Characterization of a Ca2+-dependent guanylate cyclase in the excitable ciliary membrane from Paramecium

The membraneous guanylate cyclase of cilia from Paramecium tetraurelia used MgGTP and MnGTP as substrate with Michaelis constants for GTP of 71.5 microM and 36 microM, respectively. A linear Arrhenius plot indicated that a single enzyme entity exists not sensitive to possible phase transitions of membrane lipids. Guanylate cyclase is activated by low concentrations (less than 100 microM) and inhibited by high concentrations (greater than 100 microM) of calcium, half-maximal effects were obtained with 8 microM and 500 microM Ca2+, respectively. Only strontium ions displayed partial activating and inhibiting potency, all other divalent cations tested, Ba2+, Fe2+, Co2+, Mn2+, Sn2+ and Ni2+ had no effect on guanylate cyclase activity. Ca2+ activation increased V; Km remained identical. The Ca2+ stimulated activity was not inhibited by trifluoperazine, tentatively suggesting that the stimulation may not be mediated by calmodulin. Ca2 inhibition was due to a single binding site of Ca2+ at the guanylate cyclase as evidence by a Hill coefficient h = -1 and was noncompetitive. The lanthanides La3+, Ce3+ and Tb3+ were powerful inhibitors of guanylate cyclase, with La3+ the half-maximal effect was obtained with 0.6 microM, it was kinetically a mixed-type inhibition. La3+ and CA2+ competed for the same binding site on the guanylate cyclase as determined by detailed kinetic analysis. Addition of EDTA reversed the activation and inhibition by Ca2+ and the inhibition by La3+. It is discussed that guanylate cyclase may be the initial target enzyme in the cilia for the calcium transient of the calcium-potassium action potential of Paramecium.     

Differential distribution of voltage-dependent calcium channels and guanylate cyclase in the excitable ciliary membrane from Paramecium tetraurelia

A novel method for isolation of cilia and ciliary membrane vesicles from Paramecium tetraurelia has been developed. Using a continuous Percoll gradient of low osmolarity after fragmentation of purified cilia by French Press treatment two membrane fractions with different buoyant densities were obtained. These fractions were further purified by conventional discontinuous sucrose density gradients and characterized biochemically and by electron microscopy. Guanylate cyclase, a membrane bound enzyme, was found almost exclusively in membrane vesicles of high buoyant density while the voltage-sensitive calcium-channel of the ciliary membrane was predominantly localized in low density vesicles. Examination of both fractions by SDS polyacrylamide gel electrophoresis revealed only minor differences in protein pattern in the 34 and 64 kilodaltons range. Morphologically both membrane vesicle fractions had a diameter of about 300 nm, however, the high density vesicle fraction contained a considerably larger amount of multilamellar structures with a multishell, onion-like appearance. Freeze-fracture analysis failed to detect differences in intramembrane particle content between low and high density vesicles. The possible biological relevance of the spatial separation of the calcium-sensor enzyme guanylate cyclase and the voltage-sensitive calcium-channels in the ciliary membrane is discussed in terms of a diffusion controlled mechanism for graded signal transmission.     

Regulation of ciliary adenylate cyclase by Ca2+ in Paramecium.

In the ciliated protozoan Paramecium, Ca2+ and cyclic nucleotides are believed to act as second messengers in the regulation of the ciliary beat. Ciliary adenylate cyclase was activated 20-30-fold (half-maximal at 0.8 microM) and inhibited by higher concentrations (10-20 microM) of free Ca2+ ion. Ca2+ activation was the result of an increase in Vmax., not a change in Km for ATP. The activation by Ca2+ was seen only with Mg2+ATP as substrate; with Mn2+ATP the basal adenylate cyclase activity was 10-20-fold above that with Mg2+ATP, and there was no further activation by Ca2+. The stimulation by Ca2+ of the enzyme in cilia and ciliary membranes was blocked by the calmodulin antagonists calmidazolium (half-inhibition at 5 microM), trifluoperazine (70 microM) and W-7 (50-100 microM). When ciliary membranes (which contained most of the ciliary adenylate cyclase) were prepared in the presence of Ca2+, their adenylate cyclase was insensitive to Ca2+ in the assay. However, the inclusion of EGTA in buffers used for fractionation of cilia resulted in full retention of Ca2+-sensitivity by the ciliary membrane adenylate cyclase. The membrane-active agent saponin specifically suppressed the Ca2+-dependent adenylate cyclase without inhibiting basal activity with Mg2+ATP or Mn2+ATP. The ciliary adenylate cyclase was shown to be distinct from the Ca2+-dependent guanylate cyclase; the two activities had different kinetic parameters and different responses to added calmodulin and calmodulin antagonists. Our results suggest that Ca2+ influx through the voltage-sensitive Ca2+ channels in the ciliary membrane may influence intraciliary cyclic AMP concentrations by regulating adenylate cyclase.     

Biochemical studies of the excitable membrane of Paramecium. IV. Protein kinase activities of cilia and ciliary membrane

Two protein kinases (ATP: protein phosphotransferase, EC 2.7.1.37) were detected in disrupted cilia of Paramecium tetraurelia. One of the enzymes exhibited maximum activity at pH 6.0, required 4 mM Mg2+ for its maximum activity and was stimulated by cyclic AMP and cyclic GMP. Histone was a good exogenous protein substrate for this enzyme, but protamine sulfate was not. The other protein kinase showed a peak of activity at pH 8.0, required 10 mM Mg2+ for its maximum activity and was slightly inhibited by cyclic AMP and cyclic GMP. Protamine sulfate was a good exogenous substrate for this enzyme. The pH 8.0 activity partitioned preferentially with the axonemes, but the pH 6.0 activity was divided almost equally between the axonemes and the membranes. We also found indirect evidence for the presence in cilia of phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) and adenyl cyclase (ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1) activity.     

Activation of the calcium/calmodulin-dependent guanylate cyclase from Paramecium by polypeptide antibiotics and melittin

The activity of the calcium/calmodulin-regulated guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) from Paramecium was stimulated by several polypeptides. The most potent activator was melittin (6-fold at 30 μM), followed by alamethicin, suzukacillin, trichotoxin and gramicidin S. Marginal effects were seen with herbicolin A and polymyxin B, whereas the following compounds had no effect: ionophore A23187, actinomycin C₁, destomycin A, gramicidin A, iturin A, nigericin, nonactin, Tü 1718B, valinomycin and synthetic peptide analogues of alamethicin. Guanylate cyclase activation was not related to ion-transport capacity or to the length of the α-helical segments. Rather, the degree of amphiphilicity seemed to be an important criterion. No difference in activation was seen between native guanylate cyclase and the reconstituted enzyme. Thus, in all likelihood, polypeptide stimulation requires the presence of the guanylate cyclase/calmodulin holo-enzyme. Guanylate cyclase activation was permanent. Enzyme kinetics, such as Michaelis-Menten behavior and non-cooperativity, were retained. Incubation with polypeptides at 37°C prior to substrate addition decreased enzyme stimulation. Activation of cGMP formation as enhanced at elevated incubation temperatures. The activation energy for hemolysis of erythrocytes favorably correlated with the extent of guanylate cyclase activation (r = 0.98), suggesting a similar mechanism of interaction with membrane constituents for both processes.     

Biochemical studies of the excitable membrane of Paramecium tetraurelia. V. Effects of proteases on the ciliary membrane

The swimming behavior of Paramecium is regulated by an excitable membrane that covers the body and cilia of the protozoan. In order to obtain information on the topology and function of ciliary membrane proteins, Paramecia were treated with trypsin, chymotrypsin or pronase and the effects of these proteases were analyzed using electron microscopy, gel electrophoresis of ciliary fractions and behavioral tests. At the concentrations used, trypsin and chymotrypsin had little or no effect on the cells while pronase removed the cell surface coat, visible as fuzzy material covering the cell membrane. The same pronase treatment caused the specific removal of a high molecular weight protein (250 000), as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This protein, the 'immobilization antigen', constitutes the major protein of the ciliary membrane. Although the immobilization antigen was removed (or markedly decreased), no marked and reproducible difference was observed in the swimming behavior of the treated cells. We also determined the effects of proteases on isolated ciliary fractions to explore the sidedness of ciliary membrane proteins. A set of proteins relatively resistant to protease digestion was identified; they may be intrinsic membrane proteins.     

Biochemical studies of the excitable membrane of Paramecium tetraurelia. III. Proteins of cilia and ciliary membranes

As a first step in the biochemical analysis of membrane excitation in wild-type Paramecium and its behavioral mutants we have defined the protein composition of the ciliary membrane of wild-type cells. The techniques for the isolation of cilia and ciliary membrane vesicles were refined. Membranes of high purity and integrity were obtained without the use of detergents. The fractions were characterized by electron microscopy, and the proteins of whole cilia, axonemes, and ciliary membrane vesicles were resolved by SDS polyacrylamide gel electrophoresis and isoelectric focusing in one and two dimensions. Protein patterns and EM appearance of the fractions were highly reproducible. Over 200 polypeptides were present in isolated cilia, most of which were recovered in the axonemal fraction. Trichocysts, which were sometimes present as a minor contaminant in ciliary preparations, were composed of a very distinct set of over 30 polypeptides of mol wt 11,000--19,000. Membrane vesicles contained up to 70 polypeptides of mol wt 15,000--250,000. The major vesicle species were a high molecular weight protein (the "immobilization antigen") and a group of acidic proteins with mol wt similar to or approximately 40,000. These and several other membrane proteins were specifically decreased or totally absent in the axoneme fraction. Tubulin, the major axonemal species, occurred only in trace amounts in isolated vesicles; the same was true for Tetrahymena ciliary membranes prepared by the methods described in this paper. A protein of mol wt 31,000, pI 6.8, was virtually absent in vesicles prepared from cells in exponential growth phase, but became prominent early in stationary phase in good correlation with cellular mating reactivity. This detailed characterization will provide the basis for comparison of the ciliary proteins of wild-type and behavioral mutants and for analysis of topography and function of membrane proteins. It will also be useful in future studies of trichocysts and mating reactions.     

Characterization of Ca2+- or Mg2+-ATPase of the excitable ciliary membrane from Paramecium tetraurelia: comparison with a soluble Ca2+-dependent ATPase

We have characterized divalent-cation-stimulated nucleoside triphosphate hydrolase activity of the excitable ciliary membrane and compared it with a soluble Ca2+-ATPase released upon deciliation of Paramecium. The membrane-bound activity is strongly dependent on a divalent cation; calcium stimulates the basal activity of this enzyme at least 10-fold; magnesium and manganese stimulate less well, and strontium and barium, although less effective, also give measurable stimulation. This membrane-bound activity prefers ATP and GTP as substrates but also hydrolyzes UTP and CTP at measurable rates. The maximum velocity at saturating ATP concentrations and optimal calcium concentrations is 0.3 mumol/min per mg. The pH optimum for the membrane-bound activity is broad and centers around pH 7. From the temperature dependence of ATP hydrolysis, we calculate activation energies of 14 and 11 kcal/mol for the Ca2+- and Mg2+-stimulated activities, respectively. The Arrhenius plot is linear over the temperature range of 4 to 25 degrees C. The membrane ATPase is relatively insensitive to ouabain, oligomycin, N,N'-dicyclohexylcarbodiimide, vanadate, Ruthenium red and two calmodulin antagonists. Polyclonal antisera raised against the purified soluble ATPase from the deciliation supernatant show low reactivity with the membrane-bound ATPase. We conclude from the comparison of properties of the two activities that the ciliary membrane-bound ATPase is distinct from the soluble ATPase released by deciliation.     

Investigation of the role of Ca2+ and calmodulin in the regulation of platelet guanylate cyclase activity.

Both soluble and particulate forms of human platelet guanylate cyclase were found to be sensitive to sub-micromolar concentrations of free Ca2+; soluble enzyme activity increased as Ca2+ was increased from 10 nM to 1 microM; particulate enzyme activity showed a biphasic response to Ca2+, with maximal enzyme activity between 1 and 10 nM free Ca2+ and inhibition occurring at higher Ca2+ concentrations. Neither Ca2+-sensitivity appeared to be calmodulin-dependent.     

Effect of temperature on membrane fluidity and calcium conductance of the excitable ciliary membrane from Paramecium

Fluorescence anisotropy and average fluorescence lifetime of diphenylhexatriene were measured in artificial lipid membrane vesicles. Within the temperature range investigated (15–52°C) both parameters correlate and can be used interchangeably to measure membrane fluidity. Fluorescence anisotropy of DPH in membrane vesicles of cilia from the protozoan Paramecium tetraurelia decreased slightly from 5 to 37°C, yet, no phase transition was observed. An estimated flow activation energy of approx. 2 kcal/mol indicated that the ciliary membrane is very rigid and not readily susceptible to environmental stimuli. The ciliary membrane contains two domains of different membrane fluidity as indicated by two distinct fluorescence lifetimes of diphenylhexatriene of 7.9 and 12.4 ns, respectively. Ca²⁺ flux into ciliary membrane vesicles of Paramecium as measured with the Ca²⁺ indicator dye arsenazo III showed a nonlinear temperature dependency from 5 to 35°C with a minimum around 15°C and increasing flux rates at higher and lower temperatures. The fraction of vesicles permeable for Ca²⁺ remained unaffected by temperature. The differences in temperature dependency of Ca²⁺ conductance and membrane fluidity indicate that the Ca²⁺ permeability of the ciliary membrane is a membrane property which is not directly affected by the fluidity of its lipid environment.     

Dissociation of nitric oxide from soluble guanylate cyclase and heme-nitric oxide/oxygen binding domain constructs

Regulation of soluble guanylate cyclase (sGC), the primary NO receptor, is linked to NO binding to the prosthetic heme group. Recent studies have demonstrated that the degree and duration of sGC activation depend on the presence and ratio of purine nucleotides and on the presence of excess NO. We measured NO dissociation from full-length alpha1beta1 sGC, and the constructs beta1(1-194), beta1(1-385), and beta2(1-217), at 37 and 10 degrees C with and without the substrate analogue guanosine-5'-[(alpha,beta-methylene]triphosphate (GMPCPP) or the activator 3-(5'-hydroxymethyl-3'-furyl)-1-benzylindazole (YC-1). NO dissociation from each construct was complex, requiring two exponentials to fit the data. Decreasing the temperature decreased the contribution of the faster exponential for all constructs. Inclusion of YC-1 moderately accelerated NO dissociation from sGC and beta2(1-217) at 37 degrees C and dramatically accelerated NO dissociation from sGC at 10 degrees C. The presence of GMPCPP also dramatically accelerated NO dissociation from sGC at 10 degrees C. This acceleration is due to increases in the observed rate for each exponential and in the contribution of the faster exponential. Increases in the contribution of the faster exponential correlated with higher activation of sGC by NO. These data indicate that the sGC ferrous-nitrosyl complex adopts two 5-coordinate conformations, a lower activity "closed" form, which releases NO slowly, and a higher activity "open" form, which releases NO rapidly. The ratio of these two species affects the overall rate of NO dissociation. These results have implications for the function of sGC in vivo, where there is evidence for two NO-regulated activity states.     

Biochemical studies of the excitable membrane of Paramecium aurelia. I. 45Ca2+ fluxes across resting and excited membrane.

The characteristics of Ca2+ transport across the excitable membrane of Paramecium aurelia were studied by measuring 45Ca2+ influx and efflux. The intracellular concentration of free Ca2+ in resting P. aurelia was at least ten times less than the extracellular concentration. Ca2+ influx was easily measurable at 0 degrees C, but not at 23 degrees C. The influx of 45Ca2+ was stimulated by the same conditions which cause membrane depolarization and ciliary reversal. Addition of Na+ and K+ (which stimulate ciliary reversal) resulted in a 10-fold increase in the rate of Ca2+ influx. An externally applied, pulsed, electric field (1-2 mA/cm2 of electrode surface), caused the rate of Ca2+ influx to increase 3-5 times, with the extent of stimulation dependent on the current density and the pulse width. Ca2+ influx had the characteristics of a passive transport system and was associated with the chemically or electrically triggered Ca2+ "gating" mechanism, which has been studied electrophysiologically. In contrast, Ca2+ efflux appeared to be catalyzed by an active transport system. With cells previously loaded at 0 degrees C with 45Ca2+, Ca2+ efflux was rapid at 23 degrees C, but did not occur at 0 degrees C. This active Ca2+ efflux mechanism is probably responsible for maintaining the low internal Ca2+ levels in unstimulated cells.     

Effects of K+, Ba2+ and Ca2+ on the excitable membrane in Paramecium tetraurelia

• 1.1. The effects of Ba²⁺ and K⁺ ions on the membrane currents of Paramecium tetraurelia under a voltage clamp were investigated.
• 2.2. External Ba²⁺ suppresses the inward-going K-current and the Ca-induced K-outward current and changes the activation and inactivation kinetics of transient inward current through the Ca-channel.
• 3.3. K⁺ increases the Ca-induced K-conductances but little affects the leakage conductance.
• 4.4. The resting potentials by changing those ionic concentrations shift the voltage sensitivities of all voltage sensitive channels, simultaneously.
• 5.5. The competition between ions to the channel responses was discussed.

     

Protein kinase C and calmodulin effects on the plasma membrane Ca2+-ATPase from excitable and nonexcitable cells

We have purified Ca2+-ATPase from synaptosomal membranes (SM)1 from ratcerebellum by calmodulin affinity chromatography. The enzyme was identifiedas plasma membrane Ca2+-ATPase by its interaction with calmodulin andmonoclonal antibodies produced against red blood cell (RBC) Ca2+-ATPase, andby thapsigargin insensitivity. The purpose of the study was to establishwhether two regulators of the RBC Ca2+-ATPase, calmodulin and protein kinaseC (PKC), affect the Ca2+-ATPase isolated from excitable cells and whethertheir effects are comparable to those on the RBC Ca2+-ATPase. We found thatcalmodulin and PKC activated both enzymes. There were significantquantitative differences in the phosphorylation and activation of the SMversus RBC Ca2+-ATPase. The steady-state Ca2+-ATPase activity of SMCa2+-ATPase was approximately 3 fold lower and significantly less stimulatedby calmodulin. The initial rate of PKC catalyzed phosphorylation (in thepresence of 12-myristate 13-acetate phorbol) was approximately two timesslower for SM enzyme. While phosphorylation of RBC Ca2+-ATPase approachedmaximum level at around 5 min, comparable level of phosphorylation of SMCa2+-ATPase was observed only after 30 min. The PKC-catalyzedphosphorylation resulted in a statistically significant increase inCa2+-ATPase activity of up to 20-40%, higher in the SM Ca2+-ATPase.The differences may be associated with diversities in Ca2+-ATPase functionin erythrocytes and neuronal cells and different isoforms composition.     

Biochemical studies of the excitable membrane of Paramecium tetraurelia. V. effects of proteases on the ciliary membrane

The swimming behavior of Paramecium is regulated by an excitable membrane that covers the body and cilia of the protozoan. In order to obtain information on the topology and function of ciliary membrane proteins, Paramecia were treated with trypsin, chymotrypsin or pronase and the effects of these proteases were analyzed using electron microscopy, gel electrophoresis of ciliary fractions and behavioral tests. At the concentrations used, trypsin and chymotrypsin had little or no effect on the cells while pronase removed the cell surface coat, visible as fuzzy material covering the cell membrane. The same pronase treatment caused the specific removal of a high molecular weight protein (250 000), as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This protein, the ‘immobilization antigen’, constitutes the major protein of the ciliary membrane. Although the immobilization antigen was removed (or markedly decreased), no marked and reproducible difference was observed in the swimming behavior of the treated cells. We also determined the effects of proteases on isolated ciliary fractions to explore the sidedness of ciliary membrane proteins. A set of proteins relatively resistant to protease digestion was identified; they may be intrinsic membrane proteins.     

Purification and properties of guanylate cyclase from the synaptosomal soluble fraction of rat brain.

Guanylate cyclase (GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2) was purified 2250-fold from the synaptosomal soluble fraction of rat brain. The specific activity of the purified enzyme reached 41 nmol cyclic GMP formed per min per mg protein at 37 degrees C. In the purified preparation, GTPase activity was not detected and cyclic GMP phosphodiesterase activity was less than 4% of guanylate cyclase activity. The molecular weight was approx. 480 000. Lubrol PX, hydroxylamine, or NaN3 activated the guanylate cyclase in crude preparations, but had no effect on the purified enzyme. In contrast, NaN3 plus catalase, N-methyl-N'-nitro-N-nitrosoguanidine or sodium nitroprusside activated the purified enzyme. The purified enzyme required Mn2+ for its activity; the maximum activity was observed at 3-5 mM. Cyclic GMP activated guanylate cyclase activity 1.4-fold at 2 mM, whereas inorganic pyrophosphate inhibited it by about 50% at 0.2 mM. Guanylyl-(beta,gamma-methylene)-diphosphonate and guanylyl-imidodiphosphate, analogues of GTP, served as substrates of guanylate cyclase in the purified enzyme preparation. NaN3 plus catalase or N-methyl-N'-nitro-N-nitrosoguanidine also remarkably activated guanylate cyclase activity when the analogues of GTP were used as substrates.