinity sites (n2) and raises some uncertainty in the determination of an absolute value for their number and of the ratio of n : / n l . Additional factors of variability could likely arise from possible slight differences in the sample preparation. However, even with such a high variance it is possible to assess that the number of low affinity sites is a b o u t one order of magnitude larger than the high affinity ones. An additional piece of information in this sense has been obtained from a binding experiment in which the sites were saturated with 3H-FC without any addition of the unlabeled toxin and analysed according to Eqn. 2. As reported in the experiment depicted in Fig. 3, the binding to low affinity sites is not cooperative and the high affinity sites represent <2% of the total. Obviously, such results are compatible also with a negative co-operativity and there is no way, at present time, to make one interpretation more convincing than the other one. The approach reported in this paper appears to be necessary for a quantitative determination of the parameters which characterize the binding of FC to membranes. In particular, the concentration of sites and thus of protein turns o u t to be extremely low, which is known to represent a problem in the purification of the binding sites and in the isolation of the endogenous ligands. Nevertheless, the analysis of data shows some new characteristics of
192
O
0.8
0.4
0.0
.
- o.s
,
-,.,
-u
t
,
I
-,.3
,oQ[c],
(.)
Fig. 3. Experimental binding data have been fitted according to Eqn. 2 with q = 2 and Kdl fixed according to values obtained from data reported in Fig. 1 and 2. Best fitting gave as result the same Kd2 obtained in Fig. 1 and 2 and n l H i w = 0.016 and n , / n T = 0.984. FC binding-sites, namely the high affinity sites are reproducibly present in the membranes even if t h e y may represent only a small percentage of all sites. However it must be pointed out that the physiological significance of sites with different affinities is actually not clear. In this respect useful results may be acquired using this quantitative m e t h o d of analysis of binding experiments by comparing the properties of sites present in different tissues and plants under a variety of experimental conditions that could further elucidate the characteristics of FC receptors. ACKNOWLEDGEMENTS We t h a n k Professor A. Ballio, Professor M. Brunori and Dr. M. Moloney for helpful and stimulating discussions. The authors want to t h a n k also Professor S.J. Gill and Dr. B. Richey of University of Colorado Boulder, CO for having made available the original frame of the non-linear least-squares fitting program. This research was supported by grants of the Italian Ministry of Education (Ministero della Pubblica Istruzione) and the Italian Research Council
(C.N.R.). REFERENCES 1 P. Aducci, R. Federico and A. Ballio,Phytopathol. Mediter., 19 (1980) 187. 2 U. Dorhmann, R. Hertel, P. Pesci, S. Cocucci, E. MarrY, G. Randazzo and A. Ballio, Plant Sci. Lett., 9 (1977) 291.
193 3 P. Pesci, S. Cocucci and G. Randazzo, Plant Cell Environ., 2 (1979) 295. 4 A. Ballio, Chemistry and plant growth regulating activity of Fusicoccin derivatives and analogues, in: H. Geissbiihler (Ed.), Advances in Pesticide Science, Pergamon Press, Oxford, 1979, p. 366. 5 A. Ballio, R. Federico, A. Pessi and D. Scalorbi, Plant Sci. Lett., 18 (1980) 39. 6 R.G. Stout and R.E. Cleland, Plant Physiol., 66 (1980) 353. 7 A. Ballio, M. Bottalico, A. Framondino, A. Graniti and G. Randazzo, Phytopathol. Mediter., 10 (1971) 26. 8 A. Ballio, F. Pocchiari, S. Russi and V. Silano, Physiol. Pathol., 1 (1971) 95. 9 A. BaUio, M.I. De Michelis, P. Lado and G. Randazzo, Physiol. Plant., 52 (1981) 471. 10 P. Aducci, G. Crosetti, R. Federico and A. Ballio, Planta, 148 (1980) 208. 11 P. Aducci, A. BaUio, R. Federico and L. Montesano, Studies on Fusicoccin-binding sites, in: P.F. Wareing (Ed.), Plant Growth Substances, Academic Press, London, 1982, p. 395. 12 J.G. Norby, P. Ottolenghi and J. Jorgen, Anal. Biochem., 102 (1980) 318. 13 I.M. Klotz, Science, 217 (1982) 1247. 14 A. Ballio, A. Carilli,B. Santurbano and L. Tuttobello, Ann. Ist. Super. SanitY, 4 (1968) 317. 15 G. Barisas and S.J. Gill, Biophys. Chem., 9 (1979) 235. 16 K.J. Chang, S. Jacobs and P. Cuatrecasas, Biochim. Biophys. Acta, 406 (1975) 294.
187
Elsevier Scientific Publishers Ireland Ltd.
AN IMPROVED SCATCHARD ANALYSIS OF FUSICOCCIN-BINDING TO MAIZE COLEOPTILE
MEMBRANES
P. ADUCCI a, M. COLETTA b and M. M A R R A a
aGruppo di Chimica Biologica e Strutturistica Chimiea, c/o Institute of Biological Cheniistry, University of Rome 'La Sapienza' and bC.N.R. Centro di Biologia Molecolare, c/o Institutes of Biological Chemistry and Chemistry, Faculty of Medicine, University of Rome 'La Sapienza" Rome (Italy) (Received May 5th, 1983) (Revision received July 20th, 1983) (Accepted September 2nd, 1983)
SUMMARY
A reinvestigation of fusicoccin (FC) binding properties in plasmalemmaenriched membranes of maiz? coleoptiles has been carried out. Scatchard plots of experimental binding data have been analysed by a non-linear leastsquares fitting program and are consistent with t w o classes of independent binding sites. The Ka-values obtained are Ka = 3.3 -+ 1.6 × 10 -1° M, KA = 1.3 + 0.53 X 10 M and are independent of protein concentration. They are only in partial agreement with those previously obtained by a different m e t h o d of analysis. --8
--
°
~1
o
o
~2
Key words: Fusicoccin binding-sites --Maize coleoptile -- Scatchard analysis - - Plasmamembranes INTRODUCTION
FC, the major p h y t o t o x i c metabolite of Fusicoccum amygdali Del., interacts with specific binding-sites located at the external surface of plant plasma membranes [1]. This p h e n o m e n o n is very likely responsible for the very early events in the mechanism of action of the substance. A preliminary characterization of FC binding-sites was performed on plasmalemmaenriched fractions of maize coleoptiles [2]. Further investigations on the properties of FC-binding and on the nature of FC receptors have been subsequently reported with this and other tissues [ 3--6 ]. These studies have
Abbreviations: 3H-FC, 3H-labelled dihydrofusicoccin; FC, fusicoccin. 0304-4211/84/$03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
188 been carried out using a FC derivative, dihydrofusicoccin, which displays identical phytotoxic [ 7 ] and plant growth regulation activities [ 8,9 ] as the parent c o m p o u n d and is easily obtainable in tritiated form with a very high specific activity. The importance of the role played by these sites in the regulatory mechanism of a plasma membrane pump has been also suggested by their location at the plasmalemma [ 1]. Further interest in FC binding sites has been raised by the detection in plant tissues of substances able to prevent FC-stimulated H ÷ extrusion in vivo (maize roots) and FC-binding to membranes (maize roots, maize coleoptiles, spinach leaves) [10]. Such endogenous substances might represent the physiological modulators of the H*-extruding system triggered by FC. From the studies on the characterization of FC-binding proteins the occurrence of two classes of sites has been reported along with the estimation of their respective Kd-values from Scatchard analysis [3,4,5,11]. Recent reports on c o m m o n misinterpretations of biphasic Scatchard plots [12,13] has prompted us to reinvestigate 3H-labeled dihydrofusicoccin (3H-FC) binding properties with a more reliable fitting procedure of non-linear Scatchard plots. MATERIALS AND METHODS Chemicals FC was prepared as previously reported [14]. 3H-FC (spec. act. 62 Ci/ mmol) was prepared according to Ballio [ 5]. Plant material Coleoptiles were obtained from seeds of Zea mays L., (XL 342 Italian Dekalb, Mestre, Italy) grown on vermiculite for 5 days in the dark at 26°C. Homogenization and cell fractionation Coleoptiles, precooled in ice, were gently ground in a mortar with 2 ml of ice-cold homogenization buffer (10 mM citrate buffer (pH 5.5) containing 5 mM MgSO4, 250 mM sucrose) per g of fresh tissue and then strained through a nylon cloth. The suspension was then centrifuged as previously reported [11] to obtain the crude membrane fraction. The pellets were taken up in 10 mM citrate buffer (pH 5.5) containing 5 mM MgSO4, to provide the crude membrane suspension used in binding experiments. Binding test Each sample contained: a suitable volume of crude membrane suspension to a final concentration of 0.5 g of fresh tissue in a total volume of I ml. Experiments were run in triplicate with samples incubated at 27°C for 90 min and then analysed as previously described [ 3 ]. In the experiments performed to determine the Kd-dependence on the concentration of membrane fractions, the original suspension was used as such and after a 2-, 4-, and 5-fold dilution.
189
Fitting procedure
F i t t i n g o f all data has been carried o u t with a non-linear least-squares program using a Marquardt algorithm with first derivatives [ 15]. The data were analysecl according to the following equation: [L]b = [L]f
q ni • PT Z i= 1 Kdt + [L]f
(1)
where [L]b and [ L] f are the concentrations of b o u n d and free ligand, respectively, PT is the total protein concentration, ni is the number of sites within the single molecule which bind with the same dissociation constant Kdi, and q is the total number of site classes corresponding to different affinities. As a consequence of the above definitions, ni • PT represents the total concentrations of sites i and also a maximum estimate of the protein concentration if a single site per molecule is assumed. Data in Fig. 3 have been analysed according to the following equation:
9=
q ni [ L] f Y, i= 1 n T (Kdi + [L]f)
(2)
where n T is the total number of fusicoccin-binding sites, Y is the fraction of sites occupied b y the ligand and is expressed b y the ratio between b o u n d SH-FC dpm and the maximum dpm value obtained when all sites are saturated, and all other symbols are as in Eqn. 1.
RESULTS A N D DISCUSSION The results of 3H-FC binding to maize coleoptile membrane fractions have been analysed using the fitting procedure described in Materials and Methods. The data fit satisfactorilyinto a theoretical curve which is representative of the binding behaviour when only two classes of specific sites are present. This is obviously a m i n i m u m estimate, although no further improvement of the fitting quality has be~n obtained by considering a higher number of classes. O n the other hand, when the data have been analysed assuming only one class of sites,the calculated curve could not fit satisfactorilythe experimental data (Fig. 1). The computer analysis of data from five independent binding experiments gives the following estimate for the K d of high and low affinity sites respectively: Kdi = 3.3 -+ 1.6 × 10 -~° M, Kd~ = 1.3 + 0.53 × 10 -8 M. Such values are only hi partial agreement with those previously reported (Kd, = 0.7 × 10-9 M, Kd = 0.6X lO-SM [ 4 ] ; K d = 1 . 2 . 1 0 - 9 , K d , = ND [3]}. The difference is esl~ecially remarkable in th~ case of high affinity constant. This is reasonable in view of the higher difficulty in determining this parameter
190
accurately. Thus the use of an independent non-linear least-squares fitting program represents a major advantage in the analysis of data with respect to previous procedures [3,11] where the parameter determination was accomplished by a manual line drawing through experimental points. The confidence o f the absolute values of dissociation constants is further strengthened by the observation that a 5-fold dilution of the membrane concentration did not bring a b o u t any meaningful change of the c o m p u t e d Kd (Fig. 2). Thus, it appears that the measured Kd is a true one and no stoichiometric binding is occurring. As a matter of fact it has been pointed out [ 16 ] that, if the concentration of receptor sites is not at least one order of magnitude lower than the true dissociation constant the apparent Kdvalues result incorrect because of the stoichiometric ligand binding and they vary as a linear function of the receptor concentration. That this is not the case for our data is proved also by the protein concentration values indirectly obtained from Eqn. 1 as n i PT (see Materials and Methods), which turn o u t to be always at least 10 times smaller than the high affinity dissociation constant. An additional degree of accuracy resides on the formalism used in Eqn. 1 according to which the affinity constant to a class of sites i is defined both by the slope of the transition corresponding to the specific binding process and by the upper a s y m p t o t e when [L]f ~ Kdi. Differently from Kdl and Kd~, which are very reproducible, the estimate of n2/nl ratio is somewhat variable among different experiments, ranging between a minimum of 10 to a maximum of 100. This is principally due to the formalism of Eqn. 1, according to which the slope of each transition i
[L] B
0.2
0.0 -I0.2
o
,
, -9.4
,
,
,
-8.6
, -7.8
"~ ~--7.0
,oo[L] (M) Fig. I. Experimental points have been plotted using a logarithmic scale for the independent variable [L ]f. Dashed line is the fitting curve using Eqn. 1 with q = 1. Continuous line corresponds to the best fitting with q = 2.
191
0.4
ILl. ILl, 0.2
0.0
o
' -10.2
' -9.4
' ~
'~--'--8.6
•
•
~
-7.8
"
-
~
i
nl
-7.0
(M) Fig. 2. Experimental points characterized by the symbol (o) refer to those already reported in Fig. 1. (m) refers to the same sample diluted by a factor of 5. The two curves correspond to the same Kd and Kd which were also obtained independently from the 1 2 best fitting of each separated experimental set.
corresponding to the different classes of sites, is clearly influenced b y the ratio n i P T / [ L ] f (Eqn. 1). The slope is reduced by an increase o f n i and becomes much less sensitive even to significant variations of ni; this fact occurs mostly with low affinity sites (n2) and raises some uncertainty in the determination of an absolute value for their number and of the ratio of n : / n l . Additional factors of variability could likely arise from possible slight differences in the sample preparation. However, even with such a high variance it is possible to assess that the number of low affinity sites is a b o u t one order of magnitude larger than the high affinity ones. An additional piece of information in this sense has been obtained from a binding experiment in which the sites were saturated with 3H-FC without any addition of the unlabeled toxin and analysed according to Eqn. 2. As reported in the experiment depicted in Fig. 3, the binding to low affinity sites is not cooperative and the high affinity sites represent <2% of the total. Obviously, such results are compatible also with a negative co-operativity and there is no way, at present time, to make one interpretation more convincing than the other one. The approach reported in this paper appears to be necessary for a quantitative determination of the parameters which characterize the binding of FC to membranes. In particular, the concentration of sites and thus of protein turns o u t to be extremely low, which is known to represent a problem in the purification of the binding sites and in the isolation of the endogenous ligands. Nevertheless, the analysis of data shows some new characteristics of
192
O
0.8
0.4
0.0
.
- o.s
,
-,.,
-u
t
,
I
-,.3
,oQ[c],
(.)
Fig. 3. Experimental binding data have been fitted according to Eqn. 2 with q = 2 and Kdl fixed according to values obtained from data reported in Fig. 1 and 2. Best fitting gave as result the same Kd2 obtained in Fig. 1 and 2 and n l H i w = 0.016 and n , / n T = 0.984. FC binding-sites, namely the high affinity sites are reproducibly present in the membranes even if t h e y may represent only a small percentage of all sites. However it must be pointed out that the physiological significance of sites with different affinities is actually not clear. In this respect useful results may be acquired using this quantitative m e t h o d of analysis of binding experiments by comparing the properties of sites present in different tissues and plants under a variety of experimental conditions that could further elucidate the characteristics of FC receptors. ACKNOWLEDGEMENTS We t h a n k Professor A. Ballio, Professor M. Brunori and Dr. M. Moloney for helpful and stimulating discussions. The authors want to t h a n k also Professor S.J. Gill and Dr. B. Richey of University of Colorado Boulder, CO for having made available the original frame of the non-linear least-squares fitting program. This research was supported by grants of the Italian Ministry of Education (Ministero della Pubblica Istruzione) and the Italian Research Council
(C.N.R.). REFERENCES 1 P. Aducci, R. Federico and A. Ballio,Phytopathol. Mediter., 19 (1980) 187. 2 U. Dorhmann, R. Hertel, P. Pesci, S. Cocucci, E. MarrY, G. Randazzo and A. Ballio, Plant Sci. Lett., 9 (1977) 291.
193 3 P. Pesci, S. Cocucci and G. Randazzo, Plant Cell Environ., 2 (1979) 295. 4 A. Ballio, Chemistry and plant growth regulating activity of Fusicoccin derivatives and analogues, in: H. Geissbiihler (Ed.), Advances in Pesticide Science, Pergamon Press, Oxford, 1979, p. 366. 5 A. Ballio, R. Federico, A. Pessi and D. Scalorbi, Plant Sci. Lett., 18 (1980) 39. 6 R.G. Stout and R.E. Cleland, Plant Physiol., 66 (1980) 353. 7 A. Ballio, M. Bottalico, A. Framondino, A. Graniti and G. Randazzo, Phytopathol. Mediter., 10 (1971) 26. 8 A. Ballio, F. Pocchiari, S. Russi and V. Silano, Physiol. Pathol., 1 (1971) 95. 9 A. BaUio, M.I. De Michelis, P. Lado and G. Randazzo, Physiol. Plant., 52 (1981) 471. 10 P. Aducci, G. Crosetti, R. Federico and A. Ballio, Planta, 148 (1980) 208. 11 P. Aducci, A. BaUio, R. Federico and L. Montesano, Studies on Fusicoccin-binding sites, in: P.F. Wareing (Ed.), Plant Growth Substances, Academic Press, London, 1982, p. 395. 12 J.G. Norby, P. Ottolenghi and J. Jorgen, Anal. Biochem., 102 (1980) 318. 13 I.M. Klotz, Science, 217 (1982) 1247. 14 A. Ballio, A. Carilli,B. Santurbano and L. Tuttobello, Ann. Ist. Super. SanitY, 4 (1968) 317. 15 G. Barisas and S.J. Gill, Biophys. Chem., 9 (1979) 235. 16 K.J. Chang, S. Jacobs and P. Cuatrecasas, Biochim. Biophys. Acta, 406 (1975) 294.