High-resolution structural analysis of mammalian profilin 2a complex formation with two physiological ligands: the formin homology 1 domain of mDia1 and the proline-rich domain of VASP

Profilins are small proteins capable of binding actin, poly-l-proline and other proline-rich sequences, and phosphatidylinositol (4,5)-bisphosphate. A number of proline-rich ligands for profilin have been characterised, including proteins of the Ena/VASP and formin families. We have determined the high-resolution crystal structures of mouse profilin 2a in complex with peptides from two functionally important ligands from different families, VASP and mDia1. The structures show that the binding mode of the peptide ligand is strongly affected by the non-proline residues in the sequence, and the peptides from VASP and mDia1 bind to profilin 2a in distinct modes. The high resolution of the crystallographic data allowed us to detect conserved CH-π hydrogen bonds between the peptide and profilin in both complexes. Furthermore, both peptides, which are shown to have micromolar affinity, induced the dimerisation of profilin, potentially leading to functionally different ligand-profilin-actin complexes. The peptides did not significantly affect actin polymerisation kinetics in the presence or in the absence of profilin 2a. Mutant profilins were tested for binding to poly-l-proline and the VASP and mDia1 peptides, and the F139A mutant bound proline-rich ligands with near-native affinity. Peptide blotting using a series of designed peptides with profilins 1 and 2a indicates differences between the two profilins towards proline-rich peptides from mDia1 and VASP. Our data provide structural insights into the mechanisms of mDia1 and VASP regulated actin polymerisation.     

The mammalian profilin isoforms display complementary affinities for PIP2 and proline-rich sequences.

We present a study on the binding properties of the bovine profilin isoforms to both phosphatidylinositol 4,5-bisphosphate (PIP2) and proline-rich peptides derived from vasodilator-stimulated phosphoprotein (VASP) and cyclase-associated protein (CAP). Using microfiltration, we show that compared with profilin II, profilin I has a higher affinity for PIP2. On the other hand, fluorescence spectroscopy reveals that proline-rich peptides bind better to profilin II. At micromolar concentrations, profilin II dimerizes upon binding to proline-rich peptides. Circular dichroism measurements of profilin II reveal a significant conformational change in this protein upon binding of the peptide. We show further that PIP2 effectively competes for binding of profilin I to poly-L-proline, since this isoform, but not profilin II, can be eluted from a poly-L-proline column with PIP2. Using affinity chromatography on either profilin isoform, we identified profilin II as the preferred ligand for VASP in bovine brain extracts. The complementary affinities of the profilin isoforms for PIP2 and the proline-rich peptides offer the cell an opportunity to direct actin assembly at different subcellular localizations through the same or different signal transduction pathways.     

Formation and implications of a ternary complex of profilin, thymosin beta 4, and actin

Data from affinity chromatography, analytical ultracentrifugation, covalent cross-linking, and fluorescence anisotropy show that profilin, thymosin beta(4), and actin form a ternary complex. In contrast, steady-state assays measuring F-actin concentration are insensitive to the formation of such a complex. Experiments using a peptide that corresponds to the N terminus of thymosin beta(4) (residues 6-22) confirm the presence of an extensive binding surface between actin and thymosin beta(4), and explain why thymosin beta(4) and profilin can bind simultaneously to actin. Surprisingly, despite much lower affinity, the N-terminal thymosin beta(4) peptide has a very slow dissociation rate constant relative to the intact protein, consistent with a catalytic effect of the C terminus on conformational change occurring at the N terminus of thymosin beta(4). Intracellular concentrations of thymosin beta(4) and profilin may greatly exceed the equilibrium dissociation constant of the ternary complex, inconsistent with models showing sequential formation of complexes of profilin-actin or thymosin beta(4)-actin during dynamic remodeling of the actin cytoskeleton. The formation of a ternary complex results in a very large amplification mechanism by which profilin and thymosin beta(4) can sequester much more actin than is possible for either protein acting alone, providing an explanation for significant sequestration even if molecular crowding results in a very low critical concentration of actin in vivo.     

Profilin binding to poly-L-proline and actin monomers along with ability to catalyze actin nucleotide exchange is required for viability of fission yeast

We tested the ability of 87 profilin point mutations to complement temperature-sensitive and null mutations of the single profilin gene of the fission yeast Schizosaccharomyces pombe. We compared the biochemical properties of 13 stable noncomplementing profilins with an equal number of complementing profilin mutants. A large quantitative database revealed the following: 1) in a profilin null background fission yeast grow normally with profilin mutations having >10% of wild-type affinity for actin or poly-L-proline, but lower affinity for either ligand is incompatible with life; 2) in the cdc3-124 profilin ts background, fission yeast function with profilin having only 2-5% wild-type affinity for actin or poly-L-proline; and 3) special mutations show that the ability of profilin to catalyze nucleotide exchange by actin is an essential function. Thus, poly-L-proline binding, actin binding, and actin nucleotide exchange are each independent requirements for profilin function in fission yeast.     

Nitration of profilin effects its interaction with poly (L-proline) and actin

Profilin from bovine spleen was nitrated with peroxynitrite; immunoblotting and spectrophotometric quantitation of nitrotyrosine residues suggested nitration of a single tyrosine residue in profilin with a stoichiometry of 0.6 mol of nitrotyrosine/mole of profilin. A decrease in the nitrotyrosine immunoreactivity of nitroprofilin during digestion with carboxypeptidase Y indicated that nitrotyrosine is located at the C-terminus of profilin. Nitroprofilin interaction with ligands such as phosphatidylinositol 4,5-bisphosphate, actin and poly (l-proline) was analyzed by monitoring the tryptophan fluorescence. Scatchard plot and binding isotherm data obtained revealed no significant difference in affinity of nitroprofilin to phosphatidylinositol 4,5-bisphosphate (K(d) of 4.8 +/- 0.5 muM for profilin, and K(d) of 5.7 +/- 0.6 muM for nitroprofilin), while poly (l-proline) binding studies revealed a twenty-fold increase in the affinity of profilin to poly (l-proline) upon nitration (K(d) of 21.8 +/- 1.7 muM for profilin, and K(d) of 1.1 +/- 0.1 muM for nitroprofilin). Actin polymerization studies involving pyrene-labeled actin indicated that profilin nitration inhibits the actin sequestering property of profilin. The critical actin monomer concentration (C(c)) was 150 and 250 nM in the presence of nitroprofilin and profilin, respectively. Thus, nitric oxide and free radicals produced under different conditions could alter the functions of profilin through nitration, such as its interaction with actin and poly (l-proline).     

Phosphorylation of profilin regulates its interaction with actin and poly (L-proline)

Activation of bovine platelets with thrombin and phorbol 12,13-dibutyrate (PDBu) resulted in phosphorylation of profilin on serine. The phosphorylation was inhibited when platelets were pretreated with the PI 3-kinase inhibitor, LY294002, indicating that profilin phosphorylation is a downstream event with respect to PI 3-kinase activation. Phosphorylation of profilin resulted in significant decrease in actin polymerization (16.5%), indicating an increased affinity of phosphoprofilin towards actin. The critical actin monomer concentration (Cc) increased to 260 nM in the presence of phosphoprofilin in comparison with 200 nM in the presence of profilin. The interaction of phosphoprofilin with phosphatidylinositol 4,5-bisphosphate [PI (4,5)-P2] and poly (L-proline) (PLP) was examined by monitoring the quenching of tryptophan fluorescence. Scatchard plot and binding isotherm data obtained revealed no difference in PI (4,5)-P2 binding between profilin and phosphoprofilin (Kd=20.4 microM), while poly (L-proline)-binding studies indicated a sixfold decrease (27.34 microM for profilin and 4.73 microM for phosphoprofilin) in Kd with phosphoprofilin. In vivo studies with platelets indicated an increased association of p85alpha, the regulatory subunit of PI 3-kinase with phosphoprofilin over profilin. Overall, the data presented conclude that profilin phosphorylated under in vivo conditions and phosphorylation depends upon activation of PI 3-kinase. Phosphoprofilin exhibited increased affinity to poly (L-proline) sequences both in vitro and in vivo.     

Interdependence of profilin, cation, and nucleotide binding to vertebrate non-muscle actin

The interaction of profilin and non-muscle beta,gamma-actin prepared from bovine spleen has been investigated under physiologic ionic conditions. Profilin binding to actin decreases the affinity of actin for MgADP and MgATP by about 65- and 13-fold, respectively. Kinetic measurements indicate that profilin binding to actin weakens the affinity of actin for nucleotides primarily due to an increased nucleotide dissociation rate constant, but the nucleotide association rate constant is also increased about 2-fold. Removal of the actin-bound nucleotide and divalent cation produces the labile intermediate species in the nucleotide exchange reaction, nucleotide free actin (NF-actin), and increases the affinity of actin for profilin about 10-fold. Profilin binds NF-actin with high affinity, K(D) = 0.013 microM, and slows the observed denaturation rate of NF-actin. Addition of ATP to NF-actin weakens the affinity for profilin and addition of Mg(2+) to ATP-actin further weakens the affinity for profilin. The high-affinity Mg(2+) of actin regulates binding of both nucleotide and profilin to actin and is important for actin interdomain coupling. The data suggest that profilin binding to actin weakens nucleotide binding to actin by disrupting Mg(2+) coordination in the actin central cleft.     

Dimerization of profilin II upon binding the (GP5)3 peptide from VASP overcomes the inhibition of actin nucleation by profilin II and thymosin beta4

Profilin II dimers bind the (GP5)3 peptide derived from VASP with an affinity of approximately 0.5 microM. The resulting profilin II-peptide complex overcomes the combined capacity of thymosin beta4 and profilin II to inhibit actin nucleation and restores the extent of filament formation. We do not observe such an effect when barbed filament ends are capped. Neither can profilin I, in the presence of the peptide, promote actin polymerization during its early phase consistent with a lower affinity. Since a Pro17 peptide-profilin II complex only partially restores actin polymerization, the glycine residues in the VASP peptide appear important.     

Design and physicochemical properties of new fluorescent ligands of protein kinase C isozymes focused on CH/pi interaction

Phorbol ester-type tumor promoters such as indolactam-V (IL-V, 1) bind to the C1 domains of protein kinase C (PKC) isozymes. A more convenient method to investigate the interaction between each tumor promoter and PKC C1 domain is needed. Focusing on our recent finding that the indole ring of IL-V is involved in the CH/pi interaction with Pro-11 of the PKCdelta-C1B domain, we developed new fluorescent probes (2-4) from IL-V by forming a pyrroloindazole ring. Compound 2 without a substituent at the pyrroloindazole ring bound most strongly to PKC C1 domains with a potency similar to IL-V, but its fluorescent intensity was the weakest of any of the probes. Although the binding affinity of 3 with a methyl group was significantly weaker than that of IL-V, 4 with a trifluoromethyl group showed moderate affinity and the most potent fluorescence intensity. The fluorescence intensity and emission maxima of 4 changed significantly when bound to the PKCdelta-C1B peptide in both the presence and absence of phosphatidylserine. These results suggest that 4 could be a useful probe for analyzing the interaction of tumor promoters with PKC C1 domains.     

Control of the ability of profilin to bind and facilitate nucleotide exchange from G-actin

A major factor in profilin regulation of actin cytoskeletal dynamics is its facilitation of G-actin nucleotide exchange. However, the mechanism of this facilitation is unknown. We studied the interaction of yeast (YPF) and human profilin 1 (HPF1) with yeast and mammalian skeletal muscle actins. Homologous pairs (YPF and yeast actin, HPF1 and muscle actin) bound more tightly to one another than heterologous pairs. However, with saturating profilin, HPF1 caused a faster etheno-ATP exchange with both yeast and muscle actins than did YPF. Based on the -fold change in ATP exchange rate/K(d), however, the homologous pairs are more efficient than the heterologous pairs. Thus, strength of binding of profilin to actin and nucleotide exchange rate are not tightly coupled. Actin/HPF interactions were entropically driven, whereas YPF interactions were enthalpically driven. Hybrid yeast actins containing subdomain 1 (sub1) or subdomain 1 and 2 (sub12) muscle actin residues bound more weakly to YPF than did yeast actin (K(d) = 2 microm versus 0.6 microm). These hybrids bound even more weakly to HPF than did yeast actin (K(d) = 5 microm versus 3.2 microm). sub1/YPF interactions were entropically driven, whereas the sub12/YPF binding was enthalpically driven. Compared with WT yeast actin, YPF binding to sub1 occurred with a 5 times faster k(off) and a 2 times faster k(on). sub12 bound with a 3 times faster k(off) and a 1.5 times slower k(on). Profilin controls the energetics of its interaction with nonhybrid actin, but interactions between actin subdomains 1 and 2 affect the topography of the profilin binding site.     

The Effects of ADF/Cofilin and Profilin on the Conformation of the ATP-Binding Cleft of Monomeric Actin

Actin depolymerizing factor (ADF)/cofilin and profilin are small actin-binding proteins, which have central roles in cytoskeletal dynamics in all eukaryotes. When bound to an actin monomer, ADF/cofilins inhibit the nucleotide exchange, whereas most profilins accelerate the nucleotide exchange on actin monomers. In this study the effects of ADF/cofilin and profilin on the accessibility of the actin monomer''s ATP-binding pocket was investigated by a fluorescence spectroscopic method. The fluorescence of the actin bound ɛ-ATP was quenched with a neutral quencher (acrylamide) in steady-state and time dependent experiments, and the data were analyzed with a complex form of the Stern-Volmer equation. The experiments revealed that in the presence of ADF/cofilin the accessibility of the bound ɛ-ATP decreased, indicating a closed and more compact ATP-binding pocket induced by the binding of ADF/cofilin. In the presence of profilin the accessibility of the bound ɛ-ATP increased, indicating a more open and approachable protein matrix around the ATP-binding pocket. The results of the fluorescence quenching experiments support a structural mechanism regarding the regulation of the nucleotide exchange on actin monomers by ADF/cofilin and profilin.     

Orientation of LamB signal peptides in bilayers: influence of lipid probes on peptide binding and interpretation of fluorescence quenching data.

The orientation in lipid bilayers of the signal sequence of the bacterial protein LamB was studied using binding, circular dichroism, and fluorescence quenching experiments. Measurements were made of binding modifications caused by the incorporation of lipid probes (brominated or nitroxide-labeled phospholipids) used in the parallax fluorescence quenching method of determining peptide penetration depth [Abrams, F. S., and London, E. (1992) Biochemistry 31, 5312-5322]. The signal peptide bound to a similar extent to neutral bilayers composed of either egg phosphatidylcholine (EPC) or phosphatidylcholines brominated at various positions on their acyl chains. The fluorescence of a tryptophan in either the 18 or 24 position of the peptide was quenched more by bromines in the 6 and 7 than in the 9 and 10 positions on the lipid hydrocarbon chain. Parallax calculations showed that tryptophan-18 was located only 4 A from the hydrocarbon-water interface, consistent with the peptide adopting a "hammock" configuration in the bilayer, with both termini exposed to the aqueous phase and the central alpha-helix located near the hydrocarbon-water interface. In contrast, the incorporation of 10% nitroxide-labeled lipids into EPC bilayers modified peptide binding in a manner dependent on the position of the nitroxide on the hydrocarbon chain; 7-Doxyl PC reduced the percent peptide bound by about one-half, whereas 12-Doxyl PC had little effect on binding. These binding differences modified tryptophan quenching by these probes, making parallax analysis problematical. In the presence of the positively charged LamB peptide, the incorporation of negatively charged phospholipids into EPC bilayers increased the level of peptide binding and modified tryptophan quenching by nitroxide probes. These results suggest that the nitroxide probe could be partially excluded from negatively charged lipid domains where the peptide preferentially bound. Quite different binding and quenching results were obtained with a negatively charged peptide analogue, showing that the charge on both the peptide and bilayer affects peptide-nitroxide probe interactions.     

Crystal structure of the complex between actin and human vitamin D-binding protein at 2.5 A resolution

A high-affinity complex formed between G-actin and plasma vitamin D-binding protein (DBP) is believed to form part of a scavenging system in the plasma for removing actin released from damaged cells. In the study presented here, we describe the crystal structure of the complex between actin and human vitamin D-binding protein at 2.5 A resolution. The complex contains one molecule of each protein bound together by extensive ionic, polar, and hydrophobic interactions. It includes an ATP and a calcium ion bound to actin, but no evidence of vitamin D metabolites bound to the DBP. Both actin and DBP are multidomain molecules, two major domains in actin and three in DBP. All of these domains contribute to the interaction between the molecules. DBP enfolds the end of the actin molecule, principally in actin subdomain 3 but with additional interactions in actin subdomain 1. This orientation is similar to the binding of profilin to actin, as predicted from previous studies. The more extensive interactions of DBP give an affinity for actin some 3 orders of magnitude higher than that for profilin. The larger "footprint" of DBP on actin also leads to an overlap with the actin-binding site of gelsolin domain I.     

Thymosin beta4 induces a conformational change in actin monomers

Using fluorescence resonance energy transfer spectroscopy we demonstrate that thymosin beta(4) (tbeta(4)) binding induces spatial rearrangements within the small domain (subdomains 1 and 2) of actin monomers in solution. Tbeta(4) binding increases the distance between probes attached to Gln-41 and Cys-374 of actin by 2 A and decreases the distance between the purine base of bound ATP (epsilonATP) and Lys-61 by 1.9 A, whereas the distance between Cys-374 and Lys-61 is minimally affected. Distance determinations are consistent with tbeta(4) binding being coupled to a rotation of subdomain 2. By differential scanning calorimetry, tbeta(4) binding increases the cooperativity of ATP-actin monomer denaturation, consistent with conformational rearrangements in the tbeta(4)-actin complex. Changes in fluorescence resonance energy transfer are accompanied by marked reduction in solvent accessibility of the probe at Gln-41, suggesting it forms part of the binding interface. Tbeta(4) and cofilin compete for actin binding. Tbeta(4) concentrations that dissociate cofilin from actin do not dissociate the cofilin-DNase I-actin ternary complex, consistent with the DNase binding loop contributing to high-affinity tbeta(4)-binding. Our results favor a model where thymosin binding changes the average orientation of actin subdomain 2. The tbeta(4)-induced conformational change presumably accounts for the reduced rate of amide hydrogen exchange from actin monomers and may contribute to nucleotide-dependent, high affinity binding.     

Mechanism of the interaction of human platelet profilin with actin

We have reexamined the interaction of purified platelet profilin with actin and present evidence that simple sequestration of actin monomers in a 1:1 complex with profilin cannot explain many of the effects of profilin on actin assembly. Three different methods to assess binding of profilin to actin show that the complex with platelet actin has a dissociation constant in the range of 1 to 5 microM. The value for muscle actin is similar. When bound to actin, profilin increases the rate constant for dissociation of ATP from actin by 1,000-fold and also increases the rate of dissociation of Ca2+ bound to actin. Kinetic simulation showed that the profilin exchanges between actin monomers on a subsecond time scale that allows it to catalyze nucleotide exchange. On the other hand, polymerization assays give disparate results that are inconsistent with the binding assays and each other: profilin has different effects on elongation at the two ends of actin filaments; profilin inhibits the elongation of platelet actin much more strongly than muscle actin; and simple formation of 1:1 complexes of actin with profilin cannot account for the strong inhibition of spontaneous polymerization. We suggest that the in vitro effects on actin polymerization may be explained by a complex mechanism that includes weak capping of filament ends and catalytic poisoning of nucleation. Although platelets contain only 1 profilin for every 5-10 actin molecules, these complex reactions may allow substoichiometric profilin to have an important influence on actin assembly. We also confirm the observation of I. Lassing and U. Lindberg (1985. Nature [Lond.] 318:472-474) that polyphosphoinositides inhibit the effects of profilin on actin polymerization, so lipid metabolism must also be taken into account when considering the functions of profilin in a cell.     

Functional characterization of green fluorescent protein-profilin fusion proteins.

To clarify the role of profilins in cells, fusion proteins constructed with green fluorescent protein (GFP) should be extremely helpful. As profilins are considerably smaller than the GFP fusion partner (14-17 kDa compared with 27 kDa, respectively), we characterized the fusion proteins in vitro, to ascertain their biological function. We fused mouse profilin I and II to either the C-terminus or N-terminus of GFP. These fusion proteins were expressed in Escherichia coli and affinity-purified on polyproline-Sepharose. Interaction with vasodilator-stimulated phosphoprotein, a proline-rich ligand of profilin, was investigated by ELISA, as was binding to PtdIns(4,5)P2. The affinity for actin was quantitatively determined in polymerization assays. Our results show that fusion of GFP to the C-terminus of profilin I abolishes polyproline binding. In contrast, the other fusion proteins bound to polyproline-Sepharose and VASP. Binding to PtdIns(4,5)P2 was not significantly altered. Furthermore, fusion of either isoform with GFP did not decrease the affinity for actin. In localization studies with mammalian cells, all fusion proteins showed the localization expected for profilin in areas of high actin dynamics, such as leading lamellae and ruffles induced by epidermal growth factor. However, with regard to our in vitro data, we suspect that only a minor fraction of profilin I carrying the GFP at the C-terminus can target these sites. Therefore, other constructs should be preferred for further in vivo studies.     

Fluorescent actin analogs with a high affinity for profilin in vitro exhibit an enhanced gradient of assembly in living cells

Constitutive centripetal transport of the actin-based cytoskeleton has been detected in cells spreading on a substrate, locomoting fibroblasts and keratocytes, and non-locomoting serum-deprived fibroblasts. These results suggest a gradient of actin assembly, highest in the cortex at the cytoplasm-membrane interface and lowest in the non-cortical perinuclear cytoplasm. We predicted that such a gradient would be maintained in part by phosphoinositide-regulated actin binding proteins because the intracellular free Ca2+ and pH are low and spatially constant in serum-deprived cells. The cytoplasm-membrane interface presents one surface where the assembly of actin is differentially regulated relative to the non-cortical cytoplasm. Several models, based on in vitro biochemistry, propose that phosphoinositide-regulated actin binding proteins are involved in local actin assembly. To test these models in living cells using imaging techniques, we prepared a new fluorescent analog of actin that bound profilin, a protein that interacts with phosphoinositides and actin-monomers in a mutually exclusive manner, with an order of magnitude greater affinity (Kd = 3.6 microM) than cys-374-labeled actin (Kd > 30 microM), yet retained the ability to inhibit DNase I. Hence, we were able to directly compare the distribution and activity of a biochemical mutant of actin with an analog possessing closer to wild-type activity. Three-dimensional fluorescence microscopy of the fluorescent analog of actin with a high affinity for profilin revealed that it incorporated into cortical cytoplasmic fibers and was also distributed diffusely in the non- cortical cytoplasm consistent with a bias of actin assembly near the surface of the cell. Fluorescence ratio imaging revealed that serum- deprived and migrating fibroblasts concentrated the new actin analog into fibers up to four-fold in the periphery and leading edge of these cells, respectively, relative to a soluble fluorescent dextran volume marker, consistent with the formation of a gradient of actin filament density relative to cell volume. Comparison of these gradients in the same living cell using analogs of actin with high and low affinities for profilin demonstrated that increased profilin binding enhanced the gradient. Profilin and related proteins may therefore function in part to bias the assembly of actin at the membrane-cytoplasm interface.     

纤维蛋白影响花粉肌动蛋白体外聚合分析

The effects of different ratio of native profilin on maize (Zea mays L.) pollen actin polymerization in vitro were analyzed by using ultracentrifuging sedimentation and ultraviolet absorption spectrum measurement (the molar ratio of profilin to actin was 2∶1, 1.5∶1, 1∶1, 0.5∶1, 0.1∶1 respectively). Preliminary results showed that profilin bound to G-actin and inhibited its polymerization. The inhibition of actin polymerization by profilin increased with the increasing ratio of profilin to pollen actin. The dissociation constant （Kd） value of profilin for binding to actin was (1.30±0.33) μmol/L. No stimulation effect of profilin on actin polymerization was observed, suggesting that pollen profilin may affect actin organization by sequestering the G-actin.     

Interactions among serum vitamin D binding protein, monomeric actin, profilin, and profilactin

Human serum vitamin D binding protein (hDBP), a 58-kDa inter-alpha-globulin, is known to bind, monomeric actin (G-actin) in equimolar quantities. Using monoclonal and polyclonal anti-hDBP antibodies, hDBP, and radioiodinated actin, we developed a reliable saturation assay for actin bound to hDBP. By utilizing this assay, kinetic analysis, and ultracentrifugal sedimentation in sucrose gradients, these proteins' binding affinities (Kd = 10(-9) M) were demonstrated to be 10- to 100-fold greater than earlier estimates. At 4 degrees C, hDBP has an association rate constant of 2.2 x 10(4) M-1 s-1 and a rate of dissociation displaying a t1/2 of 22 h. This high affinity binding was largely unaffected by conditions favoring actin filament formation (1 mM MgCl2 and/or 50 mM KCl), by the range of pH from 6.8 to 8.6 or by temperatures from 4 to 37 degrees C. Compared with ATP-alpha-actin, a 2-fold decrease of binding affinity was observed for the nonmuscle isoactins (beta,gamma), ADP-G-alpha-actin, and N'-ethylmaleimide-modified G-alpha-actin. The 25-hydroxyvitamin D3 and 1 alpha,25-dihydroxyvitamin D3 holo-sterol forms of hDBP bound actin in a manner indistinguishable from the apo-sterol hDBP. The common polymorphisms of hDBP (DBP1 slow, DBP1 fast, and DBP2) were shown to have an equal avidity for G-actin binding. Human platelet profilin competed with hDBP for binding to G-actin, but was 1000-fold less potent (Ki = 1.9 microM). When platelet profilactin was incubated with hDBP, profilin was liberated and hDBP-actin complexes formed. DNase I, which forms a triprotein complex with hDBP-G actin, did not alter the affinity of binding of actin by hDBP. The very high affinity binding observed, which was largely unaffected by the state of G-actin, pH, and ionic conditions, appears to support a constitutive role for plasma DBP in the sequestration of actin monomers, as well as actin from actin-profilin complexes, that are liberated during cell injury.     

Uses of fluorescent cholinergic analogues to study binding sites for cholinergic ligands in Torpedo californica acetylcholine receptor.

A series of synthetic 1,n-bis(3-aminopyridinio)-alkane fluorescent probes have been used to determine the ligand binding properties of the acetylcholine receptor purified from Torpedo californica electroplax. At equilibrium, the probes bound to a single class of sites. The binding affinity of the fluorescent decamethonium analogues increased progressively as the number of methylene groups (n) increased from 4 to 12 and decreased in the range of 16--18 such groups. The receptor bound 1,12-bis(3-aminopyridinio)dodecane and 1,14-bis(3-aminopyridinio)tetradecane with the highest affinity while related monofunctional probes such as 1-(3-amino-pyridinio)propane were bound with a substantially lower affinity. The data indicate that the receptor interacts strongly with both ends of a bifunctional probe such as 1,14-bis(3-aminopyridinio)tetradecane. Also, competition between bifunctional fluorescent probe binding and the binding of conventional cholinergic ligands, was investigated and led to the conclusion that the probes, which are antagonists, form ternary complexes in the presence of acetylcholine.