Calcium-dependent phosphatidylinositol phosphorylation in lamellibranch gill lateral cilia

Summary Pure lateral (L) cilia may be separated from the remaining (R) cilia types ofMytilus edulis gill by serotonin activation after hypertonic shock. The two classes of cilia were permeabilized with 0.012% Triton X-100 and incubated with³²P-labeled ATP at low Ca⁺⁺ (10^–7 M), where L cilia beat, or in high Ca⁺⁺ (2–20 M), where L cilia arrest but R cilia are active. The labeled cilia were separated into axoneme and membrane-matrix fractions by detergent extraction, subjected to SDS-PAGE on 5–15% gels, and autoradio-graphed. Neither cilia type undergoes Ca⁺⁺-dependent phosphorylation of specific proteins, suggesting that neither Ca⁺⁺-induced arrest in L cilia nor the Ca⁺⁺ activation of other cilia is phosphorylation-dependent. However, lipid phosphorylation in L cilia is highly Ca⁺⁺-dependent. Identified by thin-layer chromatography, the phospholipid that is phosphorylated in a Ca⁺⁺-dependent manner is phosphatidylinositol 4-phosphate (PIP), yielding the 4,5-bisphosphate (PIP₂). PIP₂ increases at least 3-fold under Ca⁺⁺-arrest conditions.Aequipecten gill lateral cilia, which require higher Ca⁺⁺ levels for arrest, show even more striking changes. In both cases, the effect is maximal at micromolar Ca⁺⁺ levels. Phosphorylation of other lipids is Ca⁺⁺-independent. In the Ca⁺⁺-insensitive or activated R cilia, PIP₂ levels are intermediate, increasing only marginally with increased [Ca⁺⁺]. The formation of PIP₂ in response to Ca⁺⁺, as opposed to its breakdown to form inositol 1,4,5-trisphosphate and diacylglycerol, may be characteristic of a Ca⁺⁺ transport system. Mechanically sensitive, the L cilia arrest as a consequence of an inward flux of Ca⁺⁺ ions, acting directly on the axoneme. After Ca⁺⁺-induced arrest, the formation of PIP₂ may be involved in sequestering Ca⁺⁺ or in augmenting Ca⁺⁺ pump activity, thus reducing Ca⁺⁺ levels so that motility may resume quickly.     

cAMP, not Ca/calmodulin, regulates the phosphorylation of acetylcholine receptor in Torpedo californica electroplax

The regulation of the phosphorylation of the acetylcholine receptor in electroplax membranes from Torpedo californica and of purified acetylcholine receptor was investigated. The phosphorylation of the membrane-bound acetylcholine receptor was not stimulated by Ca²⁺/calmodulin, nor was it inhibited by EGTA, but it was stimulated by the catalytic subunit of cAMP-dependent protein kinase, and was blocked by the protein inhibitor of cAMP-dependent protein kinase. Purified acetylcholine receptor was not phosphorylated by Ca²⁺/calmodulin-dependent protein kinase activity in electroplax membranes, nor by partially purified Ca²⁺/calmodulin-dependent protein kinases from soluble or particulate fractions from the electroplax. Of the four acetylcholine receptor subunits, termed α, β, γ and δ, only the γ- and δ-subunits were phosphorylated by the cAMP-dependent protein kinase (+cAMP), or by its purified catalytic subunits.     

Signal tranduction: regulation of cAMP concentration in cardiac muscle by calmodulin-dependent cyclic nucleotide phosphodiesterase

The bovine heart calmodulin-dependent phosphodiesterase can be phosphorylated by cAMP-dependent protein kinase, resulting in a decrease in the enzyme's affinity for calmodulin. The phosphorylation of calmodulin-dependent phosphodiesterase is blocked by Ca²⁺ and calmodulin and reversed by the calmodulin-dependent phosphatase. The dephosphorylation is accompanied by an increase in the affinity of the phosphodiesterase for calmodulin. The CaM-dependent phosphodiesterase isozymes of heart and brain are regulated by calmodulin, but the affinity for calmodulin are different. Furthermore, the bovine heart CaM-dependent phosphodiesterase isozyme in stimulated at much lower Ca²⁺ concentration than the bovine brain isozymes. Results from this study suggest that the activity of this phosphodiesterase is precisely regulated by cross-talk between Ca²⁺ and cAMP signalling pathways.     

cAMP,not Ca2+/calmodulin,regulates the phosphorylation of acetylcholine receptor in Torpedo californica electroplax

The regulation of the phosphorylation of the acetylcholine receptor in electroplax membranes from Torpedo californica and of purified acetylcholine receptor was investigated. The phosphorylation of the membrane-bound acetylcholine receptor was not stimulated by Ca²⁺/calmodulin, nor was it inhibited by EGTA, but it was stimulated by the catalytic subunit of cAMP-dependent protein kinase, and was blocked by the protein inhibitor of cAMP-dependent protein kinase. Purified acetylcholine receptor was not phosphorylated by Ca²⁺/calmodulin-dependent protein kinase activity in electroplax membranes, nor by partially purified Ca²⁺/calmodulin-dependent protein kinases from soluble or particulate fractions from the electroplax. Of the four acetylcholine receptor subunits, termed α, β, γ and δ, only the γ- and δ-subunits were phosphorylated by the cAMP-dependent protein kinase (+cAMP), or by its purified catalytic subunits.     

Adrenergic regulation of chloride secretion across the opercular epithelium: The role of cyclic AMP

Summary Activation of the -adrenergic receptors of the opercular epithelium ofFundulus heteroclitus stimulates Cl^– secretion, while activation of the -adrenergic receptors inhibits Cl^– secretion (Degnan and Zadunaisky, 1979). The possible involvement of adenosine 3, 5-monophosphate (cAMP) in these adrenergic responses was investigated. Isolated opercular epithelia incubated in Ringer, containing 10 mM theophylline, had cAMP levels ranging between 5.3 and 19.3 pmoles·mg protein^–1 (mean=9.5±1.0 pmoles·mg protein^–1). Activation of the -receptors by 10^–5 M isoproterenol increased the mean cAMP level 430% (P<0.001). Blockage of the -receptors with propranolol greatly reduced the increase in cAMP in response to isoproterenol. Activation of the -receptors by 10^–5 M arterenol stimulated the mean cAMP level 270% (P<0.01). However, when the -receptors were blocked with propranolol, arterenol had no effect on the cAMP level. The possible involvement of Ca⁺⁺ in these adrenergic responses was investigated. Neither the stimulatory effect of isoproterenol, nor the inhibitory effect of arterenol on the Cl^– secretion were diminished in the absence of extracellular Ca⁺⁺. The Ca⁺⁺ ionophore, A23187, and the calmodulin inhibitor, trifluoperazine, had no effects on the Cl^– secretion. The Ca⁺⁺-channel blocker, D600, had a significant inhibitory effect (P<0.005). Guanosine 3,5-monophosphate (cGMP) had no effect on the Cl^– secretion.The results indicate that -adrenergic stimulation of Cl^– secretion across the opercular epithelium is accompanied by an elevation in tissue cAMP levels. -adrenergic inhibition of Cl^– secretion does not involve changes in the tissue cAMP. Neither of these responses appear to require Ca⁺⁺.     

Identification of a Phosphoprotein Expressed During Somatic Embryogenesis in Wheat Leaf Base Cultures

2,4-D mediated induction of somatic embryogenesis in wheat is enhanced in the presence of Ca⁺⁺ and its removal by EGTA reduces the response significantly. Changes that occur at the polypeptide level following 2,4-D treatment were analysed. Intense cell division activity was discernable in the leaf base explants within an hour of treatment. Changes in protein profiles were prominent in the membrane fraction as compared to the soluble fraction. The protein profile of the leaf base culture with somatic embryos was distinct from the calli induced from mature embryos on a 2,4-D containing medium. The role of Ca²⁺ in the induction of somatic embryogenesis was demonstrated by the use of EGTA (a calcium chelator), verapamil, nifedipine (calcium channel blockers), W7 (calmodulin antagonist) and Li (PI inhibitor). ^{In vitro} protein phosphorylation studies showed that 2,4-D, calcium and related treatments inhibit phosphorylation of proteins. In the membrane fraction proteins, accumulation of polypeptides at the low molecular weight range was seen in samples treated with verapamil and W7, and a 30 kO polypeptide in the samples treated with calmodulin antagonist, W7. Autoradiography of membrane fraction proteins displayed the presence of a 16 kO protein phosphorylated in samples treated with verapamil, nifedipine and W7. It thus appears that 2,4-D and Ca⁺⁺ prevent the phosphorylation of this phosphoprotein. These results thus indicate the action of 2,4-D via the Ca²⁺-CaM signaling pathway in triggering the induction of somatic embryogenesis.     

Calcium- and calmodulin-regulated phosphorylation of soluble and membrane proteins from corn coleoptiles

In vitro phosphorylation of several membrane polypeptides and soluble polypeptides from corn (Zea mays var. Patriot) coleoptiles was promoted by adding Ca²⁺. Ca²⁺-promoted phosphorylation of the membrane polypeptides was further increased in the presence of calmodulin. Both Ca²⁺-stimulated and Ca²⁺- and calmodulin-stimulated phosphorylations of membrane polypeptides were inhibited by chlorpromazine, a calmodulin antagonist. Ca²⁺-stimulated phosphorylation of soluble polypeptides increased with increasing Ca²⁺ concentration. The calmodulin antagonists chlorpromazine and trifluoperazine inhibited the Ca²⁺-promoted phosphorylation of soluble polypeptides. Added calmodulin promoted the Ca²⁺-dependent phosphorylation of a 98 kilodaltons polypeptide. Both Ca²⁺-dependent and Ca²⁺-independent phosphorylations required Mg²⁺ at an optimal concentration of 5 to 10 millimolar. Cyclic AMP was found to have no stimulatory effect on protein phosphorylation. Sodium molybdate, an inhibitor of protein phosphatase, increased the net phosphorylation of several polypeptides. Rapid loss of radioactivity from the phosphorylated polypeptides following incubation in unlabeled ATP indicated the presence of phosphoprotein phosphatase activity.     

Control of ciliary orientation through cAMP-dependent phosphorylation of axonemal proteins in paramecium caudatum

Ciliary reorientations in response to cAMP do not take place after a brief digestion with trypsin in ciliated cortical sheets from Triton-glycerol-extracted Paramecium. In this study, we examined the effects of tryptic digestion on the cAMP-dependent phosphorylation of axonemal proteins to clarify the relationship between phosphorylation and ciliary reorientation. As reported for Paramecium tetraurelia, cAMP stimulated phosphorylations of the 29 kDa and 65 kDa axonemal polypeptides also in Paramecium caudatum. After a brief digestion of axonemes by trypsin, none of the cAMP-dependent phosphorylations occurred. On the other hand, the 29 kDa polypeptide still remained to be labeled after a brief digestion of axonemes that had previously been labeled with (32)P in the presence of cAMP, which indicates that this brief digestion breaks down endogenous cAMP-dependent protein kinases but not phosphorylated proteins. This must be the reason that trypsin-treated cilia on the sheets cannot reorient towards the posterior part of the cell. Our results indicate that cAMP regulates not only the beat frequency but also the ciliary orientation via phosphorylation of dynein subunits in Paramecium.     

A protein activator of the plasma membrane Ca++-ATPase of heart sarcolemma

A detergent extract of dog or beef heart sarcolemmal vesicles was prepared and found to have a stimulatory effect on the Ca⁺⁺-ATPase of plasma membranes from human erythrocyte and cardiac sarcolemma. A procedure is described which enriches the activating fraction. The protein nature of the preparation is illustrated by its sensitivity to boiling and to the proteolytic enzyme(s) trypsin and chymotrypsin. SDS polyacrylamide gels indicate that the protein(s) involved have a molecular weight of 56 and 60 kDa. The sarcolemmal activator can stimulate the Ca⁺⁺-ATPase activity of the isolated enzyme more than 100% in the presence of saturating amounts of calmodulin. The activation is calcium dependent, being greatest at approximately 10µm Ca⁺⁺, free, but does not change theK _m for Ca⁺⁺. A possible physiological role for the activator is discussed.     

A novel neuronal calcium sensor family protein, calaxin, is a potential Ca(2+)-dependent regulator for the outer arm dynein of metazoan cilia and flagella

Background information. Spermatozoa show several changes in flagellar waveform, such as upon fertilization. Ca²⁺ has been shown to play critical roles in modulating the waveforms of sperm flagella. However, a Ca²⁺‐binding protein in sperm flagella that regulates axonemal dyneins has not been fully characterized. Results. We identified a novel neuronal calcium sensor family protein, named calaxin (Ca²⁺‐binding axonemal protein), in sperm flagella of the ascidian Ciona intestinalis. Calaxin has three EF‐hand Ca²⁺‐binding motifs, and its orthologues are present in metazoan species, but not in yeast, green algae or plant. Immunolocalization revealed that calaxin is localized near the outer arm of the sperm flagellar axonemes. Moreover, it is distributed in adult tissues bearing epithelial cilia. An in vitro binding experiment indicated that calaxin binds to outer arm dynein. A cross‐linking experiment showed that calaxin binds to β‐tubulin in situ. Overlay experiments further indicated that calaxin binds the β‐dynein heavy chain of outer arm dynein in the presence of Ca²⁺. Conclusions. These results suggest that calaxin is a potential Ca²⁺‐dependent modulator of outer arm dynein in metazoan cilia and flagella.     

On the role of Ca2+-calmodulin-dependent and cAMP-dependent protein phosphorylation in the circadian rhythm ofNeurospora crassa

Summary Pulses of some Ca²⁺ channel blockers (dantrolene, Co²⁺, nifedipine) and calmodulin inhibitors (chlorpromazine) lead to medium (maximally 5–9 h) phase shifts of the circadian conidiation rhythm ofNeurospora crassa. Pulses of high Ca²⁺, or of low Ca²⁺, a Ca²⁺ ionophore (A23187) together with Ca²⁺, and other Ca²⁺ channel blockers (La³⁺, diltiazem), however, caused only minor phase shifts. The effect of these substances (A 23187) and of different temperatures on the Ca²⁺ release from isolated vacuoles was analyzed by using the fluorescent dye Fura-2. A 23187 and higher temperatures increased the release drastically, whereas dantrolene decreased the permeation of Ca²⁺ (Cornelius et al., 1989).Pulses of 8-PCTP-cAMP, IBMX and of the cAMP antagonist RP-cAMPS, also caused medium (maximally 6–9 h) phase shifts of the conidiation rhythm. The phase response curve of the agonist was almost 180° out of phase with the antagonist PRC. In spite of some variability in the PRCs of these series of experiments all showed maximal shifts during ct 0–12. The variability of the response may be due to circadian changes in the activity of phosphodiesterases: After adding cAMP to mycelial extracts HPLC analysis of cAMP metabolites showed significant differences during a circadian period with a maximum at ct 0.Protein phosphorylation was tested mainly in an in vitro phosphorylation system (with³⁵S-thio -ATP). The results showed circadian rhythmic changes predominantly in proteins of 47/48 kDa. Substances and treatments causing phase-shifts of the conidiation rhythm also caused changes in the phosphorylation of these proteins: an increase was observed when Ca²⁺ or cAMP were added, whereas a decrease occurred upon addition of a calmodulin inhibitor (TFP) or pretreatment of the mycelia with higher (42° C) temperatures.Altogether, the results indicate that Ca²⁺-calmodulin-dependent and cAMP-dependent processes play an important, but perhaps not essential, role in the clock mechanism ofNeurospora. Ca²⁺ calmodulin and the phosphorylation state of the 47/48-kDa proteins may have controlling or essential functions for this mechanism.     

Regulation of rat cardiac nuclei-associated Mg2+-NTPase by phosphorylation

A nucleoside triphosphatase (NTPase) activity appeared to be associated with a highly purified nuclear preparation from rat cardiac ventricles. Different nucleoside triphosphates (UTP > GTP > ITP > CTP) supported this enzymic activity, which was stimulated by Mg` but not by Call. The nuclear NTPase activity could be down regulated by endogenous phosphorylation of a 55,000 M_r protein. Maximal phosphorylation of the 55,000 M_r protein occurred in the presence of Mg²⁺-ATP. Addition of cAMP, cGMP, Ca²⁺, Ca²⁺/phospholipid, Ca²⁺/calmodulin, and catalytic subunit of cAMP-dependent protein kinase was not associated with any further phosphorylation of the 55,000 M_r protein. However, in the presence of Ca²⁺/calmodulin or the catalytic subunit of the cAMP-dependent protein kinase additional proteins became phosphorylated, but these had no effect on the Mg²⁺-NTPase activity. These results indicate that a protein with M_r 55,000 may be involved in the regulation the Mg²⁺-NTPase activity associated with rat cardiac nuclei.Abbreviations Hg Hemoglobin - GAR Goat Anti-Rabbit antibody - SR Sarcoplasmic Reticulum - NTP Nucleoside Triphosphate - TCA Trichloroacetic acid - PAGE Polyacrylamide gel electrophoresis     

Calmodulin in the membrane transport of Ca++

The involvement of calmodulin in a Ca⁺⁺-transporting process was demonstrated for the first time by Jarrett and Penniston and by Gopinath and Vincenzi in 1977. They observed that the Ca⁺⁺-pumping ATPase of the erythrocyte membrane was specifically activated by a soluble protein factor which shared the properties of calmodulin, and was indeed found to be identical with it. This fundamental observation has triggered a considerable amount of activity in the various areas of membrane transport of Ca⁺⁺. As a result, it is now known that calmodulin is involved in many processes of Ca⁺⁺ transport located in various membranes, and it has also become known that it plays different roles in different transport systems. The fine details of the interaction(s) between calmodulin and the various transport systems are at the moment obscure. It has become obvious, however, that some Ca⁺⁺ transporting enzymes interact with calmodulin directly, whereas some do not, and are influenced by it via intermediate (modulating) proteins. At the present initial state of knowledge, this difference offers a convenient means to classify the type of calmodulin involvment in the various Ca⁺⁺ transporting processes. It will thus be used in the synthetic survey presented in this article.     

The effect of phosphorylation of gizzard myosin on actin activation

Gizzard myosin is phosphorylated by a kinase found in chicken gizzards. The 20,000 dalton light chains are the only subunits to show an appreciable extent of ³²P incorporation. Phosphorylation requires trace amounts of Ca²⁺. The Mg²⁺-ATPase activity of gizzard myosin in the phosphorylated form is activated to an appreciable extent by skeletal actin, whereas the activation of the non-phosphorylated myosin is verylow. These results suggest that the Ca²⁺-sensitive regulatory mechanism of gizzard actomyosin is mediated via a kinase. In the presence of Ca²⁺ the onset of contraction and the resultant increase of the Mg²⁺-ATPase activity we suggest is due, at least partly, to the phosphorylation of the 20,000 dalton light chains. Whether or not Ca²⁺ binding by myosin is also essential remains to be established.     

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.     

Regulation of calcium accumulation and efflux from platelet vesicles: Possible role for cyclic-AMP-dependent phosphorylation and calmodulin

Calcium-accumulating vesicles were isolated by differential centrifugation of sonicated platelets. Such vesicles exhibit a $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ activity of about 10 nmol (min·mg)^?1 and an ATP-dependent Ca²⁺ uptake of about 10 nmol (min·mg)^?1. When incubated in the presence of Mg[γ-³²P]ATP, the pump is phosphorylated and the acyl phosphate bond is sensitive to hydroxylamine. The [³²P]phosphate-labeled Ca²⁺ pump exhibits a subunit molecular weight of 120 000 when analyzed by lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Platelet calcium-accumulating vesicles contain a 23 kDa membrane protein that is phosphorylatable by the catalytic subunit of cAMP-dependent protein kinase but not by protein kinase C. This phosphate acceptor is not phosphorylated when the vesicles are incubated in the presence of either Ca²⁺ or Ca²⁺ plus calmodulin. The latter protein is bound to the vesicles and represents 0.5% of the proteins present in the membrane fraction. Binding of ¹²⁵I-labeled calmodulin to this membrane fraction was of high affinity (16 nM), and the use of an overlay technique revealed four major calmodulin-binding proteins in the platelet cytosol ($\text{M}{}_{\mathrm{r}}\text{= 94 000, 87 000, 60 000 and 43 000}$). Some minor calmodulin-binding proteins were enriched in the membrane fractions ($\text{M}{}_{\mathrm{r}}\text{= 69 000, 57 000, 39 000 and 37 000}$). When the vesicles are phosphorylated in the presence of MgATP and of the catalytic subunit of cAMP-dependent protein kinase, the rate of Ca²⁺ uptake is essentially unaltered, while the Ca²⁺ capacity is diminished as a consequence of a doubling in the rate of Ca²⁺ efflux. Therefore, the inhibitory effect of cAMP on platelet function cannot be explained in such simple terms as an increased rate of Ca²⁺ removal from the cytosol. Calmodulin, on the other hand, was observed to have no effect on the initial rate of calcium efflux when added either in the absence or in the presence of the catalytic subunit of the cyclic AMP-dependent protein kinase, nor did the addition of 0.5 μM calmodulin result in increased levels of vesicle phosphorylation.     

Biochemical characterization of Ca2+/calmodulin dependent protein kinase from Candida albicans

A multifunctional Ca²⁺/calmodulin dependent protein kinase was purified approximately 650 fold from cytosolic extract of Candida albicans. The purified preparation gave a single band of 69 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis with its native molecular mass of 71 kDa suggesting that the enzyme is monomeric. Its activity was dependent on calcium, calmodulin and ATP when measured at saturating histone IIs concentration. The purified Ca²⁺/CaMPK was found to be autophosphorylated at serine residue(s) in the presence of Ca²⁺/calmodulin and enzyme stimulation was strongly inhibited by W-7 (CaM antagonist) and KN-62 (Ca²⁺/CaM dependent PK inhibitor). These results confirm that the purified enzyme is Ca²⁺/CaM dependent protein kinase of Candida albicans. The enzyme phosphorylated a number of exogenous and endogenous substrates in a Ca²⁺/calmodulin dependent manner suggesting that the enzyme is a multifunctional Ca²⁺/calmodulin-dependent protein kinase of Candida albicans.     

Studies on calmodulin isolated from Tetrahymena cilia and its localization within the cilium

Tetrahymena calmodulins from cilia, cell bodies and whole cells were isolated separately and compared. These calmodulins showed just the same properties: they co-migrated in SDS-polyacrylamide gel electrophoresis, had a Ca²⁺-dependent electrophoretic mobility change in alkali gel, held the same antigenic determinants in common, and activated brain cyclic nucleotide phosphodiesterase Ca²⁺-dependently with identical activation curves. Distributions of calmodulin and calmodulin-counterpart in Tetrahymena cilium were investigated by using alkali gel electrophoresis in the presence of Ca²⁺ or EGTA, and by immunoelectron microscopy. Calmodulin was detected in the membrane plus matrix fraction and outer-doublet microtubule fraction, and its Ca²⁺-dependent counterpart existed exclusively in the latter fraction. However, neither calmodulin nor its counterpart was detected in the crude dynein fraction. Immunoelectron microscopy revealed that calmodulin was localized along the longitudinal axis of outer-doublet microtubules at regular intervals of about 90 nm. The calmodulin-binding site in the ciliary axoneme was suggested to be interdoublet links.     

Regulation of axonemal Mg2+-ATPase from Paramecium cilia: effects of Ca2+ and cyclic nucleotides

Ciliary activity is regulated by Ca2+ and cyclic nucleotides, but the molecular mechanisms of the regulation are unknown. We have tested the ability of Ca2+ and cyclic nucleotides to alter ciliary Mg2+-ATPase or to stimulate phosphorylation of axonemal dynein. Mg2+-ATPase activity in cilia and axonemes from Paramecium was stimulated 2-fold by micromolar Ca2+, but this Ca2+ sensitivity was lost upon solubilization of the dyneins from the axoneme. The Ca2+-sensitive component of ciliary Mg2+-ATPase activity was inhibited by the dynein inhibitors vanadate and Zn2+, but was insensitive to the calmodulin antagonists calmidazolium and melittin. Dynein activity in the high-salt extract from axonemes was also insensitive to calmidazolium. Calmodulin did not sediment with 22 S or 12 S dyneins on sucrose gradients containing Ca2+, but it did sediment in the region from 19 S to 14 S. Mg2+-ATPase activity in ciliary fractions was unaltered in the presence of cAMP or cGMP. However, polypeptides associated with the 22 S and 12 S dyneins, as well as proteins of 19 S, 15 S, and 8 S, were substrates for endogenous ciliary kinases. High molecular weight polypeptides that sedimented at 22 S and 19 S were phosphorylated in a cyclic nucleotide-stimulated manner.     

Physarum myosin light chain one: A potential regulatory factor in cytoplasmic streaming

Summary Physarum myosin is composed of a heavy chain of about 225,000 daltons and two small polypeptides of 17,700 and 16,100 daltons, called light chain one (LC 1) and two (LC 2). Light chain one is shown to belong to the general class of regulating light chains by two independent criteria. After denaturation, purification and renaturation of thePhysarum light chains only LC 1 will combine with scallop myofibrils in which one myosin regulatory light chain has been removed. This LC 1 can restore inhibition of the ATPase activity of the myofibrils at 10^–8 M Ca⁺⁺ just as well as light chains from rabbit skeletal myosin. Secondly, this LC 1 is the only component of the myosin that is significantly phosphorylated by an endogenous kinase present in crude actomyosin. An active phosphatase is also present. Preliminary results could not detect calcium sensitivity for either kinase or phosphatase, nevertheless the importance of phosphorylation in affecting activity of biological systems suggests that LC 1 may serve some regulating function for plasmodial actomyosin.