PhotosynthesisResearch 45: 219-224, 1995. © 1995KluwerAcademicPublishers. Primedin the Netherlands. Regular paper
Intracellular localization of C A I P and C A I P phosphatase activity in leaves of Phaseolus vulgaris L. Brandon D. Moore 1, Thomas D. Sharkey2 & Jeffrey R. Seemann1 1Department of Biochemistry, University of Nevada, Reno, NV 89557, USA; 2Department of Botany, University of Wisconsin, Madison, W153706, USA Received20 June 1995;acceptedin revisedform7 August1995
Key words: nonaqueous fractionation, rubisco, carboxyarabinitol
Abstract
CA1P and CA1P phosphatase occur in the chloroplasts of leaf mesophyll cells of many species. However, whether either may occur exclusively in the chloroplast has not yet been established. To examine their intracellular distribution, mature, dark- or light-treated leaves of Phaseolus vulgaris were frozen, lyophilized and then centrifuged in density gradients of heptane and tetrachloroethylene. After gradient fractionation, both CA1P and CA1P phosphatase activity co-segregated with chloroplast material. Distribution analyses using sub-cellular compartment markers indicated that both CA1P and CA1P phosphatase do occur exclusively in leaf chloroplasts.
Abbreviations: Bicine - N,N-bis[2-hydroxyethyl]glycine; CAIP - 2-carboxyarabinitol 1-phosphate; CABP - 2carboxyarabinitol 1,5-bisphosphate; Chl - chlorophyll; DTT - dithiothreitol; EDTA - (ethylenediamine)tetraacetic acid; PEP - phosphoenolpyruvate; Tris - tris(hydroxymethyl)aminomethane
Introduction
ic sites are the same in either dark-treated leaves of
Phaseolus vulgaris or in chloroplasts isolated from CA1P is a branched-chain sugar-phosphate that accumulates in the leaves of many plant species under conditions of low irradiance (for review, see Seemann et al. 1990; Servaites 1992). CA1P resembles the reaction intermediate of the rubisco carboxylase reaction, binds tightly to carbamylated rubisco, and in a number of species can regulate the in vivo capacity for CO2 assimilation by inhibiting rubisco activity. Under conditions of increasing irradiance, CA1P is degraded by a specific phosphohydrolase, CA1P phosphatase, to produce a non-inhibitory compound, carboxyarabinitol (Holbrook et al. 1989). While there are substantial data on the associated photosynthetic physiology of CA1P, there are uncertainties as to whether CA1P occurs exclusively within the chloroplast (e.g. Portis 1992). Evidence which supports the localization of CA1P within chloroplasts can be summarized as follows: (1) Seemann et al. (1985) reported that the ratios of CA1P to rubisco catalyt-
such leaves; (2) A number of investigators have isolated a rubisco-CA1P complex from different species for use in the purification of CA1P or for other analyses (e.g. Servaites 1985; Gutteridge et al. 1986; Berry et al. 1987; Andralojc et al. 1994); and, (3) Moore and Seemann (1994) showed that in dark-treated leaves of P. vulgaris about 75% of the inactive rubisco catalytic sites in vivo are bound to CA1P. Still, whether CA1P occurs solely in the chloroplast has not been rigorously demonstrated. CA1P phosphatase is also known to be associated predominantly with the chloroplast: (1) The isolated organelle has proved useful as starting material for purification of the enzyme (Salvucci et al. 1988; Salvucci and Holbrook 1989); (2) The activity of CA1P phosphatase from stromal extracts of P. vulgaris was 2-4 fold greater than is the maximum rate of CA1P degradation that occurs in those leaves (Kobza et al. 1990); and, (3) The rate of CA1P degradation in tobac-
220 co leaves was decreased after treatment with methyl viologen, a Photosystem I electron acceptor (Salvucci and Anderson 1987). However, during the isolation of CA1P phosphatase from tobacco leaves, two peaks of activity were detected after an early purification step using ion-exchange chromatography (Salvucci and Holbrook 1989). Since only the major peak was clearly of chloroplastic origin, some CA 1P phosphatase may occur outside the chloroplast. There are a number of available methods for determining the intracellular distribution of leaf proteins and metabolites. Enzyme distributions have been studied using whole leaf homogenates (e.g. Rocha and Ting 1970) or extracts of leaf protoplasts (e.g. Spalding et al. 1979) after fractionation on sucrose density gradients. The fractionation of leaf protoplasts using rapid membrane filtration has proven very useful for sub-cellular analyses of metabolites under different physiological conditions (e.g. Gardestrom and Wigge 1988). However, such aqueous fractionation methods have inherent problems with altered metabolite levels during tissue preparation, and generally do not yield a good vacuolar fraction from most species. In contrast, the use of a non-aqueous fractionation procedure does allow the evaluation of analyte distribution between chloroplasts, cytosol, and vacuoles (Santarius and Heber 1965; Stitt et al. 1989). In this procedure, leaves are collected into liquid N2, lyophilized, and then fractionated on density gradients of non-aqueous solvents. Metabolites and proteins of a particular cellular region aggregate together and no metabolism occurs during their isolation. We successfully used a non-aqueous fractionation method previously to show that carboxyarabinitol occurs in multiple compartments within leaf cells (Moore et al. 1992). In this study, we have used this method again to examine the intracellular localization of CA1P and CA1P phosphatase in leaves of Phaseolus vulgaris, which contain relatively high levels of both analytes.
Materials and methods
Plant growth Plants of Phaseolus vulgaris L. cv. Linden were grown in a greenhouse (27/18 °C day/night), and were watered several times weekly with nutrient solution. Maximum daily irradiance was 1400 #mol quanta m -2 s -1. Fully expanded leaves from mature plants were used for experiments.
Non-aqueous fractionation Procedures were comparable to those reported elsewhere (Gerhardt and Heldt 1984; Sharkey and Vanderveer 1989). Bean leaves were collected into liquid N2 after 16 h dark (for CA1P measurements) or at mid-day (for CA1P phosphatase measurements). After removing major veins, 20 g of leaves were ground in liquid N2, using a mortar and pestle. Powdered material was transferred to cold lyophilization jars (600 ml, Labconco, Kansas City, MO), and the jars were kept in liquid N2 until the air pressure was reduced to < 25 mtorr (VirTis 10-324 freeze-dryer, VirTis Co., Gardiner, NY). Samples were dried overnight, and the powder was partitioned into five parts. Each portion was added to 20 ml of 4 °C heptane in a cut-off, 50-ml tefzel centrifuge tube (top 1/3 removed; Nalge Co., Rochester, NY), and placed in a 250-ml beaker containing heptane partially frozen in liquid N2. The sample was sonicated for 45 s with a Branson Sonitier 200 (power setting 8, continuous pulse, Branson Ultrasonics Corp., Danbury, CT), and filtered successively through 200 #m and 80 #m nylon nets (20 cm 2 each, Tetko Inc., Briarcliff Manor, NY). The pooled sample was partitioned equally into six 50-ml tefzel centrifuge tubes, and the leaf material was precipitated by centrifugation at 100 g, for 3 min. Leaf pellets were resuspended in 5 ml of 4 °C heptane, and each was loaded onto a 24-ml linear density gradient of heptane/tetrachloroethylene (J.T. Baker Inc., Phillipsburg, NJ), which ranged from 1.35 g m1-1 to 1.60 g ml -l. Samples were centrifuged at 4 °C in a swinging bucket rotor (Beckmann Instruments Inc., Fullerton, CA) at 17 000g, for 10h. All processing of gradients was done at 4 °C. Gradients were fractionated into 6 portions from the top, using a transfer pipet. Typically about 10 ml was collected as the lightest fraction, and about 3 ml each for the remaining fractions. The volumes were measured on the pooled collections. For marker assays, 0.6 ml was diluted with cold heptane in microfuge tubes, and the gradient material was precipitated by centrifugation at 10 000 g, for 5 min. The remaining gradient samples were diluted with heptane and collected by centrifugation. Residual heptane was evaporated overnight at 4 °C under reduced pressure in a vacuum jar with dessicant.
221 50
Marker assays v
Chl, PEP carboxylase, and a-mannosidase were used as markers for chloroplasts, cytosol, and vacuoles, respectively. The dried gradient fractions were resuspended in 0.6 ml of buffer containing 0.1 M Bicine pH 7.8, 5 mM MgC12, 1 mM EDTA, and 5 mM DTF. The samples were sonicated for 30 s (power setting 3, 30% duty cycle), and centrifuged at 10 000 g, 5 min. Supernatants were used for enzyme assays. Chl was measured after resuspending the pellets in 95% ethanol (Wintermanns and DeMots 1965). PEP carboxylase activity was assayed using [IaC]NaHCO3 (74 MBq mmol- l), and reaction components described by (Sharkey and Vanderveer 1989). Assays were conducted for 15 min at 25 °C, in triplicate. One blank was done for each sample, for which the reaction was terminated at 0 s. a-Mannosidase activity was assayed in duplicate for 60 min at 37 °C (Sharkey and Vanderveer 1989). Each assay was also done with replicate blanks, for which extract was added after addition of borate. Released nitrophenol was measured at A4o5 (extinction coefficient 18 500 M -1 cm-l). CA1P measurements [2-14C]CA1P was synthesized from labeled CABP (Gutteridge et al. 1989), and then used to measure sample CA1P content by isotope dilution assay (Moore et al. 1991; Moore et al. 1992). CA 1P phosphatase partial purification and activity assay CA1P phosphatase activity cannot yet be measured directly from whole leaf material due to the presence of non-specific phosphatase activity which can utilize CA1P as substrate. Therefore, gradient fractions were chromatographed using Cibacron Blue agarose (Sigma Chemical Co., St. Louis, MO) as described below to obtain samples void of such non-specific phosphatase activity but with complete recovery of CA1P phosphatase activity (Moore et al. 1995). Dried gradient fractions were resuspended in 5 ml of buffer containing 20 mM Tris-HC1 (pH 8.0), 150 mM KCI, 1 mM EDTA, 4 mM DTT, and 5/.tM leupeptin. Samples were sonicated for 30 s (power setting 3, duty cycle 30%), and clarified by centrifugation (7800 g, 10 min). The pellets were re-extracted, the solutions then clarified, and the supernatants were combined and then centrifuged at 31 000 g, 10 min.
-~ 40 C 0
E 3O
~ 2o ~- lo .< 0
l
1 Light
3
4
5
Fraction Number
6 Heavy
Fig. 1. Densitygradientfractionsshowingmarkersforcellularcompartments and CA1P phosphatase activityfrom illuminatedleaves of Phaseolus vulgaris. Chloroplastmarker- Chl, openbox; cytosol marker - PEP carboxylaseactivity, single-hatchedbox; vacuole marker - a-mannosidaseactivity,cross-hatchedbox; CAIP phosphatase activity- solidbox. The total PEP carboxylaseactivitywas 3.3/~molh- l mg- l Chl. The total a-mannosidaseactivitywas 6.4 #mol h- 1mg- l Chl.
Protein content of the supernatants was measured (BioRad Coomassie blue protein assay, Bio-Rad Laboratories, Hercules, CA), and up to 20 mg of protein from each sample was loaded onto a 2-ml column of Cibacron Blue agarose. The column was washed with 10 ml of resuspension buffer (by gravity), and the sample was eluted with five 2-ml aliquots of resuspension buffer containing 1.0 M KCI. CA 1P phosphatase activity eluted in fractions 2 and 3. In an initial experiment, the recovery of enzyme activity from fractionated material was measured. After gradient centrifugation, the Chl-containing material was partitioned into four aliquots, dried, and then extracted plus/minus partially purified CA1P phosphatase of known activity. The recovery of enzyme activity from the Cibacron Blue dye columns was 104% (data not shown). CA1P phosphatase activity was assayed in 30/11 of 100 mM Tris-HC1 (pH 8.0), 20 mM DTT, and 5 mM carboxyethylphosphonic acid (as activator; Aldrich Chemical Co., Milwaukee, WI). The reactions were initiated by addition of 0.5 mM CA1P, and stopped after 5 min by addition of 220 jul of 0.34 N HC104. Released inorganic phosphate was measured using a phosphomolybdate/malachite green assay (Kodama et al. 1986). Enzyme activity was then expressed as nmol CA1P hydrolyzed h - l m1-1, and fraction activities were calculated accordingly.
222 2.5
Table 1. Intracellular dislribution of CA1P phosphstase activity in illuminated leaves of Phaseolus vulgaris. Values are presented from separate experiments, using different collections of leaf material. Measured activities from each experiment were 0.94, 1.88, and 3.04 #tool mg- t Chl h - L In all cases, the confidence limits for the intercept values from respective graphs were not different from zero (p > 0.95)
~-~2.0
~Q-6
"~o ~_"1.5 oc® a_ 8 2 1 . 0 E_~ Q" I o o EcO . 5 0
i
0.3
0.6
i
0.9
i
i
1.2
=
1.5
Chl a-monnosidose OJg per nmol/h)
Fig. 2. Graphical analysis of the relative distribution of CAIP phosphatase activity between chloroplasts and vacuoles from one representativegradient. The enzyme activity and Chi in the gradient fractions were both expressed relative to a-mannosidase activity, and then plotted against each other as shown. In this ease, the linear regression equation was y - 1.850x + 0.005767, with the SD of the intercept equal to 0.0313. Pbosphatase activity in the chloroplast equals the slope multiplied by the amount of Chi in the gradient. Phosphatase activity in the vacuole equals the intercept multiplied by the amount of a-mannosidase activity present. For this example, the calculated ratio of phosphatase activity in the vacuole to that in the chloroplast was 0.0242. However, the 95% confidence limit for the intercept value was 0.0667 (one-tailed student's t-test, N-2 degrees of freedom), such that the calculated relative amount of pbosphatase activity in the vacuole was not significantly different than zero.
Data analysis The distributions o f CA1P and CA1P phosphatase were calculated according to a 2-compartment, graphical analysis as described by Gerhardt and Heldt (1984) and elsewhere (Sharkey and Vanderveer 1989). Since both CA1P and CA1P phosphatase are known to be largely associated with the chloroplast, we calculated from the appropriate graphs (see below) the relative distribution o f analyte between chloroplast and cytosol, and between chloroplast and vacuole. We assumed that all o f the leaf C A 1 P and CA1P phosphatase occurs in at most these three compartments. Therefore, a summarion o f the relative fractions in each compartment is equal to 1.0 (i.e. 100%). After substitution o f the relative values, we then calculated the distribution o f analyte between the three compartments.
Results and discussion In these experiments, the cell-compartment markers were Chl (chloroplasts), PEP carboxylase activi-
Sample Totalactivity Distribution of activity: number in gradient Chloroplast Cytoplasm Vacuole fractions (%) (pmol h - ]) 1 2 3
6.36 49.2 63.4
83 96 94
11 2 2
6 2 4
Table 2. Intracellulardistribution of CA1P in dark-lxeatedleaves ofPhaseolus vulgaris. Valuesare presented from separate experiments, using different collections of leaf material. Measured CA1P amounts from each experiment were 128, 113, and 119 nmol mg Chl- x. In all cases, the confidencelimits for the intercept values from respective graphs were not different from zero (p > 0.95) Sample TotalCA1P Distribution of CA1P: number in gradient Chloroplast Cytoplasm Vacuole fractions (nmol) (%) 1 2 3
482 999 1157
89 92 96
7
Intracellular localization of C A I P and C A I P phosphatase activity in leaves of Phaseolus vulgaris L. Brandon D. Moore 1, Thomas D. Sharkey2 & Jeffrey R. Seemann1 1Department of Biochemistry, University of Nevada, Reno, NV 89557, USA; 2Department of Botany, University of Wisconsin, Madison, W153706, USA Received20 June 1995;acceptedin revisedform7 August1995
Key words: nonaqueous fractionation, rubisco, carboxyarabinitol
Abstract
CA1P and CA1P phosphatase occur in the chloroplasts of leaf mesophyll cells of many species. However, whether either may occur exclusively in the chloroplast has not yet been established. To examine their intracellular distribution, mature, dark- or light-treated leaves of Phaseolus vulgaris were frozen, lyophilized and then centrifuged in density gradients of heptane and tetrachloroethylene. After gradient fractionation, both CA1P and CA1P phosphatase activity co-segregated with chloroplast material. Distribution analyses using sub-cellular compartment markers indicated that both CA1P and CA1P phosphatase do occur exclusively in leaf chloroplasts.
Abbreviations: Bicine - N,N-bis[2-hydroxyethyl]glycine; CAIP - 2-carboxyarabinitol 1-phosphate; CABP - 2carboxyarabinitol 1,5-bisphosphate; Chl - chlorophyll; DTT - dithiothreitol; EDTA - (ethylenediamine)tetraacetic acid; PEP - phosphoenolpyruvate; Tris - tris(hydroxymethyl)aminomethane
Introduction
ic sites are the same in either dark-treated leaves of
Phaseolus vulgaris or in chloroplasts isolated from CA1P is a branched-chain sugar-phosphate that accumulates in the leaves of many plant species under conditions of low irradiance (for review, see Seemann et al. 1990; Servaites 1992). CA1P resembles the reaction intermediate of the rubisco carboxylase reaction, binds tightly to carbamylated rubisco, and in a number of species can regulate the in vivo capacity for CO2 assimilation by inhibiting rubisco activity. Under conditions of increasing irradiance, CA1P is degraded by a specific phosphohydrolase, CA1P phosphatase, to produce a non-inhibitory compound, carboxyarabinitol (Holbrook et al. 1989). While there are substantial data on the associated photosynthetic physiology of CA1P, there are uncertainties as to whether CA1P occurs exclusively within the chloroplast (e.g. Portis 1992). Evidence which supports the localization of CA1P within chloroplasts can be summarized as follows: (1) Seemann et al. (1985) reported that the ratios of CA1P to rubisco catalyt-
such leaves; (2) A number of investigators have isolated a rubisco-CA1P complex from different species for use in the purification of CA1P or for other analyses (e.g. Servaites 1985; Gutteridge et al. 1986; Berry et al. 1987; Andralojc et al. 1994); and, (3) Moore and Seemann (1994) showed that in dark-treated leaves of P. vulgaris about 75% of the inactive rubisco catalytic sites in vivo are bound to CA1P. Still, whether CA1P occurs solely in the chloroplast has not been rigorously demonstrated. CA1P phosphatase is also known to be associated predominantly with the chloroplast: (1) The isolated organelle has proved useful as starting material for purification of the enzyme (Salvucci et al. 1988; Salvucci and Holbrook 1989); (2) The activity of CA1P phosphatase from stromal extracts of P. vulgaris was 2-4 fold greater than is the maximum rate of CA1P degradation that occurs in those leaves (Kobza et al. 1990); and, (3) The rate of CA1P degradation in tobac-
220 co leaves was decreased after treatment with methyl viologen, a Photosystem I electron acceptor (Salvucci and Anderson 1987). However, during the isolation of CA1P phosphatase from tobacco leaves, two peaks of activity were detected after an early purification step using ion-exchange chromatography (Salvucci and Holbrook 1989). Since only the major peak was clearly of chloroplastic origin, some CA 1P phosphatase may occur outside the chloroplast. There are a number of available methods for determining the intracellular distribution of leaf proteins and metabolites. Enzyme distributions have been studied using whole leaf homogenates (e.g. Rocha and Ting 1970) or extracts of leaf protoplasts (e.g. Spalding et al. 1979) after fractionation on sucrose density gradients. The fractionation of leaf protoplasts using rapid membrane filtration has proven very useful for sub-cellular analyses of metabolites under different physiological conditions (e.g. Gardestrom and Wigge 1988). However, such aqueous fractionation methods have inherent problems with altered metabolite levels during tissue preparation, and generally do not yield a good vacuolar fraction from most species. In contrast, the use of a non-aqueous fractionation procedure does allow the evaluation of analyte distribution between chloroplasts, cytosol, and vacuoles (Santarius and Heber 1965; Stitt et al. 1989). In this procedure, leaves are collected into liquid N2, lyophilized, and then fractionated on density gradients of non-aqueous solvents. Metabolites and proteins of a particular cellular region aggregate together and no metabolism occurs during their isolation. We successfully used a non-aqueous fractionation method previously to show that carboxyarabinitol occurs in multiple compartments within leaf cells (Moore et al. 1992). In this study, we have used this method again to examine the intracellular localization of CA1P and CA1P phosphatase in leaves of Phaseolus vulgaris, which contain relatively high levels of both analytes.
Materials and methods
Plant growth Plants of Phaseolus vulgaris L. cv. Linden were grown in a greenhouse (27/18 °C day/night), and were watered several times weekly with nutrient solution. Maximum daily irradiance was 1400 #mol quanta m -2 s -1. Fully expanded leaves from mature plants were used for experiments.
Non-aqueous fractionation Procedures were comparable to those reported elsewhere (Gerhardt and Heldt 1984; Sharkey and Vanderveer 1989). Bean leaves were collected into liquid N2 after 16 h dark (for CA1P measurements) or at mid-day (for CA1P phosphatase measurements). After removing major veins, 20 g of leaves were ground in liquid N2, using a mortar and pestle. Powdered material was transferred to cold lyophilization jars (600 ml, Labconco, Kansas City, MO), and the jars were kept in liquid N2 until the air pressure was reduced to < 25 mtorr (VirTis 10-324 freeze-dryer, VirTis Co., Gardiner, NY). Samples were dried overnight, and the powder was partitioned into five parts. Each portion was added to 20 ml of 4 °C heptane in a cut-off, 50-ml tefzel centrifuge tube (top 1/3 removed; Nalge Co., Rochester, NY), and placed in a 250-ml beaker containing heptane partially frozen in liquid N2. The sample was sonicated for 45 s with a Branson Sonitier 200 (power setting 8, continuous pulse, Branson Ultrasonics Corp., Danbury, CT), and filtered successively through 200 #m and 80 #m nylon nets (20 cm 2 each, Tetko Inc., Briarcliff Manor, NY). The pooled sample was partitioned equally into six 50-ml tefzel centrifuge tubes, and the leaf material was precipitated by centrifugation at 100 g, for 3 min. Leaf pellets were resuspended in 5 ml of 4 °C heptane, and each was loaded onto a 24-ml linear density gradient of heptane/tetrachloroethylene (J.T. Baker Inc., Phillipsburg, NJ), which ranged from 1.35 g m1-1 to 1.60 g ml -l. Samples were centrifuged at 4 °C in a swinging bucket rotor (Beckmann Instruments Inc., Fullerton, CA) at 17 000g, for 10h. All processing of gradients was done at 4 °C. Gradients were fractionated into 6 portions from the top, using a transfer pipet. Typically about 10 ml was collected as the lightest fraction, and about 3 ml each for the remaining fractions. The volumes were measured on the pooled collections. For marker assays, 0.6 ml was diluted with cold heptane in microfuge tubes, and the gradient material was precipitated by centrifugation at 10 000 g, for 5 min. The remaining gradient samples were diluted with heptane and collected by centrifugation. Residual heptane was evaporated overnight at 4 °C under reduced pressure in a vacuum jar with dessicant.
221 50
Marker assays v
Chl, PEP carboxylase, and a-mannosidase were used as markers for chloroplasts, cytosol, and vacuoles, respectively. The dried gradient fractions were resuspended in 0.6 ml of buffer containing 0.1 M Bicine pH 7.8, 5 mM MgC12, 1 mM EDTA, and 5 mM DTF. The samples were sonicated for 30 s (power setting 3, 30% duty cycle), and centrifuged at 10 000 g, 5 min. Supernatants were used for enzyme assays. Chl was measured after resuspending the pellets in 95% ethanol (Wintermanns and DeMots 1965). PEP carboxylase activity was assayed using [IaC]NaHCO3 (74 MBq mmol- l), and reaction components described by (Sharkey and Vanderveer 1989). Assays were conducted for 15 min at 25 °C, in triplicate. One blank was done for each sample, for which the reaction was terminated at 0 s. a-Mannosidase activity was assayed in duplicate for 60 min at 37 °C (Sharkey and Vanderveer 1989). Each assay was also done with replicate blanks, for which extract was added after addition of borate. Released nitrophenol was measured at A4o5 (extinction coefficient 18 500 M -1 cm-l). CA1P measurements [2-14C]CA1P was synthesized from labeled CABP (Gutteridge et al. 1989), and then used to measure sample CA1P content by isotope dilution assay (Moore et al. 1991; Moore et al. 1992). CA 1P phosphatase partial purification and activity assay CA1P phosphatase activity cannot yet be measured directly from whole leaf material due to the presence of non-specific phosphatase activity which can utilize CA1P as substrate. Therefore, gradient fractions were chromatographed using Cibacron Blue agarose (Sigma Chemical Co., St. Louis, MO) as described below to obtain samples void of such non-specific phosphatase activity but with complete recovery of CA1P phosphatase activity (Moore et al. 1995). Dried gradient fractions were resuspended in 5 ml of buffer containing 20 mM Tris-HC1 (pH 8.0), 150 mM KCI, 1 mM EDTA, 4 mM DTT, and 5/.tM leupeptin. Samples were sonicated for 30 s (power setting 3, duty cycle 30%), and clarified by centrifugation (7800 g, 10 min). The pellets were re-extracted, the solutions then clarified, and the supernatants were combined and then centrifuged at 31 000 g, 10 min.
-~ 40 C 0
E 3O
~ 2o ~- lo .< 0
l
1 Light
3
4
5
Fraction Number
6 Heavy
Fig. 1. Densitygradientfractionsshowingmarkersforcellularcompartments and CA1P phosphatase activityfrom illuminatedleaves of Phaseolus vulgaris. Chloroplastmarker- Chl, openbox; cytosol marker - PEP carboxylaseactivity, single-hatchedbox; vacuole marker - a-mannosidaseactivity,cross-hatchedbox; CAIP phosphatase activity- solidbox. The total PEP carboxylaseactivitywas 3.3/~molh- l mg- l Chl. The total a-mannosidaseactivitywas 6.4 #mol h- 1mg- l Chl.
Protein content of the supernatants was measured (BioRad Coomassie blue protein assay, Bio-Rad Laboratories, Hercules, CA), and up to 20 mg of protein from each sample was loaded onto a 2-ml column of Cibacron Blue agarose. The column was washed with 10 ml of resuspension buffer (by gravity), and the sample was eluted with five 2-ml aliquots of resuspension buffer containing 1.0 M KCI. CA 1P phosphatase activity eluted in fractions 2 and 3. In an initial experiment, the recovery of enzyme activity from fractionated material was measured. After gradient centrifugation, the Chl-containing material was partitioned into four aliquots, dried, and then extracted plus/minus partially purified CA1P phosphatase of known activity. The recovery of enzyme activity from the Cibacron Blue dye columns was 104% (data not shown). CA1P phosphatase activity was assayed in 30/11 of 100 mM Tris-HC1 (pH 8.0), 20 mM DTT, and 5 mM carboxyethylphosphonic acid (as activator; Aldrich Chemical Co., Milwaukee, WI). The reactions were initiated by addition of 0.5 mM CA1P, and stopped after 5 min by addition of 220 jul of 0.34 N HC104. Released inorganic phosphate was measured using a phosphomolybdate/malachite green assay (Kodama et al. 1986). Enzyme activity was then expressed as nmol CA1P hydrolyzed h - l m1-1, and fraction activities were calculated accordingly.
222 2.5
Table 1. Intracellular dislribution of CA1P phosphstase activity in illuminated leaves of Phaseolus vulgaris. Values are presented from separate experiments, using different collections of leaf material. Measured activities from each experiment were 0.94, 1.88, and 3.04 #tool mg- t Chl h - L In all cases, the confidence limits for the intercept values from respective graphs were not different from zero (p > 0.95)
~-~2.0
~Q-6
"~o ~_"1.5 oc® a_ 8 2 1 . 0 E_~ Q" I o o EcO . 5 0
i
0.3
0.6
i
0.9
i
i
1.2
=
1.5
Chl a-monnosidose OJg per nmol/h)
Fig. 2. Graphical analysis of the relative distribution of CAIP phosphatase activity between chloroplasts and vacuoles from one representativegradient. The enzyme activity and Chi in the gradient fractions were both expressed relative to a-mannosidase activity, and then plotted against each other as shown. In this ease, the linear regression equation was y - 1.850x + 0.005767, with the SD of the intercept equal to 0.0313. Pbosphatase activity in the chloroplast equals the slope multiplied by the amount of Chi in the gradient. Phosphatase activity in the vacuole equals the intercept multiplied by the amount of a-mannosidase activity present. For this example, the calculated ratio of phosphatase activity in the vacuole to that in the chloroplast was 0.0242. However, the 95% confidence limit for the intercept value was 0.0667 (one-tailed student's t-test, N-2 degrees of freedom), such that the calculated relative amount of pbosphatase activity in the vacuole was not significantly different than zero.
Data analysis The distributions o f CA1P and CA1P phosphatase were calculated according to a 2-compartment, graphical analysis as described by Gerhardt and Heldt (1984) and elsewhere (Sharkey and Vanderveer 1989). Since both CA1P and CA1P phosphatase are known to be largely associated with the chloroplast, we calculated from the appropriate graphs (see below) the relative distribution o f analyte between chloroplast and cytosol, and between chloroplast and vacuole. We assumed that all o f the leaf C A 1 P and CA1P phosphatase occurs in at most these three compartments. Therefore, a summarion o f the relative fractions in each compartment is equal to 1.0 (i.e. 100%). After substitution o f the relative values, we then calculated the distribution o f analyte between the three compartments.
Results and discussion In these experiments, the cell-compartment markers were Chl (chloroplasts), PEP carboxylase activi-
Sample Totalactivity Distribution of activity: number in gradient Chloroplast Cytoplasm Vacuole fractions (%) (pmol h - ]) 1 2 3
6.36 49.2 63.4
83 96 94
11 2 2
6 2 4
Table 2. Intracellulardistribution of CA1P in dark-lxeatedleaves ofPhaseolus vulgaris. Valuesare presented from separate experiments, using different collections of leaf material. Measured CA1P amounts from each experiment were 128, 113, and 119 nmol mg Chl- x. In all cases, the confidencelimits for the intercept values from respective graphs were not different from zero (p > 0.95) Sample TotalCA1P Distribution of CA1P: number in gradient Chloroplast Cytoplasm Vacuole fractions (nmol) (%) 1 2 3
482 999 1157
89 92 96
7
