苄基异喹啉类化合物与钙调素相互作用的荧光光谱研究

 苄基异喹啉类化合物拮抗钙调素(CaM)并抑制依赖CaM的环核苷酸磷酸二酯酶(CaM-PDE)的活力;用荧光测定法可检测它们与钙调素的相互作用。 Ca~(2+)存在下蝙蝙葛碱(D_1)及其衍生物(D_(14))在激发波长340nm处最大发射波长分别为463和455nm,结合CaM后荧光量子产率增加两倍多。它们同CaM的结合均依赖于Ca~(2+)。 本文制备的丹磺酰基CaM(D-CaM)结合Ca~(2+)后荧光最大发射峰值兰移(518→508nm),荧光强度增加22％。在Ca~(2+)存在下小檗胺衍生物E_6能与CaM结合并淬灭Ca~(2+)-D-CaM荧光。 单苄基异喹啉类化合物86040、86045能淬灭CaM的酪氨酸残基的特征荧光。 实验表明,CaM结合D_(14)、E_6、86040和86045的kd值分别为1.3、1.8、9.5和15.7μmol/L,所观察的化合物与CaM的亲和力的大小与它们拮抗CaM,抑制CaM-PDE的酶活力相对应。     

Effects of desethylamiodarone on thyroid hormone metabolism in rats: comparison with the effects of amiodarone

Desethylamiodarone is the principal metabolite of amiodarone. Amiodarone is a class III antiarrhythmic agent, which acts by lengthening repolarization in the myocardium, an effect that is identical to that produced by hypothyroidism. Amiodarone is known to alter thyroid hormone metabolism, and it has been suggested that the mechanism underlying its antiarrhythmic action is the induction of a myocardial but not generalized hypothyroidism. Since the serum levels of desethylamiodarone reach those of the parent compound during chronic amiodarone therapy, it has been suggested that at least part of amiodarone's pharmacological effects may be attributable to the additive effects of the metabolite. Therefore, we investigated the effects of desethylamiodarone on thyroid hormone metabolism and compared them with those of amiodarone in rats. We have shown that chronic treatment with desethylamiodarone decreased serum T3, markedly increased serum reverse T3 with no significant change in serum T4. These effects are similar to those of amiodarone. The data suggest that the chronic effects of amiodarone on thyroid hormone metabolism may be due at least in part to the actions of desethylamiodarone.     

Fluorescence studies on binding of amphiphilic drugs to isolated lamellar bodies: relevance to phospholipidosis

Lysosomal phospholipid storage disorder in lung tissue was observed during chronic treatment with amphiphilic amine drugs. The prevailing and widely accepted mechanism of phospholipidosis is that amphiphilic drugs bind to phospholipids and make the phospholipids unsuitable substrates for the action of phospholipases. We investigated hydrophobic and hydrophilic binding of fifteen drugs to the phospholipid storage organelle, lung lamellar bodies, isolated from male Sprague-Dawley rats. Hydrophobic interactions were studied using 1,6-diphenyl-1,3,5-hexatriene as a fluorescent probe and hydrophilic binding was studied using 1-anilino-8-naphthalene sulfonate as a fluorescent probe. The binding parameters were calculated using Scatchard equations. Of the fifteen drugs used, nine drugs bound to the hydrophobic moiety of lamellar bodies. The order of binding capacities was promethazine greater than chloramphenicol greater than amiodarone = desethylamiodarone greater than promazine greater than chlorpromazine greater than trimipramine greater than propranolol greater than imipramine much greater than chlorphentermine, phentermine, chloroquine, chlorimipramine, cyclizine and chlorcyclizine. Two binding affinities were calculated for all the bound drugs. Binding affinities to hydrophilic sites of lamellar bodies were calculated in terms of emission coefficients for 1-anilino-8-naphthalene sulfonate in the presence of drugs. Hydrophilic binding was in the order chlorpromazine greater than chlorimipramine greater than promazine greater than trimipramine greater than imipramine greater than chlorcyclizine greater than propranolol greater than promethazine greater than chlorphentermine greater than cyclizine greater than phentermine greater than chloroquine much greater than chloramphenicol, amiodarone and desethylamiodarone. The binding affinities of chlorinated analogs were stronger to hydrophilic sites when compared to the parent compound. Amiodarone, which is known to induce pulmonary phospholipidosis and its major non-polar metabolite, desethylamiodarone, bound strongly to lamellar bodies. These two drugs also inhibit phospholipases in vitro. The drugs with weak phospholipidosis-inducing capacity and extensive in vivo metabolism, namely, imipramine, chlorpromazine and promazine, also bound strongly to lamellar bodies with hydrophilic as well as hydrophobic interactions. On the other hand, chloroquine, which is known to induce phospholipidosis and to inhibit phospholipases, did not bind to lamellar bodies. Two major conclusions could be drawn from this study: one is that the drug interactions with isolated lamellar bodies could be studied using membrane fluorescence probes, 1,6-diphenyl-1,3,5-hexatriene and 1-anilino-8-naphthalene sulfonate; second is that the amphiphilic drugs bind to lamellar bodies, as reported for phospholipid vesicles, and the binding of drugs to lamellar bodies could be correlated with their phospholipidosis-inducing capacity only if     

A fluorescence temperature-jump study on Ca2(+)-induced conformational changes in calmodulin

Calmodulin has been shown to alter its conformation so as to interact with a number of target proteins upon Ca2+ binding. A Ca2(+)-binding study of calmodulin was performed by monitoring the fluorescence of intrinsic tyrosine residues and the probe 1-anilinonaphthalene-8-sulfonate (ANS). ANS fluorescence was shown to reflect Ca2+ binding to both high- and low-affinity sites. On the one hand, tyrosine fluorescence was sensitive only to the high-affinity Ca2+ binding. Temperature-jump investigation of the ternary complex of Ca2(+)-calmodulin-ANS in combination with monitoring of ANS fluorescence demonstrated the kinetic characteristics of the conformational change. The relaxation process was attributed to Ca2(+)-induced conformational change and the rate constants of this process were evaluated. On the basis of the rate constants of the conformational change, a rapid response of calmodulin in Ca2+ signaling is suggested.     

Effect of pH on Ca-binding properties of calmodulin and on its interaction with Ca-dependent phosphodiesterase of cyclic nucleotides

The fluorescence of dansyl immobilized on bovine brain calmodulin is sensitive to Ca2+. This effect is due to Ca2+ attachment to specific Ca2+-binding sites of calmodulin and is maintained within a wide range of pH. The native and dansyl-modified calmodulin preparations exert similar activating effects on Ca-dependent phosphodiesterase of cyclic nucleotides and have practically the same affinity for the enzyme. Using fluorescence measurements of the calmodulin--dansyl conjugate, it was shown that the decrease of pH from 9.0 down to 6.0 gradually decreases the constant of Ca2+ binding to calmodulin from 1.5 . 10(10) M-1 to 1.6 . 10(6) M-1. This decrease of pH does not affect the calmodulin affinity for phosphodiesterase. The activating effect of calmodulin on phosphodiesterase is more pronounced at acidic pH values (6.0-7.0) than at alkaline pH values (8.0-9.0).     

Calmodulin-drug interaction. A fluorescence and flow dialysis study

Various Ca2+-antagonists and related compounds were probed for possible anti-calmodulin properties. Some of them efficiently inhibit calmodulin dependent activity (the plasma membrane Ca2+-ATPase and the cyclic nucleotide phosphodiesterase). The I50-values for the most potent inhibitors varied between 15 and 30 uM. Using fluorescence spectroscopy and flow dialysis methods the stoichiometry of the binding of some of the drugs to calmodulin has been investigated. The number of Ca2+-dependent high affinity binding sites has been studied on trypsin fragments of calmodulin. Compound 12-114 was bound with high affinity in a Ca2+-dependent way to both halves of calmodulin, compound 200-737 recognized one high affinity binding site only in the C-terminal half of the molecule, whereas compound 36-079 demanded the intact protein to be able to interact with high affinity in a Ca2+-dependent manner.     

Mechanism of inhibition of Ca2+-transport activity of sarcoplasmic reticulum Ca2+-ATPase by anisodamine

The mechanism of inhibition of Ca2+-transport activity of rabbit sarcoplasmic reticulum Ca 2+-ATPase (SERCA) by anisodamine (a drug isolated from a medicinal herb Hyoscyamuns niger L) was investigated by using ANS (1-anilino-8-naphthalenesulfonate) fluorescence probe, intrinsic fluorescence quenching and Ca 2+-transport activity assays. The number of ANS binding sites for apo Ca2+-ATPase was determined as 8, using a multiple-identical binding site model. Both anisodamine and Ca2+ at millimolar level enhanced the ANS binding fluorescence intensities. Only anisodamine increased the number of ANS molecules bound by SERCA from 8 to 14. The dissociation constants of ANS to the enzyme without any ligand, with 30 mM anisodamine and with 15 mM Ca 2 were found to be 53.0 microM, 85.0 microM and 50.1 microM, respectively. Both anisodamine and Ca2+ enhanced the ANS binding fluorescenc with apparent dissociation constants of 7.6 mM and 2.3 mM, respectively, at a constant concentration of the enzyme. Binding of anisodamine significantly decreased the binding capacity of Ca2+ with the dissociation constant of 9.5 mM, but binding of Ca2+ had no obvious effect on binding of anisodamine. Intrinsic fluorescence quenching and Ca2+-transport activity assays gave the dissociation constants of anisodamine to SERCA as 9.7 and 5.4 mM, respectively, which were consistent with those obtained from ANS-binding fluorescence changes during titration of SERCA with anisodamine and anisodamine + 15 mM Ca2+, respectively. The results suggest that anisodamine regulates Ca2+-transport activity of the enzyme, by stabilizing the trans-membrane domain in an expanded, inactive conformation, at least at its annular ring region.     

Stimulation of the erythrocyte Ca2+-ATPase and of bovine brain cyclic nucleotide phosphodiesterase by chemically modified calmodulin

Chemically modified calmodulins have been used to investigate structural features which are important for the interaction of the activator with targets. Carbamoylation of lysine residues had no influence on the ability of calmodulin to stimulate the plasma membrane Ca2+-ATPase whereas the stimulation of the bovine brain cyclic-nucleotide phosphodiesterase was reduced up to 50%. Different species of carbamoylated calmodulin have been isolated but no differences were detected in their interaction with the cyclic-nucleotide phosphodiesterase. Modification of arginine residues by 1,2-cyclohexanedione had no effect of the stimulation of the phosphodiesterase but reduced by 40% the stimulation of the erythrocyte Ca2+ ATPase. Mild oxidation of methionines by N-chlorosuccinimide produced a number of differently modified calmodulins. The different species have been purified and the modified residues have been identified. They affected the two different test enzymes to different extents indicating that methionines in the central helix of calmodulin are of greater importance for the interaction with the phosphodiesterase, whereas methionines located in the C-terminal half of calmodulin are more important for the interaction with the Ca2+-ATPase.     

Response of three enzymes to oleic acid, trypsin, and calmodulin chemically modified with a reactive phenothiazine

Calmodulin was covalently modified with 10-(1-propionyloxysuccinimide)-2-trifluoromethylphenothiazine++ + to stoichiometries between 0 and 2 mol/mol in the presence of Ca2+. The modified calmodulins, oleic acid, and trypsin were assayed for their ability to activate pea plant NAD kinase, bovine brain 3',5'-cAMP phosphodiesterase, and human erythrocyte Ca2+-ATPase. All modified calmodulins activated both phosphodiesterase and Ca2+-ATPase; at the highest concentration assayed, calmodulin modified with 2 mol of reagent/mol activated phosphodiesterase and Ca2+-ATPase to 53% and 100%, respectively, of the activation obtained with unmodified calmodulin. However, higher concentrations of the modified calmodulins were required to observe the same activation; at least 900-fold and 100-fold higher concentrations were required for the two enzymes, respectively. NAD kinase was not activated by any calmodulin labeled to a stoichiometry greater than 1 mol/mol even when a concentration equal to 17,000 times the apparent dissociation constant of calmodulin for NAD kinase was assayed. Therefore, the modified protein (and not some fraction resistant to labeling) is active toward the mammalian enzymes but inactive toward plant NAD kinase. The different response of the three enzymes to the chemical modification suggests that the enzymes may utilize different binding domains on calmodulin. NAD kinase also was not activated by other known activators of the two mammalian enzymes, namely lipids and limited proteolysis. In parallel experiments using the same agents on each enzyme, NAD kinase was the only enzyme of the three that was not activated by oleic acid and several other lipids or by limited trypsin digestion. These results show that NAD kinase possesses several attributes which would not be predicted by current models of the mechanism of activation of enzymes by calmodulin.     

Ionic changes in the mitotic apparatus at the metaphase/anaphase transition

We have employed a series of permeant, nontoxic, fluorescent probes to detect changes in ionic conditions within the mitotic apparatus of living endosperm cells of Haemanthus during the transition from metaphase to anaphase. Fluorescence emission intensity measurements from the spindle for chlorotetracycline (CTC) decline before the onset of anaphase, indicating a reduction in the amount of membrane- associated Ca2+ and suggesting an efflux of Ca2+ from membrane compartments into the spindle. Subsequent to the onset of anaphase, we observe increases in fluorescence with both 8-anilino-1-naphthalene sulfonate (ANS) and 3,3'-dipentyl 2,2'-dioxacarbocyanine (diO-C5(3)), sensitive to cationic and anionic charges at membrane surfaces, respectively. The increases with ANS and diO-C5(3) suggest that redistributions of ions within the spindle accompany anaphase motion. During the metaphase/anaphase transition, spindle membrane content remains constant, as evidenced by unchanging fluorescence with the hydrophobic probe, N-phenyl-1-naphthylamine (NPN). Shifts in emission intensity from the nonspindle cytoplasm or from the spindle poles do not accompany the changes in fluorescence we observe in the spindle, suggesting that any ionic fluxes responsible for the changes in fluorescence are restricted to the spindle domain.     

Antagonism of calmodulin and phosphodiesterase by nifedipine and related calcium entry blockers

Nifedipine, a 1,4-dihydropyridine Ca2+ entry blocker, partially inhibits calmodulin-activated and, to a lesser extent, basal (non-activated) cyclic AMP phosphodiesterase activity at 10-440 microM. The inhibition of calmodulin-activated phosphodieserase does not parallel Ca2+ entry blockade, since analogs of nifedipine, which are 500-fold less potent than nifedipine as Ca2+ entry blockers (Bolger et al. (1982) Biochemical and Biophysical Research Communications 104, 1604-1609), are equal in potency to nifedipine as calmodulin-activated phosphodiesterase inhibitors. Furthermore, the inhibition of calmodulin-activated phosphodiesterase by nifedipine is about 500-fold less potent than its inhibition of Ca2+ entry blockade. It is suggested that the low affinity interaction of nifedipine and related 1,4-dihydropyridines with calmodulin and phosphodiesterase is also of low specificity and therefore is unlikely to contribute to the cardiac and vascular muscle relaxant actions of these drugs at normal pharmacological concentrations.     

Rabbit myocardial membrane Ca2+-adenosine triphosphatase activity: stimulation in vitro by thyroid hormone

The in vitro stimulation of human and rabbit erythrocyte membrane Ca2+-ATPase activity by physiological concentrations of thyroid hormone has recently been described. To extend these observations to a nucleated cell model, Ca2+-ATPase activity in a membrane preparation obtained from rabbit myocardium has been studied. Activity of 5'-nucleotidase in the preparation was increased 26-fold over that of myocardial homogenate, consistent with enrichment by sarcolemma. Mean basal enzyme activity in membranes from nine animals was 20.8 +/- 3.3 mumol Pi mg membrane protein-1 90 min-1, approximately 20-fold the activity described in rabbit red cell membranes. Exposure of heart membranes in vitro to L-thyroxine (T4) (10(-10)M) increased Ca2+-ATPase activity to 29.2 +/- 3.8 mumol Pi (P less than 0.001). Dose-response studies conducted with T4 showed that maximal stimulatory response was obtained at 10(-10) M). Hormonal stimulation was comparable for L-T4 and triiodo-L-thyronine (T3) (10(-10) M). Tetraiodothyroacetic acid was without biological activity, whereas triiodothyroacetic acid and D-T4, each at 10(-10) M, significantly decreased enzyme activity compared to control (basal) levels. The action of L-T4 on myocardial membrane Ca2+-ATPase activity was inhibited by trifluoperazine (100 microM) and the naphthalenesulfonamide W-7 (50-100 microM), compounds that block actions of calmodulin, the protein activator of membrane-associated Ca2+-ATPase. Radioimmunoassay revealed the presence of calmodulin (1.4 micrograms/mg membrane protein-1) in the myocardial membrane fraction and 0.35 micrograms/mg-1 in cytosol. Myocardial Ca2+-ATPase activity, apparently of sarcolemmal origin, is thus thyroid hormone stimulable. The hormonal responsiveness of this calcium pump-associated enzyme requires calmodulin.     

A derivative of bisbenzylisoquinoline alkaloid is a new and potential calmodulin antagonist

A new derivative of bisbenzylisoquinoline (berbamine type): 0-(4-ethoxylbutyl) berbamine (EBB) was found to possess powerful and specific calmodulin (CaM) inhibitory properties. It inhibited CaM-stimulated Ca2+-Mg2+-ATPase in human erythrocyte membrane with IC50 value of 0.35 microM compared to that of 60 microM of berbamine. CaM-independent basal Ca2+-Mg2+-ATPase, Na+-K+-ATPase and Mg2+-ATPase were not effect at 1.0 microM of EBB at which CaM-dependent Ca2+-Mg2+-ATPase was already potently inhibited. The inhibition of CaM-dependent Ca2+-Mg2+-ATPase was competitive with respect to CaM. Higher amount of CaM reversed the inhibition caused by higher concentration of EBB. Using dansyl-CaM (D-CaM), it was shown that EBB binds directly to CaM and caused a conformational change of CaM polypeptide chain. From fluorescence titration curve we obtained evidence that in the presence of Ca2+, CaM has two specific binding sites for EBB and additional unspecific binding sites. The Ca2+-dependent binding sites of EBB on CaM were novel region different from the binding sites for TFP.     

Activation of the erythrocyte Ca2+-ATPase by either self-association or interaction with calmodulin

The octaethyleneglycol mono-n-dodecyl ether solubilized Ca2+-ATPase purified from human erythrocytes has been studied to determine the physical mechanism of its activation by calmodulin. The dependence of Ca2+-ATPase activity on the enzyme concentration shows a transformation from a calmodulin-dependent to a fully active calmodulin-independent form. The transformation is cooperative with a half-maximal activation at 10-20 nM enzyme. This suggests that at higher enzyme concentrations interactions between Ca2+-ATPase polypeptide chains substitute for calmodulin-enzyme interactions, resulting in activation. In support of this interpretation, the inclusion of higher octaethyleneglycol mono-n-dodecyl ether concentrations shifts the half-maximal transformation to higher enzyme concentrations. Regardless of the detergent concentration, calmodulin decreases by about 2-fold the enzyme concentration required to observe half-maximal Ca2+-ATPase activation, without affecting the maximal velocity or cooperativity. This indicates that calmodulin facilitates interactions between enzyme molecules. The fluorescein-5'-isothiocyanate-modified Ca2+-ATPase shows an increase in fluorescence polarization which occurs over the same narrow concentration range that is seen with the Ca2+-ATPase activity, confirming association of enzyme molecules. Stimulation of the Ca2+-ATPase activity by calmodulin has revealed a stoichiometry of 0.73, with a dissociation constant of 1.6 nM calmodulin. We have demonstrated by use of calmodulin-Sepharose chromatography that both the calmodulin-dependent and independent Ca2+-ATPase forms bind calmodulin, even though stimulation of activity is seen only with the former one. Our data suggest the following two mechanisms for the Ca2+-ATPase activation: self-association of enzyme molecules or interaction with calmodulin.     

Tyrosine phosphorylation modulates the interaction of calmodulin with its target proteins.

The activation of six target enzymes by calmodulin phosphorylated on Tyr99 (PCaM) and the binding affinities of their respective calmodulin binding domains were tested. The six enzymes were: myosin light chain kinase (MLCK), 3'-5'-cyclic nucleotide phosphodiesterase (PDE), plasma membrane (PM) Ca2+-ATPase, Ca2+-CaM dependent protein phosphatase 2B (calcineurin), neuronal nitric oxide synthase (NOS) and type II Ca2+-calmodulin dependent protein kinase (CaM kinase II). In general, tyrosine phosphorylation led to an increase in the activatory properties of calmodulin (CaM). For plasma membrane (PM) Ca2+-ATPase, PDE and CaM kinase II, the primary effect was a decrease in the concentration at which half maximal velocity was attained (Kact). In contrast, for calcineurin and NOS phosphorylation of CaM significantly increased the Vmax. For MLCK, however, neither Vmax nor Kact were affected by tyrosine phosphorylation. Direct determination by fluorescence techniques of the dissociation constants with synthetic peptides corresponding to the CaM-binding domain of the six analysed enzymes revealed that phosphorylation of Tyr99 on CaM generally increased its affinity for the peptides.     

Diagnosis of familial amyloidotic polyneuropathy by recombinant DNA techniques

A calmodulin dependent cyclic nucleotide phosphodiesterase is associated with the head and tailpieces of demembranated rat caudal epididymal sperm. The phosphodiesterase was stimulated two-fold in the presence of Ca2+, while the simultaneous addition of Ca2+ and calmodulin resulted in a four-fold increase in activity. Ca2+ stimulation was abolished if demembranated sperm were extracted with EGTA and was recovered upon the addition of exogenous calmodulin. Micromolar levels of Ca2+ were required for full stimulation. Trifluoperazine inhibited the Ca2+ stimulated enzyme in a dose dependent manner (ID50 = 50 microM) but had no effect on the basal phosphodiesterase activity.     

Fluorescence studies on calmodulin binding to erythrocyte Ca2(+)-ATPase in different oligomerization states

The fluorescent spinach calmodulin derivative 2-(4-maleimidoanilino)naphthalene-6-sulfonic acid-calmodulin (MIANS-CaM) was used to investigate calmodulin interaction with the purified, detergent-solubilized erythrocyte Ca2(+)-ATPase. Previous studies have shown that the Ca2(+)-ATPase exists in equilibria between monomeric and oligomeric forms. We report here that MIANS-CaM binds to both enzyme forms in a Ca2(+)-dependent manner, with a approximately 50% fluorescence enhancement. These findings confirm our previous observation that enzyme oligomers retain their ability to bind calmodulin, even though they are fully activated in the absence of calmodulin. The Ca2+ dependence of MIANS-CaM binding to monomeric Ca2(+)-ATPase is of higher affinity (K 1/2 = 0.09 microM Ca2+) and less cooperative (nH = 1.1) than the Ca2+ dependence of enzyme activation by MIANS-CaM (K 1/2 = 0.26 microM Ca2+, nH = 2.8). These Ca2+ dependences and the order of events, in which calmodulin binding precedes enzyme activation, demonstrate that calmodulin indeed could be a physiological activator of the monomeric enzyme. The calcium dependence of calmodulin binding to oligomeric Ca2(+)-ATPase occurs at even lower levels of Ca2+ (K 1/2 = 0.04 microM Ca2+), in a highly cooperative fashion (nH = 2.3), and essentially in parallel with enzyme activation (K 1/2 = 0.05 microM Ca2+, nH = 2.9). The observed differences between monomers and oligomers suggest that the oligomerized Ca2(+)-ATPase is in a conformation necessary for efficient, cooperative calcium binding at nanomolar Ca2+, which the monomeric enzyme acquires only upon interaction with calmodulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of lipids on the binding of calmodulin with cyclic nucleotide phosphodiesterase

The effects of various lipids on calmodulin interaction with Ca-dependent phosphodiesterase were investigated. Palmitic, myristic and stearic acids increased the enzyme activity; the degree of the enzyme activation by calmodulin was decreased thereby. Oleic acid produced a weak activating effect on phosphodiesterase but completely blocked calmodulin action. The effects of the fatty acids under study were reversible, the activation constant was equal to 10(-4)-5 X 10(-4) M. In the presence of Ca2+ phosphoinositides and fatty acids changed the fluorescence intensity of dansyl-labelled calmodulin; in the absence of Ca2+ the lipids did not affect protein fluorescence. The lipids had no influence on the protein affinity for Ca2+. During chromatography of phosphodiesterase on calmodulin-Sepharose the enzyme was eluted from the column both in the presence of EGTA and palmitic acid. It was concluded that fatty acids prevent the formation of the calmodulin - phosphodiesterase complex. This effects may both be due to the lipid binding to the enzyme and to calmodulin.     

A Ca2+-sensitive cyclic-3',5'-nucleotide phosphodiesterase from sheep lung modulated by a tightly bound endogenous calmodulin

Highly purified sheep lung cyclic-3',5'-nucleotide phosphodiesterase was sensitive to Ca2+/EGTA but insensitive to exogenous calmodulin. The Ca2+-sensitivity was inhibited by trifluoperazine. Heat-treated enzyme could activate a calmodulin-deficient phosphodiesterase, suggesting the presence of endogenous calmodulin in sheep lung cyclic-3',5'-nucleotide phosphodiesterase, possibly associated with the enzyme in a Ca2+-independent manner.     

Different conformation changes induced by calcium and terbium of the porcine intestinal calcium-binding protein

The fluorescence of 1-anilino-8-naphthalenesulfonate (ANS) is progressively enhanced with increasing concentration of it, showing a proportionate blue shift of the emission maximum, by the interaction with the porcine intestinal Ca2+-binding protein (CaBP) in the absence of Ca2+. The apo-CaBP has a single binding site for ANS as determined by the fluorescence change, the apparent dissociation constant (Kd) estimated at 49.1 microM. Addition of Ca2+ or Tb3+ to the ANS-apo-CaBP system is capable of enhancing its fluorescence up to about 2- or 5-fold, respectively, causing further blue shift of the emission maximum. These metal ions do not affect the capacity of ANS binding, but Ca2+ slightly increases the Kd value. Increase of the fluorescence of the ANS-CaBP complex by increasing binding of Ca2+ to it was monophasic, while that with Tb3+ was biphasic, both saturated at the same molar ratio, 2, of added cations to the complex. Biphasic change of response has also been observed in UV absorption of the CaBP with increasing concentration of Tb3+. With a half-saturating concentration of Tb3+, Ca2+ can induce a much higher enhancement of the ANS fluorescence than excess Ca2+ alone. All these results indicate that the CaBP molecule contains a single ANS binding site and the conformation and/or microenvironment surrounding bound ANS of the protein is altered reversibly with binding of Ca2+ or Tb3+ to it and that there are differences between Ca2+- and Tb3+-induced conformation changes around the ANS-binding site and the tyrosine residue of it.