Interactions of annexins with membrane phospholipids.

The annexins are proteins that bind to membranes and can aggregate vesicles and modulate fusion rates in a Ca2(+)-dependent manner. In this study, experiments are presented that utilize a pyrene derivative of phosphatidylcholine to examine the Ca2(+)-dependent membrane binding of soluble human annexin V and other annexins. When annexin V and other annexins were bound to liposomes containing 5 mol % acyl chain labeled 3-palmitoyl-2-(1-pyrenedecanoyl)-L-alpha-phosphatidylcholine, a decrease in the excimer-to-monomer fluorescence ratio was observed, indicating that annexin binding may decrease the lateral mobility of membrane phospholipids without inducing phase separation. The observed increases of monomer fluorescence occurred only with annexins and not with other proteins such as parvalbumin or bovine serum albumin. The extent of the increase of monomer fluorescence was dependent on the protein concentration and was completely and rapidly reversible by EDTA. Annexin V binding to phosphatidylserine liposomes was consistent with a binding surface area of 59 phospholipid molecules per protein. Binding required Ca2+ concentrations ranging between approximately 10 and 100 microM, where there was no significant aggregation or fusion of liposomes on the time scale of the experiments. The polycation spermine also displaced bound annexins, suggesting that binding is largely ionic in nature under these conditions.     

Myoplasmic binding of fura-2 investigated by steady-state fluorescence and absorbance measurements.

Binding of the fluorescent Ca2+ indicator dye fura-2 by intracellular constituents has been investigated by steady-state optical measurements. Fura-2's (a) fluorescence intensity, (b) fluorescence emission anisotropy, (c) fluorescence emission spectrum, and (d) absorbance spectra were measured in glass capillary tubes containing solutions of purified myoplasmic proteins; properties b and c were also measured in frog skeletal muscle fibers microinjected with fura-2. The results indicate that more than half, and possibly as much as 85%, of fura-2 molecules in myoplasm are in a protein-bound form, and that the binding changes many properties of the dye. For example, in vitro characterization of the Ca2+-dye reaction indicates that when fura-2 is bound to aldolase (a large and abundant myoplasmic protein), the dissociation constant of the dye for Ca2+ is three- to fourfold larger than that measured in the absence of protein. The problems raised by intracellular binding of fura-2 to cytoplasmic proteins may well apply to cells other than skeletal muscle fibers.     

Characterization of (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum by laser-excited europium luminescence

The molecular environment of Ca2+ translocating sites of skeletal muscle sarcoplasmic reticulum (SR) (Ca2+ + Mg2+)-ATPase has been studied by pulsed-laser excited luminescence of Eu3+ used as a Ca2+ analogue. Interaction of Eu3+ with SR was characterized by investigating its effect on partial reactions of the Ca2+ transport cycle. In native SR vesicles, Eu3+ was found to inhibit Ca2+ binding, phosphoenzyme formation, ATP hydrolysis activity and Ca2+ uptake in parallel fashion. The non-specific binding of Eu3+ to acidic phospholipids associated with the enzyme was prevented by purifying (Ca2+ + Mg2+)-ATPase and exchanging the endogenous lipids with a neutral phospholipid, dioleoylglycerophosphocholine. The results demonstrate that the observed inhibition of Ca2+ transport by Eu3+ is due to its binding to Ca2+ translocating sites. The 7F0----5D0 transition of Eu3+ bound to these sites was monitored. The non-Lorentzian nature of the excitation profile and a double-exponential fluorescence decay revealed the heterogeneity of the two sites. Measurement of fluorescence decay rates in H2O/D2O mixture buffers further distinguished the sites. The number of water molecules in the first co-ordination sphere of Eu3+ bound at transport sites were found to be 4 and 1.5. Addition of ATP reduced these numbers to zero and 0.6. These data show that the calcium ions in translocating sites are well enclosed by protein ligands and are further occluded down to zero or one water molecule of solvation during the transport process.     

The effects of Ca2+ on lipid diffusion

The effects of Ca2+ on rotational and translational diffusion of lipids in multilamellar dimyristoylphosphatidylcholine (DMPC)-water systems were investigated by time-resolved phosphorescence anisotropy steady-state fluorescence polarization and fluorescence recovery after photobleaching (FRAP) experiments. Above the phase transition temperature (Tm), addition of Ca2+ caused a steady increase in the segmental motion of the phosphorescent probe, but resulted in slower diffusion of the fluorescent and lateral diffusion probes. The former result is attributed to changes in the structure of the lipid/water interface that affects the chromophore mobility on the phosphorescence time scale but does not reflect lipid motion. Below the phase transition temperature, slower diffusion of all probes were observed with increasing concentrations of Ca2+, with sudden large changes occurring at [Ca2+] approximately 500 mM. This behaviour is attributed to association of Ca2+ with the lipid phosphate groups and the exclusion of water molecules which results in tighter packing of lipids and smaller segmental motion, leading eventually to the immobilization of lipid molecules.     

A spin-label study of phosphatidylcholine exchange protein. Regulation of the activity by phosphatidylserine and calcium ion.

The effects of the divalent cations Mg2+, Mn2+ and Ca2+ on the Brownian rotational motion of fluorescently labeled myosin, heavy meromyosin and myosin subfragment-1 were measured by the method of time-resolved fluorescence depolarization. When Mg2+ was added to solutions of myosin or heavy meromyosin and EDTA, their rotational mobility increased. Ca2+ had no effect. Mn2+ increased the mobility of heavy meromyosin but decreased that of myosin. None of these divalent cations effected the mobility of subfragment-1. The binding of heavy meromyosin to actin was affected very little by Mg2+ or EDTA over a wide range of conditions. Divalent cations appear to change the swivel about which the heads of myosin rotate, presumably by binding to light chain 2 (also called DTNB light chain). However, the heads are still able to bind actin in nearly the same way whether Mg2+ is present or not. The concentration of free Mg2+ for the mid-point of the change in heavy meromyosin mobility is in good agreement with that for EDTA activation of ATPase activity. This suggests that EDTA activation is due to removal of Mg2+ bound to myosin itself.     

The binding of Ca2+ to actin monomer is monitored by the fluorescence of actin-bound auramine O.

The fluorescence of the cation auramine O was substantially enhanced by the presence of actin monomer. Titrations of this fluorescence enhancement indicated that actin monomer had two auramine O binding sites, each with a dissociation constant of approx. 20 microM. Calcium ions had no effect on the number of actin monomer-bound auramine O molecules or on the dissociation constant for that interaction. However, calcium ions increased the maximum change of fluorescence that occurs when actin monomer was fully saturated with auramine O. This effect of calcium was saturable and yielded a Ca2+ dissociation constant of 1.6 mM. It was concluded that auramine O bound to sites on actin monomer and independently monitored the binding of Ca2+ ion(s) to other site(s) on actin monomer. Further, the magnitude of the Ca2+ dissociation constant suggested that this Ca2+-binding site may be representative of the multiple bivalent cation-binding sites on actin monomer which are thought to be directly involved in actin polymerization. However, the exact relationship between these sites remains unclear.     

Changes in the steady-state fluorescence anisotropy of N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine attached to the specific thiol of sarcoplasmic reticulum Ca2+-ATPase throughout the catalytic cycle

Cys674 of the sarcoplasmic reticulum Ca2+-ATPase was selectively labeled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine without a loss of the catalytic activity, and the steady-state fluorescence anisotropy of this label and its total fluorescence intensity were followed throughout the catalytic cycle. At 25 degrees C, the anisotropy and the total fluorescence intensity increased by 2.1 and 9.4%, respectively, upon Ca2+ binding to the high affinity sites. Upon subsequent ATP binding to the catalytic site, the anisotropy and the total fluorescence intensity decreased by 6.8 and 23.9%, respectively. These drops likely occurred in the enzyme.ATP complex. The extents of changes upon additions of Ca2+ and ATP in the anisotropy, but not in the total fluorescence intensity, were greatly reduced by lowering the temperature. Slight drops in the anisotropy and the total fluorescence intensity occurred upon conversion of phosphoenzyme (EP) from the ADP-sensitive form to the ADP-insensitive form. The anisotropy and the total fluorescence intensity returned to the initial level when EP was hydrolyzed. Mg2+-dependent Pi-induced drops in the anisotropy and the total fluorescence intensity occurred coincidently with EP formation from Pi. These demonstrate that the ATP-induced drops in the anisotropy and the total fluorescence intensity are predominant throughout the catalytic cycle. Most probably, the changes in the anisotropy are due to changes in the rotational diffusion of the label. These findings indicate that ATP binding to the catalytic site induces a relaxed conformation in the microenvironment of the label bound to Cys674.     

Analysis of calcium binding to alpha-lactalbumin using a fluorescent calcium indicator.

A sensitive and rapid assay of Ca2+ binding to proteins was developed, based on the competition of Ca2+ binding to the protein of interest and fluo-3, a fluorescent Ca2+ indicator. Ca2+ binding to fluo-3 and bovine alpha-lactalbumin was analyzed at ten different pH values and a range of Na+ and K+ concentrations. We demonstrate that the binding constants of alpha-lactalbumin, determined by means of the competition assay and using intrinsic protein fluorescence, are the same within experimental error. The dissociation constant of the alpha-lactalbumin--Ca2+ complex in 50 mM Hepes containing 150 mM Na+ at pH 7.4 and 25 degrees C, was found to be 123 +/- 2 nM and 103 +/- 43 nM when determined by the competition assay and intrinsic protein fluorescence, respectively. Binding of Ca2+ to alpha-lactalbumin did not depend on pH in the range 6.6-8.4 and was differently affected by Na+ and K+. EDTA-agarose, a chelating chromatography material, was synthesized and used to remove Ca2+ from buffer and protein solutions. The total concentration of Ca2+ in 50 mM Hepes, containing 150 mM Na+ at pH 7.4, was lowered to 119 +/- 13 nM and the number of Ca2+ bound/molecule alpha-lactalbumin was lowered to 0.069 +/- 0.006. No interaction between fluo-3 and alpha-lactalbumin could be discerned from spectral analysis and fluorescence anisotropy measurements.     

Interaction between Ca2+ and dipalmitoylphosphatidylcholine membranes. I. Transition anomalies of ultrasonic properties

The effect of Ca2+ on a gel-to-liquid crystal transition as well as the mechanical properties of dipalmitoylphosphatidylcholine bilayers was studied by an ultrasonic technique. Transition temperature increased with increase in Ca2+ concentration, whereas the variation of ultrasonic anomalies indicated that dipalmitoylphosphatidylcholine bilayers exhibited maximum pseudocritical fluctuation at a Ca2+ concentration of about 10 mM. Hardening of dipalmitoylphosphatidylcholine membranes due to the Ca2+ binding was observed above 10 mM CaCl2, suggesting the lateral compression of the lipid bilayer by bound Ca2+. Long-range attraction between bound Ca2+ and the head groups of surrounding lipid molecules was proposed from these calcium effects.     

Binding of Eu3+ to cardiac sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase-laser excited Eu3+ spectroscopic studies.

The binding of Eu3+ with Ca2+-stimulated, Mg2+-dependent adenosine triphosphatase ([Ca2+ + Mg2+]-ATPase) of cardiac sarcoplasmic reticulum (SR) has been investigated using direct laser excited Eu3+ luminescence. Eu3+ is found to inhibit both Ca2+-dependent ATPase activity and Ca2+-uptake in a parallel manner. This is attributed to the binding of Eu3+ to the high affinity Ca2+-binding sites. The Ki for Ca2+-dependent ATPase is approximately 50 nM. The 7F0----5D0 excitation spectrum of Eu3+ in cardiac SR shows a peak at 579.3 nm, as compared to 578.8 nm in potassium-morpholino propane sulfonic acid (K-MOPS) pH 6.8. Upon binding with cardiac SR, Eu3+ shows an increase in fluorescence intensity as well as in lifetime values. The fluorescence decay of bound Eu3+ exhibits a double-exponential curve. The apparent number of water molecules in the first coordination sphere of Eu3+ in SR is 2.8 for the short component and 1.0 for the long component. In the presence of ATP, a further increase in fluorescence lifetimes is observed, and the number of water molecules in the first coordination sphere of Eu3+ is reduced further to 1.3 and 0.5. The double exponential nature of the decay curve and the different number of water molecules coordinated to Eu3+ for both decay components suggest that Eu3+ binds to two sites and that these are heterogeneous. The reduction in the number of H2O ligands in the presence of ATP shows a change in the molecular environment of the Eu3+-binding sites upon phosphoenzyme formation, with a movement of Eu3+ to an occluded site on the enzyme.     

Fluorescence anisotropy of labeled F-actin: influence of divalent cations on the interaction between F-actin and myosin heads

The interaction between F-actin and soluble proteolytic fragments of myosin, heavy meromyosin and myosin subfragment 1 without ATP, has been studied by measuring the static anisotropy and the transient anisotropy decay of the fluorescent chromophore N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl) ethylenediamine bound to F-actin. In the presence of Ca2+ ions, the mobility of the chromophore was strongly decreased by adding heavy meromyosin or myosin subfragment 1, and this conformation change of F-actin showed a strong cooperativity; that is, a very small amount of myosin heads induced the maximum anisotropy change. On the other hand, in the presence of Mg2+ ions, the addition of a small amount of myosin subfragment 1 or of heavy meromyosin increased the mobility of labeled F-actin that reached a maximum at a molar ratio of about 1/25 or 1/50, respectively. With further addition of myosin heads, the mobility of the labeled actin decreased. From these studies, one concludes that F-actin undergoes a conformation change by interacting with myosin heads, which depends on the nature of the divalent cations present in the solution.     

Polarization of chlorotetracycline fluorescence in pancreatic islet cells and its response to calcium ions and D-glucose.

Suspensions rich in pancreatic beta-cells were prepared from non-inbred ob/ob-mice, incubated with 10 micrometer-chlorotetracycline, and analysed for fluorescence polarization in a microscope. Throughout the temperature range 16--38 degrees C, fluorescence was enhanced by 5 mM-Ca2+ in the incubation medium; 20 mM-D-glucose decreased the fluorescence measured in the presence of Ca2+. Fluorescence showed a curvilinear negative regression on temperature. The curves were rectified to a virtually ideal degree by Arrhenius transformations of data. Non-parametric testing of differences between linearized regression lines forms the basis for the following conclusions. The temperature-dependence of fluorescence intensity appeared to be smaller for Ca2+-specific signals than for the background fluorescence of chlorotetracycline in Ca2+-deficient cells. D-Glucose significantly diminished the polarization of fluorescence in cells incubated with Ca2+. It is suggested that D-glucose increases the mobility of Ca2+ in beta-cell plasma membranes; this mobility increase may help to explain previously reported effects of D-glucose on 45Ca2+ fluxes and membrane electric potential.     

Characterization of the substrate-induced conformational change of N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine-labeled sarcoplasmic reticulum Ca2(+)-ATPase by using different kinds of substrate

Cys-674 of the sarcoplasmic reticulum Ca2(+)-ATPase was labeled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine without a loss of the catalytic activity, and changes in the fluorescence intensity upon addition of seven kinds of substrate were followed by the stopped-flow method. The steady-state fluorescence intensity and anisotropy were also determined. When Ca2+ was present, the fluorescence intensity and anisotropy decreased greatly upon addition of any substrate used. The observed affinity for each substrate agreed with the previously observed affinity of the catalytic site. The fluorescence drop induced by the adenine nucleotides, ATP and adenosine 5'-(beta, gamma-methylene)triphosphate (a nonhydrolyzable ATP analog), was much faster than that induced by other substrates. The ATP-induced fluorescence drop preceded phosphoenzyme formation when the ATP concentration was high, but the fluorescence drop coincided with phosphoenzyme formation when it was slowed by reducing ATP concentrations. The fluorescence drop induced by ITP or acetyl phosphate was slow even at high concentrations of the substrate, and it coincided with phosphoenzyme formation. When Ca2+ was absent, the fluorescence intensity and anisotropy decreased only slightly upon addition of any substrate other than the adenine nucleotides. They decreased substantially upon addition of the adenine nucleotides, but the kinetics of this fluorescence drop were quite different from that of the fluorescence drop induced by any substrate in the presence of Ca2+. These results show that the conformational change, which makes the bound label less constrained, is induced by substrate binding to the catalytic site of the Ca2(+)-activated enzyme. This change precedes phosphoenzyme formation in the catalytic cycle and is greatly accelerated by the adenine moiety of the substrate.     

Differential dynamic behavior of actin filaments containing tightly-bound Ca2+ or Mg2+ in the presence of myosin heads actively hydrolyzing ATP.

We have investigated how Ca2+ or Mg2+ bound at the high-affinity cation binding site in F-actin modulates the dynamic response of these filaments to ATP hydrolysis by attached myosin head fragments (S1). Rotational motions of the filaments were monitored using steady-state phosphorescence emission anisotropy of the triplet probe erythrosin-5-iodoacetamide covalently attached to cysteine 374 of actin. The anisotropy of filaments containing only Ca2+ increased from 0.080 to 0.137 upon binding S1 in a rigor complex and decreased to 0.065 in the presence of ATP, indicating that S1 induced additional rotational motions in the filament during ATP hydrolysis. The comparable anisotropy values for Mg(2+)-containing filaments were 0.067, 0.137, and 0.065, indicating that S1 hydrolysis did not induce measurable rotational motions in these filaments. Phalloidin, a fungal toxin which stabilizes F-actin and increases its rigidity, increased the anisotropy of F-actin containing either Ca2+ or Mg2+ but not the anisotropy of the 1:1 S1-actin complexes of these filaments. Mg(2+)-containing filaments with phalloidin bound also displayed increased rotational motions during S1 ATP hydrolysis. A strong positive correlation between the phosphorescence anisotropy of F-actin under specific conditions and the extent of the rotational motions induced by S1 during ATP hydrolysis suggested that the long axis torsional rigidity of F-actin plays a crucial role in modulating the dynamic response of the filaments to ATP hydrolysis by S1. Cooperative responses of F-actin to dynamic perturbations induced by S1 during ATP hydrolysis may thus be physically mediated by the torsional rigidity of the filament.     

Actin-gelsolin interactions. Evidence for two actin-binding sites

We have used a fluorescence enhancement of actin labeled with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-actin) to study the interactions between rabbit skeletal muscle G-actin and either purified platelet gelsolin or a 130-kDa binary complex of platelet actin and gelsolin that is stable in EGTA and can be purified from human platelets. We have delineated four binding reactions. The exchange of Mg2+ for Ca2+ on the divalent cation-binding site of NBD-actin gives a small fluorescence increase. Binding of monomeric NBD-actin to the binary complex results in a 2.5-fold increase in the emission at 530 nm in the presence of Ca2+ and a 2-fold increase in the presence of EGTA. Titration experiments show that, under nonpolymerizing conditions, one additional actin is bound to the 130-kDa species to form a ternary complex. This binding is Ca2+-sensitive. Purified gelsolin does not appear to bind to NBD-actin in the presence of EGTA, as determined by fluorescence enhancement, gel filtration, or sedimentation measurements, but the addition of Ca2+ promotes rapid binding with a 1.6-1.7-fold enhancement of the emission intensity. A comparison of the relative fluorescence yields/NBD-actin molecule for a binary complex of gelsolin and one NBD-actin, a ternary complex of gelsolin and two NBD-actin molecules, and a ternary complex with an unlabeled actin in the EGTA-stable site and an NBD-actin in the second site indicates that the first NBD-actin, in the EGTA-stable site, does not give a fluorescence increase on binding but the second one does. Finally, we have demonstrated that one molecule of 45Ca2+ is "trapped" when the binary complex is formed and cannot be removed by EGTA. A summary model for these reactions is presented that indicates the interaction between actin and gelsolin is not a freely reversible Ca2+-controlled reaction.     

Spectroscopic study of conformational changes in subdomain 1 of G-actin: influence of divalent cations.

Temperature dependence of the fluorescence intensity and anisotropy decay of N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine attached to Cys374 of actin monomer was investigated to characterize conformational differences between Ca- and Mg-G-actin. The fluorescence lifetime is longer in Mg-G-actin than that in Ca-G-actin in the temperature range of 5-34 degrees C. The width of the lifetime distribution is smaller by 30% in Mg-saturated actin monomer at 5 degrees C, and the difference becomes negligible above 30 degrees C. The semiangle of the cone within which the fluorophore can rotate is larger in Ca-G-actin at all temperatures. Electron paramagnetic resonance measurements on maleimide spin-labeled (on Cys374) monomer actin gave evidence that exchange of Ca2+ for Mg2+ induced a rapid decrease in the mobility of the label immediately after the addition of Mg2+. These results suggest that the C-terminal region of the monomer becomes more rigid as a result of the replacement of Ca2+ by Mg2+. The change can be related to the difference between the polymerization abilities of the two forms of G-actin.     

Nanosecond spectroscopy of retinol.

Nanosecond fluorescence spectroscopy was used to study the unique binding site of the retinol-binding protein (RBP) from human serum. At pH 7.4, the binding of retinol to RBP caused the following spectroscopic changes in the ligand: (a) an enhancement of the fluorescence decay time (gamma = 8 ns); and (b) an increase in the emission anisotropy (A = 0.29). Retinol in hexane has a fluorescent decay time of 4.2 ns and a low emission anisotropy (A = 0.02). The increase in the fluorescence decay time of bound retinol is not due to dielectric relaxation effects of polar groups, since nanosecond time-resolved emission spectra of either retinol in glycerol or retinol bound to RBP, failed to show any time-dependent shifts in emission maxima during the time period investigated 0 to 30 ns. The degree of rotational mobility of bound retinol was investigated by time emission anisotropy measurements. The observed rotational correlation time (theta = 7.2 ns) is consistent with a rigid compact macromolecule of 21,000 molecular weight.     

Purification and properties of a novel Ca2+-binding protein (10.5 kDa) from Ehrlich-ascites-tumour cells.

A novel Ca2+-binding protein (CaBP) was identified in Ehrlich-ascites-tumour cells and purified to homogeneity. The molecular mass of this protein is about 10.5 kDa as estimated by polyacrylamide-gel electrophoresis in the presence of SDS. CaBP has two Ca2+-binding sites that bind Ca2+ with a dissociation constant of about 3 x 10(-6)M. Ca2+ binding to CaBP decreases its electrophoretic mobility in urea/polyacrylamide gels, changes its u.v. spectrum, increases the intrinsic tyrosine fluorescence intensity and strengthens hydrophobic interaction with the phenyl-Sepharose matrix.     

Intensity and anisotropy decays of the tyrosine calmodulin proteolytic fragments, as studied by GHz frequency-domain fluorescence

Frequency-domain fluorescence measurements to 2 GHz were able to recover and account for essentially all of the intrinsic tyrosine anisotropy of calmodulin and its proteolytic fragments containing one or two tyrosine residues. Low-temperature measurements have detected a very rapid initial anisotropy decay in the 2-tyrosine species which may be attributed to radiationless energy transfer between the two tyrosines. The observed values of the rotational correlation times indicate that both tyrosines of calmodulin possess considerable mobility, which decreases in the presence of Ca2+ and at low temperatures.     

Influence of Mg2+ and Ca2+ bound to 1,5-IAEDANS-labeled phosphorylated and dephosphorylated heavy meromyosin complexed with F-actin on polarized fluorescence of the fluorophore

Dephosphorylated and phosphorylated heavy meromyosin, fluorescently labeled with 1,5-IAEDANS attached at the SH1 group, was introduced into myosin-free ghost fibres and the polarized fluorescence of the bound label was measured. The results depended on whether the divalent cation binding sites on heavy meromyosin were saturated with Mg2+ or Ca2+. The calculated angles of absorption and emission dipoles and the amount of random fluorophores were significantly changed, indicating that the random mobility and orientation of the fluorophores of phosphorylated and dephosphorylated heavy meromyosin heads complexed with F-actin in the ghost fibre depend on saturation of heavy meromyosin with Ca2+ or Mg2+. The presence of bound Ca2+ has an opposite effect on the polarized fluorescence of phosphorylated and dephosphorylated 1,5-IAEDANS-heavy meromyosin.