Synthesis and Accumulation of Calmodulin in Suspension Cultures of Carrot (Daucus carota L.) : Evidence for Posttranslational Control of Calmodulin Expression

The expression of calmodulin mRNA and protein were measured during a growth cycle of carrot (Daucus carota L.) cells grown in suspension culture. A full-length carrot calmodulin cDNA clone isolated from a λgt10 library was used to measure steady-state calmodulin mRNA levels. During the exponential phase of culture growth when mitotic activity and oxidative respiration rates were maximal, calmodulin mRNA levels were 4- to 5-fold higher than they were during the later stages of culture growth, when respiration rates were lower and growth was primarily by cell expansion. Net calmodulin polypeptide synthesis, as measured by pulse-labeling in vivo with [³⁵S]methionine, paralleled the changes in calmodulin steady-state mRNA level during culture growth. As a consequence, net calmodulin polypeptide synthesis declined 5- to 10-fold during the later stages of culture growth. The qualitative spectrum of polypeptides synthesized and accumulated by the carrot cells during the course of a culture cycle, however, remained largely unchanged. Calmodulin polypeptide levels, in contrast to its net synthesis, remained relatively constant during the exponential phases of the culture growth cycle and increased during the later stages of culture growth. Our data are consistent with increased calmodulin polypeptide turnover associated with periods of rapid cell proliferation and high levels of respiration.     

Calmodulin and the cell cycle: Involvement in regulation of cell-cycle progression

Calmodulin levels are elevated twofold at late G1 and/or early S phases during the growth cycle of CHO-K1 cells. These levels are maintained throughout the remainder of the cell cycle until cytokinesis. The G1 daughter cells then contain half the intracellular calmodulin level found prior to cell division. Elevation of calmodulin at the G1-S boundary is independent of the length of G1, and the increase in calmodulin appears to be related to progression into S phase. The importance of calmodulin for G1-S progression is suggested by the ability of the anticalmodulin drug W13 to elicit specific and reversible progression delays into and through S phase.     

Calmodulin activity in yeast and mycelial phases of Ceratocystis ulmi

Abstract The yeast and mycelial phases of Ceratocystis ulmi contained roughly equivalent levels of calmodulin activity as determined by their ability to stimulate calmodulin-deficient bovine brain cAMP phosphodiesterase. This stimulation was calcium-dependent and could be inhibited by either dibucaine or trifluoperazine. Also, the concentration of dibucaine necessary to achieve the mycelium-to-yeast morphological conversion was found to be 3-fold greater in the presence of exogenous calcium. A model is presented in which only 30% of the cellular calmodulin need be complexed with calcium ions for mycelial development.     

The role of calmodulin in the gravitropic response of the Arabidopsis thaliana agr-3 mutant

Calmodulin, a primary plant calcium receptor, is known to be intimately involved with gravitropic sensing and transduction. Using the calmodulin-binding inhibitors trifluoperazine, W7 and calmidazolium, gravitropic curvature of Arabidopsis thaliana (L.) Heynh, ecotype Landsberg, roots was separable into two phases. Phase I was detected at very low concentrations (0.01 μM) of trifluoperazine and calmidazolium, did not involve growth changes, accounted for about half the total curvature of the root and may represent the specific contribution of the cap to gravity sensing. Phase II commenced around 1.0 μM and involved inhibition of both growth and curvature. The agr-3 mutant exhibited a reduced gravitropic response and was found to lack phase I curvature, suggesting that the mutation alters either use or expression of calmodulin. The sequences of wild-type and agr-3 calmodulin (CaM-1) cDNAs, which are root specific were completely determined and found to be identical. Upon gravitropic stimulation, wild-type Arabidopsis seedlings increased calmodulin mRNA levels by threefold in 0.5 h. On the other hand, gravitropic stimulation of agr-3 decreased calmodulin mRNA accumulation. The possible basis of the two phases of curvature is discussed and it is concluded that agr-3 has a lesion located in a general gravity transmission sequence, present in many root cells, which involves calmodulin mRNA accumulation.     

Calmodulin in Zajdela hepatoma cell growth

Calmodulin levels were measured in Zajdela hepatoma cells growing both in vivo and in culture, with respect to the distribution of the cells into G1 and S+G2 phases of the cell cycle and growth conditions. These levels, expressed on a per-microgram of protein basis, were significantly elevated at the G1-S boundary and maintained throughout the remainder of the cell cycle. This elevation of calmodulin took place independently of the culture conditions. Taken together with previous observations, these data suggest that a threshold concentration for calmodulin is required for progression through the cell cycle, DNA synthesis and cell division.     

Calmodulin N-methyltransferase. Partial purification and characterization

The distribution, properties, and substrate specificity of S-adenosylmethionine:calmodulin (lysine) N-methyltransferse (EC 2.1.1.60, calmodulin N-methyltransferase) of the rat have been studied. This enzyme is cytosolic and is found at high levels in tissues with high levels of calmodulin and at low levels in tissues with little calmodulin. In liver, heart, and skeletal muscle, which have low levels of calmodulin and very low calmodulin N-methyltransferase activity (a low ratio of calmodulin N-methyltransferase to calmodulin), calmodulin was found to be incompletely methylated, as judged by its ability to act as a substrate for purified calmodulin N-methyltransferase. Calmodulin N-methyltransferase was purified 470-fold with a 33% yield from rat testis cytosol, using ammonium sulfate precipitation and chromatography on DEAE-cellulose, CM-Sepharose, and Sephadex G-100. At pH 7.4, calmodulin N-methyltransferase did not bind to DEAE-cellulose, but bound strongly to CM-Sepharose. The enzyme eluted from Sephadex G-100 with an apparent molecular weight of 55,000. Purified calmodulin N-methyltransferase was incubated with extracts of rat tissues, and [methyl-3H]AdoMet and methylated proteins were resolved by electrophoresis in an attempt to discover substances other than calmodulin, but this enzyme only catalyzed the methylation of calmodulin, indicating a high degree of substrate specificity. Conditions were established for the in vitro preparative methylation of des(methyl)-calmodulin from Dictyostelium discoideum. Three moles of methyl/mol of calmodulin were incorporated into lysine 115 of des(methyl)calmodulin, resulting in the formation of 1 mol of trimethyllysine at the site normally methylated in calmodulins from most species. Activation of cyclic nucleotide phosphodiesterase by des(methyl)calmodulin was indistinguishable from activation by in vitro methylated or sham methylated Dictyostelium calmodulin, indicating that methylation does not affect the ability of calmodulin to activate this enzyme.     

The role of calmodulin in the proliferation of transformed and phenotypically normal tsASV-infected rat cells

NRK cells infected with a temperature-sensitive, transformation-defective mutant of avian sarcoma virus (ASV), tsLA23, behaved as if nontransformed at a nonpermissive 40 degrees C and were rendered quiescent by serum deprivation. These serum-deprived cells were stimulated to start entering S phase about 7 hours after serum addition at 40 degrees C or about 9 hours after shifting the cultures to 36 degrees C, a temperature allowing the production of active viral pp60src and expression of the transformed phenotype. The transit of both serum- and temperature-stimulated tsLA23-NRK cells through later G1 was inhibited by the unrelated calmodulin antagonists W7 and R24571. The former drug was found to block the cells at a point in the cell cycle no more than 2 hours from the G1/S transition. The weaker calmodulin antagonist, W5, was less effective in impairing progression. Thus, calmodulin is likely required for the transit of both transformed and phenotypically normal tsLA23-NRK cells through the later stages of their G1 phases. Cells neoplastically transformed by ASV contain more calmodulin than uninfected, non-neoplastic cells. At the nonpermissive 40 degrees C, the calmodulin content of the tsLA23-NRK cells dropped to the non-neoplastic level. When these phenotypically nontransformed cells were enabled to reenter the cell cycle while still in low-serum medium by a 40 to 36 degrees C shift, they passed through the G1 and S phases and divided without a concomitant rise in the total calmodulin content. Thus, a calmodulin rise does not appear to be required for the expression of one characteristic of transformed cells, i.e., reduced requirement for exogenous growth factors.     

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.     

Calmodulin and cell proliferation

The calmodulin content of synchronized Chinese hamster ovary (CHO-K₁) cells was determined at each phase of the cell cycle. The calmodulin content was minimum in the G₁ phase, increased after the cells entered S phase and reached the maximum level at the late G₂ or early M phase. When 30 μM of W-7 (calmodulin antagonist) was added at the S phase, the cell cycle was blocked at the late G₂ or early M phase. The addition of W-7 also prevented the morphological changes caused by cholera toxin. These results suggest that calmodulin plays an important role in the phases through S to M, possibly in the initiation of DNA synthesis and in the mitosis.     

Changes in calmodulin levels during development of the silkworm,Bombyx mori

Calmodulin levels were measured in various tissues during the larval-adult development of the silkworm, Bombyx mori. In the larval period, calmodulin levels in fat body, midgut and testis were in a range of 0.3–1.7 μg/mg protein and remained almost constant during larval growth. The silk gland contained a relatively high (0.2 μg/mg protein) level of calmodulin early in the fifth instar which gradually decreased during maturation of the larva. At pupation, testis calmodulin dropped from 1.5 to 1.7 μg/mg protein to about 1 μg/mg, and remained constant thereafter. The most striking change occurred in fat body calmodulin which fell from 0.5 to 0.6 μg/mg in the larval stage to 0.01–0.03 μg/mg during pupal-adult metamorphosis. Midgut calmodulin levels were unchanged at pupation and remained constant during pupal-adult development.When expressed on per g wet weight basis, calmodulin levels in silkworm tissues were comparable to mammalian tissue levels. However, only 2–4% of the total calmodulin in silkworm tissues was in a membrane-bound form compared to 20–60% for membrane-bound calmodulin in mammals.     

Calmodulin synthesis and accumulation during oogenesis and maturation of Xenopus laevis oocytes

The calmodulin levels in stage 6 Xenopus oocytes averaged 89 +/- 24 (SD) ng/oocyte and had largely accumulated by stage 3 of oogenesis. From stage 3 to early stage 6, calmodulin levels did not increase further. However, in large stage 6 oocytes (greater than 1.25 mm diam) calmodulin levels again rose to a level as high as 121 ng/oocyte. Calmodulin levels did not change during the maturation of stage 6 oocytes and the results of measurements on animal and vegetal oocyte halves from control and mature oocytes showed no evidence of a redistribution of calmodulin during maturation. Measurements of calmodulin synthesis in stages 1 and 2 oocytes, stage 4 oocytes, and stage 6 oocytes indicated that calmodulin was being synthesized continuously during oogenesis and that the rate of synthesis increased during oogenesis. In stage 1 and 2 oocytes (combined), the synthesis rate was 3.5 pg/hr/oocyte; in stage 4 oocytes it was 48 pg/hr/oocyte, and in large stage 6 oocytes the rate had increased to 160 pg/hr/oocyte. These changes in the rates of synthesis were discussed as they relate to the pattern of calmodulin accumulation during oogenesis.     

Posttranslational Modification of Calmodulin in Rat Brain and Pituitary

The posttranslational modification of calmodulin has been studied in six brain regions and the anterior pituitary. Carboxylmethylation, calmodulin converting enzyme, and calmodulin (lysine) N-methyltransferase activities were determined. Incubation of calmodulin with cytosolic extracts of these tissues in the presence of the methyl donor [methyl-3H]-S-adenosyl-L-methionine and identification of labeled proteins by gel electrophoresis and fluorography indicated that calmodulin is a substrate for protein carboxylmethyltransferase in all tissues tested. In hippocampus, caudate nucleus, cerebral cortex, and anterior pituitary, but not in cerebellum, superior colliculus, brainstem, or diencephalon, a second methylated protein was found when calmodulin was added to incubation mixtures. This protein was shown to be identical to the previously described product of calmodulin converting enzyme. Converted calmodulin was isolated by fast protein liquid chromatography and shown to be des(Lys)calmodulin, lacking the carboxy terminal lysine residue of calmodulin. The anterior pituitary had by far the highest levels of calmodulin converting enzyme; this enzyme, in turn, was identified as a cobalt-stimulated carboxylpeptidase B. In contrast to the regional differences in these parameters, the levels of calmodulin (lysine) N-methyltransferase did not differ greatly among brain regions, although regional differences in the activity of this enzyme were statistically significant.     

Dynamics of calmodulin in cerebral structures under the action of modulated UHF electromagnetic fields]

The influence of modulated UHF-electromagnetic fields (low intensity) on calmodulin levels in several brain structures was studied. It was shown that UHF-electromagnetic fields influence calmodulin levels in the hypothalamus and sensorimotor cortex. Its effect depends on modulation regimes.     

Modulation of a neuronal calmodulin mRNA species in the rat brain stem by reserpine

Reserpine evokes transsynaptic impulse activity by depleting catecholaminergic neurotransmitters in the rat brain. Previous studies suggest a relationship between catecholaminergic activity and calmodulin concentration. In this report we employ Northern blot analysis to examine the effect of a single subcutaneous injection of reserpine on levels of calmodulin mRNA species which are preferentially expressed in neurons of the rat brain. Regional differences in mRNA levels were also investigated byin situ hybridization and drug-induced changes were noted particularly in specific regions of the rat brain stem. The riboprobe used in thein situ hybridization study recognized a 4.0 kilobase neuronal calmodulin mRNA species (NGB1), which was derived from the rat CaM1 gene. A calmodulin radio-immunoassay was utilized to demonstrate a drug-induced increased in calmodulin protein levels in a region which included the brain stem.     

Functional properties of covalent beta-endorphin peptide/calmodulin complexes. Chlorpromazine binding and phosphodiesterase activation

The 31-residue neuropeptide porcine beta-endorphin was shown to inhibit the Ca2+-dependent calmodulin activation of highly purified bovine brain cyclic nucleotide phosphodiesterase (3',5'-cyclic AMP 5'-nucleotidohydrolase, EC 3.1.4.17). Using a series of deletion peptides, the minimal inhibitory peptide sequence was found to correspond to beta-endorphin residues 14-25, confirming previously reported results for crude enzyme preparations. A correlation was found between the relative inhibitory potency of a particular beta-endorphin deletion peptide and the efficacy of cross-linking that peptide to calmodulin with bis(sulfosuccinimidyl) suberate, strongly implicating peptide binding to calmodulin as the mechanism of the observed inhibition. We found that relatively modest concentrations of chlorpromazine significantly reduced the efficiency of cross-linking beta-endorphin 14-31 to calmodulin. Chlorpromazine-Sepharose affinity chromatography of peptide/calmodulin adducts showed that a significant portion of the cross-linked beta-endorphin 14-31/calmodulin complex (stoichiometry of 1 mol/mol) retained the ability to interact with the immobilized phenothiazine in a Ca2+-dependent and calmodulin-displaceable manner. In contrast, the 2:1 (peptide:protein) product exhibited no affinity for the immobilized phenothiazine. The use of this affinity chromatographic step allowed preparation of homogeneous populations of both 1:1 and 2:1 beta-endorphin 13-31/calmodulin complexes and assessment of their functional characteristics. Equilibrium binding studies with chlorpromazine revealed that the covalent attachment of one peptide molecule to calmodulin perturbed all phases of Ca2+-dependent drug binding, but the adduct still bound significant quantities of chlorpromazine. The 2:1 complex, however, showed little detectable binding of the phenothiazine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistochemical study of calmodulin in developing mouse testis

The purpose of this study was to determine the localization of calmodulin in the developing mouse testis by the indirect immunoperoxidase method. In addition, the amount of calmodulin in pachytene spermatocytes, spermatids, and residual bodies isolated from the mouse testis and epididymal spermatozoa was quantitated by the adenylate cyclase activation assay and by enzyme immunoassay. The relative levels of calmodulin in the developing mouse testis and in the isolated testicular germ cells were confirmed by western transfer staining. The level of immunoreactive calmodulin was very low in the testes from immature animals. In testes from the mature mouse, calmodulin was found to be localized in spermatocytes and spermatids, but was not found in spermatogonia, Sertoli cells, and interstitial cells. By contrast, immunochemical staining of tubulin was extremely intense in Sertoli cells. Biochemical determinations also showed that pachytene spermatocytes, round spermatids, spermatozoa, and residual bodies contained 14.9 micrograms, 15.8 micrograms, 2.3 micrograms and 5.2 micrograms of calmodulin per mg of protein, respectively. Both the immunochemical and the biochemical studies revealed that levels of calmodulin were high in the spermatocytes and in the round spermatids, as compared to the level in spermatozoa. This fact strongly suggests that the large amount of calmodulin in mammalian testes may be associated primarily with meiotic divisions and/or spermatogenesis.     

Purification and characterization of calmodulin from porcine renal medulla

A calcium sensitive phosphodiesterase (PDE) activated by an endogenous calmodulin was identified in the cytosolic fraction of porcine renal medulla. The PDE and calmodulin were separated from each other by DEAE-cellulose column chromatography. Calmodulin was purified from a heat-treated supernatant by column chromatography with DEAE-cellulose and hydroxylapatite. The purified renal calmodulin has a molecular weight of 17,500, is heatstable, and has a pI of 4.2. Activation of the renal PDE by calmodulin was immediate and stoichiometric. The renal calmodulin and PDE cross react with bovine brain calmodulin and PDE, indicating a lack of tissue and species specificity. Thus, renal calmodulin is very similar to bovine brain calmodulin. However, renal calmodulin did not affect detergent-solubilized or membrane-bound renal adenylate cyclase or the antidiuretic hormone-stimulated activity of the enzyme. These results suggest that calmodulin may function in the renal medulla to regulate cAMP levels by stimulation of PDE but not adenylate cyclase. However, the ubiquitous distribution of calmodulin in eukaryotic cells and its effects on a number of other enzymes allow the possibility that calmodulin may have a role in renal function other than cAMP metabolism.