Mechanisms of growth inhibition of Phytomonas serpens by the alkaloids tomatine and tomatidine.

Phytomonas serpens are flagellates in the family Trypanosomatidae that parasitise the tomato plant (Solanum lycopersicum L.), which results in fruits with low commercial value. The tomato glycoalkaloid tomatine and its aglycone tomatidine inhibit the growth of P. serpens in axenic cultures. Tomatine, like many other saponins, induces permeabilisation of the cell membrane and a loss of cell content, including the cytosolic enzyme pyruvate kinase. In contrast, tomatidine does not cause permeabilisation of membranes, but instead provokes morphological changes, including vacuolisation. Phytomonas treated with tomatidine show an increased accumulation of labelled neutral lipids (BODYPY-palmitic), a notable decrease in the amount of C24-alkylated sterols and an increase in zymosterol content. These results are consistent with the inhibition of 24-sterol methyltransferase (SMT), which is an important enzyme that is responsible for the methylation of sterols at the 24 position. We propose that the main target of tomatidine is the sterols biosynthetic pathway, specifically, inhibition of the 24-SMT. Altogether, the results obtained in the present paper suggest a more general effect of alkaloids in trypanosomatids, which opens potential therapeutic possibilities for the treatment of the diseases caused by these pathogens.

Identification of a crab gill FXYD2 protein and regulation of crab microsomal Na,K-ATPase activity by mammalian FXYD2 peptide.

This investigation discloses the recognition of an FXYD2 protein in a microsomal Na,K-ATPase preparation from the posterior gills of the blue crab, Callinectes danae, by a mammalian (rabbit) FXYD2 peptide specific antibody (γC(33)) and MALDI-TOF-TOF mass spectrometry techniques. This is the first demonstration of an invertebrate FXYD2 protein. The addition of exogenous pig FXYD2 peptide to the crab gill microsomal fraction stimulated Na,K-ATPase activity in a dose-dependent manner. Exogenous pig FXYD2 also considerably increased enzyme affinity for K(+), ATP and NH(4)(+). K(0.5) for Na(+) was unaffected. Exogenous pig FXYD2 increased the V(max) for stimulation of gill Na,K-ATPase activity by Na(+), K(+) and ATP, by 30% to 40%. The crab gill FXYD2 is phosphorylated by PKA, suggesting a regulatory function similar to that known for the mammalian enzyme. The PKA-phosphorylated pig FXYD2 peptide stimulated the crab gill Na,K-ATPase activity by 80%, about 2-fold greater than did the non-phosphorylated peptide. Stimulation by the PKC-phosphorylated pig FXYD2 peptide was minimal. These findings confirm the presence of an FXYD2 peptide in the crab gill Na,K-ATPase and demonstrate that this peptide plays an important role in regulating enzyme activity.

Regulatory phosphorylation of FXYD2 by PKC and cross interactions between FXYD2, plasmalemmal Ca-ATPase and Na,K-ATPase.

FXYD2 is a regulatory peptide associated with the α-subunit of the kidney Na,K-ATPase. FXYD2 can be phosphorylated by PKA, and its phosphorylation activates Na,K-ATPase. Here we show that FXYD2 is phosphorylated by PKC (PKC-FXYD2-P), by PKA (PKA-FXYD2-P) or by PKA and PKC simultaneously (FXYD2-P(2)) modulating both the erythrocyte Na,K-ATPase and the plasma membrane Ca(2+)-ATPase (PMCA). In erythrocyte ghosts, the addition of PKA-FXYD2-P activated Na,K-ATPase by 80%, while non-phosphorylated FXYD2 (np) activated only 55%. The addition of np FXYD2 did not affect PMCA basal activity, but FXYD2-P(2) increased the basal PMCA activity by up to 200%. Calmodulin-activated PMCA activity was increased by np FXYD2 (3-fold) or FXYD2-P(2) (2.5-fold). However, PKC-FXYD2-P increased PMCA activity only by 50%. In contrast, when PMCA was treated with PKA-FXYD2-P, the ATPase activity was inhibited by 50%. The effect of all forms of FXYD2-P on calcium uptake from PMCA resembled the pattern observed in ATP hydrolysis. Our results suggest that the FXYD2 anchoring site could be conserved among the P-ATPase family permitting cross regulation. The effects of FXYD2 on calcium uptake and calcium-stimulated ATP hydrolysis suggest a novel role for FXYD2 on PMCA.

Volutin granules of Eimeria parasites are acidic compartments and have physiological and structural characteristics similar to acidocalcisomes.

The structural organization of parasites has been the subject of investigation by many groups and has lead to the identification of structures and metabolic pathways that may represent targets for anti-parasitic drugs. A specific group of organelles named acidocalcisomes has been identified in a number of organisms, including the apicomplexan parasites such as Toxoplasma and Plasmodium, where they have been shown to be involved in cation homeostasis, polyphosphate metabolism, and osmoregulation. Their structural counterparts in the apicomplexan parasite Eimeria have not been fully characterized. In this work, the ultrastructural and chemical properties of acidocalcisomes in Eimeria were characterized. Electron microscopy analysis of Eimeria parasites showed the dense organelles called volutin granules similar to acidocalcisomes. Immunolocalization of the vacuolar proton pyrophosphatase, considered as a marker for acidocalcisomes, showed labeling in vesicles of size and distribution similar to the dense organelles seen by electron microscopy. Spectrophotometric measurements of the kinetics of proton uptake showed a vacuolar proton pyrophosphatase activity. X-ray mapping revealed significant amounts of Na, Mg, P, K, Ca, and Zn in their matrix. The results suggest that volutin granules of Eimeria parasites are acidic, dense organelles, and possess structural and chemical properties analogous to those of other acidocalcisomes, suggesting a similar functional role in these parasites.

Mechanism of modulation of the plasma membrane Ca(2+)-ATPase by arachidonic acid.

The intracellular level of long chain fatty acids controls the Ca(2+) concentration in the cytoplasm. The molecular mechanisms underlying this Ca(2+) mobilization are not fully understood. We show here that the addition of low micromolar concentrations of fatty acids directly to the purified plasma membrane Ca(2+)-ATPase enhance ATP hydrolysis, while higher concentration decrease activity, exerting a dual effect on the enzyme. The effect of arachidonic acid is similar in the presence or absence of calmodulin, acidic phospholipids or ATP at the regulatory site, thereby precluding these sites as probable acid binding sites. At low arachidonic acid concentrations, neither the affinity for calcium nor the phosphoenzyme levels are significantly modified, while at higher concentrations both are decreased. The action of arachidonic acid is isoenzyme specific. The increase on ATP hydrolysis, however, is uncoupled from calcium transport, because arachidonic acid increases the permeability of erythrocyte membranes to calcium. Oleic acid has no effect on membrane permeability while linoleic acid shows an effect similar to that of arachidonic acid. Such effects might contribute to the entry of extracellular Ca(2+) following to fatty acid release.

Inhibition of plasma membrane Ca2+-ATPase by heparin is modulated by potassium.

Heparin is related to several protein receptors that control Ca2+ homeostasis. Here, we studied the effects of heparin on the plasma membrane Ca2+-ATPase from erythrocytes. Both ATP hydrolysis and Ca2+ uptake were inhibited by heparin without modification of the steady-state level of phosphoenzyme formed by ATP. Calmodulin did neither modify the inhibition nor the binding of heparin. Inhibition by heparin was counteracted by K+ but not by Li+. This effect was extended to other sulfated polysaccharides with high number of sulfate residues. Hydrolysis of p-nitrophenylphosphate was equally inhibited by heparin. No evidence for enzyme uncoupling was observed: Ca2+ uptake and ATP hydrolysis remained tightly associated at any level of heparin, and heparin did not increase the passive Ca2+ efflux of inside-out vesicles. Vanadate blocked this efflux, indicating that the main point of Ca2+ escape from these vesicles was linked to the Ca2+ pump. It is discussed that sulfated polysaccharides may physiologically increase the steady-state level of Ca2+ in the cytosol by inhibiting the Ca2+ pumps in a K+ (and tissue) regulated way. It is suggested that heparin regulates the plasma membrane Ca2+-ATPase by binding to the E2 conformer.

Inhibition of plasma membrane Ca(2+)-ATPase by CrATP. LaATP but not CrATP stabilizes the Ca(2+)-occluded state.

The bidentate complex of ATP with Cr(3+), CrATP, is a nucleotide analog that is known to inhibit the sarcoplasmic reticulum Ca(2+)-ATPase and the Na(+),K(+)-ATPase, so that these enzymes accumulate in a conformation with the transported ion (Ca(2+) and Na(+), respectively) occluded from the medium. Here, it is shown that CrATP is also an effective and irreversible inhibitor of the plasma membrane Ca(2+)-ATPase. The complex inhibited with similar efficiency the Ca(2+)-dependent ATPase and the phosphatase activities as well as the enzyme phosphorylation by ATP. The inhibition proceeded slowly (T(1/2)=30 min at 37 degrees C) with a K(i)=28+/-9 microM. The inclusion of ATP, ADP or AMPPNP in the inhibition medium effectively protected the enzyme against the inhibition, whereas ITP, which is not a PMCA substrate, did not. The rate of inhibition was strongly dependent on the presence of Mg(2+) but unaltered when Ca(2+) was replaced by EGTA. In spite of the similarities with the inhibition of other P-ATPases, no apparent Ca(2+) occlusion was detected concurrent with the inhibition by CrATP. In contrast, inhibition by the complex of La(3+) with ATP, LaATP, induced the accumulation of phosphoenzyme with a simultaneous occlusion of Ca(2+) at a ratio close to 1.5 mol/mol of phosphoenzyme. The results suggest that the transport of Ca(2+) promoted by the plasma membrane Ca(2+)-ATPase goes through an enzymatic phospho-intermediate that maintains Ca(2+) ions occluded from the media. This intermediate is stabilized by LaATP but not by CrATP.

The gamma subunit of Na+, K+-ATPase: role on ATPase activity and regulatory phosphorylation by PKA.

In kidney, Na+, K+-ATPase is an oligomer (alphabeta gamma) with equimolar amounts of essential alpha and beta subunits and one small hydrophobic FXYD protein (gamma subunit). This report describes gamma subunit as an activator of pig kidney outer medulla Na+, K+-ATPase in aqueous medium. The effects of gamma subunit on Na+, K+-ATPase were dose-dependent and preincubation-dependent. Changes in alphabeta/gamma stoichiometry did not alter Km1 for ATP, and slightly increased Km2, but Vmax was increased at both catalytic and regulatory sites. Hydroxylamine treatment of enzyme phosphorylated by ATP (E-P), in the presence of additional gamma subunit, revealed that 52% of the E-P accumulation was not via acyl-phosphate formation. The gamma subunit was phosphorylated by endogenous kinases and by commercial catalytic subunit of protein kinase A (PKA). Additionally, we demonstrated that PKA phosphorylation of gamma subunit increased its capacity to stimulate ATP hydrolysis. These results suggest that gamma subunit can act as an intrinsic Na+, K+-ATPase regulator in kidney.

Troponin C/calmodulin chimeras as erythrocyte plasma membrane Ca2+-ATPase activators.

Calmodulin (CaM) and troponin C (TnC) are EF-hand proteins that play fundamentally different roles in animal physiology. TnC has a very low affinity for the plasma membrane Ca2+-ATPase and is a poor substitute for CaM in increasing the enzyme's affinity for Ca2+ and the rate of ATP hydrolysis. We use a series of recombinant TnC (rTnC)/CaM chimeras to clarify the importance of the CaM carboxyl-terminal domain in the activation of the plasma membrane Ca2+-ATPase. The rTnC/CaM chimera, in which the carboxyl-terminal domain of TnC is replaced by that of CaM, has the same ability as CaM to bind and transmit the signal to Ca2+ sites on the enzyme. There is no further functional gain when the amino-terminal domain is modified to make the rTnC/CaM chimera more CaM-like. To identify which regions of the carboxyl-terminal domain of CaM are responsible for these effects, we constructed the chimeras rTnC/3CaM and rTnC/4CaM, where only one-half of the C-terminal domain of CaM (residues 85-112 or residues 113-148) replaces the corresponding region in rTnC. Neither rTnC/3CaM nor rTnC/4CaM can mimic CaM in its affinity for the enzyme. Nevertheless, with respect to the signal transduction process, rTnC/4CaM, but not rTnC/3CaM, shows the same behaviour as CaM. We conclude that the whole C-terminal domain is required for binding to the enzyme while Ca2+-binding site 4 of CaM bears all the requirements to increase Ca2+ binding at PMCA sites. Such mechanism of binding and activation is distinct from that proposed for most other CaM targets. Furthermore, we suggest that Ala128 and Met124 from CaM site 4 may play a crucial role in discriminating CaM from TnC.

Rotenone-sensitive mitochondrial potential in Phytomonas serpens: electrophoretic Ca(2+) accumulation.

Phytomonas sp. are flagellated trypanosomatid plant parasites that cause diseases of economic importance in plantations of coffee, oil palm, cassava and coconuts. Here we investigated Ca(2+) uptake by the vanadate-insensitive compartments using permeabilized Phytomonas serpens promastigotes. This uptake occurs at a rate of 1.13+/-0.23 nmol Ca(2+) mg x protein(-1) min(-1). It is completely abolished by the H(+) ionophore FCCP and by valinomycin and nigericin. It is also inhibited by 2 microM ruthenium red, which, at this low concentration, is known to inhibit the mitochondrial calcium uniport. Furthermore, salicylhydroxamic acid (SHAM) and propylgallate, specific inhibitors of the alternative oxidase in plant and parasite mitochondria, are also effective as inhibitors of the Ca(2+) transport. These compounds abolish the membrane potential that is monitored with safranine O. Rotenone, an inhibitor of NADH-CoQ oxidoreductase, can also dissipate 100% of the membrane potential. It is suggested that the mitochondria of P. serpens can be energized via oxidation of NADH in a pathway involving the NADH-CoQ oxidoreductase and the alternative oxidase to regenerate the ubiquinone. The electrochemical H(+) gradient can be used to promote Ca(2+) uptake by the mitochondria.

3-O-methylfluorescein phosphate as a fluorescent substrate for plasma membrane Ca2+-ATPase.

3-O-methylfluorescein phosphate hydrolysis, catalyzed by purified erythrocyte Ca2+-ATPase in the absence of Ca2+, was slow in the basal state, activated by phosphatidylserine and controlled proteolysis, but not by calmodulin. p-Nitrophenyl phosphate competitively inhibits hydrolysis in the absence of Ca2+, while ATP inhibits it with a complex kinetics showing a high and a low affinity site for ATP. Labeling with fluorescein isothiocyanate impairs the high affinity binding of ATP, but does not appreciably modify the binding of any of the pseudosubstrates. In the presence of calmodulin, an increase in the Ca2+ concentration produces a bell-shaped curve with a maximum at 50 microM Ca2+. At optimal Ca2+ concentration, hydrolysis of 3-O-methylfluorescein phosphate proceeds in the presence of fluorescein isothiocyanate, is competitively inhibited by p-nitrophenyl phosphate and, in contrast to the result observed in the absence of Ca2+, it is activated by calmodulin. In marked contrast with other pseudosubstrates, hydrolysis of 3-O-methylfluorescein phosphate supports Ca2+ transport. This highly specific activity can be used as a continuous fluorescent marker or as a tool to evaluate partial steps from the reaction cycle of plasma membrane Ca2+-ATPases.

A proton pumping pyrophosphatase in acidocalcisomes of Herpetomonas sp.

Acidocalcisomes are acidic calcium storage organelles found in several microorganisms. They are characterized by their acidic nature, high electron density, high content of polyphosphates and several cations. Electron microscopy contrast tuned images of Herpetomonas sp. showed the presence of several electron dense organelles ranging from 100 to 300 nm in size. In addition, X-ray element mapping associated with energy-filtering transmission electron microscopy showed that most of the cations, namely Na, Mg, P, K, Fe and Zn, are located in their matrix. Using acridine orange as an indicator dye, a pyrophosphate-driven H+ uptake was measured in cells permeabilized by digitonin. This uptake has an optimal pH of 6.5-6.7 and was inhibited by sodium fluoride (NaF) and imidodiphosphate (IDP), two H+-pyrophosphatase inhibitors. H+ uptake was not promoted by ATP. Addition of 50 microM Ca2+ induced the release of H+, suggesting the presence of a Ca2+/H+ countertransport system in the membranes of the acidic compartments. Na+ was unable to release protons from the organelles. The pyrophosphate-dependent H+ uptake was dependent of ion K+ and inhibited by Na+ Herpetomonas sp. immunolabeled with monoclonal antibodies raised against a Trypanosoma cruzi V-H+-pyrophosphatase shows intense fluorescence in cytoplasmatic organelles of size and distribution similar to the electron-dense vacuoles. Together, these results suggest that the electron dense organelles found in Herpetomonas sp. are homologous to the acidocalcisomes described in other trypanosomatids. They possess a vacuolar H+-pyrophosphatase and a Ca2+/H+ antiport. However, in contrast to the other trypanosomatids so far studied, we were not able to measure any ATP promoted H+ transport in the acidocalcisomes of this parasite.

Converting troponin C into calmodulin: effects of mutations in the central helix and of changes in temperature.

Calmodulin (CaM) and troponin C (TnC) are the most similar members of EF-hand family and show few differences in the primary structure. Here, we use mutants of troponin that mimic calmodulin and changes in temperature to investigate the factors that determine their specificity as regulatory proteins. Using a double mutant of troponin that resembles calmodulin in lacking both the N-terminal helix and KGK(91-93) we observe a small difference from troponin in binding to the erythrocyte Ca(2+)-ATPase, and an improvement in enzyme activation. A triple mutant, where in addition, the residues 88-90 are replaced with the corresponding sequence from calmodulin is equivalent to calmodulin in maximal activation, and it restores protein ability to increase Ca(2+) affinity for the enzyme. However, this mutant also binds less tightly (1/100) than calmodulin. Remarkably, a decrease in temperature has a more marked effect in protein binding than either mutation, reducing the difference in affinities to 18-fold, but without any improvement in their ability to increase Ca(2+) affinity for the enzyme. Spectroscopic analysis of hydrophobic domain exposure in EF-hand proteins was carried out using 8-anilino-1-naphthalenesulfonic acid (ANS). The probe shows a much higher fluorescence when bound to the complex Ca(4)-calmodulin than to Ca(4)-troponin. Decreasing the temperature exposes additional hydrophobic regions of troponin. Changing the Mg(2+) concentration does not affect their bindings to the enzyme. It is suggested that the requirements for troponin to mimic calmodulin in binding to the target enzyme, and those for activating it, are met by different regions of the protein.

Visualizing the 3D architecture of multiple erythrocytes infected with Plasmodium at nanoscale by focused ion beam-scanning electron microscopy.

Different methods for three-dimensional visualization of biological structures have been developed and extensively applied by different research groups. In the field of electron microscopy, a new technique that has emerged is the use of a focused ion beam and scanning electron microscopy for 3D reconstruction at nanoscale resolution. The higher extent of volume that can be reconstructed with this instrument represent one of the main benefits of this technique, which can provide statistically relevant 3D morphometrical data. As the life cycle of Plasmodium species is a process that involves several structurally complex developmental stages that are responsible for a series of modifications in the erythrocyte surface and cytoplasm, a high number of features within the parasites and the host cells has to be sampled for the correct interpretation of their 3D organization. Here, we used FIB-SEM to visualize the 3D architecture of multiple erythrocytes infected with Plasmodium chabaudi and analyzed their morphometrical parameters in a 3D space. We analyzed and quantified alterations on the host cells, such as the variety of shapes and sizes of their membrane profiles and parasite internal structures such as a polymorphic organization of hemoglobin-filled tubules. The results show the complex 3D organization of Plasmodium and infected erythrocyte, and demonstrate the contribution of FIB-SEM for the obtainment of statistical data for an accurate interpretation of complex biological structures.

Mechanisms of growth inhibition of Phytomonas serpens by the alkaloids tomatine and tomatidine

Phytomonas serpens are flagellates in the family Trypanosomatidae that parasitise the tomato plant (Solanum lycopersicum L.), which results in fruits with low commercial value. The tomato glycoalkaloid tomatine and its aglycone tomatidine inhibit the growth of P. serpens in axenic cultures. Tomatine, like many other saponins, induces permeabilisation of the cell membrane and a loss of cell content, including the cytosolic enzyme pyruvate kinase. In contrast, tomatidine does not cause permeabilisation of membranes, but instead provokes morphological changes, including vacuolisation. Phytomonas treated with tomatidine show an increased accumulation of labelled neutral lipids (BODYPY-palmitic), a notable decrease in the amount of C24-alkylated sterols and an increase in zymosterol content. These results are consistent with the inhibition of 24-sterol methyltransferase (SMT), which is an important enzyme that is responsible for the methylation of sterols at the 24 position. We propose that the main target of tomatidine is the sterols biosynthetic pathway, specifically, inhibition of the 24-SMT. Altogether, the results obtained in the present paper suggest a more general effect of alkaloids in trypanosomatids, which opens potential therapeutic possibilities for the treatment of the diseases caused by these pathogens.

Kinetics of pyrophosphate-driven proton uptake by acidocalcisomes of Leptomonas wallacei.

In this work, we show the kinetics of pyrophosphate-driven H+ uptake by acidocalcisomes in digitonin-permeabilized promastigotes of Leptomonas wallacei. The vacuolar proton pyrophosphatase activity was optimal in the pH range of 7.5-8.0, was inhibited by imidiodiphosphate, and was completely dependent on K+ and PPi. H+ was released with the addition of Ca2+, suggesting the presence of a Ca2+/H+ antiport. In addition, X-ray elemental mapping associated with energy-filtering transmission electron microscopy showed that most of the Ca, Na, Mg, P, K, Fe, and Zn were located in acidocalcisomes. L. wallacei immunolabeled with antibodies against Trypanosoma cruzi pyrophosphatase show intense fluorescence in cytoplasmatic organelles of size and distribution similar to the acidocalcisomes. Altogether, the results show that L. wallacei acidocalcisomes possess a H+-pyrophosphatase with characteristics of type I V-H+-PPase. However, we did not find any evidence, either for the presence of H+-ATPases or for Na+/H+ exchangers in these acidocalcisomes.

Dimethyl sulfoxide-induced conformational state of Na(+)/K(+)-ATPase studied by proteolytic cleavage.

Effects of dimethyl sulfoxide (Me(2)SO) on substrate affinity for phosphorylation by inorganic phosphate, on phosphorylation by ATP in the absence of Na(+), and on ouabain binding to the free form of the Na(+)/K(+)-ATPase have been attributed to changes in solvation of the active site or Me(2)SO-induced changes in the structure of the enzyme. Here we used selective trypsin cleavage as a procedure to determine the conformations that the Na(+)/K(+)-ATPase acquires in Me(2)SO medium. In water or in Me(2)SO medium, Na(+)/K(+)-ATPase exhibited after partial proteolysis two distinct groups of fragments: (1) in the presence of 0.1 M Na(+) or 0.1 M Na(+) + 3 mM ADP (enzyme in the E1 state) cleavage produced a main fragment of about 76 kDa; and (2) in the presence of 20 mM K(+) (E2 state) a 58-kDa fragment plus two or three fragments of 39-41 kDa were obtained. Cleavage in Me(2)SO medium in the absence of Na(+) and K(+) exhibited the same breakdown pattern as that obtained in the presence of K(+), but a 43-kDa fragment was also observed. An increase in the K(+) concentration to 0.5 mM eliminated the 43-kDa fragment, while a 39- to 41-kDa doublet was accumulated. Both in water and in Me(2)SO medium, a strong enhancement of the 43-kDa band was observed in the presence of either P(i) + ouabain or vanadate, suggesting that the 43-kDa fragment is closely related to the conformation of the phosphorylated enzyme. These results indicate that Me(2)SO acts not only by promoting the release of water from the ATP site, but also by inducing a conformation closely related to the phosphorylated state, even when the enzyme is not phosphorylated.

Conformational changes of the nucleotide site of the plasma membrane Ca2+-ATPase probed by fluorescence quenching.

Fluorescence quenching by the water-soluble ions I(-) and Cs(+) was used to probe solvent accessibility and polarity of the nucleotide/fluorescein isothiocyanate binding pocket of the purified soluble Ca(2+)-ATPase from plasma membranes. The E(1).Ca.CaM conformer was the least accessible state studied, presenting the lowest suppression constant (K(q)) for both I(-) (K(q) = 6.7 M(-)(1)) and Cs(+) (K(q) = 0.7 M(-)(1)). Accessibility to I(-) was similar for the E(2).VO(4) and E(1).Ca states (K(q) = 7.13 and 7.5 M(-)(1), respectively), whereas E(2) was slightly more accessible (K(q) = 9.1 M(-)(1)). The phosphorylated state E(2)-P presented the highest accessibility, with a K(q) of 16.5 M(-)(1), very near the K(q) of 20.3 M(-)(1) for free FITC. I(-) was unequivocally a better fluorescence quencher, being usually nearly 3-fold as efficient as Cs(+), as indicated by the K(q)(I(-))/K(q)(Cs(+)) ratio (R(q)). The advent of a positive charge cluster on the nucleotide/fluorescein binding pocket in different states was suggested by the increase in R(q), which reached a value as high as 9.5 for the E(1).Ca.CaM conformer. These results indicate (i) a very high water accessibility of the nucleotide/fluorescein pocket for E(2)-P that (ii) is more restricted on the free E(2) state and (iii) becomes rather lower for the E(1).Ca states. Additionally, a positive charge effect of amino acids on the nucleotide site, possibly related to ATP binding and phosphoryl transfer, appears in these E(1).Ca states, being absent in the phosphorylated and nonphosphorylated E(2) states.