Calmodulin-binding protein kinases in plants

Many calmodulin-binding protein kinases have been isolated from plants. Plant calmodulin-binding protein kinases are novel protein kinases that differ from calcium-dependent protein kinases in many important respects. Calmodulin-binding protein kinases are likely to be crucial mediators of responses to diverse endogenous and environmental cues in plants. In this update, we review the structure, regulation, expression and possible functions of plant calmodulin-binding protein kinases.     

Phosphorylation-regulated calmodulin binding to a prominent cellular substrate for protein kinase C

We have evaluated the possibility that a major, abundant cellular substrate for protein kinase C might be a calmodulin-binding protein. We have recently labeled this protein, which migrates on sodium dodecyl sulfate-gel electrophoresis with an apparent Mr of 60,000 from chicken and 80,000-87,000 from bovine cells and tissues, the myristoylated alanine-rich C kinase substrate (MARCKS). The MARCKS proteins from both species could be cross-linked to 125I-calmodulin in a Ca2+-dependent manner. Phosphorylation of either protein by protein kinase C prevented 125I-calmodulin binding and cross-linking, suggesting that the calmodulin-binding domain might be located at or near the sites of protein kinase C phosphorylation. Both bovine and chicken MARCKS proteins contain an identical 25-amino acid domain that contains all 4 of the serine residues phosphorylated by protein kinase C in vitro. In addition, this domain is similar in sequence and structure to previously described calmodulin-binding domains. A synthetic peptide corresponding to this domain inhibited calmodulin binding to the MARCKS protein and also could be cross-linked to 125I-calmodulin in a calcium-dependent manner. In addition, protein kinase C-dependent phosphorylation of the synthetic peptide inhibited its binding and cross-linking to 125I-calmodulin. The peptide bound to fluorescently labeled 5-dimethylaminonaphthalene-1-sulfonyl-calmodulin with a dissociation constant of 2.8 nM, and inhibited the calmodulin-dependent activation of cyclic nucleotide phosphodiesterase with an IC50 of 4.8 nM. Thus, the peptide mimics the calmodulin-binding properties of the MARCKS protein and probably represents its calmodulin-binding domain. Phosphorylation of these abundant, high affinity calmodulin-binding proteins by protein kinase C in intact cells could cause displacement of bound calmodulin, perhaps leading to activation of Ca2+-calmodulin-dependent processes.     

Calmodulin-Binding Proteins in Chromaffin Cell Plasma Membranes

Abstract: Calmodulin-binding proteins present in chromaffin cell plasma membranes were isolated and directly compared with calmodulin-binding proteins present in chromaffin granule membranes. Chromaffin cell plasma membranes were prepared using Cytodex 1 microcarriers. Marker enzyme studies on this preparation showed a nine- to 10–fold plasma membrane enrichment over cell homogenates and a low contamination of these plasma membranes by subcellular organelles. Plasma membranes prepared in this manner were solubilized with Triton X-100 and applied to a calmodulin-affinity column in the presence of calcium. Several major calmodulin-binding proteins ( 240, 105 , and 65 kilodaltons) were eluted by an EGTA-containing buffer. ¹²⁵I-Calmodulin overlay experiments on nitrocellulose sheets containing both chromaffin plasma and granule membranes showed that these two membranes have several calmodulin-binding proteins in common ( 65, 60, 53 , and 50 kilodaltons), as well as unique calmodulin-binding proteins (34 kilodaltons in granule membranes and 240 and 160 kilodaltons in plasma membranes). The 65–kilodalton calmodulin-binding protein present in both membrane types was shown to consist of two isoforms (pI 6.0 and 6.2) by two-dimensional gel electrophoresis. Previous experiments from our laboratory, using two monoclonal antibodies (mAb 30 and mAb 48) specific for a rat brain synaptic vesicle membrane protein (p65), showed that the monoclonal antibodies reacted with a 65–kilodalton calmodulin-binding protein present in at least three neurosecretory vesicles (chromaffin granules, neurohypophyseal granules, and rat brain synaptic vesicles). When these monoclonal antibodies were tested on chromaffin cell plasma membranes and calmodulin-binding proteins isolated from these membranes, they recognized a 65–kilodalton protein. These results indicate that an immunologically identical calmodulin-binding protein is expressed in both chromaffin granule membranes (as well as other secretory vesicle membranes) and chromaffin cell plasma membranes, thus suggesting a possible role for this protein in granule/plasma membrane interaction.     

Calmodulin-binding protein detection using a non-radiolabeled calmodulin fusion protein

Calmodulin-binding proteins are involved in numerous cellular signaling pathways. The biotinylated-calmodulin overlay is a nonradioactive method widely used to detect calmodulin-binding proteins in tissue and cell samples. This method has several limitations; therefore, we developed a nonradioactive calmodulin-binding protein detection overlay using an S-tag-labeled calmodulin fusion protein. An expression system was used to generate a calmodulin fusion protein with an S-tag label, a 15 amino acid sequence that binds to a 105 amino acid S-protein. The S-protein is conjugated to horseradish peroxidase for final detection with a chemiluminescent substrate. The S-tag calmodulin was compared to purified calmodulin and biotinylated calmodulin in a calmodulin-dependent phosphodiesterase assay. The results of the calmodulin-dependent phosphodiesterase assay indicate that S-tag calmodulin induces higher phosphodiesterase activity than biotinylated calmodulin and lower activity than purified calmodulin. A comparison of the biotinylated and S-tag calmodulin overlay assays indicate that S-tag calmodulin is more sensitive than biotinylated calmodulin in the detection of calcineurin, a known calmodulin-binding protein. The overlay assay results also indicate that the S-tag calmodulin and biotinylated calmodulin detect similar calmodulin-binding proteins in colon epithelial cells. In conclusion, the S-tag calmodulin overlay assay is a consistent, sensitive, and rapid nonradioactive method to detect calmodulin-binding proteins.     

Calmodulin-Binding Proteins in Human Y-79 Retinoblastoma and HTB-14 Glioma Cell Lines

The Y-79 human retinoblastoma cell line has been used as a model system for studying differentiation of primitive neuroectodermal cells into either glial-like (glial fibrillary acidic protein positive) or neuron-like (neuron-specific enolase-positive) cells. To determine whether Y-79 retinoblastoma cells express neuronotypic calmodulin-binding proteins, Y-79 cells were either treated with butyrate or dibutyryl cyclic AMP (dbcAMP) in serum-containing medium or were maintained in serum-free media. Using a biotinylated calmodulin blot overlay technique, we found that Y-79 cells treated with dbcAMP or butyrate expressed low levels of membrane-bound calmodulin-binding proteins of 150, 147, 127, and 126 kilodaltons (kDa); butyrate-treated cells also expressed a calmodulin-binding peptide of 135 kDa. Since butyrate treatment of Y-79 cells induces the expression and the secretion of interphotoreceptor retinoid-binding protein (IRBP, 140 kDa), we tested the hypothesis that the calmodulin-binding protein of 135 kDa induced by butyrate treatment was IRBP. Purified bovine IRBP did not bind calmodulin; further, the 135-kDa calmodulin binding protein was not immunoreactive with antisera directed against IRBP. Since dbcAMP and butyrate induce some glial-like characteristics in Y-79 cells, we compared the calmodulin-binding protein pattern in these cells with that seen in human HTB-14 glioma cells. The HTB-14 line did not express calmodulin-binding proteins, even after treatments with agents that induce morphologic change in these cells. Thus, we conclude that Y-79 cells express membrane-bound calmodulin-binding proteins, but in a pattern different from that seen with adult, differentiated neurons or from human HTB-14 glioma cells.     

Identification of the Major Postsynaptic Density Protein as Homologous with the Major Calmodulin-Binding Subunit of a Calmodulin-Dependent Protein Kinase

The major postsynaptic density protein (mPSDp), comprising greater than 50% of postsynaptic density (PSD) protein, is an endogenous substrate for calmodulin-dependent phosphorylation as well as a calmodulin-binding protein in PSD preparations. The results in this investigation indicate that mPSDp is highly homologous with the major calmodulin-binding subunit (p) of tubulin-associated calmodulin-dependent kinase (TACK), and that PSD fractions also contain a protein homologous with the sigma-subunit of TACK. Homologies between mPSDp and a 63,000 dalton PSD protein and the rho- and sigma-subunits of TACK were established by the following criteria: (1) identical apparent molecular weights; (2) identical calmodulin-binding properties; (3) manifestation of Ca2+-calmodulin-stimulated autophosphorylation; (4) identical isoelectric points; (5) identical calmodulin binding and autophosphorylation patterns on two-dimensional gels; (6) homologous two-dimensional tryptic peptide maps; and (7) similar phosphoamino acid-specific phosphorylation of tubulin. The results suggest that mPSDp is a calmodulin-binding protein involved in modulating protein kinase activity in the postsynaptic density and that a tubulin kinase system homologous with TACK exists in a membrane-bound form in the PSD.     

Novel bovine heart calmodulin-dependent protein kinase which phosphorylates a high molecular weight calmodulin-binding protein.

A novel calmodulin-dependent protein kinase has been isolated from bovine cardiac muscle by successive chromatography on DEAE-Sepharose 6B, Calmodulin-Sepharose 4B affinity and Sepharose 6B chromatography columns. The protein kinase was shown by gel filtration chromatography to have a molecular mass of 36,000 daltons. The highly purified protein kinase stoichiometrically phosphorylated the high molecular weight calmodulin-binding protein from cardiac muscle [Sharma RK (1990) J Biol Chem 265, 1152-1157] in a Ca2+/calmodulin-dependent manner. The phosphorylation resulted in the maximal incorporation of 1 mol of phosphate/mol of the high molecular weight calmodulin-binding protein. Other Ca2+/calmodulin-dependent protein kinases failed to phosphorylate the high molecular weight calmodulin-binding protein. The distinct substrate specificity of this protein kinase indicates that it is not related to the known calmodulin-dependent protein kinases and therefore constitutes a novel protein kinase.     

Establishment of an efficient purification method and further characterization of 32K calmodulin-binding protein in testis

A heat-stable 32K calmodulin-binding protein has been purified approximately 3,670-fold from porcine testis to apparent homogeneity as judged by both sodium dodecyl sulfate polyacrylamide gel electrophoresis and polyacrylamide gel electrophoresis under native conditions. The purification employed calmodulin-Sepharose 4B affinity chromatography; elution was performed with a free Ca2+ gradient. This provided a simple and efficient procedure, and approximately 1.62 mg of pure heat-stable calmodulin-binding protein was obtained from 390 g of porcine testis with a yield of 47% in activity. The purified protein was asymmetric (f/fo = 1.89) and consisted of a single polypeptide of Mr = 32,000. It is a highly acidic protein (pI = 3.9) with a diffusion coefficient of 5.4 X 10(-7) cm2/s, a sedimentation coefficient of 1.43 S, and a Stokes radius of 39.5 A in its free form and 41.3 A in its complex form with calmodulin. The extent of inhibition of phosphodiesterase by the calmodulin-binding protein was affected by the order of addition of the agents to the reaction mixture. The extent of inhibition was maximal when phosphodiesterase was added last, while it was minimal when the calmodulin-binding protein was added last. This protein was indistinguishable from a heat-stable calmodulin-binding protein in rat testis (Ono, T., Koide, Y., Arai, Y., & Yamashita, K. (1984) J. Biol. Chem. 259, 9011-9016).     

Participation of calcium and calmodulin in the formation of acetylcholine receptor clusters

We recently reported the detection of multiple classes of calmodulin-binding proteins in subcellular fractions of chicken embryo fibroblasts by using a gel binding procedure (Van Eldik, L.J., and W.H. Burgess, 1983, J. Biol. Chem., 258:4539-4547). In this report we identify many of these calmodulin-binding proteins and provide further evidence for the existence of multiple classes of calmodulin-binding proteins based on the interaction of these proteins with calmodulin and other calcium-modulated proteins. The fact that, in some cases, the same calmodulin-binding protein can bind troponin C and S100 alpha suggests that similar functional domains may be present in these distinct calcium-modulated proteins. We also have used protocols based on purification steps for calmodulin-binding proteins and calmodulin-regulated activities from other systems, in conjunction with enzymatic assays and various immunological methods, to identify many of the calmodulin-binding proteins in chicken embryo fibroblasts. The identities of these proteins suggest in vivo roles for calmodulin in the regulation of cell shape and motility, cyclic nucleotide metabolism, and possibly nucleic acid and protein turnover in fibroblasts.     

The binding of calmodulin to myelin basic protein and histone H2B.

1. A calmodulin-binding protein of apparent mol.wt. 19 000 has been purified from chicken gizzard. Similar proteins have been isolated from bovine uterus, rabbit skeletal muscle and rabbit liver. 2. These proteins migrated as an equimolar complex with bovine brain calmodulin on electroporesis on polyacrylamide gels in the presence of Ca2+ and 6M-urea. The complex was dissociated in the presence of EGTA. 2. The chicken gizzard calmodulin-binding protein has been shown to be identical with chicken erythrocyte histone H2B on the basis of partial amino acid sequence determination. 4. The calmodulin-binding proteins of apparent mol.wt. 22 000 isolated previously from bovine brain [Grand & Perry (1979) Biochem. J. 183, 285-295] has been shown, on the basis of partial amino-acid-sequence determination, to be identical with myelin basic protein. 5. The activation of bovine brain phosphodiesterase by calmodulin is inhibited by excess bovine uterus calmodulin-binding protein (histone H2B). 6. The phosphorylation of myelin basic protein by phosphorylase kinase is partially inhibited, whereas the phosphorylation of uterus calmodulin-binding protein (histone H2B) is unaffected by calmodulin or troponin C. 7. The subcellular distribution of myelin basic protein and calmodulin suggests that the two proteins do not exist as a complex in vivo.     

A 115 kDa calmodulin-binding protein is located in rat liver endosome fractions.

The distribution of calmodulin-binding polypeptides in various rat liver subcellular fractions was investigated. Plasma-membrane, endosome, Golgi and lysosome fractions were prepared by established procedures. The calmodulin-binding polypeptides present in the subcellular fractions were identified by using an overlay technique after transfer from gels to nitrocellulose sheets. Distinctive populations of calmodulin-binding polypeptides were present in all the fractions examined except lysosomes. A major 115 kDa calmodulin-binding polypeptide of pI 4.3 was located to the endosome subfractions, and it emerges as a candidate endosome-specific protein. Partitioning of endosome fractions between aqueous and Triton X-114 phases indicated that the calmodulin-binding polypeptide was hydrophobic. Major calmodulin-binding polypeptides of 140 and 240 kDa and minor polypeptides of 40-60 kDa were present in plasma membranes. The distribution of calmodulin in the various endosome and plasma-membrane fractions was also analysed, and the results indicated that the amounts were high compared with those in the cytosol.     

Tissue distribution of high molecular weight calmodulin-binding protein

Polyclonal antibodies raised against bovine heart high molecular weight calmodulin-binding protein were used to study the distribution of this protein in diverse bovine tissues. The high molecular weight calmodulin-binding protein, in addition to bovine heart, is also present in lung and brain at much lower levels, but not in skeletal muscle, spleen, kidney or uterus.     

A new erythrocyte membrane-associated protein with calmodulin binding activity. Identification and purification

A new protein that binds calmodulin has been identified and purified to greater than 95% homogeneity from the Triton X-100-insoluble residue of human erythrocyte ghost membranes (cytoskeletons) by DEAE chromatography and preparative rate zonal sucrose gradient sedimentation. This ghost calmodulin-binding protein is an alpha/beta heterodimer with subunits of Mr = 103,000 (alpha) and 97,000 (beta). The protein exhibits a Stokes radius of 6.9 nm and a sedimentation coefficient of 6.8 S, corresponding to a molecular weight of 197,000. Moreover, the protein is cross-linked by Cu2+/phenanthroline to a dimer of Mr = 200,000. The Mr = 97,000 beta subunit was identified as the calmodulin-binding site by photoaffinity labeling with 125I-azidocalmodulin. A 230 nM affinity for calmodulin was estimated by displacement of two different concentrations of the 125I-azidocalmodulin with unmodified calmodulin and subsequent Dixon plot analysis. This calmodulin-binding protein is present in erythrocytes at 30,000 copies/cell and is associated exclusively with the membrane. It is tightly bound to a site on red cell cytoskeletons and is totally solubilized in the low ionic strength extract derived from red cell ghost membranes. Visualization of this calmodulin-binding protein in the electron microscope by rotary shadowing, negative staining, and unidirectional shadowing indicates that it is a flattened circular molecule with a 12.4-nm diameter and a 5.4-nm height. Affinity-purified antibodies against the calmodulin-binding protein identify a cross-reacting Mr = 100,000 polypeptide(s) in brain membranes.     

Pcp1p, an Spc110p-related calmodulin target at the centrosome of the fission yeast Schizosaccharomyces pombe.

In the budding yeast Saccharomyces cerevisiae, the calmodulin-binding protein Spc110p/Nuf1p facilitates mitotic spindle formation from the fungal centrosome or spindle pole body (SPB). The human Spc110p orthologue kendrin is a centrosomal, calmodulin-binding pericentrin isoform that is specifically overexpressed in carcinoma cells. Here we establish an evolutionary and functional link between Spc110p and kendrin through identification and analysis of similar calmodulin-binding proteins in the fission yeast Schizosaccharomyces pombe (Pcp1p, pole target of calmodulin in S. pombe) and the filamentous fungus Aspergillus nidulans. Like Spc110p and kendrin, Pcp1p and the A. nidulans protein contain predicted coiled-coil secondary structure and a COOH-terminal calmodulin-binding region. Green fluorescent protein fusions of Pcp1p localize to the SPB as analyzed by fluorescence and immunoelectron microscopy. Pcp1p overexpression causes chromosome missegregation, multiple mitotic spindle fragments, and multiple abnormal SPB-like structures, a phenotype remarkably similar to that of many human carcinoma lines, which exhibit chromosome and spindle defects, and supernumerary centrosomes.     

Calcium-Dependent Calmodulin Binding to Chromaffin Granule Membranes: Presence of a 65-Kilodalton Calmodulin-Binding Protein

The presence of calmodulin-binding sites on chromaffin granule membranes has been investigated. Saturable, high-affinity 125I-calmodulin-binding sites (KD = 9.8 nM; Bmax = 25 pmol/mg protein) were observed in the presence of 10(-4) M free calcium. A second, nonsaturable, calmodulin-binding activity could also be detected at 10(-7) M free calcium. No binding occurred at lower calcium levels. When chromaffin granule membranes were delipidated by solvent extraction, calmodulin binding was observed at 10(-4) M free calcium. However no binding was detected at lower calcium concentrations. Thus it appears that a calcium concentration of 10(-7) M promotes the binding of calmodulin to some solvent-soluble components of the chromaffin granule membrane. Calmodulin-binding proteins associated with the granule membrane identified by photoaffinity cross-linking. A calmodulin-binding protein complex, of molecular weight 82K, was formed in the presence of 10(-4) M free calcium. This cross-linked product was specific because it was not detected either in the absence of calcium, in the presence of nonlabeled calmodulin, or in the absence of cross-linker activation. When solvent-treated membranes were used, a second, specific, calmodulin-binding protein complex (70K) was formed. Since the apparent molecular weight of calmodulin in our electrophoresis system was 17K, these experiments suggested the presence of two calmodulin-binding proteins, of molecular weights 65K and 53K, in the chromaffin granule membrane. This result was confirmed by the use of calmodulin-affinity chromatography. When detergent-solubilized membranes were applied on the column in the presence of calcium, two polypeptides of apparent molecular weights of 65K and 53K were specifically eluted by EGTA buffers. Since detergent treatments or solvent extractions are necessary to detect the 53K calmodulin-binding protein, it is concluded that only the 65K calmodulin-binding polypeptide may play a role in the interaction between calmodulin and secretory granules in chromaffin cells.     

A Similar Calmodulin-Binding Protein Expressed in Chromaffin, Synaptic, and Neurohypophyseal Secretory Vesicles

The presence of calmodulin-binding proteins in three neurosecretory vesicles (bovine adrenal chromaffin granules, bovine posterior pituitary secretory granules, and rat brain synaptic vesicles) was investigated. When detergent-solubilized membrane proteins from each type of secretory organelle were applied to calmodulin-affinity columns in the presence of calcium, several calmodulin-binding proteins were retained and these were eluted by EGTA from the columns. In all three membranes, a 65-kilodalton (63 kilodaltons in rat brain synaptic vesicles) and a 53-kilodalton protein were found consistently in the EGTA eluate. 125I-Calmodulin overlay tests on nitrocellulose sheets containing transferred chromaffin and posterior pituitary secretory granule membrane proteins showed a similarity in the protein bands labeled with radioactive calmodulin. In the presence of 10(-4) M calcium, eight major protein bands (240, 180, 145, 125, 65, 60, 53, and 49 kilodaltons) were labeled with 125I-calmodulin. The presence of 10 microM trifluoperazine (a calmodulin antagonist) significantly reduced this labeling, while no labeling was seen in the presence of 1 mM EGTA. Two monoclonal antibodies (mAb 30, mAb 48), previously shown to react with a cholinergic synaptic vesicle membrane protein of approximate molecular mass of 65 kilodaltons, were tested on total membrane proteins from the three different secretory vesicles and on calmodulin-binding proteins isolated from these membranes using calmodulin-affinity chromatography. Both monoclonal antibodies reacted with a 65-kilodalton protein present in membranes from chromaffin and posterior pituitary secretory granules and with a 63-kilodalton protein present in rat brain synaptic vesicle membranes. When the immunoblotting was repeated on secretory vesicle membrane calmodulin-binding proteins isolated by calmodulin-affinity chromatography, an identical staining pattern was obtained. These results clearly indicate that an immunologically identical calmodulin-binding protein is expressed in at least three different neurosecretory vesicle types, thus suggesting a common role for this protein in secretory vesicle function.     

Calmodulin-binding proteins: visualization by 125I-calmodulin overlay on blots quenched with Tween 20 or bovine serum albumin and poly(ethylene oxide)

To streamline detection of calmodulin-binding proteins, blotting techniques for the electrophoretic transfer of proteins onto nitrocellulose filters, followed by overlay with 125I-calmodulin, have been adapted. Autoradiography of the 125I-calmodulin-labeled blots allows the identification and quantitation of proteins that possess affinity for calmodulin. Five protocols for suppressing nonspecific binding and for enhancing specific interactions of 125I-calmodulin with electrophoretically separated proteins were investigated. Tween 20 and bovine serum albumin alone, as well as combinations of bovine serum albumin and poly(ethylene oxide) or hemoglobin and gelatin, were evaluated as quenching and enhancing agents. Tween 20 proved highly effective for quenching nonspecific binding and for enhancing specific 125I-calmodulin binding of a 61,000-Mr rat brain protein, which was only faintly observed on blots quenched with proteins alone. However, Tween 20 dissociated 50% of 68,000-Mr proteins and 80% of 21,000-Mr 125I-labeled protein standards from the nitrocellulose filter. An alternative, the combination of bovine serum albumin followed by incubation with 15,000- to 20,000-Mr poly(ethylene oxide), proved satisfactory for the recovery of 61,000-Mr calmodulin-binding activity and for the detection of calmodulin-binding peptides (50,000 to 14,000 Mr) produced by limited proteolysis of rat brain 51,000-Mr calmodulin-binding protein. These blotting procedures for detection of calmodulin-binding proteins are compatible with a variety of one-dimensional and two-dimensional electrophoresis systems, including a two-dimensional electrophoresis system utilizing urea and sodium dodecyl sulfate in the first dimension and nonurea sodium dodecyl sulfate electrophoresis in the second, a system which proved useful for resolving calmodulin-binding proteins displaying anomalous electrophoretic migration in the presence of urea.     

Calmodulin-binding proteins in teleost retina, rod inner and outer segments, and rod cytoskeletons

125I-calmodulin gel overlay techniques have been used to identify calmodulin-binding proteins in teleost retina, in a rod fragment preparation which contains rod inner and outer segments (RIS-ROS), and in RIS-ROS cytoskeletons. We have previously shown that teleost rods change length in response to changes in light conditions, that rod movement is mediated by the actin filaments in the rod inner segment, and that both Ca2+ and cAMP appear to be involved in regulating rod movement. We report here the development of a rod fragment preparation (RIS-ROS), which retains the movable part of the rod, for use in biochemical analysis of rod motility. Gel overlay studies indicate that isolated whole retinas have six prominent calmodulin-binding proteins, migrating at 240 K, 190 K, 150 K, 61 K and a doublet at 18/19 K. In contrast, detached RIS-ROS have three different prominent calmodulin-binding proteins, migrating at 330 K, 33 K, and 31 K. RIS-ROS cytoskeletons have been produced by extraction with Triton X-100; they contain both actin filament bundles and microtubules associated with the connecting cilium. RIS-ROS cytoskeletons have 3 prominent calmodulin-binding proteins migrating at 240 and 18/19 K. These proteins produce faint bands in gel overlays of intact RIS-ROS, but prominent bands in overlays of whole retina. The 240 K protein of RIS-ROS cytoskeletons co-migrates with the 240 K calmodulin-binding subunit of rat brain fodrin. We suggest that the rod 240 K calmodulin-binding protein may be a spectrin-like protein which participates in Ca2+- and calmodulin-regulation of rod motility.     

Calmodulin-binding proteins are developmentally regulated in gametes and embryos of fucoid algae

Calcium-binding proteins and calmodulin-binding proteins were identified in gametes and zygotes of the marine brown algae Fucus vesiculosus, Fucus distichus, and Pelvetia fastigiata using gel (SDS-PAGE) overlay techniques. A calcium current appears to be important during cell polarization in fucoid zygotes (K.R. Robinson and L.F. Jaffe, 1975, Science 187, 70-72; K.R. Robinson and R. Cone, 1980, Science 207, 77-78), but there are no biochemical data on calcium-binding proteins in these algae. By using a sensitive 45Ca2+ overlay method designed to detect high-affinity calcium-binding proteins, at least 9-11 polypeptides were detected in extracts of fucoid gametes and zygotes. All samples had calcium-binding proteins with apparent molecular weights of about 17 and 30 kDa. A 17-kDa calcium-binding protein was purified by calcium-dependent hydrophobic chromatography and was identified as calmodulin by immunological and enzyme activator criteria. A 125I-calmodulin overlay assay was used to identify potential targets of calmodulin action. Sperm contained one major calmodulin-binding protein of about 45 kDa. Eggs lacked major calmodulin-binding activity. A 72-kDa calmodulin-binding protein was prominent in zygotes from 1-65 hr postfertilization. Both calmodulin-binding proteins showed calcium-dependent binding activity. Overall, the data suggest that the appearance and distribution of certain calcium-binding and calmodulin-binding proteins are under developmental regulation, and may reflect the different roles of calcium during fertilization and early embryogenesis.