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.     

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 monoclonal antibody showing cross-reactivity toward three calmodulin-dependent enzymes

The spleen cells of a Balb/c mouse immunized with purified bovine calmodulin-dependent cyclic nucleotide phosphodiesterase were fused with nonsecreting mouse myeloma cells (P3-X63-Ag8-653). Antibody producing hybridomas were screened by the enzyme-linked immunosorbent assay using purified phosphodiesterase as the antigen. One monoclonal cell line, CR-B1, was found to produce antibodies which showed positive enzyme-linked immunosorbent assay reactions with bovine brain calcineurin and rabbit muscle phosphorylase kinase in addition to phosphodiesterase. The antibody was purified and characterized. It was shown to immunoprecipitate the calmodulin (CaM)-dependent phosphodiesterase and phosphorylase kinase activities but not those of CaM itself, CaM-independent phosphodiesterase and the catalytic unit of cAMP-dependent protein kinase. The immunoprecipitation of phosphodiesterase could be inhibited by calcineurin and phosphorylase kinase. These results suggest that the antibody interacts at a common site on these calmodulin-dependent proteins. The antigenic determinant in phosphodiesterase does not appear to reside in the calmodulin-binding domain of the enzyme since the antibody and phosphodiesterase interaction is not inhibited by calmodulin, and the calmodulin activation of phosphodiesterase is not affected by CR-B1 antibody. It is therefore suggested that the structural similarity among the three calmodulin-dependent proteins extends beyond the calmodulin-binding domains.     

The location of calmodulin in the pea plasma membrane

Plasma membrane has been prepared from pea seedlings in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA). Calmodulin has been detected in these plasma membrane preparations using calcium overlay techniques, immunoblots, quantitation with antibodies raised against spinach calmodulin, phosphodiesterase activation, mobility shift, and heat stability. EGTA-stable calmodulin represents 0.5-1% of the total plasma membrane protein, and it is the only detectable calcium-binding protein in plasma membrane isolated under these conditions. The anti-spinach calmodulin reacts only with the N-terminal region of spinach calmodulin representing residues 1-106. The positioning of EGTA-stable calmodulin in the plasma membrane has been probed with trypsin and anti-spinach calmodulin. The data suggest that the calmodulin N-terminal region representing residues 1-106 projects from the membrane and could be available for binding other proteins. Calcium-dependent calmodulin binding to the plasma membrane has also been detected. Calcium-dependent calmodulin-binding proteins have been characterized using calmodulin overlay methods. The exposure of calmodulin-binding domains of most of these proteins from the plasma membrane is further suggested by their reaction with azidoiodinated calmodulin.     

The autoantigen La/SSB is a calmodulinmbinding protein

The work reported here has been directed to the identification of new nuclear calmodulin-binding proteins. To achieve this goal, nuclei from rat hepatocytes were purified and a fraction enriched in DNA- and RNA-binding proteins was extracted using DNase I and RNase A. Calmodulin-binding proteins present in this nuclear subfraction were purified by chromatography using first a DEAE-Sephacel column and subsequently a calmodulin-Sepharose column. Four major polypeptides of 118, 107, 48 and 45 kDa were found to bind to the calmodulin column in a Ca²⁺-dependent way. [¹²⁵I]-calmodulin overlay analysis confirmed that the proteins of 118, 48 and 45 kDa are calmodulin-binding proteins. These proteins bind single-stranded and also double-stranded DNA. A partial amino acid sequence obtained from the 48 kDa protein revealed a 100% identity with the La/SSB protein, an autoantigen implicated in several autoimmune diseases, such as lupus erythematosus and Sjögren's syndrome. Two-dimensional gel electrophoresis, Western blot analysis and experiments of binding to poly(U), also supports the identity of p48 as La/SSB. CaM and La/SSB protein colocalize in the heterochromatinic regions within the nucleus of rat hepatocytes. Preincubation of La/SSB with calmodulin in the presence of Ca²⁺ resulted in an increase in the binding of ssDNA to La/SSB, suggesting that calmodulin can play a role in the regulation of the association of La/SSB with DNA.     

Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems

In response to the rapidly growing field of proteomics, the use of recombinant proteins has increased greatly in recent years. Recombinant hybrids containing a polypeptide fusion partner, termed affinity tag, to facilitate the purification of the target polypeptides are widely used. Many different proteins, domains, or peptides can be fused with the target protein. The advantages of using fusion proteins to facilitate purification and detection of recombinant proteins are well-recognized. Nevertheless, it is difficult to choose the right purification system for a specific protein of interest. This review gives an overview of the most frequently used and interesting systems: Arg-tag, calmodulin-binding peptide, cellulose-binding domain, DsbA, c-myc-tag, glutathione S-transferase, FLAG-tag, HAT-tag, His-tag, maltose-binding protein, NusA, S-tag, SBP-tag, Strep-tag, and thioredoxin.     

Production of reagents and optimization of methods for studying calmodulin-binding proteins

Owing to subtle but potentially crucial structural and functional differences between calmodulin (CaM) of different species, the biochemical study of low-affinity CaM-binding proteins from Dictyostelium discoideum likely necessitates the use of CaM from the same organism. In addition, most of the methods used for identification and purification of CaM-binding proteins require native CaM in nonlimiting biochemical quantities. The gene encoding D. discoideum CaM has previously been cloned allowing production of recombinant protein. The present study describes the expression of D. discoideum CaM in Escherichia coli and its straightforward and rapid purification. Furthermore, we describe the optimization of a complete palette of assays to detect as little as nanogram quantities of proteins binding CaM with middle to low affinities. Purified CaM was used to raise high-affinity polyclonal antibodies suitable for immunoblotting, immunofluorescence, and immunoprecipitation experiments. The purified CaM was also used to optimize a specific and sensitive nonradioactive CaM overlay assay as well as to produce a high-capacity CaM affinity chromatography matrix. The effectiveness of this methods is illustrated by the detection of potentially novel D. discoideum CaM-binding proteins and the preparatory purification of one of these proteins, a short tail myosin I.     

A plant kinesin heavy chain-like protein is a calmodulin-binding protein

Calmodulin, a calcium modulated protein, regulates the activity of several proteins that control cellular functions. A cDNA encoding a unique calmodulin-binding protein, PKCBP, was isolated from a potato expression library using protein-protein interaction based screening. The cDNA encoded protein bound to biotinylated calmodulin and ³⁵S-labeled calmodulin in the presence of calcium and failed to bind in the presence of EGTA, a calcium chelator. The deduced amino acid sequence of the PKCBP has a domain of about 340 amino acids in the C-terminus that showed significant sequence similarity with the kinesin heavy chain motor domain and contained conserved ATP- and microtubule-binding sites present in the motor domain of all known kinesin heavy chains. Outside the motor domain, the PKCBP showed no sequence similarity with any of the known kinesins, but contained a globular domain in the N-terminus and a putative coiled-coil region in the middle. The calmodulin-binding region was mapped to a stretch of 64 amino acid residues in the C-terminus region of the protein. The gene is differentially expressed with the highest expression in apical buds. A homolog of PKCBP from Arabidopsis (AKCBP) showed identical structural organization indicating that kinesin heavy chains that bind to calmodulin are likely to exist in other plants. This paper presents evidence that the motor domain has microtubule stimulated ATPase activity and binds to microtubules in a nucleotide-dependent manner. The kinesin heavy chain-like calmodulin-binding protein is a new member of the kinesin superfamily as none of the known kinesin heavy chains contain a calmodulin-binding domain. The presence of a calmodulin-binding motif and a motor domain in a single polypeptide suggests regulation of kinesin heavy chain driven motor function(s) by calcium and calmodulin.     

Identification and characterization of calmodulin-binding proteins in mammalian sperm flagella

A calcium and calmodulin-regulated cyclic nucleotide phosphodiesterase has been shown to be an integral component of both rat and bovine sperm flagella. The calcium-activated enzyme was inhibited by both trifluoperazine (ID50 = 10 microM) and [ethylene-bis(oxyethylenenitrilo)]tetraacetic acid (EGTA), and the basal activity measured in the presence of EGTA was stimulated by limited proteolysis to that observed in the presence of calcium/calmodulin. 125I-Calmodulin binding to purified rat sperm flagella has been characterized and the flagellar-associated calmodulin-binding proteins identified by a combination of gel and nitrocellulose overlay procedures and by chemical cross-linking experiments using dimethyl suberimidate. 125I-Calmodulin bound to demembranated rat sperm flagella in a time- and concentration-dependent manner. At equilibrium, 30-40% of the bound 125I-calmodulin remains associated with the flagella after treatment with EGTA or trifluoperazine. The majority of the bound 125I-calmodulin, both the Ca2+-dependent and -independent, was displaced by excess calmodulin. A 67-kDa calmodulin-binding protein was identified by both the gel and nitrocellulose overlay procedures. In both cases, binding was dependent on Ca2+ and was totally inhibited by trifluoperazine, EGTA, and excess calmodulin. On nitrocellulose overlays, the concentration of calmodulin required to decrease binding of 125I-calmodulin by 50% was between 10(-10) and 10(-11) M. Limited proteolysis resulted in the total loss of all Ca2+-dependent binding to the 67-kDa polypeptide. Chemical cross-linking experiments identified a major calcium-dependent 125I-calmodulin:polypeptide complex in the 84-90-kDa molecular mass range and a minor complex of approximately 200 kDa. Immunoblot analysis showed that the major 67-kDa calmodulin-binding protein did not cross-react with polyclonal antibodies raised against either the calcium/calmodulin-regulated cyclic nucleotide phosphodiesterase or phosphoprotein phosphatase (calcineurin) from bovine brain.     

Metabolically 35S-labeled recombinant calmodulin as a ligand for the detection of calmodulin-binding proteins

We have developed a simplified procedure for the production of metabolically labeled calmodulin. We used bacterial clones (Escherichia coli) that were found to express VU-1 calmodulin, a calmodulin that is fully active with a variety of calmodulin-regulated enzymes. VU-1 calmodulin was labeled with sulfur-35 in bacteria maintained in a sulfur-free medium. Calmodulin was then purified by chromatography on phenyl-Sepharose. Under these conditions, the specific activity of the proteins was 150 to 400 cpm/fmol of calmodulin. To demonstrate the utility of this labeled VU-1 calmodulin, we examined the calmodulin-binding proteins in aortic myocyte preparation from Day 0 and Day 15 cultures by using both the gel and the nitrocellulose overlay protocols. The results showed that calmodulin-binding proteins are easily detected by the two procedures and that the profile of these target proteins changed in myocyte with time in culture. While most of these calmodulin-binding proteins have not been identified, the relative mobility on SDS-PAGE gels suggests that myosin light chain kinase (Mr approximately 137,000) was detected by these methods. We demonstrated here that the nitrocellulose overlay was faster than the gel overlay and that this technique can be useful for the study of calmodulin-binding proteins.     

Modulation of calmodulin levels, calmodulin methylation, and calmodulin binding proteins during carrot cell growth and embryogenesis.

Carrot cell cultures were used to study the dynamics of calmodulin protein levels, calmodulin methylation, and calmodulin-binding proteins during plant growth and development. Comparisons of proliferating and nonproliferating wild carrot cells show that, while calmodulin protein levels does not vary significantly, substantial variation in post-translational methylation of calmodulin on lysine-115 is observed. Calmodulin methylation is low during the lag and early exponential stages, but increases substantially as exponential growth proceeds and becomes maximal in the postexponential phase. Unmethylated calmodulin quickly reappears within 12 h of reinoculation of cells into fresh media, suggesting that the process is regulated according to the cell growth state. Calmodulin and calmodulin-binding proteins were also analyzed during the formation and germination of domestic carrot embryos in culture. Neither calmodulin methylation nor calmodulin protein levels varied significantly during somatic embryogenesis. However, upon germination of embryos, the level of calmodulin protein doubled. By calmodulin overlay analysis, we have detected a major 54,000 M(r) calmodulin-binding protein that also increased during embryo germination. This protein was purified from carrot embryo extracts by calmodulin-Sepharose chromatography. Overall, the data suggest that calmodulin methylation is regulated depending upon the state of cell growth and that calmodulin and its target proteins are modulated during early plant development.     

Epitope mapping of antibodies against S-tagged fusion proteins and molecular weight markers

Monoclonal antibodies against S-tagged fusion proteins expressed in pET vectors were generated and further characterized. Most pET vectors contain a 15-meric S-tag as a fusion tag for the detection of recombinant proteins. Two antibodies, G18BA3 and G18BE8, recognized this S-tag in enzyme immunoassay and Western blot. Their epitopes were mapped using peptide array technology and were confirmed to be AAKFERQHMDSPD. This corresponds to the C-terminal region of the S-tag plus additional amino acids P and D, which are also present in most available pET vectors. Amino acid substitution analysis revealed several essential residues for binding. The binding motif was therefore FExxHxDxxD for G18BA3 and AxxFExxH for G18BE8. Since some commercially available protein standards are expressed in pET vectors, G18BA3 and G18BE8 were also found to detect the ladder bands of a molecular weight marker on immunoblot analysis. Both antibodies should be highly useful for the simultaneous detection of recombinant pET vector-expressed fusion proteins and protein molecular weight standards in Western blotting, especially when chemoluminescent detection systems are used.     

A nonradioactive method for isolating complementary DNAs endocing calmodulin-binding proteins

Calmodulin labeled with¹²⁵I or³⁴S has been used to screen expression libraries to isolate cDNAs encoding calmodulin-binding proteins (CBPs) from several eukaryotic systems. The use of radiolabeled calmodulin has, however, several disadvantages. We have developed a nonradiactive method to isolate cDNAs for CBPs using biotinylated calmodulin. Screening of a cDNA library in an expression vector with biotinylated calmodulin resulted in the isolation of cDNAs encoding CBPs. Avidin and biotin blocking steps, prior to incubation of the filters with biotinylated calmodulin, are found to be essential to eliminate the cDNAs that code for biotin-containing polypeptides. The cDNA clones isolated using this nonradioactive method bound calmodulin in a calcium-dependent manner. The binding of biotinylated calmodulin to these clones was completely abolished by ethylene glycolbis(\-aminoethylether)-N,N′-tetraacetic acid (EGTA), a calcium chelator. Furthermore, the isolated cDNAs were confirmed by probing the clones with³⁵S-labeled calmodulin. All the isolated clones bound to radiolabeled calmodulin in the presence of calcium but not in the presence of EGTA. The method described here is simple, fast, and does not involve preparation and handing of radiolabeled calmodulin. All the materials used in this method are commercially available; hence, this procedure should be widely applicable to isolate cDNAs encoding CBPs from any eukaryotic organism.     

Calmodulin-binding profiles for nebulin and dystrophin in human skeletal muscle

Nebulin and dystrophin are two high-molecular-mass skeletal muscle proteins that have both been associated with the defective gene in Duchenne muscular dystrophy, although the function of neither protein is known. Other high-molecular-mass, calmodulin-binding proteins have recently been implicated in regulating calcium release from skeletal muscle. Western blots of human skeletal muscle biopsy samples were probed with biotinylated calmodulin; nebulin was identified as a prominent high-molecular-mass calmodulin-binding protein but dystrophin did not bind detectable amounts of biotinylated calmodulin. Dystrophin was absent in a Duchenne muscle biopsy.     

Cyclical differentiation of Trypanosoma brucei involves changes in the cellular complement of calmodulin-binding proteins

The present study was undertaken to evaluate changes in the complement of calmodulin-binding proteins which accompany cyclical differentiation in Trypanosoma brucei. An [125I]trypanosome calmodulin overlay procedure was used to detect calmodulin-binding proteins with Mr of 126,000 and 106,000 that were present in homogenates of slender bloodstream froms but were absent in procyclic culture forms. Competition assays with unlabeled bovine brain or trypanosome calmodulins indicated that the developmentally regulated proteins associated with calmodulins from either source. Moreover, [125I]bovine brain calmodulin associated with the same proteins as trypanosome calmodulin. Homogenates of T. evansi exhibited the same pattern of calmodulin-binding activity as T. brucei slender bloodstream forms; however, T. cruzi and Leishmania tarentolae contained distinct patterns of calmodulin-binding activity. Mouse serum contained no detectable binding proteins while mouse brain contained predominantly proteins of Mr 210,000, 60,000, and 49,000 which were associated with the trypanosome calmodulin probe. The developmentally regulated calmodulin-binding proteins from T. brucei were in the 10,000g pellet. We conclude that the cellular complement of calmodulin-binding proteins varies during the trypanosome life cycle.     

Differential recognition of calmodulin-enzyme complexes by a conformation-specific anti-calmodulin monoclonal antibody

An anti-calmodulin monoclonal antibody having an absolute requirement for Ca2+ has been produced from mice immunized with a mixture of calmodulin and calmodulin-binding proteins. Radioimmune assays were developed for the determination of its specificity. the epitope for this antibody resides on the COOH-terminal half of the mammalian protein. Plant calmodulin or troponin C had little reactivity. The apparent affinity of the antibody for calmodulin was increased approximately 60-fold in the presence of heart calmodulin-dependent phosphodiesterase. The presence of heart phosphodiesterase in the radioimmune assay greatly enhanced the sensitivity for calmodulin. The intrinsic calmodulin subunit of phosphorylase kinase and calmodulin which was bound to brain phosphodiesterases was also recognized with high affinity by the antibody. The antibody reacted poorly with calmodulin which was bound to heart or brain calcineurin, skeletal muscle myosin light chain kinase, or other calmodulin-binding proteins. In direct binding experiments, most of the calmodulin-binding proteins studied were unreactive with the antibody. This selectivity allowed purification of heart and two brain calmodulin-dependent cyclic nucleotide phosphodiesterase isozymes on immobilized antibody affinity columns. Phosphodiesterase activity was adsorbed directly from crude samples and specifically eluted with EGTA. Isozyme separation was accomplished using a previously described anti-heart phosphodiesterase monoclonal antibody affinity support. The brain isozymes differed not only in reactivity with the anti-phosphodiesterase antibody, but also in apparent subunit molecular weight, and relative specificity for cAMP and cGMP as substrates. The calmodulin activation constants for the brain enzymes were 10-20-fold greater than for the heart enzyme. The data suggest that the binding of ligands to Ca2+/calmodulin induce conformation changes in calmodulin which alter reactivity with the anti-calmodulin monoclonal antibody. The differential antibody reactivity toward calmodulin-enzyme complexes indicates that target proteins either induce very different conformations in calmodulin and/or interact with different geometries relative to the antibody binding site. The anti-calmodulin monoclonal antibody should be useful for the purification of other calmodulin-dependent phosphodiesterases as well as isozymes of phosphorylase kinase.     

Calmodulin-binding proteins in brain

It is now widely accepted that actions of intracellular Ca²⁺ are mediated by a four-domain Ca²⁺-binding protein, calmodulin. Brain is especially rich in calmodulin, containing about 400 mg (24 μmol) of EGTA-extractable calmodulin per kg of brain. However, only a fraction of the above amount is required for the calmodulin-activated enzymes and most of the rest may be assigned to calmodulin-binding proteins, proteins which are apparently devoid of enzyme activities but undergo Ca²⁺-dependent associations with calmodulin. Several of such proteins have been recently discovered in brain. These include a heat-labile 80 K phosphodiesterase inhibitor protein (calcineurin), a heat-stable 70 K phosphodiesterase inhibitor protein, a 50 K protein, myelin basic protein, tubulin, microtubule τ (tau) factor, a spectrin-like doublet protein (240 plus 235 K) (calspectin; fodrin) and a particle-associated 155 K protein.Functions of these calmodulin-binding proteins have not been fully elucidated yet. Some proteins may be calmodulin-regulated enzymes catalyzing yet unknown biochemical reactions, e.g. a protein phosphatase activity was found for calcineurin. Some proteins may interact with contractile elements or cytoskeleton of the cell, e.g. τ factor and calspectin interacted with tubulin and F-actin, respectively and tubulin itself is a calmodulin-binding protein. So, interesting possibilities are the regulation of the functions of cytoskeleton by calmodulin through these calmodulin-binding proteins. Regulation of microtubule assembly by Ca²⁺-dependent binding of calmodulin to tubulin and/or τ factor and possible involvement of calspectin in the mechanism regulating axonal transport of neuronal proteins have been suggested. Thus, the exploration of the regulating functions of Ca²⁺/calmodulin in brain depends largely upon the further study of the properties of these calmodulin-binding proteins.     

Purification and characterization of 81K, heat stable calmodulin-binding protein from bovine brain

Heat stable calmodulin-binding protein has been purified from Triton X-100 soluble particulate fraction of bovine brain. Considerable purification was achieved with calmodulin coupled Sepharose 4B affinity chromatography. SDS-PAGE of the purified protein revealed the apparent homogeneity being 92% at Mr 81,000. Isoelectric focusing of purified 81K protein gave isoelectric point of 4.3. The amino acid composition was notable for high contents of acidic amino acids (15.0 mol% of glutamic acid and 8.1 mol% of aspartic acid) and 17.4 mol% of alanine. On alkaline 1 M urea gel electrophoresis, mobility of the purified 81K protein in the presence of Ca2+ and calmodulin became lower than 81K protein alone toward the anode; however, Ca2+ solely did not affect the mobility of this protein. Similarly, S-100 protein and troponin C showed the interaction with 81K protein and a decrease of mobility in the presence of Ca2+ in alkaline urea PAGE. Binding assay of 125I-labeled calmodulin revealed that 81K protein could bind to an equimolar of 125I-calmodulin as apparent dissociation constant (Kd) of 0.65 x 10(-6) M.     

Phosphorylation and characterization of bovine heart calmodulin-dependent phosphodiesterase.

Calmodulin-dependent phosphodiesterase was purified to apparent homogeneity from the total calmodulin-binding fraction of bovine heart in a single step by immunoaffinity chromatography. The isolated enzyme had significantly higher affinity for calmodulin than the bovine brain 60-kDa phosphodiesterase isozyme. The cAMP-dependent protein kinase was found to catalyze the phosphorylation of the purified cardiac calmodulin-dependent phosphodiesterase with the incorporation of 1 mol of phosphate/mol of subunit. The phosphodiesterase phosphorylation rate was increased severalfold by histidine without affecting phosphate incorporation into the enzyme. Phosphorylation of phosphodiesterase lowered its affinity for calmodulin and Ca2+. At constant saturating concentrations of calmodulin (650 nM), the phosphorylated calmodulin-dependent phosphodiesterase required a higher concentration of Ca2+ (20 microM) than the nonphosphorylated phosphodiesterase (0.8 microM) for 50% activity. Phosphorylation could be reversed by the calmodulin-dependent phosphatase (calcineurin), and dephosphorylation was accompanied by an increase in the affinity of phosphodiesterase for calmodulin.     

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).