University Agricultural Experiment Station, Baton Rouge Louisiana 70803 (M. E. T.) ABSTRACT. A protein identifiable as calmodulin has been isolated from oat ( ...
Plant Physiol. (1984) 75, 382-386 0032-0889/84/75/0382/05/$0 1.00/0
Characterization of Oat Calmodulin and Radioimmunoassay of Its Subcellular Distribution' Received for publication September 19, 1983 and in revised form January 30, 1984
RONALD L. BIRO2, SUN DAYE3, BRUCE S. SERLIN, MAURICE E. TERRY, NEERAJ DATTA, SUDHIR K. SOPORY4, AND STANLEY J. ROUX* Department of Botany, The University of Texas at Austin, Austin, Texas 78712 (R. L. B., S. D., B. S. S., N. D., S. K. S., S. J. R.); and Department ofPlant Pathology and Crop Physiology, Louisiana State University Agricultural Experiment Station, Baton Rouge Louisiana 70803 (M. E. T.) ABSTRACT A protein identifiable as calmodulin has been isolated from oat (Avena sativa, var Garry) tissues. This protein is relatively heat stable, binds to hydrophobic gels, and phenothiazines in a calcium-dependent fashion, and binds to antibody to rat testes calmodulin. Based on its migration on sodium dodecyl sulfate-polyacrylamide gels, ultraviolet absorption spectrum, and amino acid composition, oat calmodulin is essentially identical to calmodulin isolated from other higher plants. Radioimmunoassays indicate that calmodulin is associated with isolated oat protoplasts, mitochondria, etioplasts, and nuclei and also appears to be a component of oat cell wall fractions.
Each started with about 1 kg of oat tissue which had been chilled at 4°C before harvest. Method 1 (fluphenazine affinity gel method) is a combination of the methods of Charbonneau and Cormier (4) and Caldwell and Haug (3). The harvested tissue was freeze-dried (yielding approximately 100 g dry weight) and then homogenized in a Waring blender in 1.5 L ofchilled acetone (- I 5C) containing 0.25 mM PMSF.5 The homogenate was vacuum filtered repeatedly through a Whatman No. 1 filter until the liquid contained no visible suspended solids. All subsequent procedures were performed at 4°C. The acetone powder was dried under vacuum to remove any acetone, resuspended in 1.5 L of 50 mm Hepes (pH 7.0), 2 mm EDTA, 10 mM DTT, 0.25
mM PMSF, 10,000 kallikrein-inactivating units aprotinin (buffer A), and stirred for 30 min. This mixture was then centrifuged at l0,OOOg for 30 min to remove undissolved materials. The supernatant was brought to 55% ofsaturation with ammonium sulfate, stirred for I h and clarified by centrifugation at l0,OOOg for 30 Much evidence has accumulated recently to support the hy- min. The supernatant was adjusted to pH 4.1 with 2 N H2SO4 in pothesis that Ca2 plays a major role in mediating the adaptations 55% (NH4)2 SO4, stirred for 1 h and centrifuged again. The of plants to certain environmental changes (12, 22). As in ani- resulting pellets were resuspended in 20 to 30 ml 10 mM Hepes mals, at least some Ca2-imediated responses in plants are con- (pH 7.0), 0.5 mM CaCl2 (buffer B) and heated rapidly to 85°C trolled by Ca2-binding regulatory proteins. Among these, the for 2 min, cooled and centrifuged at 7500g for 20 min. The supernatant was dialyzed against buffer B for 2 to 3 h. Subsequent most studied and best characterized is calmodulin (1). was on a fluphenazine-Sepharose affinity colchromatography We have published reports suggesting a possible role for calmodulin in mediating phytochrome and gravitropic responses in umn (5 x 12 cm), as described by Charbonneau and Cormier Avena saliva (oats) (2, 21). As part of our ongoing research on (4). The calmodulin eluted from this column was pooled, dithis question, we have isolated and characterized calmodulin alyzed against water overnight, and assayed for protein content from oats and have estimated its content, both in intact tissue and ability to activate bovine brain PDE before being used further and in isolated subcellular fractions, by radioimmunoassay. Here or freeze-dried for storage. Method 2 (hydrophobic gel method) is basically a combination we report the results of these experiments. of the methods of Kakiuchi et al. (13) and Gopalakrishna and Anderson (7). For this method, I kg of fresh 4-d-old oat shoots MATERIALS AND METHODS was homogenized in 500 ml of 50 mm Hepes (pH 8.0), 1 mM Plant Material. Except where indicated, the starting material EGTA, 0.25 mM PMSF, 0.15 M NaCl, 20 mm sodium bisulfite, for all extractions was taken from the coleoptiles and primary filtered through a 100 fim nylon mesh and centrifuged at 14,000g leaves of 3- to 4-d-old dark-grown oat (A vena sativa, var. Garry) for 30 min. The supernatant was brought to a final concentration seedlings, harvested 5 to 7.5 mm above the seed. The oats were of 3% (w/v) TCA by slow addition of 30% TCA and stirred for 30 min. This mixture was centrifuged at 1 ,OOOg for 20 min and grown on water-saturated vermiculite at 27C. Calmodulin Isolation. Two extraction methods were employed. the pellet was resuspended in 300 ml of 20 mm Hepes (pH 7.6), 1 mm EGTA, 20 mm NaHSO3, 0.25 mm PMSF. This material 'Supported by grants from the National Aeronautics and Space Ad- was then rapidly heated to above 90°C for 3 min and rapidly ministration (NSG 7480), The National Science Foundation (PCM 81- cooled both by the addition of 200 ml of cold pellet-resuspension buffer and by placing the solution in an ice bath. This mixture 03429), and The Robert A. Welch Foundation (F 858) to S. J. R. 2 Wake Forest University, Biology Department, Winston-Salem, was clarified at 22,000g for 1 h and the supernatant was adjusted to 5 mm calcium with the addition of 50 mM Tris (pH 7.5), 3 North Carolina 27901. 3Hopei Teachers University, Biology Department, Peoples' Republic mM NaCl, 60 mM CaC12. The solution was further purified on a of China. 4Jawaharlal Nehru University, School of Life Sciences, New Delhi 110067, India.
I Abbreviations: PMSF, phenylmethylsulfonyl fluoride; PDE, phosphodiesterase; RIA, radioimmunoassay; PEP, P-enolpyruvate.
382
OAT CALMODULIN 1.5 x 8.8 cm phenyl-Sepharose column, as previously described (7). Calmodulin fractions, eluted by an EGTA-containing buffer, were pooled and dialyzed against distilled HO. Both methods gave approximately equal yields and purity of calmodulin. Recent attempts to purify oat calmodulin from centrifuged crude homogenates loaded directly onto fluphenazine-affinity columns have also been successful, yielding calmodulin of identical properties. However the use of this procedure results in a rapid deterioration of the affinity column. Calmodulin samples from both purification methods were routinely further purified by fractionation on a Waters uBondapak Phenyl Column using HPLC (15). Calmodulin Assay: Phosphodiesterase Activity. Biological activity of calmodulin samples was estimated by their ability to promote activity of PDE. Partially purified PDE was prepared by the methods ofCheung (5), and was further purified by passing the partially purified PDE through an Affi-Gel calmodulin column (Bio-Rad Laboratories). The activity of PDE was monitored by the spectrophotometric method of Dedman and Means (6). Organelle Isolations. Nuclei were isolated from mature oat embryos by the procedures of Luthe and Quatrano ( 14), starting with I g of embryos. Etioplasts were isolated by the method of Jacobson (I 1), using 8-d-old etiolated leaf tissue. Mitochondria were isolated from chilled 4-d-old etiolated oat tissue as described previously (24). Outer mitochondrial membranes and mitoplasts were prepared by the methods of Moreau and Lance (17). Protoplast Isolation. Protoplasts were isolated from 4-d-old coleoptile tips of oats which were incubated in 5 mm Mes (pH 5.8), 1 mM CaCl2, 0.5% (w/v) BSA, and 2% (w/v) Cellulysin (Calbiochem) as recommended by Hampp and Ziegler (9). The incubation was at 30°C for 5 to 6 h, and protoplasts were filtered through 100aum nylon mesh, pelleted by low speed centrifugation and washed twice. Isolation of Soluble Cell Wall Components. The isolation of soluble compounds from the cell wall or extracellular solution via centrifugation has been described previously by Terry and Bonner (25). The modifications to this procedure were that 7.6 to 7.9 g of 1-cm leaf sections, cut 0 to 2 cm above the coleoptile remnant of light-grown 14-d-old oats, were packed into four syringe tubes and rinsed in 10 mm KCI for 15 min followed by a 45-min rinse with 5 mM KCI, 29 mm sucrose. The sections were vacuum infiltrated with 4°C distilled H20 and centrifuged at lOOOg for 6 min. This was repeated twice. The extracellular solution from the three centrifugations was combined and analyzed for malate dehydrogenase (8, 26), PEP carboxylase (19) and calmodulin. Three to 4 grams of leaves which had been centrifuged were homogenized in a mortar and pestle with 2 g PVP (Sigma, No. P-6755) and 55 ml of 100 mM Tris (pH 8.0) containing I mM EGTA, 5 mM MgC92, and 5 mm DTT. The homogenate was clarified by centrifugation for 30 min at 40,000g. Some of the extracellular solution from separate experiments was concentrated 40-fold in an Amicon Ultrafiltration Cell Model M-3 with a 10,000 mol wt cut-off filter (Type C from Anspec). Trypsin Digests. Surface calmodulin was removed from isolated etioplasts, mitochondria, mitoplasts, and outer mitochondrial membrane by proteolysis with trypsin. Isolated organelles were exposed to 160 gg/ml trypsin for 30 min at 4°C. Following this proteolysis period the suspension was diluted with 5 ml of resuspension buffer containing 10,000 kallikrein-inactivating units aprotinin and I mM PMSF. The material was then pelleted by centrifugation and washed twice with buffer before use. RIA of Calmodulin. Calmodulin radioimmunoassay kits were purchased from CAABCO Inc. (Houston, TX). These assays were of the competitive-binding type employing 251I-labeled calmodulin and affinity purified antibody against rat testes calmodulin. The kits were used according to the enclosed instructions.
383
In addition to calibration of the assay kit by use of animal calmodulin standards (included with each kit), equivalent oat calmodulin standards were also run to determine the crossreactivity of the antibody for oat calmodulin. Electrophoresis. SDS-PAGE of calmodulin samples was performed as described in (27) on 12.5% acrylamide gels containing 1 mM EGTA or 1 mM CaC12. Gels were fixed in 20% methanol, 5% formaldehyde, 0.15 M sulfosalicylic acid and stained with Coomassie blue for visualization of the proteins.
RESULTS Figure I shows a composite picture of SDS polyacrylamide gels of calmodulin-containing fractions collected from a fluphenazine affinity column. When the gel contained small amounts of calcium these fractions normally produced only a single band (mol wt of approximately 15,000) when stained with Coomassie blue. However, when SDS-PAGE was performed either in the presence of EGTA or the absence of added calcium, either a single band with a mobility indicating a mol wt of approximately 17,000 or two bands of apparent mol wt of 17,000 and 19,000 were observed. Identical results were obtained with fractions collected from protein purified by the hydrophobic gel method. Because published reports (28, 29) indicated that the fluphenazine-affinity and hydrophobic-gel-purified calmodulin might not be as homogeneous as SDS-PAGE seemed to indicate, further purification of this material by reverse phase HPLC was performed by the methods of Marshak et al. (15). Sixty to 70% of the protein loaded onto a Waters gBondapak Phenyl HPLC column could be recovered in one sharp peak eluted at 30% acetonitrile. This material was capable of PDE activation, bound to calmodulin antibody when tested by RIA, and produced an absorbance spectrum (Fig. 2) very similar to that reported by Anderson et al. (1) for peanut calmodulin. Most of the remaining protein was eluted in the void volume or at 20% acetonitrile and was not capable of PDE activation or binding to calmodulin antibody. The HPLC-purified material was further analyzed for amino acid composition at the Amino Acid Analysis Center at the University of Texas at Austin. The results of this analysis are indicated in Table I along with values obtained by others for calmodulin from spinach, peanut, barley, and bovine brain. The composition of oat calmodulin appears to be essentially the same as that reported for spinach and is very similar to the composition reported for calmodulin from other higher plants. It has a high ratio of acidic to basic amino acids, 1 tyrosine, 1 histidine, and 1 trimethyllysine. The ability of antibody against animal calmodulin to bind oat calmodulin was also used to characterize and localize oat calmodulin. The binding affinity of the antibody is 8 to 10 times less for oat calmodulin than for rat calmodulin but is still within a useful range (Fig. 3). With this information it was possible to quantify the calmodulin content of oat tissues, protoplasts, and isolated subcellular components using purchased RIA kits for calmodulin. Table II shows the results of quantification of calmodulin by RIA from several readily available preparations used in our laboratory. Calmodulin was shown to be associated with isolated protoplasts, cell wall solutions, and all intact organelles tested. The amount of calmodulin contained in the isolated cell wall solution is greater than that which reasonably could be assumed to be a result of cellular leakage. To test for contamination by cytoplasmic enzymes, malic dehydrogenase and PEP carboxylase activities were monitored in the cell wall solution. Malate dehydrogenase activity in the cell wall solution was less than 0.05% that of a whole tissue homogenate. No PEP carboxylase activity could be detected in the cell wall solutions, even when they were concentrated 120 times.
Plant Physiol. Vol. 75, 1984
BIRO ET AL.
384
43K
_
_
25 t7
_t
124 3
I:.
:-A-
r
B
A
D
E
F
H
G3
FIG. 1. Composite SDS-polyacrylamide gel of HPLC-purified oat calmodulin. Lanes A, B, and C were run in the presence of 1 mM CaCI2. Lanes D, E, F, G, and H were run in the presence of I mM EGTA. Lanes A, D, and H contain mol wt standards (Bethesda Research Laboratories) with weights as indicated on the right. Lanes B and C contain 10 and 5 ;tg calmodulin, respectively, and lanes E, F, and G contain 10, 15, and 5 ug calmodulin, respectively.
Table I. Amino Acid Composition of Oat Calmodulin and Calmodulin from other Sources Oat Peanut' Spinachb Barley" Bovinec Amino Acid mol/16, 700g residues/
~260
240
280
300
WAV ELENGTH(nm)
FIG. 2. UV absorbance spectrum of HPLC-purified oat calmodulin
(approximately
mg/ml).
Trypsin digestion of isolated mitochondria and etioplasts to remove calmodulin from their outer surfaces did not remove all
calmodulin from these organelles. This indicated that at least some of the calmodulin associated with these organelles is internal. However, removal of the outer mitochondrial membrane from the mitochondrion resulted in loss of all detectable calmodulin in the remaining mitoplast and the outer membrane. To test for nonspecific
binding of calmodulin to isolated
organelles, '251-labeled calmodulin (20,000 cpm; 2
Mig) was added
to the oat tissues as they were homogenized for etioplasts and mitochondria. The radioactivity was followed through the isola-
molecule ND" 1/2 Cystine 22.5 Aspartic acid 8.2 Threonine 3.7 Serine 26.0 Glutamic acid 3.0 Proline 10.2 Glycine 11.2 Alanine 8.1 Valine 8.4 Methionine 5.1 Isoleucine 11.7 Leucine 1.0 Tyrosine 8.4 Phenylalanine 1.9 Trimethyllysine 8.9 Lysine 1.0 Histidine 4.9 Arginine ND Tryptophan ' Anderson et al. (1) b Schleicher et al. (23) c Watterson et al. (30) d ND, not determined.
1 27.1 9.1 5.3 28.2 2.3 11.2 10.6 6.3 7.3 5.9 11.2 0.6 8.2
1.6 7.9
1.2 4.4 0
I 24 9 4 27 2 10 11 8 8 7 11 1 9 1 9 1 5 0
ND 27.8 8.9 5.5 24.6 2.2 11.8 11.7 7.4
0 23 12 5 27 2
8.1
9 8 9 2 8 1
7.0 11.6
1.3 8.5 1.0 8.0 1.5 4.9 ND
11 11 7
7 1 6
0
tion procedures and the amounts of possible nonspecific binding to the organelles were calculated from the counts assuming worstcase possibilities for dilution and calmodulin content of the
organelles. By this method 0.03% or less of the calmodulin found associated with isolated etioplasts and mitochondria could be
accounted for by the added labeled calmodulin. Calmodulin-affinity purified PDE showed as much as a 10fold increase in activity in the presence of saturating amounts of added calmodulin (Table III). Essentially no difference could be
OAT CALMODULIN
385
Table Ill. Oat Calmodulin Activation ofBovine Brain Phosphodiesterase
a z
D
0
Test No.
Calmodulin Content
PhosAphCodietrase
z -J
0 a
0
10080
OAT
:
60
IR LL
40-
0
z w
20i
w 0-
nmoles 5'AMP/min I 0.0 15.3 2 0.13 99.4 3 0.5 143.0 4 0.7 145.0 5 0.0 +2 mMEGTA 5.6 6 0.7+2mMEGTA 10.3 a Assay mixture: 25 mm morpholinopropane sulfonic acid (Mops), 150 mm KC1, 1.5 mm Mg acetate, 5 mm 2-mercaptoethanol, 0.1 mM CaC12, 0.2 mm cyclic AMP, 5 pg 5' AMP deaminase, and 50 ug of purified PDE.
-ug
0
1
22
3
4
5
6
7
LOG CALMODULIN ( nanograms/ m )
FIG. 3. Amount of 'II-labeled rat calmodulin bound to CAABCO calmodulin antibody in the presence of various concentrations of unlabeled rat and oat calmodulin. Table II. Oat Calmodulin Associated with Oat Tissues, Cells, and Cell Fractions as Measured by Radioimmunoassay Purity of all isolated organelles was estimated to be 85% or better by light and electron microscopy. Range of Observed Values of Sample Type Calmodulin pg/mg extracted protein Whole oat coleoptiles 0.9-2.7 Isolated oat protoplasts 1.1-4.2 Oat cell wall fractions 18.4-60.6 Isolated oat mitochondria 9.2-25.0 3.8-18.0 Trypsin digested whole mitochondria Oat mitoplasts (outer membrane 0.0-0.1 removed) 0.0-0.1 Oat mitochondial outer membrane 0.8-4.0 Isolated etioplasts 0.8-1.6 Trypsin digested whole etioplasts 0.5-1.4 Isolated nuclei 0.1-0.2 Isolated nuclei (extracted in EGTA) observed between the activation of PDE by bovine brain calmodulin or by oat calmodulin.
DISCUSSION The evidence presented here indicates the presence of the protein species identifiable as calmodulin in oat tissues. This molecule binds calcium, phenothiazines, and calmodulin specific antibody. It migrates on SDS-polyacrylamide gels in a fashion identical to calmodulin from other plant species (1, 27) and, typical of calmodulin, it activates bovine brain PDE in a calciumdependent fashion, is relatively heat stable, and has increased hydrophobicity with calcium (7). Most convincing is the fact that the protein characterized here has an amino acid composition virtually indistinguishable from that of calmodulin from other plant sources (Table I).
Experience has indicated that oat tissues contain highly active proteases which can rapidly degrade calmodulin during the isolation procedure. Every effort was made to limit this proteolysis. Isolation procedures were performed quickly at low temperature and potent protease inhibitors, such as aprotinin and PMSF, were included in the isolation media. Quick-frozen, freeze-dried, and acetone-extracted tissues were used to further minimize the activity of proteases. When these stringent methods were used, the per cent of undegraded calmodulin purified from oat tissues could be as high as 20% of that estimated to be present in oat tissue by RIA. The amounts ofcalmodulin indicated to be present in isolated oat tissue, protoplasts, organelles and other fractions by RIA must be viewed in light of the possibility that at least some of the material assayed to be calmodulin may be other calciumbinding proteins. Van Eldik et al. (28) have shown that commercially prepared antibody against calmodulin can bind other proteins, resulting in an overestimate of the amount of calmodulin in spinach by RIA. Certainty as to the specificity of the RIA kit for calmodulin would require extensive further testing. However, our results from attempts to bind the calmodulin antibody to materials that initially copurified with calmodulin through affinity chromatography, and were then separated by HPLC, indicate little, if any, binding of antibody to these materials. This indicates that in oat extracts the CAABCO antibody has a high specificity for authentic calmodulin and little affinity for other calmodulin-like proteins which have a 17,000 monomer mol wt, are heat stable, and bind to phenothiazines in a calcium-dependent fashion. Our finding that calmodulin is associated with various organelle preparations is in accord with the recent findings of Muto (18), who examined barley organelie fractions, and Jarrett et al. (12) who found calmodulin in chloroplasts. Our control experiments indicate that almost no exogenous calmodulin binds to organelles during the extation steps, that trypsin digestion of intact mitochondria and etioplasts degrades only a fraction of the calmodulin associated with the organelles, and that very little of the calmodulin estimated to be associated with the purified organelles can be removed from them by EGTA washes which would be expected to disociate calmodulin from non-specific hydrophobic sites (7). These results make it seem very likely that calmodulin is an endogenous component of most isolated organelles in oats. Our assays indicate that there is calmodulin associated with material external to the plasma membrane. The original control experiments of Terry and Bonner (25) and the lack of detectable PEP carboxylase in the wall extracts we analyzed make it unlikely that these extracts were contaminated by cytoplasmic proteins. Even the trace amount of malate dehydrogenase found in the
Plant Physiol. Vol. 75, 1984 BIRO IET AL. 386 Biochim Biophys Acta 191: 303-315 wall extracts could be wall-localized enzyme (8). What function 6. DEDMAN JR, AR MEANS 1977 Characterization of a spectrophotometric assay is solution in unclear, calmodulin would have the apoplastic for cAMP phosphodieserase. J Cyclic Nucleotide Res 3: 139-152 since no mechanism for regulating the free calcium concentration 7. GOPALAKRISHNA R, WB ANDERSON 1982 Ca2?-induced hydrophobic site on calmodulin: application for purification of calmodulin by phenyl-Sepharose there within a range which would modulate calmodulin function affinity chromatography. Biochem Biophys Res Commun 104: 830-836 (-1 uM) has yet been demonstrated. However, calcium ions 8. GRoss GG 1977 Cell wall-bound malate dehydrogenase from horseradish. clearly affect several biochemical processes in the wall (22), so Phytochemistry 16: 319-321 potential roles for wall calmodulin can readily be identified and 9. HAMPP R, H ZIEGLE 1980 On the use ofAvena protoplasts to studychloroplast development. Planta 147: 485-494 tested. 0, M TOKUDA, T ITANO, H MATSUI, A Doli 1982 Purification and By our assays the mitochondria contain a relatively large 10. HATASE charcterization of calmodulin from rat liver mitochondria. Biochem Bioamount of calmodulin, in agreement with the findings of Muto phys Res Commun 104: 673-679 (18) in wheat, and Hatase et al. (10) in rat liver. Some of this 11. JACOBSON AB 1968 A procedure for isolation of proplstids from etiolated maize leaves. J Cell Biol 38: 238-244 mitochondrial calmodulin could be removed by trypsin digestion HW, CJ BROWN, CC BLACK, MJ CORmIER 1982 Evidence that of the surface of intact mitochondria. Once the outer membrane 12. JARRErr calmodulin is in the chloropla of peas and serves a regulatory role in was removed from the mitochondrion, neither the outer memphotosynthesis. J Biol Chem 257: 13795-13804 brane nor the remaining mitoplast appeared to contain signifi- 13. KAIUcHI S, K SoBuE, R YAMA7AKI, S NAGAO, S UME, Y NozAwA, M YAZAWA, K YAGI 1981 Ca2+dependent modulator proteins from Tetrahy cant amounts of calmodulin. By elimination, it appears that mena pyriformis, sea anemone and scallop and guanylate cyclase activation. some of the calmodulin is located between the inner and outer J Biol Chem 256: 19-22 membranes. Such calmodulin could be linked to phytochrome- 14. LUTHE DS, RS QUATRANO 1980 Transcription in isolated wheat (Triticum controlled calcium movements into and out of the mitochondria aestivum cultivar Yamhill) nucldei: 1. Isolation of nuclei and elimination of endogenous RNase activity. Plant Physiol 65: 305-308 (21) and calcium-modulated ATPase activity (24). DR, DW WArERSON, LJ VAN ELDIK 1981 Calcium-dependent More than half of the calmodulin associated with isolated 15. MARSHAK interaction of S100b, troponin C, and calmodulin with an immobilized nuclei can be removed by an EGTA wash, but the amount phenothiazine. Proc Natl Acad Sci USA 78: 6793-6797 remaining is still significant. Calmodulin has been shown to be 16. MASmUMOTO H, M TANIGAWA, T YAMAYA 1983 Calmodulin-ike activity associated with chromatin from pea buds. Plant Cel Physiol 24: 593-602 associated with histones and the control of protein kinases in F, C LANCE 1972 Isolement et proprietes des membranes externes et wheat leaves (20), and calmodulin-like activity has been found 17. MOREAu internes de mitochondries vegetale. Biochimie 54:1335-1348 associated with chromatin from pea buds (16). The known 18. Muro S 1982 Distribution of calmodulin within wheat leaf cells. FEBS Lett regulatory roles of calcium in mitosis and cytokinesis of plant 147: 161-164 cells are also consistent with a nuclear locale for calmodulin (22). 19. PERROr CJ, J VIDAL, A BURLE, P GADAL 1981 On the cellular lo tion of phosphoenolpyruvate carboxylase in Sorghum leaves Planta 151: 226-231 Our results, together with those of Muto (18) and Jarrett et al. GM, JR DAvIES 1982 Resolution of Ca2W.calmodulin-activated protein (12) indicate that calmodulin is a component of highly purified 20. POLYA kinase from wheat germ. FEBS Left 150: 167-171 preparations of several organelles from several different plants. 21. Roux SJ, K MCENTIRE, RDSLocum, TE CEDEL, CC HAL 1981 Phytochrome induces photoreversible calcium fluxes in purified mitochondria fractions This result is of direct significance to those studying calciumfrom oats Proc Natl Acad Sci USA 78: 283-287 regulated phenomena in isolated organelles. Our results also 22. Roux SJ, RD SLocrm 1982 Role of calcium in mediating cellular functions support the hypothesis that calmodulin is an endogenous comimportant for growth and development in higher plantL In WY Cheung, ed, ponent of at least mitochondria and etioplasts in oats. Additional Calcium and Cell Function, Vol m. Academic Press, New York, pp 408453 important data on this hypothesis should result from in situ M, TJ LuKAS, DM WArERSON 1983 Further characteization of localization studies and from investigations of calmodulin-regu- 23. SCHLCHER calmodulin from the monocotyledon barley (Hordeum vulgare). Plant Physlated enzymes in these organelles. iol 73: 666-670 Acknowledgments-We would like to thank Kerry McEntire for his instructive initial isolations and analyses of oat calmodulin and Sandra Smith for the amino acid analysis of oat calmodulin.
1.
2. 3. 4.
5.
LITERATURE CITED ANDERSON JM, H CHARDONNEAU, HP JONES, RO MCCANN, MJ CORMIER 1980 aracterization of the plant nicotinamide adenine dinucleotide kinase activator protein and its identification as calmodulin, Biochemistry 19: 3113-3120 BIRo RL, CC HALE, OF WIEGAND, SJ Roux 1982 Effects of chlorpromazine on gavitropism in Avena coleoptiles. Ann Bot 50: 735-747 CALDWELL CR, A HAUG 1981 Affinity chromatogrphic isolation of calmodulin from bovine brain acetone powder. Anal Biochem 116: 325-330 CHARBONNEAU H, MJ CORMIER 1979 Purification of plant calmodulin by fluphenazine-epharose affinity chromatography. Biochem Biophys Res Commun 90: 1039-1047 CHEUNG WY 1969 Cyclic 3',5'-nucleotide phosphodiesterase: prepaation of a parially inactive enzyme and its subsequent stimulation by snake venom.
24. SERUN BS, SK SoPORY, SJ Roux 1984 Modulation of oat mitochondrial ATPase activity by Ca2+ and phytochrome. Plant Physiol 74: 827-833 25. Timy ME, BA BONNER 1980 An examination of centrifuigton as a method of exractng an extracellular solution from peas, and its use for the study of indoleacetic acid-induced growth. Plant Physiol 66: 321-325 26. TING IP 1968 Malic dehydrogenases in corn root tips. Arch Biochem Biophys 126: 1-7 27. VAN ELDI LJ, AR GROSSMAN DB IvmwON DM WATrEoN 1980 Isolation and charcteization of calmodulin from spinach leaves and in vitro translation mixtures. Proc Natl Acad Sci USA 77: 1912-1916 28. VAN ELDIK LJ, G PIPERNO, DM WAESON 1980 Simiarities and dissimilarities between calmodulin and a Chlamydomonas flaela protein. Proc Natl Acad Sci USA 77: 47794783 29. WATFERSON DM, WG HARRELON JR, PM KELER, F SHARI, TC VANAMAN 1976 Structural similarities between the Ca2Wdependent regulatory proteins of 3':5'-cyclic nucleotide phosphodiesterase and actomycin ATPase. J Biol Chem 251: 4501-4513 30. WArFRSON DM, F SHAEF, TC VANAMAN 1980 The complete amino acid sequence of the Ca24dependent modulator protein (calmodulin) of bovine brain. J Biol Chem 255: 962-975
