- Email: [email protected]
Partial purification of plant NAD kinase by calmodulin-Sepharose affinity chromatography
Cell Calcium 1: 279-286,1980
PARTIAL PURIFICATION OF PLANT NAD KINASE BY CALMODULIN-SEPHAROSE AFFINITY CHROMATOGRAPHY Peter Dieter and Dieter Marmh Institut fiir Biologie III, University 7800 Freiburg, FRG (reprint requests to DM)
of Freiburg,
ABSTRACT Calmodulin-dependent NAD kinase has been purified more than 70fold from a crude plant(zucchini squash) homogenate by calmodulin-Sepharose affinity chromatography to a specific activity of 80 munits/mg protein. The enzyme could be activated about 8fold by calmodulin. Half-maximal activation was obtained with 6 ng of purified calmodulin from bovine brain. Together with NAD kinase other soluble plant proteins were retained specifically on the column. NaDodSO4 polyacrylamide gel electrophoresisofthe proteins which were retained by the calmodulin-Sepharose column revealed at least 7 to 8 bands. Most of the intensively stained bands on the gels obtained from the crude homogenate had disappeared. INTRODUCTION 2+ Calmodulin, a Ca -dependent regulator protein, has been found to affect many enzyme activities (1). Calmodulin originally was discovered in animals (2) and seems to occur in many (if not all) eukar otes. Plant NAD kinase activity (3) and plant microsomal Ca Y+ -transport (4) have been reported to be enhanced by calmodulin from either animal or plant sources. The mechanism by which calmodulin exerts its regulatory properties has been proposed (for review see I): Ca2+ + calmodulin (Ca*calmodulin)active
A
+ enzyme +
(Ca. calmodulin)active (Ca.calmodulin~enzyme)active
The property of calmodulin to bind proteins tightly in the presence of Ca2+ (5)and to release them when Ca2+ is with-
279
drawn can be utilized to purify calmodulin-dependent enzymes from crude plant homogenates by affinity chromatography on calmodulin-Sepharose 4B columns. This technique is also very helpful for isolating and investigating proteins which are under the control of calmodulin. Calmodulin-Sepharose 4B columns have been successfully used to purify cyclic nucleotids+phos hodiesterase (61, myosin light chain kinase (71, -Mg 2P )ATPase (8) and calmodulin-binding proteins (9). (Ca In this paper we report on the isolation and partial purification of a plant NAD kinase and at least 7 to 8 proteins from zucchini squash on a bovine brain calmodulin-Sepharose affinity column. The calmodulin-dependent stimulation of the NAD kinase is described and the electrophoretic properties of the proteins on NaDodSO4 polyacrylamide gels are demonstrated. METHODS CNBr-activated Sepharose 4B was purchased from Pharmacia, Freiburg, FRG. All other reagents were the best grades commercially available. Preparation of Calmodulin-Sepharose: Calmodulin from bovine brain was prepared to apparent electrophoretic homogeneity according to Dedman et al. (107. Calmodulin was coupled to CNBr-activated Sepharose 4B by incubating 5 mg calmodulin with 2 g CNBr-activated Sepharose 4B for 18 h at O" C in O.lM NaHCO3 buffer at pH 8.3 containing 0.5 M NaCl (coupling buffer). After extensive washing with coupling buffer the calmodulin-Sepharose was resuspended in 0.2 M glycine buffer at pH 8.0 and stirred for 2 h at room temperature to block the remaining active groups. The gel was then washed alternately with coupling buffer and 0.1 M acetate buffer at pH 4.0 containing 0.5 M NaCl and finally equilibrated with buffer A
( Cucurbita pepo L., cv. Senator, from Asgrow, Kalamazoo, Michigan, USA) were grown on vermiculite for 4.5 days at 25O C in darkness. Hypocotyls were cut and homogenized in buffer A (I g fresh weight per ml of buffer A) with an Ultra-Turrax T 45 for 2 min at full speed. The resulting brei was filtered through a single layer of nylon cloth and centrifuged at 50 000 x g for 1 h to remove the cell organelles and the membranous material. The resulting supernatant (usually 150 ml) was loaded onto the calmodulin-Sepharose column. The column was then washed extensively with buffer B (buffer A containing 0.5 M NaCl) until the absorbance at 280 nm in a cuvette of l-cm pathlength was less than 0.05. Proteins were then eluted in buffer C (buffer B without CaC12 but with 5 mM EGTA).
280
Enzyme Assays: NAD kinase activity was assayed as described by Muto and Miyachi (II). Calmodulin, CaC12 (1 mM) or EGTA (1 mM) were added as indicated. NAD kinase activity data shown in table 1 were obtained with saturating amounts of calmodulin (100 ng). Cyclic nucleotide phosphodiesterase activity was determined by a two step procedure as described by Cheung (12): 0.5 ml of reaction medium contained 100 mM imidazole at pH 7.5, 4 mM MgC12, 1 mM CaC12, 100 ng bovine brain calmodulin, 2 mM ofeachsubstrate (2':3'-c AMP, 3':5'-c GMP, 2':3'-c GMP, 3'.:5'-c AMP) and an appropriate amount of the enzyme. The mixture was incubated at 37' C for 30 min. The reaction was terminated by boiling for 5 min. The mixture was cooled down to ice-bath temperature and 5 units of alkaline phosphatase were added. The reaction was allowed to proceed at 370 C for IO min and then stopped by adding an equal volume of lO%(w/v) trichloroacetic acid. The mixture was clarified by centrifugation at 20 000 x g for 10 min. Released inorganic phosphate was determined by adding 0.4 ml of the resulting supernatant to 1 ml of 0.4% (W/V) ammoniummolybdate (in 0.5 M H2S04), lO%(w/v) ascorbic acid and lO%(w/v) trichloroacetic acid. After incubation at 370 C for 15 min the absorbance at 660nm was measured. Gel Electrophoresis: NaDodSO4 polyacrylamide gel electrophoresis was performed as previously described by Laemmli (14). RESULTS AND DISCUSSION A crude homogenate of dark grown zucchini squash seedlings was centrifuged at 50 000 x g for 1 h to remove cell organelles and most of the membranous material. The clarified supernatant which contained 0.5 mM CaC12 was loaded onto the bovine brain calmodulin-Sepharose column. The column was washed extensively at high ionic strength with buffer B until the absorbance at 280 nm was below 0.05. All the material coming through the column including the salt wash was pooled and termed the NaCl fraction. The proteins bound to the calmodulin-Sepharose column in the presence of Ca2+ were eluted by buffer C (containing 5 mM EGTA). Figure 1 shows a typical elution profile for protein and NAD kinase activity. No NAD kinase activity could be detected before elution with EGTA. The main proteins elute slightly before the NAD kinase activity. As shown in Table 1 only about 1% of the protein which was loaded onto the column was retained by the column but almost all the NAD kinase. The yield in the EGTA eluate was 80% of the total activity. The specific activity of the NAD kinase in the EGTA eluate increased more than 76fold compared to the homogenate. Pea NAD kinase activity has been shown to depend on calmodulin (3,15). Figure 2 represents the squash NAD kinase activity as 281
a function of the amount of bovine brain calmodulin added to the assay medium. The specific NAD kinase activity of the EGTA eluate in the absence of calmodulin was 80 munits/mg protein.
elutlon volumc,ml
Fig. 1 Calmodulin-Sepharose chromatography of plant NAD kinase. The enzyme activity was determined in the presence of 1 mM Ca2+ and at saturating amounts of bovine brain calmodulin. TABLE 1 NAD kinase chromatography column.
Fraction
protein (mg)
on a calmodulin-Sepharose
affinity
NAD kinase activity total specific (munits/mg protein) (munits)
50k supernatant
176.0
616.0
3.5
NaCl eluate
140.0
55.0
0.4
EGTA eluate
1.9
481 .O
282
253.0
The enzyme activity was enhanced 8fold at saturating amounts of calmodulin. Half-maximal stimulation was achieved when 6 ng calmodulin were added to 60 pg protein of the EGTA eluate in the assay medium. When Ca2+ is chelated by EGTA, no calmsdulin stimulation is observed (Fig.2, dark triangle).
5mM EGTA --without
O 1,’ 1
!
calmodulin K)
100
1000
calmodulin,ng
Fig. 2 Dose-response curve of the activation of plant NAD kinase by bovine brain calmodulin. All assays were carried out with 60 pg of EGTA eluate proteins in the presence of 1 mM Ca2+ and variable amounts of calmodulin. 5 mM EGTA was added at saturating amounts of calmodulin to inhibit the Ca*calmodulin dependent NAD kinase activation (dark triangle). In contrast to the NAD kinase only a negligible amount of squash cyclic nucleotide phosphodiesterase was found in the EGTA eluate (Table 2). Squash cyclic nucleotide phosphodiesterase as opposed to that of bovine brain (2) does not depend on calmodulin: the specific activities obtained in the presence of 100 ng calmodulin and 1 mM Ca2+ are not changed when 5 mM EGTA are added.
283
TABLE 2 Cyclic nucleotide modulin-Sepharose
phosphodiesterase affinity column.
Fraction
protein (mg)
chromatography
on a cal-
phosphodiesterase activity total specific (munits) (munits/mg protein)
50k supernatant
176.0
546.0
3.1
NaCl eluate
140.0
414.0
3.2
EGTA eluate
1.9
0.4
&
0.25
SOKsuprnatan+
_ ,EGTA
eluate
50K
supernatant
EGTA eluate marker 1
I
125
25
proteins
molecular
I
68
MO31
weight
-
Fig. 3 NaDodSO4 12.5% polyacrylamide gel electrophoresis of 50k supernatant and EGTA eluate proteins. 40 pg of proteins were applied onto each gel. The gels were scanned with a Joyce Loebl densitometer. For calibration cytochrome c (M,=12 5001, chymotrypsinogen A (M,=25 000) and bovine serum albumin (Mr= 68 000) were used.
284
The total protein of the 50k supernatant and of the EGTA eluate were solubilized in NaDodSO4 and electrophoresed on 12.5% polyacrylamid slab gels. Fig. 3 shows the photographs and the densitometric scans of two representative gels. By comparing the two gels it can be seen that predominant bands of the 50k supernatant are no longer present on the EGTA eluate gel. On the EGTA eluate gel intensively stained bands have appeared which are only barely visible in the 50k supernatant gel. Rechromatography of proteins from an EGTA eluate on the calmodulin-Sepharose affinity column yields the same protein pattern (data not shown), indicating that even a single affinity chromatographic run results in good purification. On the EGTA eluate gel at least 7 to 8 protein bands are visible. Those proteins probably represent specific calmodulin-binding proteins. The data described above suggest that affinity chromatography on calmodulin-Sepharose 4B columns facilitates the purification of calmodulin-dependent plant enzymes like NAD kinase and thus the investigation of their biochemical and regulatory properties. Furthermore this technique allows the isolation of thus far unknown calmodulin-binding proteins, the identification of which may help to understand the regulatory function of Ca2+ in plants. ACKNOWLEDGEMENTS We wish to acknowledge the excellent technical assistance of Miss A. Schafer. The authors would also like to thank Dr. B. Wakim for help in the purification of the bovine brain calmodulin. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 461, by the Stiftung Volkswagenwerk and by the Bundesminister fiir Forschung und Technologie (01 QV 318ZA/WF/WRK 0275/S). REFERENCES 1.
2. 3. 4. 5.
Wang,J.H. and Waisman,D.M. (1979). Calmodulin and its role in the second messenger system. Current Topics in Cellular Regulation 15,47-107. Cheung,W.Y. (1970). Cyclic 3',5'-nucleotide phosphodiesterase. Biochem. Biophys. Res. Commun. 38,533-538. Anderson,J.M. and Cormier,M.J. (1978). Calcium-dependent regulation of NAD kinase. Biochem. Biophys. Res. Conunun. 84,595-602. Dieter,P. and Marm&$. (1980). Calmodulin-activation of plant microsomal Ca uptake. Proc. Natl. Acad. Sci. USA, in press. Teo,T.S. and Wang,J.H. (1973). Mechanism of activation of cycl. adenosin 3' ,5'-monophosphate phosphodiesterase from bovine heart by calcium ions. J. Biol. Chem. 248, 5950-5955. 285
6. Klee,C.B.,Crouch,T.H. and Krings,M.H. (1979). Subunit structure and catalytic properties of bovine brain Ca2+dependent cyclic nucleotide phosohodiesterase. Biochemistry 18,722-729. 7. Adelstein,R.S., Conti,M.A. and Hathaway,D.R. (1978). Phosphorylation of smooth muscle myosin light chain kinase by the catalytic subunit of adenosin 3',5'-monophosphate-dependent protein kinase. J. Biol. Chem. 253,8347-8350. 8. Niggli,V.,PennisQjq,J.T. and Carafoli,E. (1979). Purification of the (Ca -Mg2+)-ATPase from human erythrocyte membranes using a calmodulin affinity column. J. Biol. Chem. 254,9955-9958. 9. Grand,R.J.A. and Perry,S.V. (1979). Calmodulin-binding proteins from brain and other tissues. Biochem. J. 183, 285-295. lO.Dedman,J.R.,Potter,J.D.,Jackson,J.D. and Means,A.R. (1977) Physiochemical oroperties of a rat testis Ca2+-dependent regulator protein of cyclic nucleotide phosnhodiesterase. J. Biol. Chem. 252,8415-8422. ll.Muto,S. and Miyachi,S. (1977). Properties of a protein activator of NAD kinase from plants. Plant Phvsiol. 59, 55-60. 12.Cheung,W.Y. (1969). Cyclic 3',5'-nucleotide phosnhodiesterase. Preparation of a partially purified inactive enzyme and its subseauent stimulation by snake venom. Biochim. Biophys. Acta 191,303-315. 13.Spector,T. (1978). Refinement of the CoomassicZBlue Method of protein quantitation. Analytical Biochem. 86,142-146. 14.Laemmli,U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227,680-684. lS.Anderson,J.M.,Charbonneau,H.,Jones,H.P.,McCann,R.O. and Cormier,M.J. (1980). Characterization of the plant NAD kinase activator protein and its identification as calmodulin. Biochemistry 19,3113-3120.
286
PARTIAL PURIFICATION OF PLANT NAD KINASE BY CALMODULIN-SEPHAROSE AFFINITY CHROMATOGRAPHY Peter Dieter and Dieter Marmh Institut fiir Biologie III, University 7800 Freiburg, FRG (reprint requests to DM)
of Freiburg,
ABSTRACT Calmodulin-dependent NAD kinase has been purified more than 70fold from a crude plant(zucchini squash) homogenate by calmodulin-Sepharose affinity chromatography to a specific activity of 80 munits/mg protein. The enzyme could be activated about 8fold by calmodulin. Half-maximal activation was obtained with 6 ng of purified calmodulin from bovine brain. Together with NAD kinase other soluble plant proteins were retained specifically on the column. NaDodSO4 polyacrylamide gel electrophoresisofthe proteins which were retained by the calmodulin-Sepharose column revealed at least 7 to 8 bands. Most of the intensively stained bands on the gels obtained from the crude homogenate had disappeared. INTRODUCTION 2+ Calmodulin, a Ca -dependent regulator protein, has been found to affect many enzyme activities (1). Calmodulin originally was discovered in animals (2) and seems to occur in many (if not all) eukar otes. Plant NAD kinase activity (3) and plant microsomal Ca Y+ -transport (4) have been reported to be enhanced by calmodulin from either animal or plant sources. The mechanism by which calmodulin exerts its regulatory properties has been proposed (for review see I): Ca2+ + calmodulin (Ca*calmodulin)active
A
+ enzyme +
(Ca. calmodulin)active (Ca.calmodulin~enzyme)active
The property of calmodulin to bind proteins tightly in the presence of Ca2+ (5)and to release them when Ca2+ is with-
279
drawn can be utilized to purify calmodulin-dependent enzymes from crude plant homogenates by affinity chromatography on calmodulin-Sepharose 4B columns. This technique is also very helpful for isolating and investigating proteins which are under the control of calmodulin. Calmodulin-Sepharose 4B columns have been successfully used to purify cyclic nucleotids+phos hodiesterase (61, myosin light chain kinase (71, -Mg 2P )ATPase (8) and calmodulin-binding proteins (9). (Ca In this paper we report on the isolation and partial purification of a plant NAD kinase and at least 7 to 8 proteins from zucchini squash on a bovine brain calmodulin-Sepharose affinity column. The calmodulin-dependent stimulation of the NAD kinase is described and the electrophoretic properties of the proteins on NaDodSO4 polyacrylamide gels are demonstrated. METHODS CNBr-activated Sepharose 4B was purchased from Pharmacia, Freiburg, FRG. All other reagents were the best grades commercially available. Preparation of Calmodulin-Sepharose: Calmodulin from bovine brain was prepared to apparent electrophoretic homogeneity according to Dedman et al. (107. Calmodulin was coupled to CNBr-activated Sepharose 4B by incubating 5 mg calmodulin with 2 g CNBr-activated Sepharose 4B for 18 h at O" C in O.lM NaHCO3 buffer at pH 8.3 containing 0.5 M NaCl (coupling buffer). After extensive washing with coupling buffer the calmodulin-Sepharose was resuspended in 0.2 M glycine buffer at pH 8.0 and stirred for 2 h at room temperature to block the remaining active groups. The gel was then washed alternately with coupling buffer and 0.1 M acetate buffer at pH 4.0 containing 0.5 M NaCl and finally equilibrated with buffer A
( Cucurbita pepo L., cv. Senator, from Asgrow, Kalamazoo, Michigan, USA) were grown on vermiculite for 4.5 days at 25O C in darkness. Hypocotyls were cut and homogenized in buffer A (I g fresh weight per ml of buffer A) with an Ultra-Turrax T 45 for 2 min at full speed. The resulting brei was filtered through a single layer of nylon cloth and centrifuged at 50 000 x g for 1 h to remove the cell organelles and the membranous material. The resulting supernatant (usually 150 ml) was loaded onto the calmodulin-Sepharose column. The column was then washed extensively with buffer B (buffer A containing 0.5 M NaCl) until the absorbance at 280 nm in a cuvette of l-cm pathlength was less than 0.05. Proteins were then eluted in buffer C (buffer B without CaC12 but with 5 mM EGTA).
280
Enzyme Assays: NAD kinase activity was assayed as described by Muto and Miyachi (II). Calmodulin, CaC12 (1 mM) or EGTA (1 mM) were added as indicated. NAD kinase activity data shown in table 1 were obtained with saturating amounts of calmodulin (100 ng). Cyclic nucleotide phosphodiesterase activity was determined by a two step procedure as described by Cheung (12): 0.5 ml of reaction medium contained 100 mM imidazole at pH 7.5, 4 mM MgC12, 1 mM CaC12, 100 ng bovine brain calmodulin, 2 mM ofeachsubstrate (2':3'-c AMP, 3':5'-c GMP, 2':3'-c GMP, 3'.:5'-c AMP) and an appropriate amount of the enzyme. The mixture was incubated at 37' C for 30 min. The reaction was terminated by boiling for 5 min. The mixture was cooled down to ice-bath temperature and 5 units of alkaline phosphatase were added. The reaction was allowed to proceed at 370 C for IO min and then stopped by adding an equal volume of lO%(w/v) trichloroacetic acid. The mixture was clarified by centrifugation at 20 000 x g for 10 min. Released inorganic phosphate was determined by adding 0.4 ml of the resulting supernatant to 1 ml of 0.4% (W/V) ammoniummolybdate (in 0.5 M H2S04), lO%(w/v) ascorbic acid and lO%(w/v) trichloroacetic acid. After incubation at 370 C for 15 min the absorbance at 660nm was measured. Gel Electrophoresis: NaDodSO4 polyacrylamide gel electrophoresis was performed as previously described by Laemmli (14). RESULTS AND DISCUSSION A crude homogenate of dark grown zucchini squash seedlings was centrifuged at 50 000 x g for 1 h to remove cell organelles and most of the membranous material. The clarified supernatant which contained 0.5 mM CaC12 was loaded onto the bovine brain calmodulin-Sepharose column. The column was washed extensively at high ionic strength with buffer B until the absorbance at 280 nm was below 0.05. All the material coming through the column including the salt wash was pooled and termed the NaCl fraction. The proteins bound to the calmodulin-Sepharose column in the presence of Ca2+ were eluted by buffer C (containing 5 mM EGTA). Figure 1 shows a typical elution profile for protein and NAD kinase activity. No NAD kinase activity could be detected before elution with EGTA. The main proteins elute slightly before the NAD kinase activity. As shown in Table 1 only about 1% of the protein which was loaded onto the column was retained by the column but almost all the NAD kinase. The yield in the EGTA eluate was 80% of the total activity. The specific activity of the NAD kinase in the EGTA eluate increased more than 76fold compared to the homogenate. Pea NAD kinase activity has been shown to depend on calmodulin (3,15). Figure 2 represents the squash NAD kinase activity as 281
a function of the amount of bovine brain calmodulin added to the assay medium. The specific NAD kinase activity of the EGTA eluate in the absence of calmodulin was 80 munits/mg protein.
elutlon volumc,ml
Fig. 1 Calmodulin-Sepharose chromatography of plant NAD kinase. The enzyme activity was determined in the presence of 1 mM Ca2+ and at saturating amounts of bovine brain calmodulin. TABLE 1 NAD kinase chromatography column.
Fraction
protein (mg)
on a calmodulin-Sepharose
affinity
NAD kinase activity total specific (munits/mg protein) (munits)
50k supernatant
176.0
616.0
3.5
NaCl eluate
140.0
55.0
0.4
EGTA eluate
1.9
481 .O
282
253.0
The enzyme activity was enhanced 8fold at saturating amounts of calmodulin. Half-maximal stimulation was achieved when 6 ng calmodulin were added to 60 pg protein of the EGTA eluate in the assay medium. When Ca2+ is chelated by EGTA, no calmsdulin stimulation is observed (Fig.2, dark triangle).
5mM EGTA --without
O 1,’ 1
!
calmodulin K)
100
1000
calmodulin,ng
Fig. 2 Dose-response curve of the activation of plant NAD kinase by bovine brain calmodulin. All assays were carried out with 60 pg of EGTA eluate proteins in the presence of 1 mM Ca2+ and variable amounts of calmodulin. 5 mM EGTA was added at saturating amounts of calmodulin to inhibit the Ca*calmodulin dependent NAD kinase activation (dark triangle). In contrast to the NAD kinase only a negligible amount of squash cyclic nucleotide phosphodiesterase was found in the EGTA eluate (Table 2). Squash cyclic nucleotide phosphodiesterase as opposed to that of bovine brain (2) does not depend on calmodulin: the specific activities obtained in the presence of 100 ng calmodulin and 1 mM Ca2+ are not changed when 5 mM EGTA are added.
283
TABLE 2 Cyclic nucleotide modulin-Sepharose
phosphodiesterase affinity column.
Fraction
protein (mg)
chromatography
on a cal-
phosphodiesterase activity total specific (munits) (munits/mg protein)
50k supernatant
176.0
546.0
3.1
NaCl eluate
140.0
414.0
3.2
EGTA eluate
1.9
0.4
&
0.25
SOKsuprnatan+
_ ,EGTA
eluate
50K
supernatant
EGTA eluate marker 1
I
125
25
proteins
molecular
I
68
MO31
weight
-
Fig. 3 NaDodSO4 12.5% polyacrylamide gel electrophoresis of 50k supernatant and EGTA eluate proteins. 40 pg of proteins were applied onto each gel. The gels were scanned with a Joyce Loebl densitometer. For calibration cytochrome c (M,=12 5001, chymotrypsinogen A (M,=25 000) and bovine serum albumin (Mr= 68 000) were used.
284
The total protein of the 50k supernatant and of the EGTA eluate were solubilized in NaDodSO4 and electrophoresed on 12.5% polyacrylamid slab gels. Fig. 3 shows the photographs and the densitometric scans of two representative gels. By comparing the two gels it can be seen that predominant bands of the 50k supernatant are no longer present on the EGTA eluate gel. On the EGTA eluate gel intensively stained bands have appeared which are only barely visible in the 50k supernatant gel. Rechromatography of proteins from an EGTA eluate on the calmodulin-Sepharose affinity column yields the same protein pattern (data not shown), indicating that even a single affinity chromatographic run results in good purification. On the EGTA eluate gel at least 7 to 8 protein bands are visible. Those proteins probably represent specific calmodulin-binding proteins. The data described above suggest that affinity chromatography on calmodulin-Sepharose 4B columns facilitates the purification of calmodulin-dependent plant enzymes like NAD kinase and thus the investigation of their biochemical and regulatory properties. Furthermore this technique allows the isolation of thus far unknown calmodulin-binding proteins, the identification of which may help to understand the regulatory function of Ca2+ in plants. ACKNOWLEDGEMENTS We wish to acknowledge the excellent technical assistance of Miss A. Schafer. The authors would also like to thank Dr. B. Wakim for help in the purification of the bovine brain calmodulin. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 461, by the Stiftung Volkswagenwerk and by the Bundesminister fiir Forschung und Technologie (01 QV 318ZA/WF/WRK 0275/S). REFERENCES 1.
2. 3. 4. 5.
Wang,J.H. and Waisman,D.M. (1979). Calmodulin and its role in the second messenger system. Current Topics in Cellular Regulation 15,47-107. Cheung,W.Y. (1970). Cyclic 3',5'-nucleotide phosphodiesterase. Biochem. Biophys. Res. Commun. 38,533-538. Anderson,J.M. and Cormier,M.J. (1978). Calcium-dependent regulation of NAD kinase. Biochem. Biophys. Res. Conunun. 84,595-602. Dieter,P. and Marm&$. (1980). Calmodulin-activation of plant microsomal Ca uptake. Proc. Natl. Acad. Sci. USA, in press. Teo,T.S. and Wang,J.H. (1973). Mechanism of activation of cycl. adenosin 3' ,5'-monophosphate phosphodiesterase from bovine heart by calcium ions. J. Biol. Chem. 248, 5950-5955. 285
6. Klee,C.B.,Crouch,T.H. and Krings,M.H. (1979). Subunit structure and catalytic properties of bovine brain Ca2+dependent cyclic nucleotide phosohodiesterase. Biochemistry 18,722-729. 7. Adelstein,R.S., Conti,M.A. and Hathaway,D.R. (1978). Phosphorylation of smooth muscle myosin light chain kinase by the catalytic subunit of adenosin 3',5'-monophosphate-dependent protein kinase. J. Biol. Chem. 253,8347-8350. 8. Niggli,V.,PennisQjq,J.T. and Carafoli,E. (1979). Purification of the (Ca -Mg2+)-ATPase from human erythrocyte membranes using a calmodulin affinity column. J. Biol. Chem. 254,9955-9958. 9. Grand,R.J.A. and Perry,S.V. (1979). Calmodulin-binding proteins from brain and other tissues. Biochem. J. 183, 285-295. lO.Dedman,J.R.,Potter,J.D.,Jackson,J.D. and Means,A.R. (1977) Physiochemical oroperties of a rat testis Ca2+-dependent regulator protein of cyclic nucleotide phosnhodiesterase. J. Biol. Chem. 252,8415-8422. ll.Muto,S. and Miyachi,S. (1977). Properties of a protein activator of NAD kinase from plants. Plant Phvsiol. 59, 55-60. 12.Cheung,W.Y. (1969). Cyclic 3',5'-nucleotide phosnhodiesterase. Preparation of a partially purified inactive enzyme and its subseauent stimulation by snake venom. Biochim. Biophys. Acta 191,303-315. 13.Spector,T. (1978). Refinement of the CoomassicZBlue Method of protein quantitation. Analytical Biochem. 86,142-146. 14.Laemmli,U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227,680-684. lS.Anderson,J.M.,Charbonneau,H.,Jones,H.P.,McCann,R.O. and Cormier,M.J. (1980). Characterization of the plant NAD kinase activator protein and its identification as calmodulin. Biochemistry 19,3113-3120.
286