Reversible binding kinetics of a cytoskeletal protein at the erythrocyte submembrane.

Reversible binding among components of the cellular submembrane cytoskeleton and reversible binding of some of these components with the plasma membrane likely play a role in nonelastic morphological changes and mechanoplastic properties of cells. However, relatively few studies have been devoted to investigating directly the kinetic aspects of the interactions of individual components of the membrane skeleton with the membrane. The experiments described here investigated whether one component of the erythrocyte membrane cytoskeleton, protein 4.1, binds to its sites on the membrane reversibly and if so, whether the different 4.1-binding sites display distinct kinetic behavior. Protein 4.1 is known to stabilize the membrane and to mediate the attachment of spectrin filaments to the membrane. Protein 4.1 previously has been shown to bind to integral membrane proteins band 3, glycophorin C, and to negatively charged phospholipids. To examine the kinetic rates of dissociation of carboxymethyl fluorescein-labeled 4.1 (CF-4.1) to the cytofacial surface of erythrocyte membrane, a special preparation of hemolyzed erythrocyte ghosts was used, in which the ghosts became flattened on a glass surface and exposed their cytofacial surfaces to the solution through a membrane rip in a distinctive characteristic pattern. This preparation was examined by the microscopy technique of total internal reflection/fluorescence recovery after photobleaching (TIR/FRAP). Four different treatments were employed to help identify which membrane binding sites gave rise to the multiplicity of observed kinetic rates. The first treatment, the control, stripped off the native spectrin, actin, 4.1, and ankyrin. About 60% of the CF-4.1 bound to this control binded irreversibly (dissociation time > 20 min), but the remaining approximately 40% binded reversibly with a range of residency times averaging approximately 3 s. The second treatment subjected these stripped membranes to trypsin, which presumably removed most of the band 3. CF-4.1 binded significantly less to these trypsinized membranes and most of the decrease was a loss of the irreversibly binding sites. The third treatment simply preserved the native 4.1 and ankyrin. CF-4.1 binded less to this sample too, and the loss involved both the irreversible and reversible sites. The fourth treatment blocked the gycophorin C sites on the native 4.1-stripped membranes with an antibody. CF-4.1 again binded less to this sample than to a nonimmune serum control, and almost all of the decrease is a loss of irreversible sites.(ABSTRACT TRUNCATED AT 400 WORDS)     

Actin dynamics at the living cell submembrane imaged by total internal reflection fluorescence photobleaching

Although reversible chemistry is crucial to dynamical processes in living cells, relatively little is known about relevant chemical kinetic rates in vivo. Total internal reflection/fluorescence recovery after photobleaching (TIR/FRAP), an established technique previously demonstrated to measure reversible biomolecular kinetic rates at surfaces in vitro, is extended here to measure reversible biomolecular kinetic rates of actin at the cytofacial (subplasma membrane) surface of living cells. For the first time, spatial imaging (with a charge-coupled device camera) is used in conjunction with TIR/FRAP. TIR/FRAP imaging produces both spatial maps of kinetic parameters (off-rates and mobile fractions) and estimates of kinetic correlation distances, cell-wide kinetic gradients, and dependences of kinetic parameters on initial fluorescence intensity. For microinjected rhodamine actin in living cultured smooth muscle (BC3H1) cells, the unbinding rate at or near the cytofacial surface of the plasma membrane (averaged over the entire cell) is measured at 0.032 +/- 0.007 s(-1). The corresponding rate for actin marked by microinjected rhodamine phalloidin is very similar, 0.033 +/- 0.013 s(-1), suggesting that TIR/FRAP is reporting the dynamics of entire filaments or protofilaments. For submembrane fluorescence-marked actin, the intensity, off-rate, and mobile fraction show a positive correlation over a characteristic distance of 1-3 microm and a negative correlation over larger distances greater than approximately 7-14 microm. Furthermore, the kinetic parameters display a statistically significant cell-wide gradient, with the cell having a "fast" and "slow" end with respect to actin kinetics.     

Phosphatidylserine binding sites in erythroid spectrin: location and implications for membrane stability

The erythrocyte membrane is a composite structure consisting of a lipid bilayer tethered to the spectrin-based membrane skeleton. Two complexes of spectrin with other proteins are known to participate in the attachment. Spectrin has also been shown to interact with phosphatidylserine (PS), a component of the lipid bilayer, which is confined to its inner leaflet. That there may be multiple sites of interaction with PS in the spectrin sequence has been inferred, but they have not hitherto been identified. Here we have explored the interaction of PS-containing liposomes with native alpha- and beta-spectrin chains and with recombinant spectrin fragments encompassing the entire sequences of both chains. We show that both alpha-spectrin and beta-spectrin bind PS and that sites of high affinity are located within 8 of the 38 triple-helical structural repeats which make up the bulk of both chains; these are alpha8, alpha9-10, beta2, beta3, beta4, beta12, beta13, and beta14, and PS affinity was also found in the nonhomologous N-terminal domain of the beta-chain. No other fragments of either chain showed appreciable binding. Binding of spectrin and its constituent chains to mixed liposomes of PS and phosphatidylcholine (PC) depended on the proportion of PS. Binding of spectrin dimers to PS liposomes was inhibited by single repeats containing PS binding sites. It is noteworthy that the PS binding sites in beta-spectrin are grouped in close proximity to the sites of attachment both of ankyrin and of 4.1R, the proteins engaged in attachment of spectrin to the membrane. We conjecture that direct interaction of spectrin with PS in the membrane may modulate its interactions with the proteins and that (considering also the known affinity of 4.1R for PS) the formation of PS-rich lipid domains, which have been observed in the red cell membrane, may be a result.     

The linkage of catalysis and regulation in enzyme action: oxidative diversion in the hysteretically regulated yeast pyruvate decarboxylase.

The reaction catalyzed by the thiamin-diphosphate-dependent yeast pyruvate decarboxylase, which is hysteretically regulated by pyruvate, undergoes paracatalytic oxidative diversion by 2,6-dichlorophenolindophenol, which traps a carbanionic intermediate and diverts the product from acetaldehyde to acetate (Christen, P. Meth. Enzymol. 1977, 46, 48). This reaction is now shown to exhibit an oxidant on-rate constant somewhat faster than that for pyruvate in the normal catalytic cycle and a product off-rate constant about 60-fold smaller than that for acetaldehyde. Both on-rates and off-rates exhibit an inverse solvent isotope effect of 1.5-2, observed in normal catalysis as a signal of sulfhydryl addition to the keto group of pyruvate at the allosteric regulatory site. The findings are consistent with a model for regulation in which the sulfhydryl-addition process mediates access to a fully catalytically competent active site, the oxidative-diversion reaction being forced to make use of the normal entry exit machinery.     

Two-dimensional kinetics regulation of alphaLbeta2-ICAM-1 interaction by conformational changes of the alphaL-inserted domain

The leukocyte integrin alphaLbeta2 mediates cell adhesion and migration during inflammatory and immune responses. Ligand binding of alphaLbeta2 is regulated by or induces conformational changes in the inserted (I) domain. By using a micropipette, we measured the conformational regulation of two-dimensional (2D) binding affinity and the kinetics of cell-bound intercellular adhesion molecule-1 interacting with alphaLbeta2 or isolated I domain expressed on K562 cells. Locking the I domain into open and intermediate conformations with a disulfide bond increased the affinities by approximately 8000- and approximately 30-fold, respectively, from the locked closed conformation, which has similar affinity as the wild-type I domain. Most surprisingly, the 2D affinity increases were due mostly to the 2D on-rate increases, as the 2D off-rates only decreased by severalfold. The wild-type alphaLbeta2, but not its I domain in isolation, could be up-regulated by Mn2+ or Mg2+ to have high affinities and on-rates. Locking the I domain in any of the three conformations abolished the ability of divalent cations to regulate 2D affinity. These results indicate that a downward displacement of the I domain C-terminal helix, induced by conformational changes of other domains of the alphaLbeta2, is required for affinity and on-rate up-regulation.     

Selected-fit versus induced-fit protein binding: kinetic differences and mutational analysis

The binding of a ligand molecule to a protein is often accompanied by conformational changes of the protein. A central question is whether the ligand induces the conformational change (induced-fit), or rather selects and stabilizes a complementary conformation from a pre-existing equilibrium of ground and excited states of the protein (selected-fit). We consider here the binding kinetics in a simple four-state model of ligand-protein binding. In this model, the protein has two conformations, which can both bind the ligand. The first conformation is the ground state of the protein when the ligand is off, and the second conformation is the ground state when the ligand is bound. The induced-fit mechanism corresponds to ligand binding in the unbound ground state, and the selected-fit mechanism to ligand binding in the excited state. We find a simple, characteristic difference between the on- and off-rates in the two mechanisms if the conformational relaxation into the ground states is fast. In the case of selected-fit binding, the on-rate depends on the conformational equilibrium constant, whereas the off-rate is independent. In the case of induced-fit binding, in contrast, the off-rate depends on the conformational equilibrium, while the on-rate is independent. Whether a protein binds a ligand via selected-fit or induced-fit thus may be revealed by mutations far from the protein's binding pocket, or other "perturbations" that only affect the conformational equilibrium. In the case of selected-fit, such mutations will only change the on-rate, and in the case of induced-fit, only the off-rate.     

Associations of erythrocyte membrane proteins. Binding of purified bands 2.1 and 4.1 to spectrin

Specific associations of spectrin with Bands 2.1 and 4.1 have been examined by measuring the binding of purified 125I-Band 2.1 and 125I-Band 4.1 to [32P]spectrin in solution. Binding of Bands 2.1 and 4.1 to spectrin was measured as 125I radioactivity precipitated by an anti-spectrin.Staphylococcus aureus complex. The association between spectrin and Band 2.1 is characterized by relatively high affinity (Kd congruent to 10(-7) M at pH 7.6) and saturation of available binding sites at a molar ratio of 1:1 (Band 2.1/spectrin heterodimer). Band 4.1 binding to spectrin is characterized by a similar affinity (Kd congruent to 10(-7) M at pH 7.6) with saturation of available sites occurring at a stoichiometric ration of 2:1 (Band 4.1/spectrin heterodimer). Scatchard plots of Band 4.1 binding to spectrin are curvilinear and consistent with a positively cooperative interation. Bands 2.1 and 4.1 bind to different sites on the spectrin molecule: unlabeled Band 4.1 does not competitively displace 125 I-Band 2.1 from spectrin in solution, and low angle rotary-shadowed platinum-carbon replicas of these polypeptides reveal two discrete binding sites.     

Ankyrin and synapsin: spectrin-binding proteins associated with brain membranes

Brain membranes contain an actin-binding protein closely related in structure and function to erythrocyte spectrin. The proteins that attach brain spectrin to membranes are not established, but, by analogy with the erythrocyte membrane, may include ankyrin and protein 4.1. In support of this idea, proteins closely related to ankyrin and 4.1 have been purified from brain and have been demonstrated to associate with brain spectrin. Brain ankyrin binds with high affinity to the spectrin beta subunit at the midregion of spectrin tetramers. Brain ankyrin also has binding sites for the cytoplasmic domain of the erythrocyte anion channel (band 3), as well as for tubulin. Ankyrins from brain and erythrocytes have a similar domain structure with protease-resistant domains of Mr = 72,000 that contain spectrin-binding activity, and domains of Mr = 95,000 (brain ankyrin) or 90,000 (erythrocyte ankyrin) that contain binding sites for both tubulin and the anion channel. Brain ankyrin is present at about 100 pmol/mg membrane protein, or about twice the number of copies of spectrum beta chains. Brain ankyrin thus is present in sufficient amounts to attach spectrin to membranes, and it has the potential to attach microtubules to membranes as well as to interconnect microtubules with spectrin-associated actin filaments. Another spectrin-binding protein has been purified from brain membranes, and this protein cross-reacts with erythrocyte 4.1. Brain 4.1 is identical to the membrane protein synapsin, which is one of the brain's major substrates for cAMP-dependent and Ca/calmodulin-dependent protein kinases with equivalent physical properties, immunological cross-reaction, and peptide maps. Synapsin (4.1) is present at about 60 pmol/mg membrane protein, and thus is a logical candidate to regulate certain protein linkages involving spectrin.     

N-isonicotinoyl-(L)-4-aminophenylalanine derivatives as tight binding VLA-4 antagonists

A series of isonicotinoyl-(L)-aminophenylalanine derivatives was prepared and evaluated as VLA-4 antagonists. These compounds exhibit subnanomolar binding affinity to VLA-4 and significant off-rates. The interplay between off-rate, protein binding and pharmacokinetics is discussed.     

Antibody specific for the peptide.major histocompatibility complex. Is it T cell receptor-like?

Antibodies recognizing peptide bound to a major histocompatibility complex (MHC) protein usually have a higher affinity for the composite peptide.MHC (pMHC) ligand than T cell receptors (TCR) with the same specificity. Because the solvent-accessible peptide area constitutes only a small portion of the contacting pMHC surface, we hypothesized that the contribution of the MHC moiety to the TCR-pMHC complex stability is limited, ensuring a small increment of the binding energy delivered by the peptide to be distinguishable by the TCR or the peptide-specific antibody. This suggests that the gain in affinity of the antibody-pMHC interaction can be achieved through an increase in the on-rate without a significant change in the off-rate of the interaction. To test the hypothesis, we have analyzed the binding of an ovalbumin peptide (pOV8) and its variants associated with soluble H-2Kb protein to the 25-D1.16 monoclonal antibody and compared it with the binding of the same pMHC complexes to the OT-1 TCR. This comparison revealed a substantially higher on-rate of the antibody-pMHC interaction compared with the TCR-pMHC interaction. In contrast, both the antibody and the TCR-pMHC complexes exhibited comparably fast off-rates. Sequencing of the 25-D1.16 VH and VL genes showed that they have very few somatic mutations and those occur mainly in framework regions. We propose that the above features constitute a signature of the recognition of MHC-bound peptide antigens by TCR and TCR-like antibodies, which could explain why the latter are rarely produced in vivo.     

Investigation of binding mechanisms of nuclear proteins using confocal scanning laser microscopy and FRAP

Fluorescence recovery after photobleaching (FRAP) measurements offer an important tool towards analysing diffusion processes within living biological cells. A model is presented that aims to provide a rigorous theoretical framework from which binding information of proteins from FRAP data can be extracted. A single binding reaction is considered and a set of mathematical equations is introduced that incorporates the concentration of free proteins, vacant binding sites and bound complexes in addition to the on- and off-rates of the proteins. To allow a realistic FRAP model, characteristics of the instruments used to perform FRAP measurements are included in the equation. The proposed model has been designed to be applied to biological samples with a confocal scanning laser microscope (CSLM) equipped with the feature to bleach regions characterised by a radially Gaussian distributed profile. Binding information emerges from FRAP simulations considering the diffusion coefficient, radial extent of the bleached volume and bleach constant as parameters derived from experimental data. The proposed model leads to FRAP curves that depend on the on- and off-rates. Analytical expressions are used to define the boundaries of on- and off-rate parameter space in simplified cases when molecules can move on an infinite domain. A similar approach is ensued when movement is restricted in a compartment with a finite size. The theoretical model can be used in conjunction to experimental data acquired by CSLM to investigate the biophysical properties of proteins in living cells.     

Membrane interaction of erythroid spectrin: surface-density-dependent high-affinity binding to phosphatidylethanolamine

Density-dependent spectrin binding to dimyristoylphosphatidylcholine/dimyristoylphosphatidylethanolamine (DMPC/DMPE) small uni-lamellar vesicles (SUVs) has been directly evaluated in this work from the increase in the extent of quenching of the tryptophan fluorescence of spectrin at two different temperatures, above and below the main phase transition temperatures (Tm). Results from the binding studies of spectrin to phospholipid SUVs indicated that the binding dissociation constant Kd, increased from 45 +/- 7 nM in pure DMPC SUVs to 219 +/- 20 nM in DMPC/DMPE (50:50) SUVs, both in the gel and liquid crystalline phase. However, in pure DMPE SUVs the Kd decreased drastically to 0.7 +/- 0.2 nM in the gel phase at 18 degrees C and to 2.6 +/- 0.7 nM in the fluid phase at 55 degrees C indicating a high affinity binding of spectrin for the bilayer-forming DMPE. The maximum extent of phospholipid-induced quenching and the number of spectrin molecules associated with one SUV particle, evaluated in the present work, led to a model in DMPC/DMPE bilayer membranes indicating the PE-binding site of spectrin to localize at one of the terminal domains of the dimeric spectrin. A direct evidence of the localization of the PE-binding site at one of the terminal ends of the spectrin dimer also came from electron microscopic observation in fluid membranes made of bovine brain PE.     

Investigations of spectrin-lipid interactions using fluoresceinphosphatidylethanolamine as a membrane probe

The binding of human erythrocyte spectrin to large unilamellar vesicles (LUVET) formed by the extrusion technique has been studied using fluoresceinphosphatidylethanolamine (FPE) as a reporter of electrostatic membrane potential. Spectrin aliquots were added to a suspension of FPE-labelled LUVETs to elucidate both the type of charge involved and the dissociation constants for spectrin binding to various lipids. All binding experiments showed serial increases in FPE fluorescence intensity upon serial additions of spectrin, indicative of increasing positive charge at the membrane surface. This proves for the first time that although exhibiting an overall net negative charge, spectrin binds to lipid surfaces by presenting positive charges to the lipid surface. Binding curves were obtained from the change in fluorescence intensity upon each spectrin addition and analysed to determine dissociation constants. A K(d) of 0.14+/-0.12 microM was found for spectrin binding to FPE-labelled phosphatidylcholine/phosphatidylserine (PC/PS) LUVETs at 22 degrees C in high salt conditions. A similar K(d) of 0.17+/-0.11 microM was obtained for spectrin binding to neutral LUVETs composed of PC. However, binding was found to be much weaker for PC/PS LUVETs under low salt conditions with a K(d) of 1.22+/-0.48 microM.     

Membrane interaction of erythroid spectrin: Surface-density-dependent high-affinity binding to phosphatidylethanolamine

Density-dependent spectrin binding to dimyristoylphosphatidylcholine/dimyristoylphosphatidylethanolamine (DMPC/DMPE) small uni-lamellar vesicles (SUVs) has been directly evaluated in this work from the increase in the extent of quenching of the tryptophan fluorescence of spectrin at two different temperatures, above and below the main phase transition temperatures (T_m). Results from the binding studies of spectrin to phospholipid SUVs indicated that the binding dissociation constant K_d, increased from 45±7 nM in pure DMPC SUVs to 219±20 nM in DMPC/DMPE (50:50) SUVs, both in the gel and liquid crystalline phase. However, in pure DMPE SUVs the K_d decreased drastically to 0.7±0.2 nM in the gel phase at 18°C and to 2.6±0.7 nM in the fluid phase at 55°C indicating a high affinity binding of spectrin for the bilayer-forming DMPE. The maximum extent of phospholipid-induced quenching and the number of spectrin molecules associated with one SUV particle, evaluated in the present work, led to a model in DMPC/DMPE bilayer membranes indicating the PE-binding site of spectrin to localize at one of the terminal domains of the dimeric spectrin. A direct evidence of the localization of the PE-binding site at one of the terminal ends of the spectrin dimer also came from electron microscopic observation in fluid membranes made of bovine brain PE.     

Correlation between binding rate constants and individual information of E. coli Fis binding sites

Individual protein binding sites on DNA can be measured in bits of information. This information is related to the free energy of binding by the second law of thermodynamics, but binding kinetics appear to be inaccessible from sequence information since the relative contributions of the on- and off-rates to the binding constant, and hence the free energy, are unknown. However, the on-rate could be independent of the sequence since a protein is likely to bind once it is near a site. To test this, we used surface plasmon resonance and electromobility shift assays to determine the kinetics for binding of the Fis protein to a range of naturally occurring binding sites. We observed that the logarithm of the off-rate is indeed proportional to the individual information of the binding sites, as predicted. However, the on-rate is also related to the information, but to a lesser degree. We suggest that the on-rate is mostly determined by DNA bending, which in turn is determined by the sequence information. Finally, we observed a break in the binding curve around zero bits of information. The break is expected from information theory because it represents the coding demarcation between specific and nonspecific binding.     

Forcing Switch from Short- to Intermediate- and Long-lived States of the αA Domain Generates LFA-1/ICAM-1 Catch Bonds

Binding of lymphocyte function-associated antigen-1 (LFA-1) to intercellular adhesion molecule-1 (ICAM-1) mediates leukocyte adhesion under force. Using a biomembrane force probe capable of measuring single bond interactions, we showed ICAM-1 binding to LFA-1 at different conformations, including the bent conformation with the lowest affinity. We quantify how force and conformations of LFA-1 regulate its kinetics with ICAM-1. At zero-force, on-rates were substantially changed by conditions that differentially favor a bent or extended LFA-1 with a closed or open headpiece; but off-rates were identical. With increasing force, LFA-1/ICAM-1 bond lifetimes (reciprocal off-rates) first increased (catch bonds) and then decreased (slip bonds). Three states with distinct off-rates were identified from lifetime distributions. Force shifted the associated fractions from the short- to intermediate- and long-lived states, producing catch bonds at low forces, but increased their off-rates exponentially, converting catch to slip bonds at high forces. An internal ligand antagonist that blocks pulling of the α₇-helix suppressed the intermediate-/long-lived states and eliminated catch bonds, revealing an internal catch bond between the αA and βA domains. These results elucidate an allosteric mechanism for the mechanochemistry of LFA-1/ICAM-1 binding.     

Ankyrin-independent membrane protein-binding sites for brain and erythrocyte spectrin

Brain spectrin reassociates in in vitro binding assays with protein(s) in highly extracted brain membranes quantitatively depleted of ankyrin and spectrin. These newly described membrane sites for spectrin are biologically significant and involve a protein since (a) binding occurs optimally at physiological pH (6.7-6.9) and salt concentrations (50 mM), (b) binding is abolished by digestion of membranes with alpha-chymotrypsin, (c) Scatchard analysis is consistent with a binding capacity of at least 50 pmol/mg total membrane protein, and highest affinity of 3 nM. The major ankyrin-independent binding activity of brain spectrin is localized to the beta subunit of spectrin. Brain membranes also contain high affinity binding sites for erythrocyte spectrin, but a 3-4 fold lower capacity than for brain spectrin. Some spectrin-binding sites associate preferentially with brain spectrin, some with erythrocyte spectrin, and some associate with both types of spectrin. Erythrocyte spectrin contains distinct binding domains for ankyrin and brain membrane protein sites, since the Mr = 72,000 spectrin-binding fragment of ankyrin does not compete for binding of spectrin to brain membranes. Spectrin binds to a small number of ankyrin-independent sites in erythrocyte membranes present in about 10,000-15,000 copies/cell or 10% of the number of sites for ankyrin. Brain spectrin binds to these sites better than erythrocyte spectrin suggesting that erythrocytes have residual binding sites for nonerythroid spectrin. Ankyrin-independent-binding proteins that selectively bind to certain isoforms of spectrin provide a potentially important flexibility in cellular localization and time of synthesis of proteins involved in spectrin-membrane interactions. This flexibility has implications for assembly of the membrane skeleton and targeting of spectrin isoforms to specialized regions of cells.     

Anthrax toxin protective antigen: inhibition of channel function by chloroquine and related compounds and study of binding kinetics using the current noise analysis

Protective antigen (PA) of the tripartite anthrax toxin binds to a cell surface receptor and mediates the transport of two enzymatic components, edema factor and lethal factor, into the cytosol of host cells. Here recombinant PA(63) from Bacillus anthracis was reconstituted into artificial lipid bilayer membranes and formed ion permeable channels. The heptameric PA(63)-channel contains a binding site for 4-aminoquinolones, which block ion transport through PA in vitro. This result allowed a detailed investigation of ligand binding and the stability constants for the binding of chloroquine, fluphenazine, and quinacrine to the binding site inside the PA(63)-channel were determined using titration experiments. Open PA(63)-channels exhibit 1/f noise in the frequency range between 1 and 100 Hz, whereas the spectral density of the ligand-induced current noise was of Lorentzian type. The analysis of the power density spectra allowed the evaluation of the on- and off-rate constants (k(1) and k(-1)) of ligand binding. The on-rate constants of ligand binding were between 10(6) and 10(8) M(-1) s(-1) and were dependent on the ionic strength of the aqueous phase, sidedness of ligand addition, as well as the orientation and intensity of the applied electric field. The off-rates varied between approximately 10 s(-1) and 2600 s(-1) and depended mainly on the structure of the ligand.     

Ca2(+)-dependent regulation of the spectrin/actin interaction by calmodulin and protein 4.1

The Ca2(+)-dependent regulation of the erythroid membrane cytoskeleton was investigated. The low-salt extract of erythroid membranes, which is mainly composed of spectrin, protein 4.1, and actin, confers a Ca2+ sensitivity on its interaction with F-actin. This Ca2+ sensitivity is fortified by calmodulin and antagonized by trifluoperazine, a potent calmodulin inhibitor. Additionally, calmodulin is detected in the low-salt extract. These results suggest that calmodulin is the sole Ca2(+)-sensitive factor in the low-salt extract. The main target of calmodulin in the erythroid membrane cytoskeleton was further examined. Under native conditions, calmodulin forms a stable and equivalent complex with protein 4.1 as determined by calmodulin affinity chromatography, cross-linking experiments, and fluorescence binding assays with an apparent Kd of 5.5 x 10(-7) M irrespective of the free Ca2+ concentration. Domain mapping with chymotryptic digestion reveals that the calmodulin-binding site resides within the N-terminal 30-kDa fragment of protein 4.1. In contrast, the interaction of calmodulin with spectrin is unexpectedly weak (Kd = 1.2 x 10(-4) M). Given the content of calmodulin in erythrocytes (2-5 microM), these results imply that the major target for calmodulin in the erythroid membrane cytoskeleton is protein 4.1. Low- and high-shear viscometry and binding assays reveal that an equivalent complex of calmodulin with protein 4.1 regulates the spectrin/actin interaction in a Ca2(+)-dependent manner. At a low Ca2+ concentration, protein 4.1 potentiates the actin cross-linking and the actin binding activities of spectrin. At a high Ca2+ concentration, the protein 4.1-potentiated actin cross-linking activity but not the actin binding activity of spectrin is suppressed by Ca2+/calmodulin. The Ca2(+)-dependent regulation of the spectrin/protein 4.1/calmodulin/actin interaction is discussed.     

Multiple interconvertible affinity states for the delta opioid agonist-receptor complex

Previously we demonstrated that rates of dissociation of [3H-D-Ala2-D-Leu5]enkephalin [( 3H]DADL) from bovine hippocampal synaptic plasma membranes (SPMs) varied depending upon association time, suggesting a multistep binding process. To characterize different kinetic intermediates, we examined the effects of guanine nucleotide on dissociation rate. Control off-rates were compared to those obtained when guanyl-5'-yl-imidodiphosphate (Gpp(NH)p) (50 microM) was added either coincident with the radioligand at association or with 1 microM unlabeled DADL which initiated dissociation. delta site selectivity of [3H]DADL was ensured by addition of 20 nM unlabeled [D-Ala2-Me-Phe4-Gly-Ol5]enkephalin which suppressed mu site cross-reactivity in this preparation. Addition of Gpp(NH)p at the onset of dissociation increased the off-rate to a much greater extent after steady state binding was reached (60 min) compared to that following an association time of only 7 or 20 min. A slowly formed high affinity state appeared to be rapidly converted to a lower affinity state under these conditions. When Gpp(NH)p was present throughout the association period, the slowly dissociating state was no longer observed. Also, the off-rate following a 7-min association is linear and much faster than control, suggesting that Gpp(NH)p may affect an initial intermediate state as well as the high affinity complex. Pretreatment of the membranes with N-ethylmaleimide (NEM) eliminated the association time-dependent dissociation rates, apparently preventing time-dependent formation of a high affinity state. This state is thought to be possibly a ligand-receptor complex interacting with a GTP binding protein. However, the rate of dissociation from NEM-treated membranes was accelerated by addition of Gpp(NH)p and the effect was not association time-dependent. NEM treatment resulted in an increased potency for Gpp(NH)p inhibition of [3H]DADL steady state binding. These results suggest the occurrence of at least three steps in the association of DADL to bovine hippocampal synaptic membranes.