Biochemical SocietyTransactions ( 1 992) 20
Cyclic Nucleotide Phosphodiesterase Activity of Medicago satita L. PATRICK S. ROBINSON, CAROL J. COOKE, RUSSELL P. NEWTON, TERENCE J. WALTON and CHRISTOPHER J. SMITH. Biochemistry Research Group, School of Biological Sciences, University College Swansea, Swansea SA2 8PP, Wales, U.K. The hypersensitve response (HR) is an expression of resistance in plants that occurs at the point of infection by a microorganism. It involves expression of a number of active defence responses and synthesis of defence related molecules. Phytoalexin synthesis is one such defence response, and its occurrence in Medicago satjva (lucerne) tissues is the subject of our present studies. Phytoalexin synthesis induced in lucerne by the phytopathogen Verticillium albo-atrum involves interaction of a component (elicitor) from the fungus with a receptor in the host and results in increased synthesis of enzymes from the biosynthetic pathway. Previously we have identified some elements of the signal transduction mechanism that mediate the response in lucerne and adenosine 3’,5’-cyclic monophosphate (CAMP) was implicated as a second messenger [l]. CAMP is known to bring about its effects by regulating the activities of intracellular kinases and ion channels, however, as a second messenger it does not always operate in isolation and its interaction with its targets can be regulated by fluxes in intracellular Ca2’. For example, Ca” is known to act directly on enzymes such as cAh4P-phosphodiestere, or indirectly via the calcium binding protein calmodulin. e.g. via adenylyl cyclase [2]. As part of our examination of the role of CAMP in the phytoalexin response in lucerne we report here the presence of CAMP-phosphodiestere activity and evidence of its regulation by Ca” and calmodulin. Cells from 4-day suspension cultures of lucerne were harvested by centrifugation (800g, 6 min) and disrupted with a pestle and mortar with the aid of a little acid washed sand, in 50 mM TrisMCI buffer, pH 7.5 (0.5ml buffer/g. fresh wt.), containing lOmM Bmercaptoethanol, 1 mM EDTA, 1 pm leupeptin, 1 pM pepstatin A and 0.5 mM phenylmethylsulphonyl fluoride (PMSF). The homogenate was filtered through 2 layers of Miracloth (Calbiochem), centrifuged (31,OOOg, 10 min) and the supernatant dialysed for 16 hours at 4 “C against 20 volumes of homogenising buffer without EDTA. The dialysate was freeze-dried, and resuspended in the same buffer at a concentration of 6.76 mg proteidml. Cyclic nucleotide phosphodiesterase activity was determined by incubation of the extract (100-200 pg protein.) in 50 mM Tris/HCI pH 7.5 containing ImM CaCI,: 10 mM MgCI2; and either lOmM 3’,5’-cyclic AMP or lOmM 2’,3’-cyclic AMP as substrate, in a total volume of 600 ul. At the end of 10 min at 37 “C, the reaction was terminated by heating for 2 min at 90 “C. After cooling, the reaction mixture was incubated with 0.01 U (equivalent to 0.01 mmol substrate transformed per min) 5’-nucleotidase (Crotalus adamantus venom-Sigma) and/or 0.01U 3’-nucleotidase(Rye Grass -Sigma), for 1 hour at 37°C. The reaction was terminated by heating at 90 “C for 2 min. The phosphate content of a 600p1 aliquot of the sample was determined following centrifugation by incubation with 150 p1 of freshly made malachite green solution [3]. The absorbance was measured after 10 min at 630 nm. A standard curve was used to determine the concentration of phosphate. Lucerne extract, under the assay conditions described and in the presence of 10 mM 3’,5’-cyclic AMP, was found to contain phosphodiesterase activity equivalent to 36 nmol phosphatehg proteidmin Activity was not restricted to the 3 ’ 3 ’ nucleotide, however. When lOmM 2’,3’-cyclic AMP was employed as substrate an activity of 1586 nmol phosphate/mg proteidmin. was
355s
observed. Repeating the incubation with the 3’,5’-nucleotide but leaving out either the 3’- or 5’- nucleotidase indicated the ratio of 3 ’- to 5’-AMP products to be 1: 1. The two activities may represent a single enzyme with least two separate multisubstrate specificity or at phosphodiesterases. To establish their dependency on Ca”, the effect of 2mM EGTA on activity was determined. The results, (Table 1) show that chelating Ca” had more effect upon 3’,5’activity (44.9% inhibition) than on the 2’,3’-activity (23.6%). _ _ _ _ ~.~ ~ _
Table - e -1
. .
A n h
Substrate
Actmty.
+EGTA
Control
(10 mM) (nmol phosphatdmg/proteidmin). 3’-5’-CAMP 35.9 19.8 2’-3’-cAMP 1585.6 1210.9 ~~~~~~
~~~~~
44.9 23.6
~~~
~~~
~~~~~
Ca2’ may affect activity directly or by way of a Ca” binding protein such as calmodulin. Addition of the calmodulin antagonist Compound R24571 (I-[bis(pdichloro-B-benzyloxy)-phenethyllimidazoliniumchloride),to the assay mixture resulted in differential inhibition of the two activities, the data of Table 2 indicating that that the 3’,5’-activity but not 2’,3’-activity is regulated, at least in part, by calmodulin. _
_
~~~
~
~
~
~~~~~~
~~~~
~~
~
~.
~~
Table 2 &csphodiesterase Activity. . .. Control +0.5yM R24571 % Inhib1tW (nmol phosphate/mg proteidmin). (10 mM) 3’,5’-cAMP 35.9 18.6 48.2 2’,3’-cAMP 1585.6 1430.9 9.8
Substrate
.- .~
~~
~~
In order to further distinguish the two activities their responsiveness to calmodulin was determined by inclusion of 100 units of bovine calmodulin (Sigma-I unit of calmodulin stimulates to 50% of maximum the activity of 0.016 units of cyclicAMP phosphodiesterase) to the assay. This led to a 25% stimulation of 3’,5’-activity, the 2’,3’-activity remaining unaffected, (Table 3). The calcium requirement for calmodulin stimulation is shown by inhibition of the stimulated 3’,5’-activity by 2 mM EGTA. An inhibition by 0.5 pM of the calmodulin antagonist R24571 is further evidence that the stimulation is calmodulin specific. ~~~~~~~
Table 3 The Effect of Calmodulin and Calmodulin Antagonists on PhosDhodiesterase.Activity, SubstControl +CaM + CaM+EGTA + CaM+R24571 (nmol phosphate/mg protein/min). (IOmM) 3’-5’-cAMP 5 1.2 64.0 25.6 41.3 1181.3 1430.5 2’-3’-cAMP 1585.6 1632.5 ~~
~
The involvement of cAMP in phytoalexin induction in lucerne has been demonstrated in response to elicitor. [4]. Fluxes in Ca2’ have been implicated in the induction process [ I ] and regulation of 3’,5’-phosphodiesterase activity by calmodulin indicates that Ca” could affect the duration of a cAMP mediated process by its effect on the rate of degradation of CAMP. The lack of responsivness of the 2’J-activity to Ca2’ and calmodulin suggest a role outside signal transduction, not involving activation by Ca”, and that the two phosphodiesterase activities may be indicative of distinct enzymes. I . Smith C.J. (1991) Proc. of the Phytochem. Soc. Eur. (Smith, C.J. ed.)vol. 32, pp. 255-269, Oxford Science Publications. 2. Barrit, G.J. (1992) Communication within Animal Cells, pp ,127155, Oxford Science Publications. 3. Baykov, A.A., Evtushenko, O.A. & Avaeva, S.M. (1980) Biochem. Biophys. Acta. 623,257-270. 4. Cooke, C.J., Newton, R.P., Smith, C.J. & Walton, T.J. (1989) Biochem. Soc.Trans. 17,919-20.
Cyclic Nucleotide Phosphodiesterase Activity of Medicago satita L. PATRICK S. ROBINSON, CAROL J. COOKE, RUSSELL P. NEWTON, TERENCE J. WALTON and CHRISTOPHER J. SMITH. Biochemistry Research Group, School of Biological Sciences, University College Swansea, Swansea SA2 8PP, Wales, U.K. The hypersensitve response (HR) is an expression of resistance in plants that occurs at the point of infection by a microorganism. It involves expression of a number of active defence responses and synthesis of defence related molecules. Phytoalexin synthesis is one such defence response, and its occurrence in Medicago satjva (lucerne) tissues is the subject of our present studies. Phytoalexin synthesis induced in lucerne by the phytopathogen Verticillium albo-atrum involves interaction of a component (elicitor) from the fungus with a receptor in the host and results in increased synthesis of enzymes from the biosynthetic pathway. Previously we have identified some elements of the signal transduction mechanism that mediate the response in lucerne and adenosine 3’,5’-cyclic monophosphate (CAMP) was implicated as a second messenger [l]. CAMP is known to bring about its effects by regulating the activities of intracellular kinases and ion channels, however, as a second messenger it does not always operate in isolation and its interaction with its targets can be regulated by fluxes in intracellular Ca2’. For example, Ca” is known to act directly on enzymes such as cAh4P-phosphodiestere, or indirectly via the calcium binding protein calmodulin. e.g. via adenylyl cyclase [2]. As part of our examination of the role of CAMP in the phytoalexin response in lucerne we report here the presence of CAMP-phosphodiestere activity and evidence of its regulation by Ca” and calmodulin. Cells from 4-day suspension cultures of lucerne were harvested by centrifugation (800g, 6 min) and disrupted with a pestle and mortar with the aid of a little acid washed sand, in 50 mM TrisMCI buffer, pH 7.5 (0.5ml buffer/g. fresh wt.), containing lOmM Bmercaptoethanol, 1 mM EDTA, 1 pm leupeptin, 1 pM pepstatin A and 0.5 mM phenylmethylsulphonyl fluoride (PMSF). The homogenate was filtered through 2 layers of Miracloth (Calbiochem), centrifuged (31,OOOg, 10 min) and the supernatant dialysed for 16 hours at 4 “C against 20 volumes of homogenising buffer without EDTA. The dialysate was freeze-dried, and resuspended in the same buffer at a concentration of 6.76 mg proteidml. Cyclic nucleotide phosphodiesterase activity was determined by incubation of the extract (100-200 pg protein.) in 50 mM Tris/HCI pH 7.5 containing ImM CaCI,: 10 mM MgCI2; and either lOmM 3’,5’-cyclic AMP or lOmM 2’,3’-cyclic AMP as substrate, in a total volume of 600 ul. At the end of 10 min at 37 “C, the reaction was terminated by heating for 2 min at 90 “C. After cooling, the reaction mixture was incubated with 0.01 U (equivalent to 0.01 mmol substrate transformed per min) 5’-nucleotidase (Crotalus adamantus venom-Sigma) and/or 0.01U 3’-nucleotidase(Rye Grass -Sigma), for 1 hour at 37°C. The reaction was terminated by heating at 90 “C for 2 min. The phosphate content of a 600p1 aliquot of the sample was determined following centrifugation by incubation with 150 p1 of freshly made malachite green solution [3]. The absorbance was measured after 10 min at 630 nm. A standard curve was used to determine the concentration of phosphate. Lucerne extract, under the assay conditions described and in the presence of 10 mM 3’,5’-cyclic AMP, was found to contain phosphodiesterase activity equivalent to 36 nmol phosphatehg proteidmin Activity was not restricted to the 3 ’ 3 ’ nucleotide, however. When lOmM 2’,3’-cyclic AMP was employed as substrate an activity of 1586 nmol phosphate/mg proteidmin. was
355s
observed. Repeating the incubation with the 3’,5’-nucleotide but leaving out either the 3’- or 5’- nucleotidase indicated the ratio of 3 ’- to 5’-AMP products to be 1: 1. The two activities may represent a single enzyme with least two separate multisubstrate specificity or at phosphodiesterases. To establish their dependency on Ca”, the effect of 2mM EGTA on activity was determined. The results, (Table 1) show that chelating Ca” had more effect upon 3’,5’activity (44.9% inhibition) than on the 2’,3’-activity (23.6%). _ _ _ _ ~.~ ~ _
Table - e -1
. .
A n h
Substrate
Actmty.
+EGTA
Control
(10 mM) (nmol phosphatdmg/proteidmin). 3’-5’-CAMP 35.9 19.8 2’-3’-cAMP 1585.6 1210.9 ~~~~~~
~~~~~
44.9 23.6
~~~
~~~
~~~~~
Ca2’ may affect activity directly or by way of a Ca” binding protein such as calmodulin. Addition of the calmodulin antagonist Compound R24571 (I-[bis(pdichloro-B-benzyloxy)-phenethyllimidazoliniumchloride),to the assay mixture resulted in differential inhibition of the two activities, the data of Table 2 indicating that that the 3’,5’-activity but not 2’,3’-activity is regulated, at least in part, by calmodulin. _
_
~~~
~
~
~
~~~~~~
~~~~
~~
~
~.
~~
Table 2 &csphodiesterase Activity. . .. Control +0.5yM R24571 % Inhib1tW (nmol phosphate/mg proteidmin). (10 mM) 3’,5’-cAMP 35.9 18.6 48.2 2’,3’-cAMP 1585.6 1430.9 9.8
Substrate
.- .~
~~
~~
In order to further distinguish the two activities their responsiveness to calmodulin was determined by inclusion of 100 units of bovine calmodulin (Sigma-I unit of calmodulin stimulates to 50% of maximum the activity of 0.016 units of cyclicAMP phosphodiesterase) to the assay. This led to a 25% stimulation of 3’,5’-activity, the 2’,3’-activity remaining unaffected, (Table 3). The calcium requirement for calmodulin stimulation is shown by inhibition of the stimulated 3’,5’-activity by 2 mM EGTA. An inhibition by 0.5 pM of the calmodulin antagonist R24571 is further evidence that the stimulation is calmodulin specific. ~~~~~~~
Table 3 The Effect of Calmodulin and Calmodulin Antagonists on PhosDhodiesterase.Activity, SubstControl +CaM + CaM+EGTA + CaM+R24571 (nmol phosphate/mg protein/min). (IOmM) 3’-5’-cAMP 5 1.2 64.0 25.6 41.3 1181.3 1430.5 2’-3’-cAMP 1585.6 1632.5 ~~
~
The involvement of cAMP in phytoalexin induction in lucerne has been demonstrated in response to elicitor. [4]. Fluxes in Ca2’ have been implicated in the induction process [ I ] and regulation of 3’,5’-phosphodiesterase activity by calmodulin indicates that Ca” could affect the duration of a cAMP mediated process by its effect on the rate of degradation of CAMP. The lack of responsivness of the 2’J-activity to Ca2’ and calmodulin suggest a role outside signal transduction, not involving activation by Ca”, and that the two phosphodiesterase activities may be indicative of distinct enzymes. I . Smith C.J. (1991) Proc. of the Phytochem. Soc. Eur. (Smith, C.J. ed.)vol. 32, pp. 255-269, Oxford Science Publications. 2. Barrit, G.J. (1992) Communication within Animal Cells, pp ,127155, Oxford Science Publications. 3. Baykov, A.A., Evtushenko, O.A. & Avaeva, S.M. (1980) Biochem. Biophys. Acta. 623,257-270. 4. Cooke, C.J., Newton, R.P., Smith, C.J. & Walton, T.J. (1989) Biochem. Soc.Trans. 17,919-20.
