Calmodulin in starfish oocytes: II. Trypsin treatment suppresses the trifluoperazine-sensitive step

The 1-methyladenine-induced oocyte maturation in starfish is reversibly inhibited by the anticalmodulin drug, trifluoperazine (TFP). However, when oocytes are exposed for 10 min to trypsin, they lose their sensitivity to TFP. Trypsin does not alter the length of the hormone-dependent period (1-methyladenine minimal contact time) or the 1-methyladenine concentration requirements. Trypsin-treated oocytes remain sensitive to other maturation inhibitors such as procaine, theophylline, caffeine, and D-600. Trypsin exposure modifies the protein pattern composition of the oocyte cortex (breakdown of a 140-kDa protein). TFP binding site localization was studied using fluorescence microscopy: in addition to a general diffuse fluorescence, staining is localized to probably acidic granules located in the cortex. Results are discussed in relation to calmodulin and plasma membrane calmodulin-dependent enzyme involvement in the stimulation of starfish oocyte maturation.     

Induction of starfish oocyte maturation by the beta gamma subunit of starfish G protein and possible existence of the subsequent effector in cytoplasm.

beta gamma subunits of G proteins were purified from starfish oocytes, and their role in the induction of oocyte maturation by 1-methyladenine was investigated. When injected into starfish oocytes, the purified beta gamma subunit of the starfish G protein induced germinal vesicle breakdown (GVBD) faster than that of bovine brain G protein. Injection of the starfish beta gamma into cytoplasm near the germinal vesicle (GV) induced GVBD earlier than when injected into the GV or the cytoplasm near the plasma membrane. Fluorescent-labeled beta gamma was retained in the injected area even after GVBD. Injected beta gamma also induced the formation of maturation-promoting factor as well as an increase of histone H1 kinase activity. These results suggest that beta gamma dissociates from alpha-subunit by the stimulation of 1-methyladenine and interacts with a cytoplasmic effector, which results in formation of active cdc2 kinase.     

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.     

Extraction and preliminary characterization of maturation-promoting factor from starfish oocytes

Cytoplasm of maturing starfish oocytes possesses a factor which induces maturation upon injection into immature oocytes. Such maturation-promoting factor (MPF) was extracted from maturing oocytes of Asterina pectinifera and characterized preliminarily. After 1-methyladenine (1-MeAde) treatment, maturing oocytes were packed in a centrifuge tube to remove jelly and excess medium, and then crushed by centrifugation. The turbid supernatant was homogenized with a buffer containing NaF, Na-beta-glycerophosphate, ATP, EGTA and leupeptin, followed by centrifugation. MPF extracted in the supernatant was purified partially by ammonium sulfate precipitation, hydrophobic chromatography on pentyl-agarose and gel filtration on Sephacryl S-300. The final material induced maturation in the recipient starfish oocytes when 0.5 ng of protein was injected in a volume of 400 pl. The maturation response included germinal vesicle breakdown, and formation of polar bodies and egg pronucleus. Such MPF preparation induced maturation in oocytes of Xenopus laevis as well. Further, starfish MPF was found to be a heat-labile protein; its molecular weight (MW) was estimated as 300 X 10(3) D by gel filtration and its sedimentation coefficient value as 5S by centrifugation on sucrose density gradients.     

Generality of the action of various maturation-promoting factors

It has been known in amphibians and starfishes that a cytoplasmic factor called maturation-promoting factor (MPF), produced in maturing oocytes under the influence of the maturation-inducing hormones, can induce germinal vesicle breakdown (GVBD) and the subsequent process of meiotic maturation. The present study revealed that injection of cytoplasm of maturing starfish oocytes (starfish MPF) into immature sea cucumber oocytes brought about maturation of the recipients. Amphibian MPF obtained from mature oocytes of Xenopus laevis or Bufo bufo was found to induce maturation of starfish oocytes following injection. Cytoplasm taken from cleaving starfish blastomeres induced maturation when injected into immature starfish oocytes. The maturation-inducing activity of cytoplasm of starfish blastomeres changed along with the mitotic cell cycle during 1- to 4-cell stages so far tested and reached a peak just before cleaving. Furthermore, an extract of mammalian cultured cells, CHO or V-79, synchronized in M phase, induced GVBD in starfish oocytes following injection, whereas S phase extract had little activity. These facts suggest that MPF generally brings about nuclear membrane breakdown in both meiosis and mitosis, and that the nature of MPF is very similar among vertebrates and invertebrates.     

Calmodulin triggers the resumption of meiosis in amphibian oocytes

The calcium-binding protein, calmodulin, has been purified from Xenopus laevis oocytes. This 18,500-dalton protein, pl 4.3, has two high-affinity calcium-binding sites per mole protein having a dissociation constant of 2.8 x 10(-6) M. Full-grown Xenopus oocytes, arrested in late G2 of the meiotic cell cycle, resumed meiosis when microinjected with 60-80 ng (3-4 pmol) of calmodulin in the form of a calcium-calmodulin complex. The timing of the meiotic events in these recipient oocytes was the same as that normally induced by progesterone. Xenopus ovarian calmodulin stimulated bovine brain phosphodiesterase (PDE) 3- to 10-fold in a calcium-dependent manner, but it had no apparent effect on ovarian PDE activity. A calcium-calmodulin-dependent protein kinase has been isolated from Xenopus oocytes using a calmodulin-Sepharose 4B affinity column. The possible role for this kinase in regulating the G2-M transition in oocytes has been discussed.     

Plasma Membrane Ca-ATPase of Radish Seedlings : II. Regulation by Calmodulin

The effect of calmodulin on the activity of the plasma membrane Ca-ATPase was investigated on plasma membranes purified from radish (Raphanus sativus L.) seedlings. Calmodulin stimulated the hydrolytic activity and the transport activity of the plasma membrane Ca-ATPase to comparable extents in a manner dependent on the free Ca²⁺ concentration. Stimulation was marked at low, nonsaturating Ca²⁺ concentrations and decreased increasing Ca²⁺, so that the effect of calmodulin resulted in an increase of the apparent affinity of the enzyme for free Ca²⁺. The pattern of calmodulin stimulation of the plasma membrane Ca-ATPase activity was substantially the same at pH 6.9 and 7.5, in the presence of ATP or ITP, and when calmodulin from radish seeds was used rather than that from bovine brain. At pH 6.9 in the presence of 5 micromolar free Ca²⁺, stimulation of the plasma membrane Ca-ATPase was saturated by 30 to 50 micrograms per milliliter bovine brain calmodulin. The calmodulin antagonist calmidazolium inhibited both basal and calmodulin-stimulated plasma membrane Ca-ATPase activity to comparable extents.     

Mode of activation of bovine brain inositol 1,4,5-trisphosphate 3-kinase by calmodulin and calcium.

The effect of Ca2+ and calmodulin (CaM) on the activation of purified bovine brain Ins(1,4,5)P3 kinase was quantified and interpreted according to the model of sequential equilibria generally used for other calmodulin-stimulated systems. Two main conclusions can be drawn. (i) CaM.Ca3 and CaM.Ca4 together are the biologically active species in vitro, as is the case for the great majority of other calmodulin targets. (ii) These species bind in a non-co-operative way to the enzyme with an affinity constant of 8.23 x 10(9) M-1, i.e. approx 10-fold higher than for most calmodulin-activated target enzymes. The dose-response curve of the activation of Ins(1,4,5)P3 kinase by calmodulin is not significantly impaired by melittin and trifluoperazine, whereas under very similar assay conditions the half-maximal activation of bovine brain cyclic AMP phosphodiesterase requires over 30-50-fold higher concentrations of CaM when 1 microM melittin or 20 microM-trifluoperazine is present in the assay medium. Similarly, 1 microM of the anti-calmodulin peptides seminalplasmin and gramicidin S, as well as 20 microM of N-(6-aminohexyl)-5-chloro-1-naphthalene-sulphonamide (W7), do not inhibit the activation process. These data suggest that binding and activation of Ins(1,4,5)P3 kinase require surface sites of calmodulin which are different from those involved in the binding of most other target enzymes or of model peptides.     

Free intracellular cations in echinoderm oocytes and eggs

The concentrations of Ca²⁺, Na⁺ and H⁺ in echinoderm oocytes and eggs were measured during maturation and activation using ion-selective microelectrodes. In both oocytes and eggs, from three species of starfish and two species of sea urchin, the resting level of cytosolic Ca²⁺ was about 10^-7 M. We did not detect any change in Ca²⁺ concentration either during hormone-induced oocyte maturation (starfish) or during egg activation (starfish and sea urchin) induced by spermatozoa or chemical agents. During 1-methyl-adenine induced maturation of starfish oocytes the intracellular level of Na⁺ increased from 12–35 mM to 40–90 mM, while the pH changed from 6.6–6.8 to 7.0–7.2 Aged oocytes, with intact germinal vesicles, also had elevated levels of Na⁺ and pH.     

Correlation between Calmodulin Activity and Gravitropic Sensitivity in Primary Roots of Maize

Recent evidence indicates a role for calcium and calmodulin in the gravitropic response of primary roots of maize (Zea mays, L.). We examined this possibility by testing the relationship between calmodulin activity and gravitropic sensitivity in roots of the maize cultivars Merit and B73 × Missouri 17. Roots of the Merit cultivar require light to be gravitropically competent. The gravitropic response of the Missouri cultivar is independent of light. The occurrence of calmodulin in primary roots of these maize cultivars was tested by affinity gel chromatography followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with bovine brain calmodulin as standard. The distribution of calmodulin activity was measured using both the phosphodiesterase and NAD kinase assays for calmodulin. These assays were performed on whole tissue segments, crude extracts, and purified extracts. In light-grown seedlings of the Merit cultivar or in either dark- or light-grown seedlings of the Missouri cultivar, calmodulin activity per millimeter of root tissue was about 4-fold higher in the apical millimeter than in the subtending 3 millimeters. Calmodulin activity was very low in the apical millimeter of roots of dark-grown (gravitropically nonresponsive) seedlings of the Merit cultivar. Upon illumination, the calmodulin activity in the apical millimeter increased to a level comparable to that of light-grown seedlings and the roots became gravitropically competent. The time course of the development of gravitropic sensitivity following illumination paralleled the time course of the increase in calmodulin activity in the apical millimeter of the root. The results are consistent with the suggestion that calmodulin plays an important role in the gravitropic response of roots.     

Triterpene Biosynthesis in the Latex of Euphorbia lathyris: Effect of Calmodulin Antagonists and Chlorinated Phenoxy Compounds

Recognized calmodulin antagonists and chlorinated phenoxyalkylamines were tested as inhibitors of mevalonate incorporation into triterpenols and their fatty acid esters in a centrifuged pellet from the latex of Euphorbia lathyris. The calmodulin antagonists, chlorpromazine (II), fluphenzine, and trifluoperazine were good inhibitors; I₅₀ values for II and trifluoperazine were 150 and 55 micromolar, respectively. Inhibition by the phenoxyalkylamines increased with increasing chlorine substitution, and I₅₀ for 2-(pentachlorophenoxy)ethyl N,N-diethylamine (IX) was 35 micromolar. The calmodulin-stimulated phosphodiesterase catalyzed hydrolysis of cAMP was used as an assay to quantitate the calmodulin antagonism of the tested compounds. Compounds II and IX were calmodulin antagonists over a concentration range similar to their effective range in the biosynthesis of triterpenes. The antagonism of the chlorinated phenoxy compounds increased in parallel to their inhibitory effect upon triterpene biosynthesis.     

Calmodulin stimulates DNA synthesis by rat liver cells

Incubation in low (0.02 mM)-calcium medium prevented T51B rat liver cells from initiating DNA synthesis. Raising the calcium concentration in the medium from 0.02 to 1.25 mM caused these arrested cells to initiate DNA synthesis 1–2 hours later. The possibility of this rapid DNA-synthetic response to calcium addition being mediated through Ca-calmodulin complexes was suggested by the following observations: It was blocked by the putative Ca-calmodulin blockers chlorpromazine and trifluoperazine; the trifluoperazine-inhibited cells were stimulated by purified rat calmodulin; and purified rat calmodulin itself (10^?7 to 10^?6 moles/l) mimicked calcium action, unless the already low ionic calcium concentration in the calcium-deficient medium was reduced further by adding the specific calcium chelator EGTA.     

Role of germinal vesicle material in producing maturation-promoting factor in starfish oocyte

In starfish, oocyte maturation is induced by 1-methyladenine (1-MeAde). 1-MeAde acts on the oocyte surface to produce a cytoplasmic maturation-promoting factor (MPF), which in turn brings about germinal vesicle breakdown and subsequent process of oocyte maturation. The participation of germinal vesicle material in the production of MPF was investigated with oocytes of the starfish, Asterina pectinifera. When enucleated oocytes or oocyte fragments without germinal vesicles were treated with 1-MeAde, MPF was found to be produced. However, the amount of MPF produced was small as compared with that in the case of intact oocytes with germinal vesicles. The capacity of the enucleated oocytes to produce MPF was restored when germinal vesicle material was injected. On the other hand, it has been known that the amount of MPF increases when MPF is injected into intact oocytes (amplification of MPF). However, in the case of enucleated oocytes such increase of MPF was no longer observed, suggesting that germinal vesicle material is required for MPF amplification.     

一种同时纯化钙谓神经磷酸酶和钙调素的有效方法

本文报导了一种能同时纯化钙调神经磷酸酶和钙调素的有效方法。牛脑粗提液经DE-52纤维素层析分段洗脱:0.5mol/L NaCl缓冲液洗脱峰经phenyl-sepharose亲和柱和G75 sephadex制得电泳纯钙调素。0.18mol/L KCl缓冲液洗脱峰经Affigel-Blue层析,硫酸铵盐析,钙调素亲和层析,G-200 Sephadex凝胶过滤制得电泳纯钙调神经磷酸酶。     

Calmodulin-like activity in Mucor rouxii

Abstract A modulator factor with properties similar to those of calmodulin was found and partially purified from the soluble fraction of Mucor rouxii . These properties include: heat-stability, stimulation in a Ca²⁺-dependent fashion of cyclic nucleotide phosphodiesterase from bovine brain, and inhibition of this process by trifluoperazine. This calmodulin-like activity was detected in the soluble fraction of both mycelium and yeast-like cells of the fungus.     

Calmodulin, a ganglioside-binding protein. Binding of gangliosides to calmodulin in the presence of calcium.

Ca(2+)-dependent ganglioside-binding protein was isolated from a soluble cytosol fraction of mouse brains using a ganglioside affinity column prepared with a mixture of bovine brain gangliosides. It was identified as calmodulin based on the following features identical with those of calmodulin: molecular weight, pI, chromatographic profile and amino acid sequences of lysyl-endopeptidase digests, and ability to activate cyclic nucleotide phosphodiesterase. Bovine brain calmodulin derivatized with 5-dimethylaminonaphthalene-1-sulfonyl (dansyl-calmodulin), tetramethylrhodamine isothiocyanate, or biotin was also shown to bind to the ganglioside affinity column Ca2+ dependently and elute with gangliosides GD1a, GD1b, GT1b, GQ1b, GM1, and GM2, melittin, and trifluoperazine but not with GgOse4Cer and oligosaccharides of GM1, GD1a, and GT1b. Modification of the Lys94 residue of calmodulin by biotinylation drastically reduced the capacity for ganglioside binding. Ganglioside GD1b caused a blue shift and increase in intensity of the fluorescence emission spectrum of dansyl-calmodulin in the presence of Ca2+. The increment in fluorescence was proportional to the amount of GD1b added and was maximal at the molar ratio of GD1b to calmodulin, approximately 7.8. Gangliosides are thus shown to specifically bind to calmodulin, and this binding may be a general mechanism for regulating calmodulin-dependent enzymes with consequent cellular response, such as cell differentiation.     

Calmodulin activates bovine-cardiac myosin light-chain kinase by increasing the affinity for myosin light-chain 2

The basic mechanism by which calmodulin activates bovine-cardiac muscle myosin light-chain kinase was investigated using highly purified preparations of mixed bovine-cardiac myosin light chains or isolated myosin light chain 2. The apparent contamination of these substrate proteins by calmodulin, as detected by activation of calmodulin-sensitive phosphodiesterase, was less than 4 parts/million and was undetectable by antibodies against calmodulin. The apparent KA for calmodulin was 2 nM and 20 nM in the presence of isolated myosin light-chain 2 and mixed myosin light chains, respectively. Purified bovine cardiac troponin C activated myosin light-chain kinase by about 10% at a concentration of 2 microM. Mixed myosin light chains were phosphorylated in the absence and presence of calmodulin and in the presence of calcium with a V of 11.1 and 11.0 mumol phosphate transferred min-1 (mg enzyme)-1, respectively. The apparent Km values for mixed myosin light chains were 8.0 and 0.35 mg/ml in the absence and presence of calmodulin, respectively. Similarly calmodulin lowered the Km value for isolated myosin light-chain 2 over 20-fold and increased the V value only about 1.5-fold. Activity observed in the absence of calmodulin was dependent on the presence of calcium and was suppressed by chelating free calcium either before or during a phosphorylation reaction. The apparent KA for calcium was 1.2 microM and 0.4 microM in the absence and presence of calmodulin. Activity in the absence of calmodulin was inhibited at very high concentrations of the 'specific' calmodulin antagonists W-7, trifluoperazine and R24571 with apparent IC50 values of 0.3 mM, 0.2 mM and 0.02 mM. Antibiotics raised against calmodulin suppressed completely the kinase activity in the presence of calmodulin but had no effect on the activity measured in its absence. These results suggest that calmodulin stimulates the activity of bovine-cardiac myosin light-chain kinase by increasing over 20-fold the affinity for its substrate myosin light-chain 2.     

Calmodulin

Summary Ca²⁺ as an important cellular regulator has long been recognized. Calmodulin is unique among several proteins considered to be Ca²⁺ receptors in its ubiquitous distribution in eukaryotic cells and in its multiple effects through interaction with different enzymes and proteins. Apparently, calmodulin is the major Ca²⁺ receptor in most of these cells and most of metabolic active Ca²⁺ exists as a Ca²⁺-calmodulin complex.The importance of calmodulin as a Ca²⁺ mediator is also indicated by its role as the Ca²⁺-sensor in the regulation of Ca²⁺ pump which effectively maintains a low steady level of intracellular free Ca²⁺. The participation of calmodulin in the regulation of intracellular Ca²⁺ level suggests the desire for the cell to maintain adequate steady levels of metabolic active Ca²⁺. A low calmodulin concentration may in effect slow down the Ca²⁺ pump allowing a higher concentration of intracellular free Ca²⁺, but may also require higher Ca²⁺ threshold for Cat+ effects. A prominent difference in calmodulin contents of different eukaryotic cells has been noted and this difference may reflect the difference in the extents and the types of Ca²⁺-mediated reactions that operate in the cells. It is also possible that calmodulin concentration may fluctuate in response to different metabolic conditions. The evident for such possibility has been provided by the observations that cAMP-dependent protein kinase and ATP together with cAMP or neurotransmitters that stimulate cAMP synthesis cause the release of calmodulin from synaptic membranes (139, 140). However, the cytosolic calmodulin increased as the result of its release from the membranes is unlikely to be sufficient for eliciting calmodulin-mediated Ca²⁺ effects without a concomitant significant increase of intracellular Ca²⁺. The calmodulin release, in effect, may decrease the Ca²⁺ threshold of these effects.The manifestation of calmodulin-mediated Ca²⁺ effects in a particular type of cells appears determined mainly by the calmodulin-regulated enzymes existing in the cells. Within the same cells, however, the particular species of Ca²⁺-calmodulin complex serving as the active calmodulin, the affinity of the enzyme for the active calmodulin and the localization of the enzyme in the cells may determine the circumstance under which particular reactions are expressed.During the past years, substantial progress has been made in understanding calmodulin in terms of primary structure and molecular properties and in discovering many Ca²⁺-dependent, calmodulin-regulated enzymes and cellular activities. Our understanding of calmodulin and its relation to the wide range of Ca²⁺-dependent enzymes and activities has provided a framework for comprehending Ca²⁺ functions in the cells at the molecular level. Further works, however, are required to unravel fully the detailed mechanisms and properties that govern the calmodulin-enzyme interactions and to narrow further the gaps between Ca²⁺-elicited cellular expressions and the molecular events that lead to such expressions.     

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.