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PubMed2003

Stereoblock copolymers and tacticity control in controlled/living radical polymerization.

Three controlled/living radical polymerization processes, atom transfer radical polymerization (ATRP), reversible addition-fragmentation transfer (RAFT) polymerization, and nitroxide-mediated polymerization (NMP), were investigated for the polymerization of N,N-dimethylacrylamide in the presence of Lewis acids known to enhance isotacticity, such as yttrium trifluoromethanesulfonate (Y(OTf)(3)) and ytterbium trifluoromethanesulfonate (Yb(OTf)(3)). Poly(N,N-dimethylacrylamide) with controlled molecular weight, low polydispersity (M(w)/M(n) < 1.2), and a high proportion of meso dyads ( approximately 85%) was prepared by ATRP (with initiating system methyl 2-chloropropionate/CuCl/Me(6)TREN) and RAFT (with cumyl dithiobenzoate transfer agent) in the presence of Y(OTf)(3). The combination of NMP (using N-tert-butyl-1-diethylphosphono-2,2-dimethylpropyl nitroxide, SG1) and a Lewis acid complexation technique led to less precise control over chain architecture and microstructure ( approximately 65% meso dyads), as compared to RAFT/Y(OTf)(3) or ATRP/Y(OTf)(3). The latter two systems were used for the first one-pot synthesis of stereoblock copolymers by radical polymerization. Well-defined stereoblock copolymers, atactic-b-isotactic poly(N,N-dimethylacrylamides), were obtained by adding Y(OTf)(3) at a given time to either RAFT or ATRP polymerizations, initially started without the presence of the Lewis acid.

Acrylamides↗
PubMed2003

Syndiospecific living propylene polymerization catalyzed by titanium complexes having fluorine-containing phenoxy-imine chelate ligands.

The propylene polymerization behavior of a series of Ti complexes featuring fluorine-containing phenoxy-imine chelate ligands is reported. The Ti complexes combined with methylalumoxane (MAO) can be catalysts for living and, at the same time, stereospecific polymerization of propylene at room temperature or above. DFT calculations suggest that the attractive interaction between a fluorine ortho to the imine nitrogen and a beta-hydrogen of a growing polymer chain is responsible for the achievement of room-temperature living propylene polymerization. Although the Ti complexes possess C(2) symmetry, they are capable of producing highly syndiotactic polypropylenes. (13)C NMR is used to demonstrate that the syndiotacticity is governed by a chain-end control mechanism and that the polymerization is initiated exclusively via 1,2-insertion followed by 2,1-insertion as the principal mode of polymerization. (13)C NMR spectroscopy also elucidated that the polypropylenes produced with the Ti complexes possess regio-block structures. Substitutions on the phenoxy-imine ligands have profound effects on catalytic behavior of the Ti complexes. The steric bulk of the substituent ortho to the phenoxy oxygen plays a decisive role in achieving high syndioselectivity for the chain-end controlled polymerization. Over a temperature range of 0-50 degrees C, Ti complex having a trimethylsilyl group ortho to the phenoxy oxygen forms highly syndiotactic, nearly monodisperse polypropylenes (94-90% rr) with extremely high peak melting temperatures (T(m) = 156-149 degrees C). The polymerization behavior of the Ti complexes can be explained well by the recently proposed site-inversion mechanism for the formation of syndiotactic polypropylene by a Ti complex having a pair of fluorine-containing phenoxy-imine ligands.

Journal Article↗
PubMed2003

Photoinitiated polymerization of columnar stacks of self-assembled trialkyl-1,3,5-benzenetricarboxamide derivatives.

A disk-shaped molecule, N-(5-sorbyl-pentyl)-N',N"-di(n-octyl)benzene-1,3,5-tricarboxamide (1), has been synthesized and assembled into a columnar stack in cyclohexane. Using a photoinitiated process, we polymerized monomer 1 in its self-assembled state and analyzed the resulting poly-1. On cooling a boiling cyclohexane solution of 1, the molecules aggregate via amide hydrogen bonds, as supported by the position of the N-H stretching band in FT-IR spectroscopy. Evidence of helical columnar stacking of 1 in the aggregate is provided by an induced CD effect upon blending 1 with a chiral side-chain homologue 3 in a so-called "sergeants and soldiers" experiment. The columnar assembly in cyclohexane was polymerized by UV light (365 nm) irradiation in the presence of 2,2-dimethoxy-2-phenylacetophenone as radical photoinitiator. The polymerization occurs selectively to give 1,4-polymer with isolated trans C=C bonds, as shown by FT-IR, and (1)H and (13)C NMR spectroscopy. Lack of polymerization of methyl sorbate (4) under identical conditions, and low incorporation of 4 in copolymerizations with 1 in cyclohexane, suggests that the polymerization preferentially occurs within the columnar assembly. Size exclusion chromatography shows that the degree of polymerization (X(n)()) based on number average molecular weight is approximately 65. Incorporation of small amounts of nonpolymerizable derivatives 2 and 3 into the columns has no effect on X(n)() and conversion. Molecular models show that the polymerizable sidearm of 1 is long enough to span the distance between the monomers in the chiral stack. Under the atomic force microscope (AFM), purified samples of polymerized 1 displayed nanometer-sized fibrous morphologies with a high-axial-ratio (>150), uniform width (60 nm), and a thickness (1.0 nm) which corresponds to the width of the benzene tricarboxamide core of 1, whereas aggregates of nonpolymerized 1 yield a featureless image due to their instability.

Journal Article↗
PubMed2003

Nucleation-elongation polymerization under imbalanced stoichiometry.

As a result of the helical structure of the polymeric product, the folding-driven polymerization of oligo(m-phenyleneethynylene) imines in solution should inherently show nucleation-elongation in chain growth. Here, we present evidence for this behavior based on results of polymerizations conducted under conditions of imbalanced stoichiometry. Because the polymerization proceeds via imine metathesis between a pair of bifunctional monomers of types A-A and B-B, the molar ratio of the polymerizing functional groups can be arbitrarily varied. Alternatively, stoichiometry can be controlled by the addition of a monofunctional oligomer. Similar results were obtained in both cases whereby the molecular weight distribution was significantly different from that expected for classical step-growth polymerizations. At equilibrium, high molecular weight polymers were observed to coexist with the monomer in excess. Thermodynamic equilibrium was established by showing that the same distribution was reached starting either from a monomer mixture or from high polymers to which one monomer was added. These results are in great contrast to the low molecular weight oligomers that were produced when the reaction was conducted by melt condensation of bifunctional aldehyde and amine monomers, a polymerization that apparently proceeds without the nucleation event. An equilibrium model that captures the features of nucleation-elongation under conditions of imbalanced stoichiometry qualitatively supports the monomer-polymer distribution observed experimentally.

Journal Article↗
PubMed1999

Structural specificity of chloroquine-hematin binding related to inhibition of hematin polymerization and parasite growth.

Considerable data now support the hypothesis that chloroquine (CQ)-hematin binding in the parasite food vacuole leads to inhibition of hematin polymerization and parasite death by hematin poisoning. To better understand the structural specificity of CQ-hematin binding, 13 CQ analogues were chosen and their hematin binding affinity, inhibition of hematin polymerization, and inhibition of parasite growth were measured. As determined by isothermal titration calorimetry (ITC), the stoichiometry data and exothermic binding enthalpies indicated that, like CQ, these analogues bind to two or more hematin mu-oxo dimers in a cofacial pi-pi sandwich-type complex. Association constants (K(a)'s) ranged from 0.46 to 2.9 x 10(5) M(-1) compared to 4.0 x 10(5) M(-1) for CQ. Remarkably, we were not able to measure any significant interaction between hematin mu-oxo dimer and 11, the 6-chloro analogue of CQ. This result indicates that the 7-chloro substituent in CQ is a critical structural determinant in its binding affinity to hematin mu-oxo dimer. Molecular modeling experiments reinforce the view that the enthalpically favorable pi-pi interaction observed in the CQ-hematin mu-oxo dimer complex derives from a favorable alignment of the out-of-plane pi-electron density in CQ and hematin mu-oxo dimer at the points of intermolecular contact. For 4-aminoquinolines related to CQ, our data suggest that electron-withdrawing functional groups at the 7-position of the quinoline ring are required for activity against both hematin polymerization and parasite growth and that chlorine substitution at position 7 is optimal. Our results also confirm that the CQ diaminoalkyl side chain, especially the aliphatic tertiary nitrogen atom, is an important structural determinant in CQ drug resistance. For CQ analogues 1-13, the lack of correlation between K(a) and hematin polymerization IC(50) values suggests that other properties of the CQ-hematin mu-oxo dimer complex, rather than its association constant alone, play a role in the inhibition of hematin polymerization. However, there was a modest correlation between inhibition of hematin polymerization and inhibition of parasite growth when hematin polymerization IC(50) values were normalized for hematin mu-oxo dimer binding affinities, adding further evidence that antimalarial 4-aminoquinolines act by this mechanism.

Animals↗
PubMed1998

Intestinal absorption of nutrients is not influenced by soy fiber and does not differ between oligomeric and polymeric enteral diets.

Enteric feeding is often associated with diarrhea. To avoid this side effect, isoosmotic and fiber-supplemented enteral diets are recommended. The aims of this study were to determine whether supplementing enteral diets with soy fiber influences nutrient absorption and whether in enteric feeding absorption of nutrients and water fluxes differ between hyperosmotic oligomeric and isoosmotic polymeric diets. In mini pigs intestinal absorption and water fluxes were measured by perfusing a 150-cm length of jejunum. Six noncommercial iso- and hyperosmotic oligomeric and polymeric diets and six commercial polymeric diets, either fiber-free or supplemented with soy fiber, were used. Pancreatic enzymes were infused concomitantly with the polymeric diets. The absorption of nutrients and energy did not differ between oligomeric and polymeric diets. Oligomeric diets of high energy density produced a pronounced secretion of water. Despite lower initial osmolality, polymeric diets produced a similar secretion of water due to rapid pancreatic hydrolysis. Supplementing diets with largely insoluble soy fiber increased viscosity only between 4.6 and 14.5 mPa x sec. Soy fiber did not influence absorption of nutrients and energy and had also no effects on luminal transit and flow rate. The lack of effects was not due to dilution of chyme by intestinal secretion of water because no differences existed between isoosmotic and hyperosmotic oligomeric diets. In conclusion, supplementing enteral diets with soy fiber does not impair the absorption of nutrients. Enteric feeding with isoosmotic polymeric diets provides no advantage compared with hyperosmotic oligomeric diets with respect to absorption of nutrients and secretion of water.

Animals↗
PubMed2002

Melanosomal proteins--role in melanin polymerization.

Melanin, the major determinant of skin colour, is a tyrosine-based heteropolymer of indeterminate molecular weight. In vivo, melanin synthesis occurs within highly specialized organelles called melanosomes. Coated vesicles encapsulating the enzyme tyrosinase and tyrosinase related proteins, fuse with premelanosomes that contain structural proteins to form mature melanosomes. Coated vesicles and premelanosomes have been shown to have only melanin monomers but not the polymer. Our earlier results have clearly shown that the presence of proteins other than tyrosinase are critical for the post-tyrosinase steps of melanin polymerization at acidic pH. Proteins in melanosomes are difficult to purify because of their firm association with melanin. Thus, with progressive melanization, melanoproteins become progressively insoluble. In this paper, we discuss the isolation and purification of melanosomal proteins and their role in melanin polymerization. We have hypothesized that the initiation of polymerization and the binding of melanin to proteins are two discrete events and we have developed assays to quantify these events. Purified melanosomal proteins differ in their ability to polymerize melanin monomers. Further, we have also shown that two polypeptides (28 and 45 kDa) purified from melanosomes inhibit melanin polymerization but can bind preformed melanin. In conclusion, melanosomal proteins regulate melanin polymerization and differ in their ability to bind melanin. Polymerization and binding abilities of melanosomal proteins are specific to each protein and melanin-protein interaction is not nonspecific.

Animals↗
PubMed2004

Chemical polymerization of m-phenylenediamine, in the presence of glucose oxidase, produces an enzyme-retaining electrooxidisable polymer used to produce a biosensor for amperometric detection of glucose from brain dialysate.

A new procedure involving chemical polymerization of a monomer of m-phenylenediamine (m-ppd) containing glucose oxidase (GOx) and subsequent electro-synthesis of the functional GOx containing polymer onto platinum needle electrodes (PTNE) was used for the amperometric analysis of glucose concentration in brain dialysates. Monomer solutions of o-phenylenediamine (o-ppd) and m-ppd were polymerized by low concentrations of glutaraldehyde (GA) and precipitated from solution. The 1,3 position of the amines on the benzene was amenable to stable polymerization by GA but polymerization of o-ppd (1,2 position) by GA was unstable and degraded. Polymerization of m-ppd appears to proceed by dehydration synthesis. GA induced polymerization of m-ppd polymer in the presence of GOx produced a polymer with strongly bound, functional GOx. This GOx-m-ppd polymer formed a stable matrix that was effectively employed in flow injection analysis (FIA) of glucose. If maintained under O(2) free atmosphere after chemical polymerization, the GOx-m-ppd polymer retained the ability to be electropolymerized. PTNE coated with GOx-m-ppd polymer by repeated dip/amperometry produced stable, sensitive amperometric glucose sensors with good interference exclusion properties and long shelf life. Scanning EM demonstrated that amperometry modified the structure of the GOx-m-ppd on the PTNE surface. GOx-m-ppd PTNE glucose sensors and bare PTNE were placed in a radial flow cell and FIA was employed for the simultaneous measurement of glucose and ascorbic acid, respectively, from dialysates of brain tissue.

Biosensing Techniques↗
PubMed1973

Biosynthesis of immunoglobulin A (IgA) and immunoglobulin M (IgM). Requirement for J chain and a disulphide-exchanging enzyme for polymerization.

Mouse myeloma cells secreting 19S IgM (immunoglobulin M) (MOPC 104E and TEPC 183) or monomer and polymer IgA (immunoglobulin A) (MOPC 315) were incubated with radioactive leucine and the intracellular and secreted immunoglobulins and immunoglobulin subunits were prepared by preparative sucrose-density-gradient centrifugation. Samples were reduced in the presence or absence of isolated J chain, passed over Sephadex G-25 and then incubated at 37 degrees C for 30min with or without a source of disulphide-interchange enzyme. The extent of reassembly of reduced subunits was then evaluated by electrophoresis in polyacrylamide gels. Provided that J chain and the disulphide-interchange enzyme were supplied, both IgM and IgA could be assembled from their respective subunits, obtained by reductive cleavage of polymeric forms. Under similar conditions, assembly of polymeric forms from intracellular or secreted 7S monomer subunits also occurred. Under these conditions polymerization was total, there being no residue of the monomeric form. Reassembly did not occur in the absence of either J chain or the enzyme. All of the J chain released from IgM by reductive cleavage was incorporated back into the reassembled polymer. The J chain is therefore likely to be an essential structural requirement for polymeric immunoglobulins. A variety of controls ruled out non-specific interactions, and further suggested that the amino acid sequence of polypeptide chains determines the specificity of polymerization. The fact that intracellular IgA and IgM monomer subunits known to be deficient in galactose and fucose can be completely polymerized suggests that the addition of carbohydrate does not control polymerization.

Animals↗
PubMed1997

Mutation of recombinant complement component C9 reveals the significance of the N-terminal region for polymerization.

Complement component C9 binds to C5b-8 sites on target cells and polymerizes to form the membrane attack complex (MAC). The aim of the work reported here was to discover which region within C9 was responsible for protecting the globular protein against self-polymerization. Computer prediction modelling highlighted the domain at the N-terminus of C9, which was then investigated by site-directed mutagenesis. The mutated proteins were expressed using insect cells infected with baculovirus. Removal of 16, 20 or 23 amino acids at the N-terminus of C9 resulted in inactivation due to self-polymerization. In contrast, removal of 4, 8 or 12 amino acids resulted in a C9 that did not polymerize spontaneously, had two to threefold enhanced lytic activity on erythrocytes, and had increased binding to C5b-8 sites on rat neutrophils. These results suggest that the domain within the first 16 amino acids at the N-terminus of C9 is crucial in preventing the self-polymerization of the globular protein. We have also found that C9 contains a motif (27WSEWS31) common to a family of cytokine receptors that is similar to a tryptophan-rich motif (WEWWR) of the membrane pore formers, thiol-activated cytolysins. Mutation of this motif in C9 resulted in polymerized protein, consistent with this site keeping the N-terminus in a protected conformation and preventing premature self-polymerization.

Amino Acid Sequence↗
PubMedDate not supplied

Preparation and in vitro characteristics of polymerized pyridoxylated hemoglobin.

Stroma-free hemoglobin solutions have been the subject of extensive studies as potential acellular oxygen carriers. Oncotic pressure considerations limit the hemoglobin concentration of the solutions (6-8 g/dl) to one-half the normal whole blood values. Furthermore, the free hemoglobin has a short plasma half-life (2-4 hours). In principle, polymerization offers a means of normalizing the oxygen-carrying capacity as well as extending the plasma half-life. Pyridoxylation of hemoglobin prior to polymerization provides an acceptable P50. Pyridoxylated hemoglobin (14-16 g/dl) was polymerized with a 12.5 percent glutaraldehyde solution. Since the goal was to obtain a 15 g per dl solution iso-oncotic with plasma, the polymerization reaction was monitored by the drop in colloid osmotic pressure. The reaction was quenched with 1.3 M lysine when the colloid osmotic pressure reached normal values (20-25 torr). The polymerization yield was 80 percent, with molecular weights ranging from 120,000 to 600,000 Dalton. The polymerized hemoglobin had a binding coefficient of 1.32, a P50 of 16 torr, a Bohr coefficient of -0.12, and a Hill coefficient of 1.7. The viscosity of the solution was 4.5 centipoise. The methemoglobin levels were comparable to that of unpolymerized stroma-free hemoglobin. Polymerized hemoglobin solutions provide a normal oxygen-carrying capacity with a P50 comparable to that of unpolymerized stroma-free hemoglobin solutions.

Blood Substitutes↗
PubMed2006

Thermal conductivity of a polymerizing liquid.

Thermal conductivity kappa of seven polymerizing liquids has been measured in real time at different temperatures, and calorimetry and dielectric spectroscopy of one liquid are performed to help interpret the results. As a covalently bonded linear chain or a network structure in the liquid grows, kappa of the Debye equation initially increases with the polymerization time t(polym) as the molecular weight, density, and sound velocity increase, as on cooling a liquid. The measured kappa reaches a maximum and then decreases, thus showing a peak at a certain t(polym) and finally becomes constant, which is not the true behavior of steady state kappa. The dielectric relaxation time of the covalently bonded structure at the t(polym) for the kappa peak is less than 5 s and the extent of polymerization is below the vitrification plateau value. The peak height increases when the pulse time for kappa measurement is increased. An increase in the liquid's temperature shifts the kappa peak to a shorter t(polym). Liquid compositions polymerizing rapidly show a similar shift, and those polymerizing slowly or whose viscosity does not reach a high enough value show a small kappa peak or none. The kappa peak may be an artifact of the time dependence of heat capacity during the pulse time used for the kappa measurement, as proposed for glasses and supercooled liquids, similar to the changes in other properties observed as an artifact of kinetic freezing/unfreezing. For a polymerizing liquid, the peak may additionally arise when the rate of increase in the elastic modulus becomes equal to the rate of decrease in equilibrium Cp. In either case, its appearance does not distinguish the Brownian motions' slowing on polymerization from that on cooling or compressing a liquid.

Journal Article↗
PubMed1980

Cellulose biogenesis: Polymerization and crystallization are coupled processes in Acetobacter xylinum.

Calcofluor White ST, stilbene derivative used commerically as an optical brightener for cellulose, increased the rate of glucose polymerization into cellulose by resting cells of the gram-negative bacterium Acetobacter xylinum. This bacterium normally produces a ribbon of cellulose that is a composite of crystalline microfibrils. In concentrations above 0.1 mM, Calcofluor disrupts the assembly of crystalline cellulose I microfibrils and their integration into a composite ribbon by stoichiometric binding to glucose residues of newly polymerized glucan chains. Under these conditions, the rate of glucose polymerization increases up to 4 times the control rate, whereas oxygen uptake increases only 10-15%. These observed effects are readily reversible. If free Calcofluor is washed away or depleted below the threshold value by binding to cellulose as polymerization continues, ribbon production and the normal rate of polymerization resume. It is concluded that polymerization and crystallization are cell-directed, coupled processes and that the rate of crystallization determines the rate of polymerization. It is suggested that coupling must be maintained for biogenesis of crystalline cellulose I.

Journal Article↗
PubMed1984

Localization of a fibrin gamma-chain polymerization site within segment Thr-374 to Glu-396 of human fibrinogen.

Fibrinogen fragment D1 was converted to fragment D3 by plasmic digestion. This conversion eliminates the ability of the fragment to interact with thrombin-exposed sites on fibrin monomer. Peptides released during this plasmic digestion were assayed for the presence of a polymerization site by affinity chromatography on fibrin monomer-Sepharose. We found that a 33-residue peptide, corresponding to gamma-chain Thr-374 to Lys-406, binds to immobilized fibrin monomer. This peptide is a shorter variant of a previously isolated 38-residue peptide (gamma-chain Thr-374 to Val-411) that contains a polymerization site [Olexa, S. A. & Budzynski, A. Z. (1981) J. Biol. Chem. 256, 3544-3549]. The peptide mixture derived from fragment D1 was digested further with Staphylococcus aureus protease V8, and a 23-residue peptide, gamma-chain Thr-374 to Glu-396, carrying a polymerization site, was isolated by affinity chromatography. This 23-residue peptide inhibits the polymerization of desA-fibrinogen. We conclude that a polymerization site complementary to the site exposed by removal of fibrinopeptide A is present in this segment. The localization of the polymerization site within the gamma-chain segment 374-396 implies that the polymerization site does not overlap with segments of the gamma-chain that are responsible for platelet aggregation and for Staphylococcus clumping (residues 400-411 and 397-411, respectively) or with the residues involved in factor XIIIa-catalyzed fibrin crosslinking (Gln-398 and Lys-406).

Amino Acid Sequence↗
PubMed1996

Effect of constitutive 70-kDa heat shock protein polymerization on its interaction with protein substrate.

Constitutive 70-kDa heat shock protein (hsc70) is a mixture of monomers and oligomers in ADP, while in ATP it is monomeric unless certain DnaJ homologs are present which induce hsc70 to form large polymers in an ATP-dependent reaction. A key question regarding polymerized hsc70 is whether it is able to bind protein substrates. Polymerized BiP, the hsc70 present in the endoplasmic reticulum, has been found to bind substrates in vitro although substrates appear to bind only to monomeric BiP in vivo. In this study, we investigated whether substrate binds to polymerized cytoplasmic hsc70 in vitro. Although both stoichiometric ATP and high concentrations of cytochrome c peptide monomerized hsc70, direct binding studies provided no evidence that cytochrome c peptide binds to polymerized hsc70. Furthermore, the time course of cytochrome c peptide and clathrin binding to hsc70 suggested that rather than binding to polymerized hsc70, they monomerized it by reducing free monomer, thereby shifting the monomer-polymer equilibrium toward monomer. We conclude that peptide and protein substrates bind at least an order of magnitude more weakly to polymerized hsc70 than to monomer, suggesting that polymerization of hsc70 in vivo, perhaps by DnaJ homologs, may store it in an inactive form.

Adenosine Triphosphate↗
PubMed1997

The polymerization pocket "a" within the carboxyl-terminal region of the gamma chain of human fibrinogen is adjacent to but independent from the calcium-binding site.

The carboxyl-terminal region of the gamma chain of fibrinogen is involved in calcium binding, fibrin polymerization, factor XIIIa-mediated cross-linking, and binding to the platelet fibrin(ogen) receptor. Protein fragments encoding amino acids Val143 to Val411 (rFbggammaC30) or Val143 to Leu427 (gamma'C30) from the carboxyl end of the gamma or gamma' chains, respectively, of human fibrinogen were expressed in yeast (Pichia pastoris) and characterized as to their cross-linking by factor XIIIa, polymerization pocket, and calcium-binding site. rFbggammaC30 and gamma'C30 were both readily cross-linked by factor XIIIa, but only rFbggammaC30 was capable of inhibiting thrombin-induced platelet aggregation. Two mutants, gammaC30-Q329R and gammaC30-D364A, which were based on the three-dimensional structure of the polymerization pocket within rFbggammaC30 and on information derived from naturally occurring mutant fibrinogens, were also expressed and characterized. rFbggammaC30 inhibited (desAA)fibrin polymerization in a dose-dependent manner, while the two mutant forms did not. Similarly, rFbggammaC30 and gamma'C30 were protected from plasmin degradation by the presence of Ca2+ or the peptide Gly-Pro-Arg-Pro, indicating that a functional Ca2+-binding site and polymerization pocket are contained within each of these fragments. The mutant fragments, however, were protected from plasmin only by metal ions, while no protective effect was conferred by GPRP or by any other peptide tested. These results indicate that the polymerization pocket "a", which binds the peptide GPRP, functions independently from the nearby calcium-binding site and that amino acids Gln329 and Asp364 play a crucial role in fibrin polymerization.

Amino Acid Sequence↗
PubMed2000

Tropomyosin-dependent filament formation by a polymerization-defective mutant yeast actin (V266G,L267G).

A major function of tropomyosin (TPM) in nonmuscle cells may be stabilization of F-actin by binding longitudinally along the actin filament axis. However, no clear evidence exists in vitro that TPM can significantly affect the critical concentration of actin. We previously made a polymerization-defective mutant actin, GG (V266G, L267G). This actin will not polymerize alone at 25 degrees C but will in the presence of phalloidin or beryllium fluoride. With beryllium fluoride, but not phalloidin, this polymerization rescue is cold-sensitive. We show here that GG-actin polymerizability was restored by cardiac tropomyosin and yeast TPM1 and TPM2 at 25 degrees C with rescue efficiency inversely proportional to TPM length (TPM2 > TPM1 > cardiac tropomyosin), indicating the importance of the ends in polymerization rescue. In the presence of TPM, the apparent critical concentration of actin is 5.5 microm, 10-15-fold higher than that of wild type actin but well below that of the GG-actin alone (>20 microm). Non N-acetylated TPMs did not rescue GG-actin polymerization. The TPMs did not prevent cold-induced depolymerization of GG F-actin. TPM-dependent GG-actin polymerization did not occur at temperatures below 20 degrees C. Polymerization rescue may depend initially on the capture of unstable GG-F-actin oligomers by the TPM, resulting in the strengthening of actin monomer-monomer contacts along the filament axis.

Actins↗
PubMed2001

F-actin-like ATPase activity in a polymerization-defective mutant yeast actin (V266G/L267G).

Polymerization increases a low level G-actin ATPase activity yielding ADP-P(i) F-actin and then ADP F-actin following release of P(i). By monitoring P(i) release, we explored the relationship between the ATPase activity and polymerization characteristics of a mutant yeast actin, GG. In this mutant, two hydrophobic residues at the tip of a proposed hydrophobic plug between actin subdomains 3 and 4, Val(266) and Leu(267), were mutated to Gly. Although GG-actin does not polymerize by itself in vitro, GG cells are viable. We show that GG-actin ATPase activity increases under normal polymerization conditions, although stable filaments do not form. A plot of P(i) release rate versus actin concentration yields an apparent critical concentration, like that seen for actin polymerization, of approximately 8 microm for Mg(2+) GG-actin and 11 microm for Ca(2+) GG-actin. In contrast to WT-actin, P(i) release from GG-actin is cold-sensitive, reflecting the temperature sensitivity associated with mutations that decrease hydrophobicity in this region. Thus, under polymerization conditions, GG-actin exhibits a continuous F-actin-like ATPase activity resulting from the temperature-sensitive formation of unstable cycling F-actin oligomers. Tropomyosin limits the extent and rate of this activity and restores polymerization by capturing and stabilizing these oligomers rather than enhancing filament nucleation.

Actins↗