Calcium-induced protein phosphorylation and changes in levels of calmodulin and calreticulin in maize sperm cells

 To examine possible calcium (Ca²⁺)-mediated prefertilization events in male gametes of higher plants, we studied protein phosphorylation and the Ca²⁺-binding proteins, calmodulin and calreticulin, in sperm cells isolated from maize (Zea mays L.) pollen in the presence and absence of Ca²⁺. Using immunoblotting, we detected calmodulin and calreticulin and Ca²⁺-induced variations. Exposure of sperm cells to 1 mM Ca²⁺ for 1 h increased calmodulin content by 136% compared with the control. Ca²⁺ had little effect on calreticulin at 1 h, but induced a 34% increase after 3 h. Phosphorylation of proteins was low in 1 h-control and Ca²⁺-treated cells. However, a 13-fold increase in phosphorylation of a 18-kDa protein was found at 12 h in the presence of Ca²⁺. Ca²⁺-induced changes in calmodulin, calreticulin and protein phosphorylation observed in maize sperm cells may reflect prefertilization changes in vivo that facilitate sperm cell fusion with egg and central cells. Received: 26 July 1996 / Revision accepted: 7 February 1997     

A Calcium Aspect of the Effect of Noradrenaline on the Resting Membrane Potential in Somatic Muscle Cells of the Earthworm

A hyperpolarizing effect of noradrenaline (NA) on muscle cells of the earthworm caused by activation of the membrane ion pumps is eliminated in a Ca-free medium, in the case of replacement of Na⁺ by Mn²⁺, or when verapamil or chlorpromazine have been added to the bath solution. A decrease or an increase in the Ca²⁺ concentration in the solution, as well as caffeine application, do not influence the resting membrane potential (RMP) of muscle cells. It is supposed that signal transmission from the membrane adrenoreceptors to the ion pump of earthworm muscle cells by NA is provided via entry of extracellular Ca²⁺ ions into the cell with subsequent involving of Ca²⁺ acceptor proteins similar to calmodulin in vertebrate animals.     

Nature of the repair process associated with the recovery of Escherichia coli after exposure to Cd2+.

When different strains of $\text{Escherichia coli}$ are exposed to Cd²⁺, the cells accommodate after a long lag and proliferate. The time required for this response depends on the nature of the strain and the supplements in the growth medium. Immediately after exposure to Cd²⁺, considerable single strand breaks in the DNA are observed but the DNA is repaired prior to the initiation of cell proliferation. The finding that accommodation occurs in DNA polymerase I-deficient mutant cells suggests that DNA polymerase I may not be required for repair of damaged DNA in Cd²⁺-exposed cells. The recovery of Cd²⁺-exposed cells in a temperature-sensitive DNA ligase mutant cells at the permissive temperature (30° C) and failure to recover at the non-permissive temperature (42° C) indicates, however, that DNA ligase is involved in the repair of the single strand breaks associated with Cd²⁺-induced damage.     

Regulation of DNA replication by serum and the transforming function in cultured rat fibroblasts transformed by Rous sarcoma virus.

The onset and rate of semiconservative DNA replication were measured in stimulated cultured rat fibroblasts and their Rous sarcoma virus-transformed derivatives after a period of serum deprivation. Rat-1 (tsLA24/RSV) cells initiated DNA synthesis following a shift to the permissive temperature or addition of serum at the non-permissive temperature. Their rate of DNA replication was unaffected by the presence of serum at the permissive temperature, however, there was a serum requirement at the non-permissive temperature. The transition probability was less at the permissive temperature, independent of serum, than at the non-permissive temperature in the presence of serum. The amount of DNA induced to replicate by addition of serum at the non-permissive temperature or by a shift to the permissive temperature was similar. Using the untransformed Rat-1 cells and these cells transformed by wild-type RSV (Rat-1 (wt/RSV)), it was confirmed that the rate of entry into S phase (transition probability) was always lower in the transformed cell line at both 39° and 35°. In both cell lines the rate of DNA replication was independent of temperature, but the onset was delayed at the lower temperature. These results indicate that in the cell lines examined, (1) serum was able to commit the cells to replicate DNA (alter the transition probability) in both transformed and untransformed cells, but the transforming function was able to supplant a serum-dependent process during G₁ necessary for the initiation of DNA replication, and (2) the effects of the transforming function and serum factor(s) on the alteration of the transition probability are not additive, suggesting that the transforming function initiates a process which acts at the level of the commitment to DNA replication which may render the normal serum-related control mechanisms ineffective in the regulation of growth.     

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.     

Genetic study of the role of calcium ions in the cell division cycle of Saccharomyces cerevisiae: a calcium-dependent mutant and its trifluoperazine-dependent pseudorevertants

Summary A cal1-1 mutant of the yeast Saccharomyces cerevisiae showing Ca²⁺-dependent growth was isolated. Its growth continued exponentially in Ca²⁺-rich medium, but stopped in Ca²⁺-poor medium at 37°C. Mg²⁺ ions could not replace Ca²⁺ ions. In Ca²⁺-poor medium, the mutant cells stopped growing homogeneously at the stage of cell division cycle with a tiny bud. The nucleus in these arrested cells was in the G2 stage, judging from observation after nuclear staining and determination of the DNA content. Trifluoperazine-dependent pseudorevertants, which could grow in the presence of 20 M to 80 M trifluoperazine in Ca²⁺-poor medium at 37°C, were obtained from this cal1-1 mutant. The suppressor mutation, tfrl, itself conferred trifluoperazine resistance. Other calmodulin inhibitors structurally unrelated to trifluoperazine had similar effects to trifluoperazine on these pseudorevertants. These results suggest that Ca²⁺ ions and a calmodulin play important roles in the yeast cell division cycle at the stage of bud growth and nuclear division.Abbreviations Tfp trifluoperazine - DAP1 46-diamidino-2-phenylindole - EMS ethyl methanesulfonate - PD parental ditype - NPD nonparental ditype - T tetratype     

Calcium depletion alters energy transfer and prevents state changes in intact Anacystis

A time-dependent loss of Photosystem II (PS II) activity seen in Anacystis nidulans grown without Ca²⁺ was paralleled by a loss in chlorophyll (Chl) a fluorescence of variable yield which reflects inhibition of Q reduction and of state changes. Both inhibitions were fully reversed by the addition of Ca²⁺ to the growth medium. The lack of state changes in Ca²⁺-depleted cells was confirmed in 77 K fluorescence difference spectra of light versus dark-adapted cells.Absorption spectra of control and of Ca²⁺-depleted cells were identical whether measured at room temperature or at 77 K. Fluorescence emission spectra measured at 39°C (cell growth temperature) demonstrated higher yields in Ca²⁺-depleted cells compared to controls. Fluorescence emission spectra at 77 K also produced higher yields in Ca²⁺-depleted cells but the increased fluorescence at this temperature occurred principally at 683 nm. The increased relative fluorescence yield in Ca²⁺-depleted samples results from light absorbed by phycocyanin (PC), but not from light absorbed almost exclusively by Chl. The 683 run fluorescence peak probably represents increased allophycocyanin (APC) emission as intact phycobilisomes become energetically disassociated from the photosynthetic apparatus. This inferred disassociation occurred only after PSII activity was mostly inhibited in Ca²⁺-depleted cells, and was not fully reversible.Abbreviations APC Allophycocyanin - Chl chlorophyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - EDTA ethylenediaminotetraacetic acid - PC phycocyanin - PS photosystem - Q primary quinone electron acceptor of Photosystem II also a quencher of Chl a fluorescence DPB-CIW Publ. No. 817     

Glutamate triggers elevation of intracellular Ca2+ concentration in neural precursor cells

Both neurons and glial cells are derived from neuralprecursor cells in the ventricular zone during braindevelopment. The fate of the neural precursor cells isaffected by neurotransmitters such as glutamate. Inthis study, we examined glutamate-triggeredintracellular Ca²⁺ signaling in neural precursorcell lines by the calcium digital imaging method. Whenimmortalized primary-cultured neural precursor cellswere treated with glutamate, a subpopulation of thesecells showed an increase in intracellular Ca²⁺concentration. In an effort to determine the role ofthe glutamate-triggered intracellular Ca²⁺ signalin neural precursor cells, we tried to cultureimmortalized basal ganglial and hippocampal neuralprecursor cell lines in glutamate-free medium. Thehippocampal (MHP-2) cells became adapted to theglutamate-free medium, and when treated with glutamatethe adapted subline (MHP-2-E1) showed an increase inintracellular Ca²⁺ concentration. In contrast,the basal ganglial neural precursor cell lines failedto become adapted to the glutamate-free medium. Theseresults suggest that hippocampal and basal ganglialneural precursor cells differ in their cellularresponse to glutamate as an exogenous stimulus.     

Calcium-dependent peripheral localization of 4.1-like proteins and fodrin in cultured human keratinocytes

Summary— Recently, several proteins immunologically related to erythrocyte membrane skeletal proteins, such as protein 4.1 and fodrin (non-erythroid spectrin), have been found in keratinocytes. In the present study, in order to investigate the roles of these proteins in cell-cell contact, we analyzed the distribution of non-erythroid protein 4.1, β-fodrin and actin in cultured human keratinocytes at low (0.15 mM) and standard (1.85 mM) Ca²⁺ concentrations. Immunofluorescence microscopy revealed that immunoreactive forms of protein 4.1, β-fodrin and actin filaments were present in the cytoplasm of cells cultured in low Ca²⁺ medium, while in cells in the standard Ca²⁺ medium, these proteins were localized at the cell boundary and partially in the cytoplasm. When cells in the low-Ca²⁺ medium were treated with 100 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) for 1 h, these proteins were also present at the cell boundary. Increasing extracellular Ca²⁺ concentration from low to standard in the medium induces cell-cell contact among the cultured human keratinocytes, accompanied by the translocation of protein 4.1 and β-fodrin from the cytoplasm to the membrane. On the basis of the present study, movement of membrane skeletal proteins from the cytosol to the membrane suggests that either these proteins or the membrane skeletal lattice plays an important role in the regulation of cell-cell intergigitations in response to changes in the Ca²⁺ concentrations in culture medium, and that phosphorylation of these skeletal proteins might be involved in the regulation of the membrane skeletal proteins of keratinocytes in response to Ca²⁺.     

Rational method in the repetitive calcium oscillation measurement in wild type human epithelial kidney cells

Cells stimulated with physiological stimuli usually exhibit oscillations in cytosolic Ca²⁺ concentration ([Ca²⁺]_i), a signal playing central roles in regulation of various cellular processes. For explicating their unknown mechanisms, studies are commonly conducted in single cells from several cell lines, in particular the human epithelial kidney (HEK293) cell line. However, [Ca²⁺]_i oscillating responses to agonists in vitro are found difficult to be induced and varied with different types of cells and agonists. This study shows that treatment of the wild type HEK293 cells with low concentrations of carbachol (1–10 μM), an agonist of the muscarinic receptor, resulted in non-oscillated but sustained [Ca²⁺]_i increase by loading the cells with 1 μM fura2/AM. However, repetitive and long lasting [Ca²⁺]_i oscillations could be induced in 31.1% of the tested cells loaded with 0.1 μM fura2/AM. Additionally, the occurrence of the typical Ca²⁺ spikes further increased to 47.2% and 60.7% when the Ca²⁺ concentration in the bathing medium was decreased from 1.8 mM to 1.5 mM and the medium temperature was set to 35 ± 1°C from 22 ± 2°C. Therefore, this study provides a useful approach for measuring [Ca²⁺]_i oscillatory response to relevant physiological stimulation in a wild type cell line through the adjustments of the concentrations adopted for the Ca²⁺ indicator and extracellular medium Ca²⁺ and of the temperature set for the experiment.     

Specific protein phosphorylation occurs in molluscan red blood cell ghosts in response to hypoosmotic stress

Summary The regulation of cellular volume upon exposure to hypoosmotic stress is accomplished by specific plasma membrane permeability changes that allow the efflux of certain intracellular solutes (osmolytes). The mechanism of this membrane permeability regulation is not understood; however, previous data implicate Ca²⁺ as an important component in the response. The regulation of protein phosphorylation is a pervasive aspect of celllular physiology that is often Ca²⁺ dependent. Therefore, we tested for osmotically induced protein phosphorylation as a possible mechanism by which Ca²⁺ may mediate osmotically dependent osmolyte efflux. We have found a rapid increase in³²P_i incorporation into two proteins in clam blood cell ghosts after exposure of the intact cells to a hypoosmotic medium. The osmotic component of the stress, not the ionic dilution, was the stimulus for the phosphorylations. The osmotically induced phosphorylation of both proteins was significantly inhibited when Ca²⁺ was omitted from the medium, or by the calmodulin antagonist. chlorpromazine. These results correlate temporally with cell volume recovery and osmolyte (specifically free amino acid) efflux. The two proteins that become phosphorylated in response to hypoosmotic stress may be involved in the regulation of plasma membrane permeability to organic solutes, and thus. contribute to hypoosmotic cell volume regulation.     

Possible involvement of miRNAs in tropism of Parvovirus B19

Human Parvovirus B19 (PVB19) is one of the most important pathogens that targets erythroid lineage. Many factors were mentioned for restriction to erythroid progenitor cells (EPCs). Previous studies showed that in non-permissive cells VP1 and VP2 (structural proteins) mRNAs were detected but could not translate to proteins. A bioinformatics study showed that this inhibition might be due to specific microRNAs (miRNAs) present in non-permissive cells but not in permissive EPCs. To confirm the hypothesis, we evaluated the effect of miRNAs on VP expression. CD34⁺ HSCs were separated from cord blood. Then, CD34⁺ cells were treated with differentiation medium to obtain CD36⁺ EPCs. To evaluate the effect of miRNAs on VP expression in MCF7 and HEK-293 cell lines (non-permissive cells) and CD36⁺ EPCs, dual luciferase assay was performed in presence of shRNAs against Dicer and Drosha to disrupt miRNA biogenesis. QRT-PCR was performed to check down-regulation of Dicer and Drosha after transfection. All measurements were done in triplicate. Data means were compared using one-way ANOVAs. MicroRNA prediction was done by the online microRNA prediction tools. No significant difference was shown in luciferase activity of CD36⁺ EPCs after co-transfection with shRNAs, while it was significant in non-permissive cells. Our study revealed that miRNAs may be involved in inhibition of VP expression in non-permissive cells, although further studies are required to demonstrate which miRNAs exactly are involved in regulation of PVB19 replication.     

Differential Modulation of 7-Ketocholesterol Toxicity Against PC12 Cells by Calmodulin Antagonists and Ca<Superscript>2+</Superscript> Channel Blockers

The present study assessed the influence of intracellular Ca²⁺ and calmodulin against the neurotoxicity of oxysterol 7-ketocholesterol in relation to the mitochondria-mediated cell death process and oxidative stress in PC12 cells. Calmodulin antagonists calmidazolium and W-7 prevented the 7-ketocholesterol-induced mitochondrial damage, leading to caspase-3 activation and cell death, whereas Ca²⁺ channel blocker nicardipine, mitochondrial Ca²⁺ uptake inhibitor ruthenium red, and cell permeable Ca²⁺ chelator BAPTA-AM did not reduce it. Exposure of PC12 cells to 7-ketocholesterol caused elevation of intracellular Ca²⁺ levels. Unlike cell injury, calmodulin antagonists, nicardipine, and BAPTA-AM prevented the 7-ketocholesterol-induced elevations of intracellular Ca²⁺ levels. The results show that the cytotoxicity of 7-ketocholesterol seems to be modulated by calmodulin rather than changes in intracellular Ca²⁺ levels. Calmodulin antagonists may prevent the cytotoxicity of 7-ketocholesterol by suppressing the mitochondrial permeability transition formation, which is associated with the increased formation of reactive oxygen species and the depletion of GSH.     

The effect of calcium ion concentration on myotube formation in vitro

The relationship of Ca^a+ concentration in in vitro tissue culture medium to the ability of the medium to support lizard and chick myotube formation was studied. Lizard myogenic cells (from lines established from the regenerating tail of the lizard, Anolis caroliensis) do not fuse in media with a Ca²⁺ concentration of below 650 μM; good fusion occurs at 1 750μM; and large anastomosing tubes result in media with concentrations of 2 750,μM. Chick myogenic cells from the limb of 11 day embryos do not fuse at Ca²⁺ concentrations below 260,μM, fuse well at 1 000 μM, and produce large anastomosing myotubes at concentrations above 1 700 μM. Colonies of myogenic cells from established lines plated at clonal densities in a medium with a 1 750 μM Ca²⁺ concentration grow more rapidly than those at 650 μM Ca²⁺; however, there is no increase in plating efficiency. Regardless of the Ca²⁺ concentration, lizard myogenic cells do not fuse until a large percentage of the cells in a colony have withdrawn from the mitotic cycle in Gl and entered GO. The similarities between in vivo and in vitro lizard myogenesis are discussed.     

The src gene product (pp60 src) of avian sarcoma virus rapidly induces DNA synthesis and proliferation of calcium-deprived rat cells

A general characteristic of neoplastic cells, but not their non-neoplastic counterparts, is the ability to proliferate in calcium-deficient medium. NRK cells infected with the transformation-defective, temperature-sensitive, ASV mutant, tsLA23, were unable to proliferate in calcium-deficient medium at the non-permissive 40°C, but they very rapidly initiated DNA synthesis (within 1 hour) and resumed proliferation in this medium after being shifted to 36°C, a temperature permissive for the production of active pp60^src and for neoplastic transformation. These observations suggest that activated pp60^src acts near the $\text{G1}\text{S}$ transition point in the cell cycle to bypass or stimulate a calcium-dependent mechanism required for the initiation of DNA synthesis, which enables the cells to display the neoplastic property of proliferating in calcium-deficient medium.     

Plasma membrane Ca2+ transport: Antagonism by several potential inhibitors

Summary Inside-out vesicles prepared from human red blood cells took up Ca²⁺ by an active transport process. Membranes from the same red blood cells displayed Ca²⁺-activated, Mg²⁺-dependent adenosine triphosphatase activity. Both the initial rate of Ca²⁺ transport and the (Ca²⁺+Mg²⁺)-adenosine triphosphatase activity were increased approximately twofold by the calcium binding protein, calmodulin. Activities in the absence of added calmodulin were termed basal activities. Calmodulin-activated Ca²⁺ transport and adenosine triphosphatase activities could be antagonized in a relatively selective fashion by the phenothiazine tranquilizer drug, trifluoperazine. High concentrations of trifluoperazine also inhibited basal Ca²⁺ transport and adenosine triphosphatase activity. By contrast, calmodulin binding protein from beef brain selectively antagonized the effect of calmodulin on Ca²⁺ transport with no inhibition of basal activity. Ruthenium red antagonized calmodulin-activated and basal activity with equal potency. The results demonstrate that although phenothiazines can act as relatively selective antagonists of calmodulin-induced effects, other effects are possible and cannot be ignored. Calmodulin-binding protein may be a useful tool in the analysis of calmodulin functions. Ruthenium red probably interacts with Ca²⁺ pump adenosine triphosphatase at a site not related to calmodulin.     

Transforming NRK cells with avian sarcoma virus reduces the extracellular Ca requirement without affecting the calcicalmodulin requirement for the G1/S transition

NRK rat cells infected with a transformation-defective, temperature-sensitive (ts) mutant of the avian sarcoma virus could not proliferate in Ca²⁺-deficient medium at a nonpermissive temperature (40 °C) that inactivated the viral pp60^v-scr-transforming product and rendered the cells phenotypically untransformed. However, these arrested cells were stimulated to initiate DNA replication with little or no delay while still in the Ca²⁺-deficient medium, either by adding Ca²⁺ or calmodulin at 40 °C or by reducing the temperature to 36 °C which restored the transformed phenotype by rapidly reactivating pp60^v-src. The G1/S transition triggered by restoring the transformed phenotype was suppressed by three different anticalmodulin drugs (R24571, trifluoperazine, W7). The suppression by one of these drugs, trifluoperazine, was overcome by adding calmodulin. Thus, neoplastic transformation by the avian sarcoma virus sharply reduces the extracellular Ca²⁺ requirement for the initiation of DNA replication without bypassing a calcical-modulin-dependent mechanism also needed for the G1/S transition.     

Evidence for Ca2+-dependent ATPase activity,stimulated by decapacitation factor and calmodulin,in mouse sperm

Membrane preparations from mouse sperm heads and tails were used in a γ³²P-ATP hydrolysis assay to investigate Ca²⁺-dependent ATPase activity. In membranes from sperm heads, but not tails, a Ca²⁺-dependent ATPase that was further stimulated by calmodulin was detected. The addition of partially purified mouse sperm decapacitation factor (DF) to head membrane preparations significantly stimulated Ca²⁺-ATPase activity, this effect being further increased in the presence of DF plus calmodulin; in contrast, no response was observed when the same treatment was applied to tail membranes. Sperm preincubated in the presence of trifluoperazine (TFP), a calmodulin antagonist, were significantly more fertile than cells from the same males incubated in the absence of TFP, indicating that inhibition of calmodulin accelerates capacitation. When sperm cells were preincubated briefly, then gently centrifuged to remove DF and resuspended in medium containing ⁴⁵Ca²⁺ ± DF, their ability to accumulate ⁴⁵Ca²⁺ was significantly lower in the early stages after resuspension in the presence of DF than in its absence. These data correlated with chlortetracycline analysis of the sperm functional state. When cells were centrifuged and resuspended in medium only, there was a noticeable shift from the F pattern (characteristic of uncapacitated cells) to the B pattern (characteristic of capacitated cells), but the reintroduction of DF caused a significant reversion to the F pattern. Finally, using a monoclonal antibody to somatic cell Ca²⁺-ATPase, we have obtained evidence that the enzyme is particularly localized to the postacrosomal region of the mouse sperm head; specific binding was observed only in permeabilized cells, indicating that the epitope involved in the binding has an intracellular location. Based on these various pieces of evidence, we propose that when present on mouse sperm, DF stimulates calmodulin-sensitive Ca²⁺-ATPase activity and thus ensures maintenance of a low intracellular Ca²⁺ concentration. As capacitation proceeds, DF is lost and Ca²⁺-ATPase activity declines, allowing intracellular Ca²⁺ to rise and promoting capacitation-related changes. The fact that inhibitors of Ca²⁺-ATPase and calmodulin appear to accelerate capacitation in several mammalian species, as determined by chlortetracycline analysis, suggests that Ca²⁺-ATPase activity may play an important role in modulating capacitation in many or even all mammals. © 1996 Wiley-Liss, Inc     

Calcium and the control of muscle-specific creatine kinase accumulation during skeletal muscle differentiation in vitro.

A polyacrylamide gel separation method for creatine kinase (CPK) isoenzymes is described, and its use to determine muscle-specific CPK (M-CPK) levels in skeletal muscle cultures is illustrated. In cultures in which cell fusion has been prevented by very low Ca²⁺ concentrations, the increases in M-CPK after 96 hr are similar to those in control cultures. Slightly higher concentrations of Ca²⁺, however, inhibit both cell fusion and M-CPK accumulation. As the calcium concentration is gradually increased further, cell fusion is permitted, followed, at even higher Ca²⁺ levels, by M-CPK accumulation. These effects can be obtained both by adding EGTA to the culture medium and by using Ca²⁺-free culture medium and varying the Ca²⁺ concentration directly. The latter method has the advantage that deleterious effects of EGTA on cell attachment and cell numbers do not occur, even at the lowest Ca²⁺ concentrations. By revealing dramatic effects on CPK levels of small changes in external Ca²⁺ concentrations, these observations may resolve conflicting data in the literature on the question of whether cell fusion is a prerequisite for muscle-specific protein synthesis. Possible mechanisms for the two effects of Ca²⁺ on CPK specific activity (permissive at very low, but inhibitory at intermediate, concentrations) are considered, including membrane mediation, mediation by changes in ionized cytoplasmic Ca²⁺ levels, and possible involvement of cyclic nucleotides.     

Ca2+ calmodulin-dependent protein kinase activity in the ascomycetes Neurospora crassa

DEAE-cellulose column chromatography of Neurospora crassa soluble mycelial extracts leads to the resolution of three major protein kinase activity peaks designated PKI, PKII, and PKIII.PKII activity is stimulated by Ca²⁺ and Neurospora or brain calmodulin. Maximal stimulation was observed at 2 µM-free Ca²⁺ and 1 µg/ml of the modulator. The stimulatory effect of the Ca²⁺-calmodulin complex was blocked by EGTA and by some calmodulin antagonists such as phenothiazine drugs or compound 48/80.PKII phosphorylates different proteins, among which histone II-A at a low concentration and CDPKS, the synthetic peptide specific for Ca²⁺-calmodulin dependent protein kinases, are the best substrates. Some phosphorylation can be detected in the absence of any exogenous acceptor. PKII activity assayed in the presence of histone II-A or in the absence of exogenous phosphate acceptor (autophosphorylation) co-elute in a DEAE-cellulose column at 0.28 M NaCl. As result of the autophosphorylation reaction of the purified enzyme a main phosphorylated component of 70 kDa was resolved by SDS-polyacrylamide gel electrophoresis. It is possible that this component is an active part of this enzyme.