Dependent Electrophoretic Mobility Research Articles

Regulatory Phosphorylation of Bacterial-Type PEP Carboxylase by the Ca2+-Dependent Protein Kinase RcCDPK1 in Developing Castor Oil Seeds

Phosphoenolpyruvate carboxylase (PEPC) is a tightly controlled cytosolic enzyme situated at a crucial branch point of central plant metabolism. In developing castor oil seeds (Ricinus communis) a novel, allosterically desensitized 910-kD Class-2 PEPC hetero-octameric complex, arises from a tight interaction between 107-kD plant-type PEPC and 118-kD bacterial-type (BTPC) subunits. The native Ca2+-dependent protein kinase (CDPK) responsible for in vivo inhibitory phosphorylation of Class-2 PEPC's BTPC subunit's at Ser-451 was highly purified from COS and identified as RcCDPK1 (XP_002526815) by mass spectrometry. Heterologously expressed RcCDPK1 catalyzed Ca2+-dependent, inhibitory phosphorylation of BTPC at Ser-451 while exhibiting: (i) a pair of Ca2+ binding sites with identical dissociation constants of 5.03 μM, (ii) a Ca2+-dependent electrophoretic mobility shift, and (iii) a marked Ca2+-independent hydrophobicity. Pull-down experiments established the Ca2+-dependent interaction of N-terminal GST-tagged RcCDPK1 with BTPC. RcCDPK1-Cherry localized to the cytosol and nucleus of tobacco bright yellow-2 cells, but colocalized with mitochondrial-surface associated BTPC-enhanced yellow fluorescent protein when both fusion proteins were coexpressed. Deletion analyses demonstrated that although its N-terminal variable domain plays an essential role in optimizing Ca2+-dependent RcCDPK1 autophosphorylation and BTPC transphosphorylation activity, it is not critical for in vitro or in vivo target recognition. Arabidopsis (Arabidopsis thaliana) CPK4 and soybean (Glycine max) CDPKβ are RcCDPK1 orthologs that effectively phosphorylated castor BTPC at Ser-451. Overall, the results highlight a potential link between cytosolic Ca2+ signaling and the posttranslational control of respiratory CO2 refixation and anaplerotic photosynthate partitioning in support of storage oil and protein biosynthesis in developing COS.

The ionic strength dependent zeta potential at the surface of hexadecane droplets in water and the corresponding interfacial adsorption of surfactants.

An anomalous maximum in the ionic strength dependent electrophoretic mobility curves has been observed in previous reports from particles dispersed in colloids. This maximum has been considered anomalous because it is contradictory with the Gouy-Chapman model. The existence of such a maximum has been attributed to specific ionic adsorption, a hairy layer at the surface, or the effect of the anomalous change of surface conductivity in different studies. It was also pointed out that the O'Brien-White approach based on the Gouy-Chapman model could be used to understand this maximum in electrophoretic mobility curves and lead to understandable zeta potential curves. This implied that the observed maximum was actually not "anomalous". In this work we report our simulation of ionic strength dependent zeta potential curves based on the O'Brien-White approach and experimental studies of the ionic strength dependent electrophoretic mobility of the hexadecane droplets in the hexadecane-water emulsions at different pH or in the presence of sodium dodecyl sulphate at varied concentrations. In some cases, the simulation shows that the calculation with the O'Brien-White approach does change the trend in the concerned ionic strength dependent curves. However, the simulation in some other cases also leads to similar trends in the ionic strength dependent electrophoretic mobility curves and zeta potential curves. In the experiments, both the existence and non-existence of such a maximum were observed and demonstrated to be system dependent. The corresponding molecular structure of the oil-water interface was then discussed with the analyses of the zeta potential curves and second harmonic generation signals recorded at the hexadecane-water interface.

Complex Temperature Dependent Equilibria Dictate DNA Polymerase Exchange Processes during Synthesis

Most organisms encode for multiple DNA polymerases with similar substrate affinities, but vastly different fidelities. Proper genomic maintenance by the high fidelity (PolB1) and lesion bypass polymerases (PolY) from Sulfolobus solfataricus involves a complex solution equilibria of protein complexes and specific recognition of appropriate DNA substrates. Using isothermal titration calorimetry (ITC) and temperature dependent electrophoretic mobility shift assays (tEMSAs), we have found differences in oligomeric assemblies of Dpo1 and Dpo4 on DNA that include unusually strong temperature dependence changes in heat capacity (ΔCp), which switch from positive to negative values as temperature increases over a 60°C range. The thermodynamic data suggests that binding of PolB1 to DNA is favored over PolY by changes in solution multiequilibria (monomer-oligomer) with temperature that influence ΔCp values. We have also followed polymerase exchange events between PolB1 and PolY and themselves and with the sliding clamp, PCNA, during active DNA synthesis using ensemble kinetic and fluorescence resonance energy transfer (FRET) assays. Surprisingly, the assembled PolB1 holoenzyme complex synthesizes DNA distributively and with low processivity, unlike most other well-characterized DNA polymerase holoenzyme complexes. Exchange between polymerases is temperature and concentration dependent process that is orchestrated by several contacts with PCNA. Simultaneous binding of PolB1 and PolY1 to PCNA allows for dynamic exchange of polymerase active sites during replication and lesion bypass synthesis. Taken together, our results distinguish between specific thermodynamic parameters including temperature dependent coupled equilibria and structural complementary; kinetic processes; and protein contacts that direct binding for uninterrupted but dynamic DNA replication and repair processes at high temperatures.

AtCML8, a calmodulin-like protein, differentially activating CaM-dependent enzymes in Arabidopsis thaliana

Plants express many calmodulins (CaMs) and calmodulin-like (CML) proteins that sense and transduce different Ca(2+) signals. Previously, we reported divergent soybean (Glycine max) CaM isoforms (GmCaM4/5) with differential abilities to activate CaM-dependent enzymes. To elucidate biological functions of divergent CaM proteins, we isolated a cDNA encoding a CML protein, AtCML8, from Arabidopsis. AtCML8 shows highest identity with GmCaM4 at the protein sequence level. Expression of AtCML8 was high in roots, leaves, and flowers but low in stems. In addition, the expression of AtCML8 was induced by exposure to salicylic acid or NaCl. AtCML8 showed typical characteristics of CaM such as Ca(2+)-dependent electrophoretic mobility shift and Ca(2+) binding ability. In immunoblot analyses, AtCML8 was recognized only by antiserum against GmCaM4 but not by GmCaM1 antibodies. Interestingly, AtCML8 was able to activate phosphodiesterase (PDE) but did not activate NAD kinase. These results suggest that AtCML8 acts as a CML protein in Arabidopsis with characteristics similar to soybean divergent GmCaM4 at the biochemical levels.

Measurement of the Field Dependent Electrophoretic Mobility of Surface Modified Silica/AOT Suspensions

We have investigated the electrophoretic mobility of silica spheres in a silica/AOT/paraffin ternary system as a function of the applied electric field and observed a large discrepancy with data published on a similar system ( Jin, F. H. ; Davis, H. T. ; Evans, D. F. Int. Conf. Digital Printing Technol. 1998, 206-209 ). We attribute the discrepancy to an artifact in the measurement technique used to obtain that published data. We believe the artifact is caused by high velocity particles being "swept" from the measurement volume, thereby biasing the result toward lower mobilities. Thus, the published measurements appear to indicate a high field plateau in mobility data around 500 kV/m. Our results indicate that the silica/AOT/paraffin solution is reaching dielectric breakdown prior to a conclusive high field plateau being unambiguously measured. At low field the mobility is in general agreement with the prior work ( Jin, F. H. ; Davis, H. T. ; Evans, D. F. Int. Conf. Digital Printing Technol. 1998, 206- 209 ). Any proposed models of apparent charging of particles which utilize the presence of a high field plateau are inappropriate unless the high field plateau is clearly established. Our work indicates that caution is required regarding the measurement of electrophoretic mobility at high electric fields.

P19 detected in the rat retina and pineal gland is a guanylyl cyclase-activating protein (GCAP).

The Ca(2+)-dependent activation of retina-specific guanylyl cyclase (retGC) is mediated by guanylyl cyclase-activating proteins (GCAPs). Here we report for the first time detection of a 19 kDa protein (p19) with GCAP properties in extracts of rat retina and pineal gland. Both extracts stimulate synthesis of cGMP in rod outer segment (ROS) membranes at low (30 nM) but not at high (1 microM) concentrations of Ca(2+). At low Ca(2+), immunoaffinity purified p19 activates guanylyl cyclase(s) in bovine ROS and rat retinal membranes. Moreover, p19 is recognized by antibodies against bovine GCAP1 and, similarly to other GCAPs, exhibits a Ca(2+)-dependent electrophoretic mobility shift.

P19 detected in the rat retina and pineal gland is a guanylyl cyclase-activating protein (GCAP).

The Ca(2+)-dependent activation of retina-specific guanylyl cyclase (retGC) is mediated by guanylyl cyclase-activating proteins (GCAPs). Here we report for the first time detection of a 19 kDa protein (p19) with GCAP properties in extracts of rat retina and pineal gland. Both extracts stimulate synthesis of cGMP in rod outer segment (ROS) membranes at low (30 nM) but not at high (1 microM) concentrations of Ca(2+). At low Ca(2+), immunoaffinity purified p19 activates guanylyl cyclase(s) in bovine ROS and rat retinal membranes. Moreover, p19 is recognized by antibodies against bovine GCAP1 and, similarly to other GCAPs, exhibits a Ca(2+)-dependent electrophoretic mobility shift.

A rapid induction by elicitors of the mRNA encoding CCD-1, a 14kDa Ca2+ -binding protein in wheat cultured cells.

Intracellular Ca(2+) has been implicated in the signal transduction processes during the development of the plant defense system against fungal pathogens. From wheat cultured cells that had been treated with the elicitor derived from Typhula ishikariensis, the ccd-1 gene encoding a 14 kDa Ca(2+)-binding protein with an acidic amphiphilic feature was isolated. The ccd-1-encoded protein (CCD-1) shares homology to the C-terminal half domain of centrin, a Ca(2+)-binding protein conserved in eukaryotes. Unlike typical eukaryotic centrins, CCD-1 contains only one Ca(2+)-binding loop, which corresponds to the one in the fourth EF-hand from the N-terminus of centrin. The recombinant CCD protein expressed in Escherichia coli bound to a phenyl-Sepharose column in the presence of Ca(2+) and was eluted out by EGTA. It also showed a Ca(2+)-dependent electrophoretic mobility shift on the non-denaturing polyacrylamide gel. The ccd-1 mRNA expression was rapidly induced by treatment with fungal and chitosan oligosaccharide elicitors, implying that it might have a role in transducing Ca(2+) signals provoked by the elicitors. The expression of the ccd-1 mRNA was induced by treatment with A23187, and the induction was suppressed by La(3+) or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). This study suggests the involvement of intracellular Ca(2+) in the elicitor-induced mRNA expression of a novel class of Ca(2+)-binding proteins conserved in higher plants.

Cloning of a cDNA encoding SjIrV1, a Schistosoma japonicum calcium-binding protein similar to calnexin, and expression of the recombinant protein in Escherichia coli

Membrane-associated proteins were isolated from adult Philippine strain Schistosoma japonicum by partitioning into the detergent phase of Triton X-114. A rabbit polyclonal antiserum raised against these proteins was used to screen an S. japonicum expression cDNA library. Positive clones were identified which encoded the species orthologue of SmIrV1, a Schistosoma mansoni protein which was initially identified by screening with sera from mice protectively vaccinated with irradiated cercariae [Hawn et al., J. Biol. Chem. 268 (1993) 7692–7698]. The S. japonicum molecule, which we term SjIrV1, is 83% identical to SmIrV1 at the predicted amino acid level and is a member of the calreticulin family of non-EF-hand, calcium-binding proteins. The Chinese strain S. japonicum orthologue of SjIrV1 was obtained by screening with the radiolabelled insert of the Philippine strain clone. Northern blot analysis revealed a single message of around 2.4 kb and gave no indication of alternative splicing. Southern blot analysis gave a simple pattern, indicating a single-copy gene, and showed a single restriction fragment length polymorphism between the genomes of Chinese and Philippine strains of S. japonicum. Recombinant, full-length SjIrV1 was expressed with a hexahistidine tag in Escherichia coli and the recombinant protein isolated by nickel-chelate chromatography. Recombinant SjIrV1 was shown to exhibit calcium-dependent, differential electrophoretic migration and to bind ruthenium red in the absence but not in the presence of calcium ions. The presence of conserved Ca 2+-binding motifs predicted from the primary sequence, together with the Ca 2+-dependent electrophoretic mobility of recombinant SjIrV1, confirmed that SjIrV1 was a functional calcium-binding protein.

Molecular cloning of a novel Ca2+-binding protein that is induced by NaCl stress.

Plant responses to high salt stress have been studied for several decades. However, the molecular mechanisms underlying these responses still elude us. In order to understand better the molecular mechanism related to NaCl stress in plants, we initiated the cloning of a large number of NaCl-induced genes in Arabidopsis. Here, we report the cloning of a cDNA encoding a novel Ca2+-binding protein, named AtCP1, which shares sequence similarities with calmodulins. AtCP1 exhibits, in particular, a high degree of amino acid sequence homology to the Ca2+-binding loops of the EF hands of calmodulin. However, unlike calmodulin, AtCP1 appears to have only three Ca2+-binding loops. We examined Ca2+ binding of the protein by a Ca2+-dependent electrophoretic mobility shift assay. A recombinant AtCP1 protein that was expressed in Escherichia coli did show a Ca2+-dependent electrophoretic mobility shift. To gain insight into the expression of the AtCP1 gene, northern blot analysis was carried out. The AtCP1 gene had a tissue-specific expression pattern: high levels of expression in flower and root tissues and nearly undetectable levels in leaves and siliques. Also, the expression of the AtCP1 gene was induced by NaCl treatment but not by ABA treatment. Finally, subcellular localization experiments using an AtCP1:smGFP fusion gene in soybean suspension culture cells and tobacco leaf protoplasts indicate that AtCP1 is most likely a cytosolic protein.

Identification of a Novel Divergent Calmodulin Isoform from Soybean Which Has Differential Ability to Activate Calmodulin-dependent Enzymes

Calmodulin plays pivotal roles in the transduction of various Ca(2+)-mediated signals and is one of the most highly conserved proteins in eukaryotic cells. In plants, multiple calmodulin isoforms with minor amino acid sequence differences were identified but their functional significances are unknown. To investigate the biological function of calmodulins in the regulation of calmodulin-dependent enzymes, we cloned cDNAs encoding calmodulins in soybean. Among the five cDNAs isolated from soybean, designated as SCaM-1 to -5, SCaM-4 and -5 encoded very divergent calmodulin isoforms which have 32 amino acid substitutions from the highly conserved calmodulin, SCaM-1 encoded by SCaM-1 and SCaM-3. SCaM-4 protein produced in Escherichia coli showed typical characteristics of calmodulin such as Ca(2+)-dependent electrophoretic mobility shift and the ability to activate phosphodiesterase. However, the extent of mobility shift and antigenicity of SCaM-4 were different from those of SCaM-1. Moreover, SCaM-4 did not activate NAD kinase at all in contrast to SCaM-1. Also there were differences in the expression pattern of SCaM-1 and SCaM-4. Expression levels of SCaM-4 were approximately 5-fold lower than those of SCaM-1 in apical and elongating regions of hypocotyls. In addition, SCaM-4 transcripts were barely detectable in root whereas SCaM-1 transcripts were as abundant as in apical and elongating regions of hypocotyls. In conclusion, the different biochemical properties together with differential expression of SCaM-4 suggest that this novel calmodulin may have different functions in plant cells.

Characterization of the human calmodulin-like protein expressed in Escherichia coli.

The protein-coding region of an intronless human calmodulin-like gene [Koller, M., & Strehler, E. E. (1988) FEBS Lett. 239, 121-128] has been inserted into a pKK233-2 expression vector, and the 148-residue, M(r) = 16,800 human protein was purified to apparent homogeneity by phenyl-Sepharose affinity chromatography from cultures of Escherichia coli JM105 transformed with the recombinant vector. Several milligrams of the purified protein were obtained from 1 L of bacterial culture. A number of properties of human CLP were compared to those of bacterially expressed human calmodulin (CaM) and of bovine brain CaM. CLP showed a characteristic Ca(2+)-dependent electrophoretic mobility shift on SDS-polyacrylamide gels, although the magnitude of this shift was smaller than that observed with CaM. CLP was able to activate the 3',5'-cyclic nucleotide phosphodiesterase to the same Vmax as normal CaM, albeit with a 7-fold higher Kact. In contrast, the erythrocyte plasma membrane Ca(2+)-ATPase could only be stimulated to 62% of its maximal CaM-dependent activity by CLP. CLP was found to contain four Ca(2+)-binding sites with a mean affinity constant of 10(5) M-1, a value about 10-fold lower than that for CaM under comparable conditions. The highly tissue-specifically-expressed CLP represents a novel human Ca(2+)-binding protein showing characteristics of a CaM isoform.

A soluble calcium-binding protein (scbp) present in drosophila melanogaster and calliphora erythrocephala muscle cells

1. 1. Soluble calcium binding proteins (SCBP) were isolated from homogenates of whole flies, from the thorax and from muscles of Drosophila melanogaster and Calliphora erythrocephala. 2. 2. Crude preparations were obtained by extraction at low ionic strength, acid and heat treatment. The Drosophila protein was purified by gel filtration, hydrophobic interaction and ion exchange chromatography. In contrast to calmodulin the Drosophila SCBP did not bind to phenyl-Sepharose in a Ca 2+-dependent way. 3. 3. Both the Drosophila and Calliphora protein revealed identical properties. 4. 4. The apparent molecular mass of the SCBP is 24 kDa. Separation in urea-PAGE demonstrated the existence of two isoforms. 5. 5. The calcium-binding property was assured by a calcium dependent electrophoretic mobility shift and autoradiography of 45Ca 2+-incubated Western blots. 6. 6. The proteins are abundant in the thorax and were even detectable in crude extracts of various mascles (leg muscles and the extracoxal depressor). In contrast, in power muscles and in the thoracic ganglion the proteins could not be observed.

Stimulation of calmodulin synthesis in proliferating myoblasts by 1,25-dihydroxy-vitamin D 3

Myoblasts contain a receptor specific for 1,25-dihydroxy-vitamin D 3. Morphological data have indicated that the hormone stimulates both myoblast proliferation and fusion. The synthesis of myoblast proteins in response to the sterol was studied during the proliferating stage of the cells. Chick embryo myoblast primary cultures (precultured for 24 h in the presence of low levels of 1,25-dihydroxy-vitamin D 3 after isolation) were used. Labelling (2 h) of cells incubated in the absence and presence of 1,25-dihydroxy-vitamin D 3 (10 −10 M) for 1–12 h with [ 14C]leucine and [ 3H]leucine, respectively, followed by coelectrophoresis of double-labelled proteins on sodium dodecyl sulfate polyacrylamide gels showed that the sterol initially stimulates the synthesis of proteins of 60 kDa (1.2 h), 70 kDa (2.4 h) and 80 kDa (4 h). These changes were transient and between 6 and 12 h a protein of 19 kDa was induced. This protein was identified as calmodulin on the basis of its isoelectric point (p I 4.1), Ca 2+-dependent electrophoretic mobility, ability to bind 45Ca and to interact with an immobilized phenothiazine in a Ca 2+-dependent manner, and by means of immunoblotting with a specific anti-calmodulin antibody and 3′,5′-cyclic AMP phosphodiesterase activation assays. In agreement with these results, hybridization analysis with a specific cDNA probe showed increased calmodulin mRNA levels in myoblasts treated for 4–12 h with 1,25-dihydroxy-vitamin D 3. These changes were paralleled by a stimulation of [ 3H]thymidine incorporation into DNA suggesting that they may be involved in the mitogenic action of the hormone.

Rapid changes in the expression of a gene encoding a calcium-binding protein in Schistosoma mansoni

Genes expressed in a stage-specific manner may help us understand the molecular events controlling the complex life cycle of schistosomes. cDNA and genomic clones encoding a calcium-binding protein (CaBP) were obtained from cercariae and their sequence determined. The encoded protein (69 amino acids long) shows clear resemblance to the domain structure and organization of CaBP molecules. It contains two typical calcium-binding loops, the distance between which is identical to the length conserved in other CaBP molecules. In addition, the schistosome CaBP shows Ca 2+-dependent electrophoretic mobility (increased with Ca 2+-ions and decreased with EGTA). Northern blots revealed expression of the CaBP gene in cercariae but not in sporocyst or worm (developmental stages preceding and following cercaria). The preferential expression of this CaBP in the cercaria raises questions as to what cercaria-specific function(s) it performs. The structure of the gene is similar to that in other eukaryotes, and one intron interrupts the coding sequence. The region of the cap site was determined, and there was no evidence of the spliced leader sequence found in the mRNAs of other parasites. The CaBP reveals a rapid change in gene expression, since the mRNA is missing in the parasite residing in infected snails, but is readily detected in cercariae 1 h after shedding. We identified other genes which are turned on (like the CaBP) or shut off within the short period of transition from cercariae in the snail to free-swimming cercariae.

Identification of calcium binding proteins in two-dimensional gel electrophoretic pattern of Drosophila thorax and their distribution in two types of muscles.

Ten Drosophila thorax proteins (six myosin light chains and four proteins called a, b, c, d) were found to have high affinities with Ca2+. This was proved after subjecting the total Drosophila thorax proteins to two-dimensional (2D) transblot, followed by 45Ca2+ autoradiography. Three proteins (a, c, d) showed Ca2+ dependent electrophoretic mobility changes. To know their tissue-specific localization, fibrillar and tubular type muscle fibers were individually dissected from freeze-dried flies and separately subjected to 2D gel electrophoresis. Fibrillar type muscle had protein b and a small amount of protein a. Tubular type muscle had proteins c, d and a very large amount of protein a. Protein d was characterized to be calmodulin.

A soluble calcium-binding protein from the terrestrial annelidLumbricus terrestris L.

Soluble calcium-binding proteins (SCBP) considerably different from calmodulin were purified from the body wall muscle of the earthwormLumbricus terrestris. Three isoforms were obtained with similar UV absorption spectra and amino acid compositions and an apparent molecular weight close to 20 kDa. They can be distinguished by their histidine and proline content and by their peptide maps. The tissue content, as determined by quantitative ELISA varies individually from 0.1 to 0.3 mmol kg−1. The calcium-binding property can be demonstrated by Ca2+-dependent electrophoretic mobility shift and45Ca2+ autoradiography on nitrocellulose sheets. The apparentKD values for the SCBP-Ca2+ complex is approximately 10−7 mol l−1 as revealed by euquilibrium and flow dialysis experiments. In the presence of 1 mmol l−1 MgCl2 the maximum binding capacity of SCBP was determined to be either 2 mol Ca2+ mol−1 protein (SCBP2) or 3 mol Ca2+ mol−1 protein (SCBP3). Preliminary studies concerning the functional role of SCBP indicate that it facilitates the diffusion of Ca2+ ions by a factor of 2 and is capable of inhibiting the ATPase of isolated body wall muscle actomyosin. The results reveal that earthworm SCBP are similar to vertebrate parvalbumin and to SCBP characterized from aquatic invertebrates.

Vitamin D-3 induces the de novo synthesis of calmodulin in Phaseolus vulgaris root segments growing in vitro

Vitamin D-3 affects growth and Ca 2+ transport in Phaseolus vulgaris roots by a mechanism dependent on de novo protein synthesis. The objective of this work was to identify the protein(s) induced by the sterol. Phaseolus vulgaris root segments cultured in vitro were used. Protein extracts of control cultures and cultures treated with 10 −9 M vitamin D-3 for 2 h labelled with [ 14C]leucine and [ 3H]leucine, respectively, were mixed and electrophoresed on sodium dodecyl sulfate polyacrylamide gels. Examination of 3H: 14C ratios in slices of gels revealed that vitamin D-3 stimulates production of a protein which exhibited Ca 2+-dependent electrophoretic mobility. The apparent molecular weight was 14500 in the presence of 1 mM Ca 2+ and 18 000 in the presence of 5 mM EGTA. This protein was heat- and acid-stable, bound 45Ca 2+ and had an isoelectric pH of 3.8-4.1. These data are consistent with the properties of calmodulin. In agreement with these observations, higher levels of calmodulin were detected in roots treated with vitamin D-3 than in control roots by means of assays based on the activation of calmodulin-dependent enzymes. Moreover, estimation of calmodulin relative synthesis and degradation rates in roots cultures in the absence and presence of vitamin D-3 indicated that the sterol increases calmodulin synthesis without affecting its degradation. In addition, the results suggest that the synthesis of other low-molecular-weight Ca 2+-binding proteins may be affected by vitamin D-3.

Bacterial expression and characterization of proteins derived from the chicken calmodulin cDNA and a calmodulin processed gene.

Both normal chicken calmodulin (CaM) and a CaM-like mutant protein have been expressed in bacteria, isolated and evaluated with respect to several physical and biological properties. The mutant CaM is derived from a CaM-like gene that lacks intervening sequences and probably evolved from a CaM-processed gene (Stein, J. P., Munjaal, R. P., Lagacé, L., Lai, E. C., O'Malley, B. W., and Means, A. R. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 6485-6489). The mutant CaM protein contains 16 of the 19 amino acids encoded by the CaM-like gene. Normal chicken CaM produced in bacteria is identical to rat CaM by all criteria tested except that it is not trimethylated. The protein product of the CaM-like gene has been termed CaML and exhibits properties which are very similar to CaM despite the presence of 16 amino acid substitutions. CaML binds Ca2+ as evidenced by Ca2+-dependent binding to phenothiazine- and phenyl-Sepharose affinity resins and a Ca2+-dependent electrophoretic mobility shift which is similar to but distinct from CaM. CaML cross-reacts with a monospecific CaM antibody and has an immunodilution curve which is identical to bacterially synthesized CaM. Finally, CaML can maximally activate rat brain phosphodiesterase but with altered kinetic parameters as compared to CaM. These data suggest that the nucleotide substitutions in the putative CaM processed gene are not random but are selected to retain CaM-like functions in the encoded protein. Such a mechanism may exist for other processed genes.

Identification of calmodulin in the green alga Mougeotia and its possible function in chloroplast reorientational movement

A soluble protein was isolated from Mougeotia by chloropromazine-sepharose 4 B affinity chromatography. The protein matches the properties of calmodulin in terms of heat stability, Ca(2+)-dependent electrophoretic mobility in sodium-dodecyl-sulfate polyacrylamide gels, and its ability to activate cyclic nucleotide phosphodiesterase in a Ca(2+)-dependent manner. Phytochrome-mediated chloroplast reorientational movement in Mougeotia was inhibited by the calmodulin antagonist trifluoperazine, a hydrophobic compound, or N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a hydrophilic compound; 50% inhibition (IC50) of chloroplast movement is caused by 20-50 μmol l(-1) trifluoperazine or 100 μmol l(-1) W-7. The Ca(2+)-calmodulin may act as an intermediate in the chloroplast reorientational response in Mougeotia governed by phytochrome.