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PubMed1994

The (DD)E complex is maintained by a composite fibrin polymerization site.

The (DD)E complex is the major cross-linked fibrin degradation fragment. Structural components required for maintenance of the (DD)E complex were examined in order to better understand clot structure and the contribution of specific polypeptide chain segments in the process of polymerization. First, the (DD)E complex was reversibly dissociated by peptides derived from the alpha-chain NH2-terminus of fibrin having a minimal sequence of GPR (alpha 17-19). In addition, the complex was partially dissociated by peptide beta 40-54, while beta 50-55 and peptides derived from the fibrin beta-chain NH2-terminus had no effect. Second, monoclonal antibody (mAb) 1B6, specific for the alpha-chain NH2-terminus of fibrin, reacted rapidly with fragment E1, but did not recognize the corresponding epitope on the (DD)E complex. On the other hand, mAb 59D8, specific for GHRPL at the beta-chain NH2-terminus of fibrin, reacted with the (DD)E complex in a dose-dependent manner. Third, the (DD)E complex was irreversibly dissociated by proteolytic cleavage of fragment E1 by either thrombin, which removed GPR from the alpha-chain NH2-terminus, or Crotalus atrox protease III, which released beta 15-42. It has been concluded that fragment E1 contains a composite polymerization site consisting at least of residues alpha 17-19 and beta 20-49, which together maintain the (DD)E complex. These results illustrate that the complex is kept together by complementary binding sites which form a nucleus of linear fibrin polymerization sites. The (DD)E complex can thus be considered as a soluble model of fibrin clot.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1983

Kinetic evidence for a monomer activation step in actin polymerization.

We measured the time course of skeletal muscle actin polymerization at different actin concentrations. In 0.1 M KCl with 1 mM Mg2+, log/log plots of the rate of the early, slow phase of polymerization vs. actin concentration were linear with slopes from 1.0 to 1.3. Computer-assisted calculations of similar curves from theoretical models with different sizes for the nucleus showed that no simple model gave a log/log plot with a slope less than 1.5. Addition of a first-order, monomer activation step before nucleation allowed models of any reasonable nucleus size to have a slope of 1. This is the first evidence that such a step is part of the kinetic pathway for actin polymerization. In 0.1 M KCl with 0.2 mM Ca2+, log/log plots of the rate of the slow phase vs. actin concentration were linear with slopes from 2.0 to 2.5. Monomer activation was not necessary to account for this slope. However, fits of kinetic curves calculated from theoretical models to experimental kinetic curves showed that filament fragmentation was important to achieve a good fit, confirming the finding of Wegner and Savko [Wegner, A., & Savko, P. (1982) Biochemistry 21, 1909-1913]. Our fit procedure also allowed us to estimate the size of the nucleus and the rate constants for activation, nucleation, and fragmentation. In 0.1 M KCl with 1 mM Mg2+, the nucleus was a dimer or trimer, and nucleation was fast. In 0.1 M KCl with 2.0 mM Ca2+, the nucleus was a trimer, and nucleation was slow.

Actins↗
PubMed1984

Modulation of actin polymerization by the spectrin-band 4.1 complex.

The effect of human erythrocyte spectrin dimer and band 4.1 on the polymerization of actin was studied by two independent methods: by following the increase in fluorescence of actin covalently conjugated to N-pyrenyl-iodoacetamide (pyrenylactin) and by following the increase in light scattered by actin polymers. Both techniques indicated that the complex of spectrin dimer and band 4.1, but neither spectrin nor band 4.1 alone, stimulates the rate of nucleation (decreases the lag phase of polymerization) and stabilizes oligomers of F-actin. While the band 4.1-spectrin complex, but not spectrin alone, had no immediate effect on the rate of polymerization after the lag phase, it eventually decreases the rate of actin filament growth when the molecular ratio of actin-spectrin-band 4.1 approaches the physiological range. The complex has no detectable effect on the critical actin concentration and does not significantly alter the apparent order of the nucleation reaction.

Actins↗
PubMed1985

The membrane attack complex of complement: lipid insertion of tubular and nontubular polymerized C9.

The membrane-restricted photoactivatable carbene generator 3-(trifluoromethyl)-3-(m-[125I]-iodophenyl)diazirine [Brunner, J., & Semenza, G. (1981) Biochemistry 20, 7174-7182] was used to label the subunits of the membrane attack complex of complement (C5b-9). C5b-9 complexes either were assembled from serum on erythrocyte membranes or were reconstituted from purified components on liposomes. After irradiation, most of the probe is bound to C9 independent of the membrane system used, indicating that the wall of the transmembrane channel is predominantly composed of C9. No difference was observed whether polymerized C9 was in the tubular or nontubular form [Podack, E. R., & Tschopp, J. (1983) J. Biol. Chem. 257, 15204-15212], showing that tubule closure is not essential for successful lipid insertion. The same label distribution between the two forms of polymerized C9 was obtained by analyzing zinc-polymerized C9 in the absence of C5b-8. Since the photoreactive probe reacted with at least two distinct polypeptide segments within C9, lipid interaction does not occur via a single segment of hydrophobic amino acids.

Animals↗
PubMed1986

Effect of temperature on the mechanism of actin polymerization.

The rate of the Mg2+-induced polymerization of rabbit skeletal muscle G-actin has been measured as as function of temperature at pH 8 by using various concentrations of Mg2+, Ca2+, and G-actin. A polymerization mechanism similar to that proposed at this pH [Frieden, C. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6513-6517] was found to fit the data from 10 to 35 degrees C. From the kinetic data, no evidence for actin filament fragmentation was found at any temperature. Dimer formation is the most temperature-sensitive step, with the ratio of forward and reverse rate constants changing 4 orders of magnitude from 10 to 35 degrees C. Over this temperature change, all other ratios of forward and reverse rate constants change 7-fold or less, and the critical concentration remains nearly constant. The reversible Mg2+-induced isomerization of G-actin monomer occurs to a greater extent with increasing temperature, measured either by using N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine-labeled actin or by simulation of the full-time course of the polymerization reaction. This is partially due to Mg2+ binding becoming tighter, and Ca2+ binding becoming weaker, with increasing temperature. Elongation rates from the filament-pointed end, determined by using actin nucleated by plasma gelsolin, show a temperature dependence slightly larger than that expected for a diffusion-limited reaction.

Actins↗
PubMed1981

Change of reactivity of lysine residues upon actin polymerization.

The reactivity of lysine residues of actin was measured by a surface labeling method--limited reductive methylation. After labeling, actin was subjected to CNBr and enzymatic cleavage, and all lysines were obtained either singly in a peptide or as a free residue. The specific activity of each lysine was taken as the measure of its reactivity. In actin denatured in 8 M urea, the reactivity of each lysine residue is approximately equal whereas those in G-actin fall into three categories: Lys-61 and Lys-113 are the most reactive ones; Lys-18, -213, -215, -314, and -358 are hardly reactive; the remainder, including Lys-50, -68, -84, -118, -191, -237, -283, -290, -325, -327, -335, and -372, are moderately reactive. The least reactive ones are probably buried in the native G-actin and all the others are most likely on the surface. Upon actin polymerization the reactivities of Lys-61, -68, -113, and -283 are significantly reduced while that of Lys-335 is strikingly enhanced. The decrease in reactivity could be readily explained if these residues were located in the monomer-monomer contact area although a polymerization-induced conformational change cannot be excluded. Such a conformational change may be invoked to explain the increase in the reactivity of Lys-335. Alternatively, the latter may be interacting with the bound ATP of G-actin, and the increased reactivity might be directly attributable to the loss of gamma-P for ATP accompanying polymerization.

Actins↗
PubMed1981

Different polymeric forms of actin detected by the fluorescent probe terbium ion.

The interaction of actin with Tb3+ was studied by following the fluorescence emitted at 545 nm when the protein was excited at 285 nm in the presence of Tb3+. It was shown that, at low ionic strength, each actin monomer binds, at saturation, six Tb3+ with an association constant of 0.8 microM-1. In the presence of 0.1 M KCl the association constant decreases to 0.15 and 0.24 microM-1 at subcritical and overcritical actin concentrations, respectively; the number of the binding sites remains six. When polymeric actin is formed by the addition of 2 mM MgCl2, the association constant drops to 0.008 micro M-1 and the number of the binding sites to four. The lower number of the Tb3+ binding sites (four) in the actin polymerized by MgCl2 as compared to the number of binding sites (six) of the actin polymerized by KCl is taken as evidence of a looser structure of this latter polymer. We have also shown that Tb3+ does not replace 45Ca2+ at the single, "high-affinity" site of G-actin. Removal of this Ca2+, in the presence of Tb3+, destroys the typical G- and F-actin structures.

Actins↗
PubMed1982

Effects of organic acids on tubulin polymerization and associated guanosine 5'-triphosphate hydrolysis.

We have examined the effects of a number of organic anions, which stabilize tubulin, on tubulin polymerization, associated GTP hydrolysis, and polymer morphology. While microtubule-associated proteins, as well as glycerol, induced formation of typical microtubules in a reaction coupled to GTP hydrolysis at an initial 1:1 stoichiometry, the organic anions had varying effects. Only 2-(N-morpholino)ethanesulfonate induced formation of structures with the morphology of microtubules. With glutamate, fructose 1,6-bisphosphate, piperazine-N-N'-bis(2-ethanesulfonate), glutarate, and glucose 1-phosphate, the predominant structures formed were sheets of parallel protofilaments rather than microtubules. Creatine phosphate induced the formation of clusters of rings. GTP hydrolysis was closely coupled to polymerization only with glutamate. With creatine phosphate, there was minimal GTP hydrolysis. With all other organic anions, GTP hydrolysis substantially exceeded polymerization at all time points, with the onset of hydrolysis significantly preceding the onset of turbidity development. Nevertheless, the rate of GTP hydrolysis was a sigmoidal function of tubulin concentration under all conditions examined, suggesting that tubulin-tubulin interactions are required for hydrolysis. All anion-induced reactions were temperature dependent and cold reversible, but only the creatine phosphate induced reaction was not inhibited by GDP, CA2+, or colchicine and did not require GTP.

Animals↗
PubMed1979

High concentration active enzyme centrifugation: analysis of active polymeric forms at up to 10 000-fold higher concentrations than with conventional methods.

This paper describes the theoretical basis, experimental technique, and experimental evaluation of a new method of analysis called "high concentration active enzyme centrifugation". It extends by up to four orders of magnitude the upper concentration limits at which the technique of "active enzyme centrifugation" can be used for analysis of enzyme structure. This new theory is largely based on certain properties of Gaussian curves which we have described in previous publications [Wei, G.J., & Deal, W.C., Jr. (1976) Anal. Biochem. 75, 113-121; Anal. Biochem. (1978) 87, 433-446]. One of the most important aspects of this development is that it extends the concentration range upward so that experiments can be performed on enzymes in the active polymeric forms corresponding to their in vivo states. Furthermore, this expansion includes the range in which most enzymes go through all their association-dissociation transitions from one polymeric form to another. Hence, the method can be used to define the various concentration-dependent transitions and also to ascertain which of the various polymeric forms of an enzyme are active, under various conditions. This method also retains the many favorable characteristics inherent in the active enzyme centrifugation technique. In studies with lactate dehydrogenase, the results from this method of band sedimentation were identical within experimental error (about 1.5%) with results from conventional boundary sedimentation velocity studies.

Animals↗
PubMed1978

Sulfhydryls of platelet tubulin: their role in polymerization and colchicine binding.

Sulfhydryls and disulfides of platelet tubulin have been quantified, their accessibility and reactivity measured, and their role in polymerization and colchicine binding evaluated. Platelet tubulin isolated by two cycles of temperature-dependent polymerization--depolymerization was found to contain 12 free sulfhydryl groups per tubulin monomer all of which reacted rapidly with p-chloromercuribenzoate. One sulfhydryl was inaccessible to dithiobis(nitrobenzoic acid). Under anaerobic conditions of tubulin extraction, one intrachain disulfide bridge was found per tubulin monomer. Polymerization of tubulin reduced the number of sulfhydryls by one which were able to react with p-chloromercuribenzoate or dithiobis(nicotinic acid) but did not affect the disulfide bridge. Polymerizability of platelet tubulin was very sensitive to blocking of free sulfhydryl groups. Complete inhibition of microtubule assembly was obtained when the number of free sulfhydryls per tubulin was reduced by 3 but could be reversed by the addition of dithiothreitol. Colchicine binding, on the other hand, was only minimally influenced by blocking of sulfhydryls.

Blood Platelets↗
PubMed1977

Synthesis and template-directed polymerization of adenylyl(3'-5')adenosine cyclic 2', 3'-phosphate.

Adenylyl(3'-5')adenosine cyclic 2',3'-phosphate (A-A greater than p) was synthesized and its polymerization was attempted under various conditions inthe presence of poly(uridylic acid) and1,3-propanediamine. Reaction at -20 degrees C for 16 days gave polymerized products (up to the 8-mer) in 15% yield and was proved to be dependent on the template. Reaction at 0 degrees C for 16 days gave more extensive (up to the 10-mer) and more efficient (35%) polymerization. The newly formed phosphodiester linkage was exclusively 2'-5'. These results are discussed in comparison with the monomer-condensation reaction.

Adenosine Monophosphate↗
PubMed2002

Hydrolysis of ATP by polymerized actin depends on the bound divalent cation but not profilin.

Growing evidence suggests that the nucleotide bound to actin filaments serves as a timer to control actin filament turnover during cell motility (Pollard, T. D., Blanchoin, L., and Mullins, R. D. (2000) Annu. Rev. Biophys. Biomol. Struct. 29, 545-576). We re-examined the hydrolysis of ATP by polymerized actin using mechanical quenched-flow methods to improve temporal resolution. The rate constant for ATP hydrolysis by polymerized Mg actin is 0.3 s(-1), 3-fold faster than that measured manually. The ATP hydrolysis rate is similar when Mg ATP actin elongates either the pointed end or the barbed end of filaments. Polymerized Ca actin hydrolyzes ATP at 0.05 s(-1). Mg ATP actin saturated with profilin can elongate barbed ends at >60 s(-1), 2 orders of magnitude faster than ATP hydrolysis (0.3 s(-1)). Given that profilin binds to a surface on actin that is buried in the Holmes model of the actin filament, we expect that profilin will block subunit addition at the barbed end of a filament. Profilin must move from this site at rates much faster than it dissociates from monomers (4 s(-1)). ATP hydrolysis is not required for this movement.

Actins↗
PubMed2005

Effect of arginine loss in myelin basic protein, as occurs in its deiminated charge isoform, on mediation of actin polymerization and actin binding to a lipid membrane in vitro.

Myelin basic protein (MBP) binds to negatively charged lipids on the cytosolic surface of oligodendrocyte membranes and is most likely responsible for adhesion of these surfaces in the multilayered myelin sheath. It can also polymerize actin, bundle F-actin filaments, and bind actin filaments to lipid bilayers through electrostatic interactions. MBP consists of a number of posttranslationally modified isoforms of varying charge, including C8, in which six arginines are deiminated to the uncharged residue citrulline. The deiminated form decreases with development, but is increased in patients with the demyelinating disease multiple sclerosis. Here we investigate the effect of decreased net positive charge of MBP on its interaction with actin in vitro by comparing a recombinant murine form, rmC1, of the most highly charged unmodified isoform, C1, and a recombinant analogue of C8 in which six basic residues are converted to glutamine, rmC8. The dissociation constant of the less charged isoform rmC8 for actin was a little greater than that of rmC1, and rmC8 had somewhat reduced ability to polymerize actin and bundle F-actin filaments than rmC1. Moreover, rmC8 was more readily dissociated from actin by Ca(2+)-calmodulin than rmC1, and the ability of the deiminated isoform to bind actin to lipid bilayers was reduced. These results indicate that electrostatic forces are the primary determinant of the interaction of MBP with actin. The spin labeled side chains of a series of rmC1 and rmC8 variants containing single Cys substitutions at seven sites throughout the sequence all became motionally restricted to a similar degree on binding F-actin, indicating that the entire sequence is involved in interacting with actin filaments or is otherwise structurally constrained in actin bundles. Thus, this posttranslational modification of MBP, which occurs early in life and is increased in multiple sclerosis, attenuates the ability of MBP to polymerize and bundle actin, and to bind it to a negatively charged membrane.

Actin Cytoskeleton↗
PubMed1997

Horseradish peroxidase oxidation of tyrosine-containing peptides and their subsequent polymerization: a kinetic study.

Tyrosine-containing model peptides were oxidized by horseradish peroxidase (HRP). This led to a peptide polymerization via condensation of the aromatic rings. Dimers, trimers, and tetramers (depending on the peptide length and on the position of the tyrosine in the sequence) were identified by electron spray mass spectroscopy. The second-order rate constants of the second step of the HRP reduction (CII --> E) was decreased by the presence of a positively charged amino group in the vicinity of the aromatic ring as determined by stopped flow measurements [k3 = 19 398 M-1 s-1 and k3 = 1016 M-1 s-1 for N-acetyltyrosine (NAT) and l-Tyr oxidations, respectively]. High-performance liquid chromatography was used to follow the kinetics of polymerization of some model peptides after their enzymatic oxidation. The first polymerization products exhibited a strong inhibitory effect toward further oxidation by HRP. This effect was not observed when using manganese-dependent peroxidase (MnP) which does not bind directly to the tyrosine residue but rather acts as a "distant catalyst". Saturation of the HRP was achieved with Pro-Gln-Gln-Pro-Tyr (kcat = 58 s-1, = 2.1 mM), NAT (kcat = 94 s-1, = 5.6 mM), and Gly-Tyr (kcat = 175 s-1, = 10.8 mM). Analysis of steady state kinetics of the reaction showed that the dimers formed initially behaved like competitive inhibitors. The value of the dissociation constant between HRP and dimers was 20 microM. A simplified model which accounts for these observations, including the formation of a Michaelis-Menten-like complex involving the donor and enzyme, is proposed and discussed.

Chromatography, High Pressure Liquid↗
PubMed2000

In vitro polymerization and copolymerization of 3-hydroxypropionyl-CoA with the PHB synthase from Ralstonia eutropha.

The poly(3-hydroxybutyrate) (PHB) synthase of Ralstonia eutropha, which was produced by a recombinant strain of Escherichia coli and purified in one step with a methyl-HIC column to a purity of more than 90%, was used to polymerize 3-hydroxypropionyl-CoA (3HPCoA) and to copolymerize 3HPCoA with 3-hydroxybutyryl-CoA (3HBCoA). A Km of 189 microM and a kcat of 10 s-1 were determined for the activity of the enzyme in the polymerization reaction of 3HPCoA based on the assumption that the dimer form of PHB synthase was the active form. Free coenzyme A was found to be a very effective competitive inhibitor for the polymerization of 3HPCoA with a Ki of 85 microM. The maximum degree of conversion of 3HPCoA to polymer was less than 40%. In the simultaneous copolymerization reactions of these two monomers, both the turnover number for the copolymerization reaction and the maximum degree of conversion of 3HPCoA and 3HBCoA to copolymers increased with an increase in the amount of 3HBCoA in the monomer mixture. However, the maximum conversion of 3HPCoA to copolymer was always less than 35%, regardless of the ratio of 3HPCoA to 3HBCoA. Block copolymers were obtained by the sequential copolymerization of the two monomers and these copolymers had a much narrower molecular weight distribution than those obtained by the simultaneous copolymerization for the same molar ratio of 3HPCoA to 3HBCoA.

Acyltransferases↗
PubMedDate not supplied

Protease-catalyzed regioselective polymerization and copolymerization of glutamic acid diethyl ester.

Protease-catalyzed polymerization and copolymerization of L-glutamic acid diethyl ester hydrochloride (1) have been performed in a buffer of high concentration. Papain and bromelain showed high catalytic activity toward the polymerization. H-H COSY NMR analysis of the product showed the exclusive formation of poly(alpha-peptide), which was further confirmed by comparison with NMR spectra of poly(alpha-methyl gamma-L-glutamate). The papain-catalyzed polymerization of gamma-methyl L-glutamate did not occur under the similar reaction conditions, supporting the regioselective production of the polymer having an alpha-peptide linkage from 1. The effects of the reaction parameters have been systematically investigated. The copolymerization of 1 with various amino acid esters took place by the papain catalyst to give peptide copolymers.

Amino Acids↗
PubMedDate not supplied

Enzymatic surface-initiated polymerization: a novel approach for the in situ solid-phase synthesis of biocompatible polymer poly(3-hydroxybutyrate).

A novel system for surface-initiated enzymatic polymerization of a film of polyhydroxyalkanoate (PHA) on solid surfaces has been developed and characterized. PHAs are aliphatic polyesters produced by a variety of microorganisms as a reserve of carbon and energy, and their properties range from elastomers to thermoplastics, depending on their monomeric composition. The PHA synthase from Ralstonia eutropha H16 was expressed as a poly-histidine fusion in Escherichia coli and immobilized onto several solid substrates through a transition-metal complex, Ni(2+)-nitrilotriacetic acid. The immobilized PHA synthase catalyzed the surface-initiated polymerization of 3-(R)-hydroxybutyryl-CoA, forming a polymer film with a uniform thickness on the surface. In this work, we describe the patterned immobilization of the intact enzyme on silicon and subsequent enzymatic polymerization. The immobilized enzyme had a lower specific activity and did not exhibit a lag phase as compared to the soluble enzyme.

Acyltransferases↗
PubMedDate not supplied

Aggregation during coniferyl alcohol polymerization in pectin solution: a biomimetic approach of the first steps of lignification.

Coniferyl alcohol was polymerized in pectin solution in order to mimic the lignification that is the final step of biosynthesis of plant cell wall. Dehydrogenated polymers (DHP = coniferyl alcohol polymers = synthetic lignin) interact with pectin to form hydrophobic clusters as monitored by pyrene fluorescence spectroscopy. The structure of these clusters was studied during the polymerization of synthetic lignin by static and quasielastic light scattering and small angle neutron scattering experiments. We show that synthetic lignin and pectin contribute to the same clusters, but the inner structure of these clusters is very heterogeneous and displays three phases. One observes a segregation between well separated pectin and lignin rich phases at length scales below approximately 30 nm. As a corollary of this segregation, clusters embody a large amount of solvent. On average, the density of the polymer rich phase (lignin plus pectin) inside clusters increases while its specific surface area decreases throughout the polymerization process. These results are discussed with respect to in vivo lignification of the plant cell wall.

Biomimetic Materials↗