N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin antagonist,also inhibits phospholipid sensitive calcium-dependent protein kinase

N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), commonly regared as a calmodulin antagonist, inhibted phospholipid-sensitive Ca²⁺-dependent protein kinase and to a lesser extent cyclic GMP- and cyclic AMP-dependent protein kinases. Kinetic studies of the inhibition of the homogenous spleen phospholipid-sensitive Ca²⁺-dependent protein kinase indicated that W-7 inhibited the enzyme activity competitively with respect to phospholipid (K_i = 60 μM). N-(6-Aminohexyl)-1-naphthalenesulfonamide (W-5) was found to be musch less potent than W-7. The findings indicate that W-6 was able to inhibit a variety of protein kinases, in addition to those requiring calmodulin previously reported.     

Effect of calmodulin antagonists on auxin-induced elongation

Coleoptile segments of oat (Avena sativa var Cayuse) and corn (Zea mays L. var Patriot) were incubated in different concentrations of calmodulin antagonists in the presence and absence of α-naphthaleneacetic acid. The calmodulin antgonists (chlorpromazine (CP), trifluoperazine, and fluphenazine) inhibited the auxin-induced elongation at 5 to 50 micromolar concentrations. Chlorpromazine sulfoxide, an analog of chlorpromazine, did not have significant effect on the elongation of oat and corn coleoptiles. A specific inhibitor of calmodulin N-(6-aminohexyl)5-chloro-1-naphthalenesulfonamide hydrochloride (W-7, a naphthalenesulfonamide derivative) inhibited coleoptile elongation, while its inactive analog N-(6-aminohexyl)-1-naphthalenesulfonamide hydrochloride (W-5) was ineffective at similar concentrations. During a 4-hour incubation period, coleoptile segments accumulated significant quantities of ³H-CP. About 85 to 90% of auxin-induced growth was recovered after 4 hours of preincubation with CP or 12 hours with W-7 and transferring coleoptiles to buffer containing NAA. Leakage of amino acids from coleoptiles increased with increasing concentration of CP, showing a rapid and significant increase above 20 micromolar CP. The amount of amino acids released in the presence of W-7 and W-5 was significantly lower than the amount released in the presence of CP. Both W-5 and W-7 increased amino acid release but only W-7 inhibited auxin-induced growth. Calmodulin activity measured by phosphodiesterase activation did not differ significantly between auxin-treated and control coleoptile segments. These results suggest the possible involvement of calmodulin in auxin-induced coleoptile elongation.     

Involvement of Calmodulin and Calmodulin-Dependent Myosin Light Chain Kinase in Blue Light-Dependent H Pumping by Guard Cell Protoplasts from Vicia faba L

Signal transduction processes involved in blue light-dependent proton pumping were investigated using guard cell protoplasts from Vicia faba. N-[2-(Methylamino)ethyl]-5-isoquinolinesulfonamide, an inhibitor of cyclic AMP- and cyclic GMP-dependent protein kinases, had no effect. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H-7) and calphostin C, inhibitors of protein kinase C, produced slight inhibition of the blue light-dependent proton pumping. 1-[N, O-Bis(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl] -4-phenylpiperazine, a specific inhibitor of Ca²⁺/calmodulin (CaM)-dependent protein kinase II, did not inhibit the proton pumping, but 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine and 1-(5-chloro-naphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9), inhibitors of Ca²⁺/CaM-dependent myosin light chain kinase, strongly suppressed the proton pumping. A CaM antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), inhibited blue light-dependent proton pumping, whereas its less active structural analog, N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5), had little effect on the response. Other CaM antagonists, trifluoperazine, compound 48/80, prenylamine, and 3-(2-benzothiazolyl)-4,5-dimethoxy-N-[3-(4-phenyl-piperidinyl)- propylbenzenesulfonamide inhibited the proton pumping. In accord with these results, light-induced stomatal opening in the epidermis of Commelina benghalensis ssp. was inhibited by ML-9 and W-7, but not by H-7 and W-5. Thus, it is concluded that CaM and Ca²⁺/CaM-dependent myosin light chain kinase are the components of the signal transduction process in blue light-dependent proton pumping in guard cells.     

Possible involvement of calmodulin and the cytoskeleton in electrofusion of plant protoplasts

Calmodulin antagonists, trifluoperazine, chlorpromazine, calmidazolium, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), strongly inhibited the electrofusion of barley (Hordeum vulgare L. cv Moor) protoplasts with a marked increase of broken fusion products, after 60 minutes of incubation. W-5, a dechlorinated analog of W-7, was found less effective for the inhibition than W-7. Ethyleneglycol-bis(β- aminoethylether)-N,N′-tetraacetic acid a Ca²⁺ chelator, La³⁺, a surface Ca²⁺ antagonist, and verapamil, a Ca²⁺ channel blocker, also inhibited electrofusion. Dielectrophoresis was inhibited by La³⁺. A microtubule inhibitor, vinblastine, inhibited electrofusion strongly while colchicine, slightly. A microfilament inhibitor, cytochalasin B, promoted fused cells to become spherical while phalloidin did not affect electrofusion.     

Evidence for calmodulin inter-domain compaction in solution induced by W-7 binding

Small-angle X-ray scattering and nuclear magnetic resonance were used to investigate the structural change of calcium-bound calmodulin (Ca²⁺/CaM) in solution upon binding to its antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). The radius of gyration was 17.4±0.3 Å for Ca²⁺/CaM-W-7 with a molar ratio of 1:5 and 20.3±0.7 Å for Ca²⁺/CaM. Comparison of the radius of gyration and the pair distance distribution function of the Ca²⁺/CaM-W-7 complex with those of other complexes indicates that binding of two W-7 molecules induces a globular shape for Ca²⁺/CaM, probably caused by an inter-domain compaction. The results suggest a tendency for Ca²⁺/CaM to form a globular structure in solution, which is inducible by a small compound like W-7.     

Evidence for the involvement of calmodulin in the operation of Ca-activated K channels in mouse fibroblasts

Summary The oscillation of membrane potential in fibroblastic L cells is known to result from periodic stimulation of Ca²⁺-activated K⁺ channels due to the oscillatory increase in the intracellular Ca²⁺ concentration. These repeated hyperpolarizations were inhibited by putative calmodulin antagonists, trifluoperazine (TFP), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and promethazine (PMZ), and the concentrations required for half-maximal inhibition were 25, 30 and 300 m, respectively. These doses were lower than those for reducing the membrane resistance due to nonspecific cell damages. Another calmodulin antagonist, chlorpromazine (CPZ), was also effective, but CPZ-sulfoxide was not. Intracellular pressure injections of calmodulin-interacting divalent cations, Ca²⁺, Sr²⁺, Mn²⁺ and Ni²⁺, elicited slow hyperpolarizations, whereas Mg²⁺ and Ba²⁺, which are known to be essentially inert for calmodulin, failed to evoke any responses. The injection of purified calmodulin also brought about a similar hyperpolarization. Quinine, an inhibitor of Ca²⁺-activated K⁺ channels, abolished both Ca²⁺-and calmodulin-induced hyperpolarizations. TFP prevented Ca²⁺-induced hyperpolarizations. The TFP effect was partially reversed by the calmodulin injection. It is concluded that calmodulin is involved in the operation of Ca²⁺-activated K⁺ channels in fibroblasts.     

ATP-dependent Ca2+ transport and Ca2+-ATPase activities in an enriched plasma membrane fraction from gypsy moth larval midgut tissue

A subcellular fraction enriched in plasma membranes was obtained from gypsy moth (Lymantria dispar) larval midgut tissue. Using [⁴⁵Ca]²⁺ as a tracer, Ca²⁺ transport activity by membrane vesicles in the enriched fraction was measured and shown to be ATP-dependent, with a very high affinity for Ca²⁺ (apparent K_m for [Ca²⁺ _free]

1 Abbreviations used: [Ca²⁺free] = concentration of free (unbound) calcium ion;CaM = calmodulin; F = fraction; IOV = inside-out membrane vesicles; W-5 = N-(6-aminohexyl)-1-naphthalenesulfonamide; W-7 = N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide.

= 22 nM). Ca²⁺ transport was abolished upon addition of the calcium ionophore, A23187. Ca²⁺-stimulated, Mg²⁺-dependent ATPase activity peaked between 100 and 200 nM Ca²⁺_free. Ca²⁺-Mg²⁺-ATPase activity was inhibited by vanadate, 2 phenothiazine drugs (trifluoperazine and chlorpromazine), and the naphthalene sulfonamide, W-7; the related compound, W-5, and ouabain had a negligible effect. These results suggest the presence of a high affinity plasma membrane Ca²⁺ pump in gypsy moth larval midgut cells and are discussed in light of earlier work involving calcium transport in isolated midguts of larval Hyalophora cecropia. Ionic and other conditions that characterize the midgut physiology of larval Lepidoptera (e.g., luminal pH; electrochemical gradient for Ca²⁺; effect of certain ions and inhibitors on Ca²⁺ transport) contrast significantly with those found in adult Diptera. The implications that these differences may have for calcium regulation are discussed. © 1992 Wiley-Liss, Inc.     

Effects of W-7 on catecholamine release and 45Ca2+ uptake in cultured adrenal chromaffin cells.

Effects of N-(6-aminohexyl)-5-chloro-1-naph-thalenesulfonamide (W-7), a calmodulin antagonist, on catecholamine (CA) release and ⁴⁵Ca²⁺ uptake were studied using cultured bovine adrenal chromaffin cells. W-7 inhibited the carbamylcholine (CCh)-evoked CA release and ⁴⁵Ca²⁺ uptake in a concentration-dependent manner. The inhibitory effect of W-7 on CCh-evoked CA release was not overcome either by an increase in extracellular calcium or CCh concentration. Although W-7 inhibited the high K⁺-evoked CA release and ⁴⁵Ca²⁺ uptake, potency of the drug was approximately 50–100 fold less than when inhibiting the CCh-evoked CA release and ⁴⁵Ca²⁺ uptake. The inhibitory effects of W-7 were observed both in norepinephrine release and epinephrine release. Moreover, W-7 inhibited the CCh-evoked ⁴⁵Ca²⁺ efflux. These results suggest that the inhibition of CA release by W-7 in adrenal chromaffin cells is mainly due to its inhibition of calcium uptake. W-7 may influence the linkage between acetylcholine-receptor and calcium uptake with higher potency than depolarization-dependent calcium entry.     

K+-Dependent Stimulation of Dopamine Synthesis in Striatal Synaptosomes Is Mediated by Protein Kinase C

Dopamine synthesis rate was measured in striatal synaptosomes. Removal of Na⁺ increased synthesis rate; this was blocked in Ca²⁺-free medium and by addition of the Ca²⁺/calmodulin inhibitor N-6-aminohexyl-5-chloro-1-naphthalenesulfonamide (W7). The increase in dopamine synthesis rate caused by the addition of the phorbol ester 12-O-tetradecanoylphorboI-13-acetate (TPA) was blocked by the protein kinase C inhibitor polymyxin B. K⁺-stimulated synthesis was unchanged in Ca²⁺-free medium or by addition of W7; it was blocked by polymyxin B. The effect of 50 mM K⁺ was additive with that of 8-Br cyclic AMP and of Na⁺ removal; the combined effect of 50 mM K⁺ and TPA was no greater than that of either alone. These results suggest that stimulation of dopamine synthesis in striatal synaptosomes by 50 mM K⁺ is mediated by protein kinase C.     

Calmodulin regulates the Ca2+-dependent slow-vacuolar ion channel in the tonoplast of Chenopodium rubrum suspension cells

The patch-clamp technique was applied to vacuoles isolated from a photoautotrophic suspension cell culture of Chenopodium rubrum L. and vacuolar clamp currents, which are predominantly carried by the previously identified Ca²⁺-dependent slow vacuolar (SV) ion channels, were recorded. These currents, which were activated by 1-s voltage pulses of -100 mV (vacuolar interior negative) in the presence of 100 M Ca²⁺ (cytosolic side), could be blocked completely and reversibly by the calmodulin antagonist W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide] and its chlorine-deficient analogue W-5; half-maximum inhibition was found at approx. 6 M for W-7 and 70 M for W-5. Inhibition was reversed by addition of 1 g · ml^–1 calmodulin purified from Chenopodium cell suspensions; reversal by bovine brain calmodulin was scarcely appreciable. We conclude that cytosolic calmodulin mediates the Ca²⁺ dependence of the SV-channel in the Chenopodium tonoplast.Abbreviations SV-channel slowly activated, vacuolar ion channel - W-5 N-(6-aminohexyl)-1-naphthalenesulfonamide - W-7 N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide We acknowledge support by the Deutsche Forschungsgemeinschaft and the Bundesminister für Forschung und Technologie, Bonn, and by the Justus-Liebig-Universität Giessen (to W.B.)     

Influx of extracellular Ca2+ involved in jasmonic-acid-induced elevation of [Ca2+]cyt and JR1 expression in Arabidopsis thaliana

The changes in cytosolic Ca²⁺ levels play important roles in the signal transduction pathways of many environmental and developmental stimuli in plants and animals. We demonstrated that the increase in cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) of Arabidopsis thaliana leaf cells was induced by exogenous application of jasmonic acid (JA). The elevation of [Ca²⁺]_cyt was detected within 1 min after JA treatment by the fluorescence intensity using laser scanning confocal microscopy, and the elevated level of fluorescence was maintained during measuring time. With pretreatment of nifedipine (Nif), a nonpermeable L-type channel blocker, the fluorescence of [Ca²⁺]_cyt induced by JA was inhibited in a dose-dependent manner. In contrast, verapamil, another L-type channel blocker, had no significant effect. Furthermore, Nif repressed JA-induced gene expression of JR1 but verapamil did not. JA-induced gene expression could be mimicked by higher concentration of extracellular Ca²⁺. W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide], an antagonist of calmodulin (CaM), blocked the JA induction of JR1 expression while W-5 [N-(6-aminohexyl)-1-naphthalenesulfonamide], its inactive antagonist, had no apparent effect. These data provide the evidence that the influx of extracellular Ca²⁺ through Nif sensitive plasma membrane Ca²⁺ channel may be responsible for JA-induced elevation of [Ca²⁺]_cyt and downstream gene expression, CaM may be also involved in JA signaling pathway.     

The effects of several ion channel blockers and calmodulin antagonists on fertilization-induced acid release and 45Ca2+ uptake in sea urchin eggs

The effects of calcium antagonists, diltiazem and verapamil, and calmodulin antagonists, chlorpromazine, N-(6-aminohexyl)-1-naphthalenesulfonamide hydrochloride (W-5) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), were tested on two responses of the sea urchin egg to insemination: (1) H+ release; (2) Ca2+ uptake. It was found that calcium antagonists inhibited both processes, while calmodulin antagonists only inhibited H+ release but not Ca2+ uptake. Verapamil and diltiazem were effective to inhibit H+ release when added to the egg suspension up to 120 sec and W-7 was effective around 150 sec after insemination. Calcium antagonists became ineffective earlier than W-7 in inhibiting H+ release. A calmodulin-dependent step may thus occur linking the Ca2+ uptake and H+ release. 4,4'-Diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), an anion channel blocker, also inhibited both Ca2+ uptake and H+ release. This result suggests that an uptake of anion(s) occurs along with Ca2+ uptake.     

A rapid separation of bovine brain S-100a and S-100b proteins and related conformation studies

S-100 protein absorbs to the calmodulin antagonist W-7 coupled to epoxy-activated Sepharose 6B in the presence of Ca²⁺ and is eluted by ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid buffer. S-100a and S-100b were separated and isolated by Ca²⁺-dependent affinity chromatography on W-7 Sepharose. The Ca²⁺-induced conformational changes of S-100a and S-100b were examined using circular dichroism, ultraviolet difference spectra, and a fluorescence probe. Differences in Ca²⁺-dependent conformational changes between S-100a and S-100b became apparent. Circular dichroism studies revealed that both S-100a and S-100b undergo a conformational change upon binding of Ca²⁺ in the aromatic and far-uv range. In the presence or absence of Ca²⁺, the aromatic CD spectrum of S-100a differed completely from that of S-100b, possibly due to the single tryptophan residue of S-100a. Far-uv studies indicate that α-helical contents of both S-100a and S-100b decreased with addition of Ca²⁺. Ca²⁺-induced conformational changes of S-100a and S-100b were also detected by uv difference spectra. The spectrum of S-100a also differed from that of S-100b. Fluorescence studies using 2-p-toluidinylnaphthalene-6-sulfonate (TNS), a hydrophobic probe for protein, revealed a slight difference in conformational changes of these two components. The interaction of TNS and S-100b was observed with concentrations above 3 μm Ca²⁺; on the other hand, S-100a required concentrations above 8 μm. This finding was supported by the difference in the binding affinities of S-100a and S-100b to the W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide)-Sepharose column; both S-100a and S-100b bound the column in the presence of Ca²⁺ but S-100a was eluted prior to S-100b. These results suggest that S-100a and S-100b differ in their dependence on Ca²⁺ and that the affinity-chromatographic separation of S-100a from S-100b on the W-7-Sepharose column makes feasible a rapid purification of these two components.     

Effects of calmodulin antagonists on auxin-stimulated proton extrusion in Avena sativa coleoptile segments

The effect of the 5 calmodulin (CaM) antagonists trifluoperazine (TFP). compound 48/80, N-(6-aminohexyl)-naphthalenesulfonamtde (W-5), N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), and calmidazolium on auxin-dependent medium acidification was investigated in abraded segments of Avena sativa L. cv. Victory I. Buffering capacity, Asn content, and changes in pH of bathing solutions were measured in the presence of these inhibitors. When coleoptiles were treated with TFP or compound 48/80, the Asn content and the buffering capacity increased, thus suggesting that plasma membrane permeability was modified. On the contrary. the effect of calmidazolium, W-5. and W-7 on Asn release and buffering capacity was rather low; only small effects being observable at the highest concentration employed. Calmidazolium and W-7 strongly inhibited auxin-dependent medium acidification. W-5 did not affect medium acidification. The specificity of these CaM antagonists and their effects on medium acidification are discussed. The data adduced is consistent with the working hypothesis which postulates an essential role for the Ca²⁺-CaM system on auxin-dependent medium acidification.     

Delayed activation of plasma membrane Ca pump in human neutrophils

The interplay between Ca²⁺ efflux mechanisms of the plasma membrane (PM) and transient changes of the cytosolic concentration of ionized calcium ([Ca²⁺]_i) was studied in suspensions of human neutrophils loaded with the [Ca²⁺]_i indicator, Fura-2. To reveal Ca²⁺ efflux through PM the interference of intracellular Ca stores was prevented by preincubating the cells in the presence of EGTA, thapsigargin, and ionomycin. Addition of econazole prevented varying entry of divalent cations regulated by the filling state of Ca stores. The preincubation seemed to empty and permeabilize virtually all Ca stores, ensuring that the monitored changes of [Ca²⁺]_i were caused exclusively by PM Ca²⁺ transporters. Following preincubation, the addition of CaCl₂ induced, mediated by ionomycin, a transient rise of [Ca²⁺]_i, a spike, eventually decreasing to an intermediary [Ca²⁺]_i level. The ATP-dependent decrease of [Ca²⁺]_i terminating the spike was abolished by the calmodulin antagonist, N-(6-aminohexyl)-1-naphthalenesulfonamide (W-7), but not by the protein kinase C inhibitor, staurosporine, nor by Na⁺-free medium, suggesting that neither activity of protein kinase C nor exchange was necessary for generation of the Ca²⁺ spike. In conclusion, the PM Ca²⁺ pump was responsible for the Ca²⁺ spike by responding to the rapid rise of [Ca²⁺]_i by a delayed activation, possibly involving calmodulin. This characteristic feature of the PM pump may be important for the generation of cellular [Ca²⁺]_i spikes in general.     

Calcium/calmodulin-dependent inhibition of microtubule assembly by brain synaptic junction

The effect of synaptic junction (SJ) on microtubule assembly was examined. After preincubation with ATP at 37°C, rat SJ decreased the initial velocity and the extent of the porcine brain microtubule assembly (initiated by the addition of GTP) in a Ca²⁺/calmodulin (CaM)-dependent manner. The degree of the inhibition reached 35% of the control assembly (0-min preincubation) after 20-min preincubation with ATP. There was no inhibition either with heat-treated SJ, at 0°C, or in the presence of EGTA or W-7 (CaM antagonist). The inhibition was due neither to protease(s) nor CaM contaminating the preparations. Free Ca²⁺ concentration level required for the inhibition of microtubule assembly was 10^–6 M. Phosphorylation of microtubule proteins was inhibited by SJ in a Ca²⁺/CaM-dependent manner, and the inhibition occurred in a physiological increase range of intracellular Ca²⁺ concentration (10^–6M) The heat-treated SJ caused no inhibition. The result suggested that the microtubule assembly in the postsynaptic region was regulated by a Ca²⁺/CaM-dependent protein kinase associated with SJ; i. e., major postsynaptic density protein.Abbreviations used CaM calmodulin - DTT dithiothreitol - MAPs microtubule-associated proteins - MES 2-(N-morphorino)ethanesulfonic acid - mPSDp major postsynaptic density protein - PSD postsynaptic density - SDS PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - W-7 N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride     

Breakdown of Phosphatidylinositol during the Elicitation of Phytoalexin Production in Cultured Carrot Cells

Elicitor-induced production of the phytoalexin, 6-methoxymellein, in cultured carrot cells was appreciably depressed by the calmodulin inhibitors N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and trifluoperazine. An inhibitor of Ca²⁺-phospholipid dependent protein kinase (protein kinase C), 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, also inhibited the phytoalexin production in carrot. Both phorbol ester and synthetic diacylglycerol, activators of protein kinase C, showed an ability to induce 6-methoxymellein production even in the absence of elicitor. Phosphatidylinositol-degrading phospholipase activity increased in elicitor-treated carrot cells without a notable lag, and a product of this reaction, inositol trisphosphate, appeared to increase in parallel with the phospholipase activity. These results suggest that breakdown of phosphatidylinositol takes place in the elicitor-treated carrot cells. The messengers liberated from the phospholipid in the plasma membrane may participate in the elicitation process by controlling the activity of protein kinase C-like enzyme(s) and Ca²⁺-mediated processes including calmodulin.     

Partial Purification and Characterization of a Ca2+-Dependent Protein Kinase from the Green Alga, Dunaliella salina

A calcium-dependent protein kinase was partially purified and characterized from the green alga Dunaliella salina. The enzyme was activated at free Ca²⁺ concentrations above 10⁻⁷ molar. and half-maximal activation was at about 3 × 10⁻⁷ molar. The optimum pH for its Ca²⁺-dependent activity was 7.5. The addition of various phospholipids and diolein had no effects on enzyme activity and did not alter the sensitivity of the enzyme toward Ca²⁺. The enzyme was inhibited by calmodulin antagonists, N-(6-aminohexyl)-1-naphthalene sulfonamide and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide in a dose-dependent manner while the protein kinase C inhibitor, sphingosine, had little effect on enzyme activity up to 800 micromolar. Immunoassay showed some calmodulin was present in the kinase preparations. However, it is unlikely the kinase was calmodulin regulated, since it still showed stimulation by Ca²⁺ in gel assays after being electrophoretically separted from calmodulin by two different methods. This gel method of detection of the enzyme indicated that a protein band with an apparent molecular weight of 40,000 showed protein kinase activity at each one of the several steps in the purification procedure. Gel assay analysis also showed that after native gel isoelectric focusing the partially purified kinase preparations had two bands with calcium-dependent activity, at isoelectric points 6.7 and 7.1. By molecular weight, by isoelectric point, and by a comparative immunoassay, the Dunaliella kinase appears to differ from at least some of the calcium-dependent, but calmodulin and phospholipid independent kinases described from higher plants.     

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

A soluble protein was isolated from Mougeotia by chloropromazine-sepharose 4 B affinity chromatography. The protein matches the properties of calmodulin in terms of heat stability, Ca²⁺-dependent electrophoretic mobility in sodium-dodecyl-sulfate polyacrylamide gels, and its ability to activate cyclic nucleotide phosphodiesterase in a Ca²⁺-dependent manner. Phytochrome-mediated chloroplast reorientational movement in Mougeotia was inhibited by the calmodulin antagonist trifluoperazine, a hydrophobic compound, or N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a hydrophilic compound; 50% inhibition (IC₅₀) of chloroplast movement is caused by 20–50 mol l^-1 trifluoperazine or 100 mol l^-1 W-7. The Ca²⁺-calmodulin may act as an intermediate in the chloroplast reorientational response in Mougeotia governed by phytochrome.Abbreviations EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - SDS sodium dodecyl sulfate - W-7 N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide