Planta (1985)165:493 501
P l a n t a 9 Springer-Verlag1985
Quantitative changes in calmodulin and NAD kinase during early cell development in the root apex of Pisum sativum L. E. Allan* and A. Trewavas Botany Department, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JH, UK
Abstract. Calmodulin and N A D kinase were extracted from serial developmental sections of the pea root apex. Highly purified samples of calmodulin were assayed by NAD-kinase activation, and whole-cell extracts were examined by two-dimensional polyacrylamide gel electrophoresis. Calmodulin was found to vary 17-fold in concentration over the apical 2 mm, being high in the region of the root cap and meristem, falling rapidly at the base of the meristem during early stages of rapid cell elongation. The rate of decline was different between stele and cortex. Except for a minor increase in concentration 2.5-5 mm from the apex, which coincides with the region of localised meristematic activity during initiation of lateral root primordia, the concentration of calmodulin remained at the lower level throughout the more basal sections of the apical 10 ram. In-vitro N A D kinase activity was found to increase 17-fold per cell over the apical 30 ram, almost entirely as the result of an increase in calmodulin-dependent activity. Quantitative estimates of both calmodulin and N A D kinase were found to be highly dependent on extraction procedures. Key words: Calmodulin - N A D kinase - Pisum (calmodulin, N A D kinase) - R o o t development.
Introduction The calcium-binding regulatory protein calmodulin was originally discovered as the activator of Department of Botany, University of Massaschusetts, Amherst, MA 01003, USA
* Present address:
Abbreviation: EGTA = ethylene glycol-bis (fl-aminoethyl ether)N,N,N',N'-tetraacetic acid
bovine brain cyclic nucleotide phosphodiesterase (Cheung 1967), and is well established as a major mediator of the calcium signal in animal cells (for reviews, see Cheung 1980; Klee et al. 1980). Many processes in plant cells are known to be dependent on calcium; however, its mechanism of action is poorly understood in plant systems. There is, nevertheless, an increasing amount of evidence to link calmodulin with several calcium-dependent activities in plant cells. Calmodulin has been isolated from several plant species (Charbonneau and Cormier 1979; Anderson et al. 1980), and is known to regulate in-vitro activity of isoenzymes of protein kinase, Ca 2 § ATPase and N A D kinase, while certain inhibitors of calmodulin will arrest cytoplasmic streaming and geotropism. It may also regulate calcium distribution by activation of calciumtransport pumps (Roux and Slocum 1982). It is generally accepted that several calciumdependent processes involved in cell division, expansion and differentiation are present in the embryonic region of the root apex. In order to investigate the potential involvement of calmodulin in root development we have attempted to identify and quantitate calmodulin and a calmodulin-dependent enzyme, N A D kinase, during cell development. Potentially there are two methods for the quantitative estimation of incompletely purified calmodulin. These are radio-immune assay or the controlled activation of calmodulin-dependent enzymes. We have chosen to use the latter, more laborious, procedure because we consider it more selective and specific and because it estimates biologically active calmodulin. We have used pea N A D kinase as the activatable enzyme since it has a 50-fold greater affinity for calmodulin than any other enzyme tested and unlike a variety of animal enzymes is specific for calmodulin (Jarrett et al.
494
E. Allan and A. Trewavas: Calmodulin and N A D kinase in the pea root apex
1982) and thus represents potentially a very sensitive system. In addition, we have identified pea calmodulin in two-dimensional gel separations and used this as a check on our above enzymatic assays for calmodulin variation in developing pea root cells.
Material and methods Chemicals. All reagents used were the best commercial grade available. Trifluoperazine was a gift of Smith, Kline and French, Welwyn, Hefts., U K and fluphenazine was a gift of E.R. Squibb and Sons, Twickenham, Middlesex, U K . Glucose6-phosphate dehydrogenase (Type XV) was obtained from Sigma Chemical Co., London, U K . Plant material. Calmodulin and N A D kinase were purified from pea seedlings (Pisum sativum L. cv. Feltham 1st). Seeds were surface-sterilised and then germinated in trays of sterilised vermiculite in the dark at 22-24 ~ C. To obtain root tissue, the seedlings were harvested after 65 h, and roots 2.5-3.5 cm long were selected for study. When necessary, they were sectioned into serial segments approx. 1.2 m m in length. To obtain etiolated shoot tissue, the seedlings were grown a further 8 d under the same conditions. To obtain green shoots, the plants were transferred to a greenhouse for the final 4 d. Extraction of calmodulin from the root apex. In order to avoid potentially interfering substances in the calmodulin assay system (such as coenzymes, calmodulin-binding and calmodulinlike proteins) calmodulin and N A D kinase were partially purified. Published methods for extraction of calmodulin (e.g. Charbonneau and Cormier (1979)), however, gave extremely low or negligible yields from both shoot or root. Considerable experimentation showed the necessity in pea root tissue of modifying both a m m o n i u m sulphate concentrations and pH conditions for precipitation and also the necessity for separating calmodulin from C a 2 +-calmodulin-binding proteins and for solubilising substantial amounts of calmodulin from membranes (Allan 1984). The following procedure gave final yields of between 67-84 lag calmodulin g 1 fresh weight of root tips representing about 0.5% of the total extractable protein and a yield two orders of magnitude higher than the original C h a r b o n n e a u and Cormier (1979) method. Experiments in which known amounts of bovine calmodulin were added to homogenates showed 105% recovery, thus indicating calmodulin was obtained in a biologically intact state. A 300-mg sample of the appropriate segments of approx. 300 pea roots was homogenised in a Potter homogeniser (Potter-Elvehjem, Zurich, Switzerland) in 40 vol. of buffer containing 1 mol dm -3 KCI; 2 mmol dm -3 MgCI2 ; 4 mmol dm -3 ethylene glycol bis(fl-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) ; 50 mmol d m - 3 2-amino-2-(hydroxymethyl)-1,3propanediol p H 7 . 4 ; 2.5% (w/v) polyvinylpolypyrrolidone (PVPP). After centrifugation for 30 min at 20000 g, the supern a t a n t was taken to 60% saturation with a m m o n i u m sulphate, and adjusted to pH 4 with t mol dm -3 H2SO4 containing ammonium sulphate at 60% saturation. After stirring at 4 ~ C for 12 h, the solution was centrifuged at 42000 g for 30 min, and the pellet resuspended in and dialysed against 20 mmol d i n - 3 Tris pH 8, 1 mmol dm -3 E G T A for a further 12 h. The sample was heated to 100~ for 2 rain and the denatured protein removed by centrifugation. The supernatant was then dialysed against 20 mmol dm -3 Tris pH for 42 h, and the samples were
assayed for calmodulin. Cahnodulin prepared by this procedure was almost completely free of polypeptide contamination as determined by two-dimensional polyacrylamide gel electrophoresis.
Separation of ealmodulin-dependent and calmodulin-independent ?CAD kinase for the assay of calmodulin. Calmodulin-deficient, calmodulin-dependent N A D kinase activity was separated from calmodulin-independent activity, and the calmodulin-dependent form used to assay calmodulin. Light-grown pea shoot tissue (50 g) was homogenised in four volumes of buffer A (1 tool d m - 3 KCI, 50 mmol d m - 3 Tris pH 7.4, 2 mmol dm -3 MgCI 2, 1 mmol dm -3 EGTA, 0.5 1yffnol d m -3 phenylmethylsulfonyl fluoride (PMSF) containing 2.5% PVPP (Anderson et al. 4980). The homogenate was filtered, and then centrifuged at 12000 g for 30 rain. The supernatant was diluted 4:1 with distilled water, and loaded onto an anion-exchange column of 100 ml diethylaminoethyl (DEAE)-Sephacel equilibrated with buffer A diluted 1:1. The void volume was collected and a m m o n i u m sulphate was immediately added to 50% saturation. The 12000-g pellet was resuspended in a minimum volume of buffer A diluted 1:9 with distilled water. After dialysis against the same buffer for 12 h, the dialysate was clarified by centrifugation at 24000 g for 1 h. The supernatant was then loaded onto a second DEAE-Sephacel column equilibrated with buffer A diluted 1:9, and was eluted with this buffer. The N A D kinase eluting at this salt concentration was used to assay calmodulin. When the absorbance of the column eluate at 280 nm had returned nearly to the baseline value, protein more strongly bound to the column was eluted with buffer A diluted 1:9 with the addition of KC1 to a final concentration of 0.4 mol dan- 3. All procedures were performed at 4 ~ C. Calmodulin activated the purified N A D kinase over 100-fold compared with 1.3-fold in the crude homogenate.
Assay of calmodulin. Calmodulin was assayed by its ability to activate the calmodulin-dependent calmodulin-deficient N A D kinase, obtained as above. The N A D kinase was assayed by the procedure of M n t o and Miyachi (1977), modified as follows: 50 lal of N A D kinase were incubated in a final volume of 0.5 ml containing 3 m m o l d m -3 ATP, 1 0 m m o l d m -3 MgCI 2, 4 0 m m o l d m - 3 T r i s p H 8 , l m m o l d m - 3 C a C I z, 2 mmol d i n - 3 ethylenediaminetetracetic acid (EDTA), 2 mmol d m -3 N A D at 37 ~ C. The reaction was initiated by addition of NAD, and was terminated after 30 min by addition of 0.1 ml of I mol d m - 3 HC1. The solution was neutralised with 0.1 ml of 1 mol dm -3 N A D H , and N A D P formed by N A D kinase activity was determined. The NADP solution was preincubated for 2• min with 30 lag 2,6-dichlorophenolindophenol, 20 gg phenazine methosulphate, and glucose-6-phosphate to a final concentration of 2 mmol d m - 3 in the complete reaction mix. To initiate the reaction, 1 unit of NADP-dependent glucose-6-phosphate dehydrogenase was added, bringing the reaction mix to a final volume of 1 ml. The rate of decrease in absorbance at 600 n m was recorded, and the initial velocity over the first 2 rain was used to estimate N A D P concentration. One unit of N A D kinase is defined as the amount required to convert 1 lamol of N A D to N A D P / m i n when fully activated. Owing to the instability of N A D kinase, all calmodulin assays were carried out 27-30 h after harvesting, and the enzyme was recalibrated with bovine calmodulin for each experiment. The amount of calmodulin in a sample was estimated by comparison with activation of N A D kinase by a known amount of pure bovine brain calmodulin prepared from brain acetone powder by the method of Caldwell and Haug (1984 a). Bovine brain cahnodulin migrated as a single polypeptide on
E. Allan and A. Trewavas: Calmodulin and NAD kinase in the pea root apex two-dimensional polyacrylamide gels (see Fig. 4 later) and was estimated by E276 values (E276 1 % = 1.8 f o r mammalian calmodulin; Anderson et al. 1980). The values for pea calmodulin were then adjusted on the assumption that plant calmodulin is sevenfold more effective in half-maximal activation of NAD kinase than mammalian calmodulin (Cormier et al. 1982). This assay system detected as little as 50 ng bovine calmodulin (about 7 ng plant calmodulin) and had an upper limit of 4 gg bovine calmodulin. The concentration of calmodulin in pea root tissue was then estimated on the assumption that the toolecular weight of calmodulin was 17000 Da, and that 1 mm 3 of tissue was equivalent to 1 gl.
Assay of pea root NAD kinase. A 200-mg sample of 1.2 mm serial sections of root tissue was homogenised in 20 vol. of 1 tool dm- 3 KC1, 1 mmol din- 3 EGTA, 50 mmol dm- 3 Tris pH 7.4, 2 mmol dm 3 MgCI2, 0.5 mmol dm -3 PMSF, 2.5% (w/v) PVPP, and centrifuged at 25 000 g for 30 min. The supernatants were dialysed against 50mmoldm-3Tris pH7.4, 2 mmol dm-3 MgCI2, 1 mmol dm-3 EGTA, 0.2 mol dm-3 KC1, 0.5 mmol dm -3 PMSF for 2 h to remove interfering factors in the extract, particularly pyridine nucleotides. The NAD kinase was assayed 4 h after harvesting by a modification of the procedure of Muto and Miyachi (1977) as described above. Calmodulin-independent NAD kinase activity was estimated as the activity of NAD kinase when incubated in the presence of 0.1 mmol dm 3 trifluoperazine or 2 mmol dm -3 EGTA. Calmodulin-dependent activity was estimated by subtraction of the value obtained for calrnodulin-independentactivity from that obtained for NAD kinase fully saturated with calmodulin and calcium.
Two-dimensional polyacrylamide gel electrophoresis of pea root proteins. Protein was extracted in 9.5 tool dm -3 urea, 2% (w/v) NP40, 5% (v/v) 2-mercaptoethanol, ampholines pH 3-10, and either 15 mmol dm -3 EGTA or 10 mmol dm -3 CaCI2, and was separated on two-dimensional polyacrylamide gels essentially according to the method of O'Farrell (1975). First-dimension gels were eleetrophoresed under non-equilibrium pH-gradient conditions (NEPHGE) (O'Farrell et al. 1977) for 5 h at 400 V, or under isoelectricfocusing conditions for 30 min at 200 V, 30 rain at 300 V and 20 h at 400 V. Electrophoresis was from the anode (0.2% H2SO4) to the cathode (0.5% ethanolamine). Proteins were separated in the second dimension in a 10-15% exponential acrylamide gradient containing 0.1% sodium dodecyl sulfate (SDS) and, when required, 1.5 mmol dm -3 EGTA. Proteins were stained with silver using a modification of the method of Switzer et al. (1979). Radioactively labelled proteins from seedlings incubated for 2 h in 1.8 MBq [35S]methionine were visualised by exposure to Kodak (London) Regulix BB5 film for six weeks. The film was developed in Ilford (London) Phen-X developer.
One-dimensional polyacrylamide gel electrophoresis. Proteins were solubilised in 2.3% SDS, 5% mercaptoethanol, 62.5 mmol dm 3 Tris pH 6.8, 2% Ficoll and either 15 mmol dm -3 EGTA or 10 mmol dm 3 CaC12 at 100~ C for 2 rain, and were electrophoresed according to Laemmli (1970). Proteins were stained with Coomassie Blue.
495 0 -TFP A .+TFP
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32 fraction number
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P l a n t a 9 Springer-Verlag1985
Quantitative changes in calmodulin and NAD kinase during early cell development in the root apex of Pisum sativum L. E. Allan* and A. Trewavas Botany Department, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JH, UK
Abstract. Calmodulin and N A D kinase were extracted from serial developmental sections of the pea root apex. Highly purified samples of calmodulin were assayed by NAD-kinase activation, and whole-cell extracts were examined by two-dimensional polyacrylamide gel electrophoresis. Calmodulin was found to vary 17-fold in concentration over the apical 2 mm, being high in the region of the root cap and meristem, falling rapidly at the base of the meristem during early stages of rapid cell elongation. The rate of decline was different between stele and cortex. Except for a minor increase in concentration 2.5-5 mm from the apex, which coincides with the region of localised meristematic activity during initiation of lateral root primordia, the concentration of calmodulin remained at the lower level throughout the more basal sections of the apical 10 ram. In-vitro N A D kinase activity was found to increase 17-fold per cell over the apical 30 ram, almost entirely as the result of an increase in calmodulin-dependent activity. Quantitative estimates of both calmodulin and N A D kinase were found to be highly dependent on extraction procedures. Key words: Calmodulin - N A D kinase - Pisum (calmodulin, N A D kinase) - R o o t development.
Introduction The calcium-binding regulatory protein calmodulin was originally discovered as the activator of Department of Botany, University of Massaschusetts, Amherst, MA 01003, USA
* Present address:
Abbreviation: EGTA = ethylene glycol-bis (fl-aminoethyl ether)N,N,N',N'-tetraacetic acid
bovine brain cyclic nucleotide phosphodiesterase (Cheung 1967), and is well established as a major mediator of the calcium signal in animal cells (for reviews, see Cheung 1980; Klee et al. 1980). Many processes in plant cells are known to be dependent on calcium; however, its mechanism of action is poorly understood in plant systems. There is, nevertheless, an increasing amount of evidence to link calmodulin with several calcium-dependent activities in plant cells. Calmodulin has been isolated from several plant species (Charbonneau and Cormier 1979; Anderson et al. 1980), and is known to regulate in-vitro activity of isoenzymes of protein kinase, Ca 2 § ATPase and N A D kinase, while certain inhibitors of calmodulin will arrest cytoplasmic streaming and geotropism. It may also regulate calcium distribution by activation of calciumtransport pumps (Roux and Slocum 1982). It is generally accepted that several calciumdependent processes involved in cell division, expansion and differentiation are present in the embryonic region of the root apex. In order to investigate the potential involvement of calmodulin in root development we have attempted to identify and quantitate calmodulin and a calmodulin-dependent enzyme, N A D kinase, during cell development. Potentially there are two methods for the quantitative estimation of incompletely purified calmodulin. These are radio-immune assay or the controlled activation of calmodulin-dependent enzymes. We have chosen to use the latter, more laborious, procedure because we consider it more selective and specific and because it estimates biologically active calmodulin. We have used pea N A D kinase as the activatable enzyme since it has a 50-fold greater affinity for calmodulin than any other enzyme tested and unlike a variety of animal enzymes is specific for calmodulin (Jarrett et al.
494
E. Allan and A. Trewavas: Calmodulin and N A D kinase in the pea root apex
1982) and thus represents potentially a very sensitive system. In addition, we have identified pea calmodulin in two-dimensional gel separations and used this as a check on our above enzymatic assays for calmodulin variation in developing pea root cells.
Material and methods Chemicals. All reagents used were the best commercial grade available. Trifluoperazine was a gift of Smith, Kline and French, Welwyn, Hefts., U K and fluphenazine was a gift of E.R. Squibb and Sons, Twickenham, Middlesex, U K . Glucose6-phosphate dehydrogenase (Type XV) was obtained from Sigma Chemical Co., London, U K . Plant material. Calmodulin and N A D kinase were purified from pea seedlings (Pisum sativum L. cv. Feltham 1st). Seeds were surface-sterilised and then germinated in trays of sterilised vermiculite in the dark at 22-24 ~ C. To obtain root tissue, the seedlings were harvested after 65 h, and roots 2.5-3.5 cm long were selected for study. When necessary, they were sectioned into serial segments approx. 1.2 m m in length. To obtain etiolated shoot tissue, the seedlings were grown a further 8 d under the same conditions. To obtain green shoots, the plants were transferred to a greenhouse for the final 4 d. Extraction of calmodulin from the root apex. In order to avoid potentially interfering substances in the calmodulin assay system (such as coenzymes, calmodulin-binding and calmodulinlike proteins) calmodulin and N A D kinase were partially purified. Published methods for extraction of calmodulin (e.g. Charbonneau and Cormier (1979)), however, gave extremely low or negligible yields from both shoot or root. Considerable experimentation showed the necessity in pea root tissue of modifying both a m m o n i u m sulphate concentrations and pH conditions for precipitation and also the necessity for separating calmodulin from C a 2 +-calmodulin-binding proteins and for solubilising substantial amounts of calmodulin from membranes (Allan 1984). The following procedure gave final yields of between 67-84 lag calmodulin g 1 fresh weight of root tips representing about 0.5% of the total extractable protein and a yield two orders of magnitude higher than the original C h a r b o n n e a u and Cormier (1979) method. Experiments in which known amounts of bovine calmodulin were added to homogenates showed 105% recovery, thus indicating calmodulin was obtained in a biologically intact state. A 300-mg sample of the appropriate segments of approx. 300 pea roots was homogenised in a Potter homogeniser (Potter-Elvehjem, Zurich, Switzerland) in 40 vol. of buffer containing 1 mol dm -3 KCI; 2 mmol dm -3 MgCI2 ; 4 mmol dm -3 ethylene glycol bis(fl-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) ; 50 mmol d m - 3 2-amino-2-(hydroxymethyl)-1,3propanediol p H 7 . 4 ; 2.5% (w/v) polyvinylpolypyrrolidone (PVPP). After centrifugation for 30 min at 20000 g, the supern a t a n t was taken to 60% saturation with a m m o n i u m sulphate, and adjusted to pH 4 with t mol dm -3 H2SO4 containing ammonium sulphate at 60% saturation. After stirring at 4 ~ C for 12 h, the solution was centrifuged at 42000 g for 30 min, and the pellet resuspended in and dialysed against 20 mmol d i n - 3 Tris pH 8, 1 mmol dm -3 E G T A for a further 12 h. The sample was heated to 100~ for 2 rain and the denatured protein removed by centrifugation. The supernatant was then dialysed against 20 mmol dm -3 Tris pH for 42 h, and the samples were
assayed for calmodulin. Cahnodulin prepared by this procedure was almost completely free of polypeptide contamination as determined by two-dimensional polyacrylamide gel electrophoresis.
Separation of ealmodulin-dependent and calmodulin-independent ?CAD kinase for the assay of calmodulin. Calmodulin-deficient, calmodulin-dependent N A D kinase activity was separated from calmodulin-independent activity, and the calmodulin-dependent form used to assay calmodulin. Light-grown pea shoot tissue (50 g) was homogenised in four volumes of buffer A (1 tool d m - 3 KCI, 50 mmol d m - 3 Tris pH 7.4, 2 mmol dm -3 MgCI 2, 1 mmol dm -3 EGTA, 0.5 1yffnol d m -3 phenylmethylsulfonyl fluoride (PMSF) containing 2.5% PVPP (Anderson et al. 4980). The homogenate was filtered, and then centrifuged at 12000 g for 30 rain. The supernatant was diluted 4:1 with distilled water, and loaded onto an anion-exchange column of 100 ml diethylaminoethyl (DEAE)-Sephacel equilibrated with buffer A diluted 1:1. The void volume was collected and a m m o n i u m sulphate was immediately added to 50% saturation. The 12000-g pellet was resuspended in a minimum volume of buffer A diluted 1:9 with distilled water. After dialysis against the same buffer for 12 h, the dialysate was clarified by centrifugation at 24000 g for 1 h. The supernatant was then loaded onto a second DEAE-Sephacel column equilibrated with buffer A diluted 1:9, and was eluted with this buffer. The N A D kinase eluting at this salt concentration was used to assay calmodulin. When the absorbance of the column eluate at 280 nm had returned nearly to the baseline value, protein more strongly bound to the column was eluted with buffer A diluted 1:9 with the addition of KC1 to a final concentration of 0.4 mol dan- 3. All procedures were performed at 4 ~ C. Calmodulin activated the purified N A D kinase over 100-fold compared with 1.3-fold in the crude homogenate.
Assay of calmodulin. Calmodulin was assayed by its ability to activate the calmodulin-dependent calmodulin-deficient N A D kinase, obtained as above. The N A D kinase was assayed by the procedure of M n t o and Miyachi (1977), modified as follows: 50 lal of N A D kinase were incubated in a final volume of 0.5 ml containing 3 m m o l d m -3 ATP, 1 0 m m o l d m -3 MgCI 2, 4 0 m m o l d m - 3 T r i s p H 8 , l m m o l d m - 3 C a C I z, 2 mmol d i n - 3 ethylenediaminetetracetic acid (EDTA), 2 mmol d m -3 N A D at 37 ~ C. The reaction was initiated by addition of NAD, and was terminated after 30 min by addition of 0.1 ml of I mol d m - 3 HC1. The solution was neutralised with 0.1 ml of 1 mol dm -3 N A D H , and N A D P formed by N A D kinase activity was determined. The NADP solution was preincubated for 2• min with 30 lag 2,6-dichlorophenolindophenol, 20 gg phenazine methosulphate, and glucose-6-phosphate to a final concentration of 2 mmol d m - 3 in the complete reaction mix. To initiate the reaction, 1 unit of NADP-dependent glucose-6-phosphate dehydrogenase was added, bringing the reaction mix to a final volume of 1 ml. The rate of decrease in absorbance at 600 n m was recorded, and the initial velocity over the first 2 rain was used to estimate N A D P concentration. One unit of N A D kinase is defined as the amount required to convert 1 lamol of N A D to N A D P / m i n when fully activated. Owing to the instability of N A D kinase, all calmodulin assays were carried out 27-30 h after harvesting, and the enzyme was recalibrated with bovine calmodulin for each experiment. The amount of calmodulin in a sample was estimated by comparison with activation of N A D kinase by a known amount of pure bovine brain calmodulin prepared from brain acetone powder by the method of Caldwell and Haug (1984 a). Bovine brain cahnodulin migrated as a single polypeptide on
E. Allan and A. Trewavas: Calmodulin and NAD kinase in the pea root apex two-dimensional polyacrylamide gels (see Fig. 4 later) and was estimated by E276 values (E276 1 % = 1.8 f o r mammalian calmodulin; Anderson et al. 1980). The values for pea calmodulin were then adjusted on the assumption that plant calmodulin is sevenfold more effective in half-maximal activation of NAD kinase than mammalian calmodulin (Cormier et al. 1982). This assay system detected as little as 50 ng bovine calmodulin (about 7 ng plant calmodulin) and had an upper limit of 4 gg bovine calmodulin. The concentration of calmodulin in pea root tissue was then estimated on the assumption that the toolecular weight of calmodulin was 17000 Da, and that 1 mm 3 of tissue was equivalent to 1 gl.
Assay of pea root NAD kinase. A 200-mg sample of 1.2 mm serial sections of root tissue was homogenised in 20 vol. of 1 tool dm- 3 KC1, 1 mmol din- 3 EGTA, 50 mmol dm- 3 Tris pH 7.4, 2 mmol dm 3 MgCI2, 0.5 mmol dm -3 PMSF, 2.5% (w/v) PVPP, and centrifuged at 25 000 g for 30 min. The supernatants were dialysed against 50mmoldm-3Tris pH7.4, 2 mmol dm-3 MgCI2, 1 mmol dm-3 EGTA, 0.2 mol dm-3 KC1, 0.5 mmol dm -3 PMSF for 2 h to remove interfering factors in the extract, particularly pyridine nucleotides. The NAD kinase was assayed 4 h after harvesting by a modification of the procedure of Muto and Miyachi (1977) as described above. Calmodulin-independent NAD kinase activity was estimated as the activity of NAD kinase when incubated in the presence of 0.1 mmol dm 3 trifluoperazine or 2 mmol dm -3 EGTA. Calmodulin-dependent activity was estimated by subtraction of the value obtained for calrnodulin-independentactivity from that obtained for NAD kinase fully saturated with calmodulin and calcium.
Two-dimensional polyacrylamide gel electrophoresis of pea root proteins. Protein was extracted in 9.5 tool dm -3 urea, 2% (w/v) NP40, 5% (v/v) 2-mercaptoethanol, ampholines pH 3-10, and either 15 mmol dm -3 EGTA or 10 mmol dm -3 CaCI2, and was separated on two-dimensional polyacrylamide gels essentially according to the method of O'Farrell (1975). First-dimension gels were eleetrophoresed under non-equilibrium pH-gradient conditions (NEPHGE) (O'Farrell et al. 1977) for 5 h at 400 V, or under isoelectricfocusing conditions for 30 min at 200 V, 30 rain at 300 V and 20 h at 400 V. Electrophoresis was from the anode (0.2% H2SO4) to the cathode (0.5% ethanolamine). Proteins were separated in the second dimension in a 10-15% exponential acrylamide gradient containing 0.1% sodium dodecyl sulfate (SDS) and, when required, 1.5 mmol dm -3 EGTA. Proteins were stained with silver using a modification of the method of Switzer et al. (1979). Radioactively labelled proteins from seedlings incubated for 2 h in 1.8 MBq [35S]methionine were visualised by exposure to Kodak (London) Regulix BB5 film for six weeks. The film was developed in Ilford (London) Phen-X developer.
One-dimensional polyacrylamide gel electrophoresis. Proteins were solubilised in 2.3% SDS, 5% mercaptoethanol, 62.5 mmol dm 3 Tris pH 6.8, 2% Ficoll and either 15 mmol dm -3 EGTA or 10 mmol dm 3 CaC12 at 100~ C for 2 rain, and were electrophoresed according to Laemmli (1970). Proteins were stained with Coomassie Blue.
495 0 -TFP A .+TFP
'E ~ ~ i~ ~ ~ ~ ~ Q_ z ~< z
3
l- ~l
[]
,N
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m
9
16
32 fraction number
,,'
