Monomer-polymer Equilibrium Research Articles

High Molecular Weight Semicrystalline Substituted Polycyclohexene From Alternating Copolymerization of Butadiene and Methacrylate and Its Ambient Depolymerization.

Cyclohexene cannot be polymerized via ring-opening polymerization under any conditions due to its lack of ring strain. A hypothetical polycyclohexene would therefore have a strong thermodynamic driving force to depolymerize to monomer if a metathesis catalyst were provided while otherwise having thermal and hydrolytic stability under normal conditions because of its hydrocarbon backbone. We envisioned access to this otherwise unattainable family of polymers via the alternating polymerization of a diene and an alkene. Ethyl aluminum chloride was found to promote highly alternating polymerization of butadiene and methacrylate when radically initiated at room temperature, resulting in formal polycyclohexene structures. Ultrahigh molecular weight (up to 1750 kDa) polymers can be synthesized at the decagram scale in high monomer conversions. The resulting presumably atactic copolymers exhibited semicrystallinity, leading to high toughness. In the presence of a small amount of the Grubbs catalyst, the generated polycyclohexene can be fully depolymerized at ambient temperatures into pure constituent cyclohexene. The strategy of using orthogonal chemistry for the polymerization and depolymerization processes allows access to polymer structures with subambient ceiling temperatures without using ultralow temperature synthesis or relying on the monomer-polymer equilibrium.

Reversibly growing crosslinked polymers with programmable sizes and properties

Growth constitutes a powerful method to post-modulate materials’ structures and functions without compromising their mechanical performance for sustainable use, but the process is irreversible. To address this issue, we here report a growing-degrowing strategy that enables thermosetting materials to either absorb or release components for continuously changing their sizes, shapes, compositions, and a set of properties simultaneously. The strategy is based on the monomer-polymer equilibrium of networks in which supplying or removing small polymerizable components would drive the networks toward expansion or contraction. Using acid-catalyzed equilibration of siloxane as an example, we demonstrate that the size and mechanical properties of the resulting silicone materials can be significantly or finely tuned in both directions of growth and decomposition. The equilibration can be turned off to yield stable products or reactivated again. During the degrowing-growing circle, material structures are selectively varied either uniformly or heterogeneously, by the availability of fillers. Our strategy endows the materials with many appealing capabilities including environment adaptivity, self-healing, and switchability of surface morphologies, shapes, and optical properties. Since monomer-polymer equilibration exists in many polymers, we envision the expansion of the presented strategy to various systems for many applications.

“Like Recycles Like”: Selective Ring‐Closing Depolymerization of Poly(L‐Lactic Acid) to L‐Lactide

AbstractChemical recycling of poly(L‐lactic acid) to the cyclic monomer L‐lactide is hampered by low selectivity and by epimerization and elimination reactions, impeding its use on a large scale. The high number of side reactions originates from the high ceiling temperature (Tc) of L‐lactide, which necessitates high temperatures or multistep reactions to achieve recycling to L‐lactide. To circumvent this issue, we utilized the impact of solvent interactions on the monomer–polymer equilibrium to decrease theTcof L‐lactide. Analyzing the observedTcin different solvents in relation to their Hildebrand solubility parameter revealed a “like recycles like” relationship. The decreasedTc, obtained by selecting solvents that interact strongly with the monomer (dimethyl formamide or the green solvent γ‐valerolactone), allowed chemical recycling of high‐molecular‐weight poly(L‐lactic acid) directly to L‐lactide, within 1–4 h at 140 °C, with >95 % conversion and 98–99 % selectivity. Recycled L‐lactide was isolated and repolymerized with high control over molecular weight and dispersity, closing the polymer loop.

"Like Recycles Like": Selective Ring-Closing Depolymerization of Poly(L-Lactic Acid) to L-Lactide.

Chemical recycling of poly(L‐lactic acid) to the cyclic monomer L‐lactide is hampered by low selectivity and by epimerization and elimination reactions, impeding its use on a large scale. The high number of side reactions originates from the high ceiling temperature (T c) of L‐lactide, which necessitates high temperatures or multistep reactions to achieve recycling to L‐lactide. To circumvent this issue, we utilized the impact of solvent interactions on the monomer–polymer equilibrium to decrease the T c of L‐lactide. Analyzing the observed T c in different solvents in relation to their Hildebrand solubility parameter revealed a “like recycles like” relationship. The decreased T c, obtained by selecting solvents that interact strongly with the monomer (dimethyl formamide or the green solvent γ‐valerolactone), allowed chemical recycling of high‐molecular‐weight poly(L‐lactic acid) directly to L‐lactide, within 1–4 h at 140 °C, with >95 % conversion and 98–99 % selectivity. Recycled L‐lactide was isolated and repolymerized with high control over molecular weight and dispersity, closing the polymer loop.

Cytoskeletal Drugs Modulate Off-Target Protein Folding Landscapes Inside Cells.

The dynamic cytoskeletal network of microtubules and actin filaments can be disassembled by drugs. Cytoskeletal drugs work by perturbing the monomer-polymer equilibrium, thus changing the size and number of macromolecular crowders inside cells. Changes in both crowding and nonspecific surface interactions ("sticking") following cytoskeleton disassembly can affect the protein stability, structure, and function directly or indirectly by changing the fluidity of the cytoplasm and altering the crowding and sticking of other macromolecules in the cytoplasm. The effect of cytoskeleton disassembly on protein energy landscapes inside cells has yet to be observed. Here we have measured the effect of several cytoskeletal drugs on the folding energy landscape of two FRET-labeled proteins with different in vitro sensitivities to macromolecular crowding. Phosphoglycerate kinase (PGK) was previously shown to be more sensitive to crowding, whereas variable major protein-like sequence expressed (VlsE) was previously shown to be more sensitive to sticking. The in-cell effects of drugs that depolymerize either actin filaments (cytochalasin D and latrunculin B) or microtubules (nocodazole and vinblastine) were compared. The crowding sensor protein CrH2-FRET verified that cytoskeletal drugs decrease the extent of crowding inside cells despite also reducing the overall cell volume. The decreased compactness and folding stability of PGK could be explained by the decreased extent of crowding induced by these drugs. VlsE's opposite response to the drugs shows that depolymerization of the cytoskeleton also changes sticking in the cellular milieu. Our results demonstrate that perturbation of the monomer-polymer cytoskeletal equilibrium, for example, during natural cell migration or stresses from drug treatment, has off-target effects on the energy landscapes of proteins in the cell.

Polymerization regulates hexokinase activity

Cell Biology The yeast hexokinase Glk1 is an actin-fold protein that forms polymers in response to binding its substrates and products. Stoddard et al. now show that Glk1 polymers are structurally distinct from actin filaments and suggest that polymerization of Glk1 evolved independently of the polymerization of other actin-like polymers. Glk1 polymerization inhibits its hexokinase activity, and the monomerpolymer equilibrium appears to set a maximum rate for the entire enzyme pool rather than a maximum rate per enzyme. This inhibition was found to be important for cell viability in the context of nutrient shifts, allowing yeast cells to modulate their metabolism rapidly in response to stochastic changes in the environment. Science , this issue p. [1039][1] [1]: /lookup/doi/10.1126/science.aay5359

Polymerization in the actin ATPase clan regulates hexokinase activity in yeast.

The actin fold is found in cytoskeletal polymers, chaperones, and various metabolic enzymes. Many actin-fold proteins, such as the carbohydrate kinases, do not polymerize. We found that Glk1, a Saccharomyces cerevisiae glucokinase, forms two-stranded filaments with ultrastructure that is distinct from that of cytoskeletal polymers. In cells, Glk1 polymerized upon sugar addition and depolymerized upon sugar withdrawal. Polymerization inhibits enzymatic activity; the Glk1 monomer-polymer equilibrium sets a maximum rate of glucose phosphorylation regardless of Glk1 concentration. A mutation that eliminated Glk1 polymerization alleviated concentration-dependent enzyme inhibition. Yeast containing nonpolymerizing Glk1 were less fit when growing on sugars and more likely to die when refed glucose. Glk1 polymerization arose independently from other actin-related filaments and may allow yeast to rapidly modulate glucokinase activity as nutrient availability changes.

Role of torsional strain in the ring-opening polymerisation of low strain [n]nickelocenophanes.

Ring-opening polymerisation (ROP) of strained [1]- and [2]metallocenophanes and related species is well-established, and the monomer ring-strain is manifest in a substantial tilting of the cyclopentadienyl ligands, giving α angles of ∼14-32°. Surprisingly, tetracarba[4]nickelocenophane [Ni(η5-C5H4)2(CH2)4] (2) undergoes ROP (pyridine, 20 °C, 5 days) to give primarily insoluble poly(nickelocenylbutylene) [Ni(η5-C5H4)2(CH2)4] n (12), despite the lack of significant ring-tilt. The exoenthalpic nature of the ROP was confirmed by DFT calculations involving the cyclic precursor and model oligomers (ΔH0ROP = -14 ± 2 kJ mol-1), and is proposed to be a consequence of torsional strain present in the ansa bridge of 2. The similarly untilted disila-2-oxa[3]nickelocenophanes [Ni(η5-C5H4)2(SiMe2)2O] (13) and [Ni(η5-C5H4)2(SiMePh)2O] (14) were found to lack similar torsional strain and to be resistant to ROP under the same conditions. In contrast, 1-methyltricarba[3]nickelocenophane {Ni(η5-C5H4)2(CH2)2[CH(CH3)]} (15) with a significant tilt angle (α ∼ 16°) was found to undergo ROP to give soluble polymer {Ni(η5-C5H4)2(CH2)2[CH(CH3)]} n (18). The reversibility of the process in this case allowed for the effects of temperature and reaction concentration on the monomer-polymer equilibrium to be explored and thereby thermodynamic data to be elucidated (ΔH0ROP = -8.9 kJ mol-1, ΔG0ROP = -3.1 kJ mol-1). Compared to the previously described ROP of the unsubstituted analogue [Ni(η5-C5H4)2(CH2)3] (1) (ΔH0ROP = -10 kJ mol-1, ΔG0ROP = -4.0 kJ mol-1), the presence of the additional methyl substituent in the ansa bridge appears to marginally disfavour ROP and leads to a corresponding decrease in the equilibrium polymer yield.

Abstract 1586: Developing novel inhibitors of S100A4 for neuroendocrine (NE) and metastatic prostate cancer: systematic testing using relevant models and drug development techniques

Abstract S100A4, a calcium binding protein has been well studied as a marker of fibrosis and metastasis. We recently showed that in addition of being a metastatic marker, S100A4 is in fact an oncogene that plays an important role in the development of prostate cancer (CaP) and is amenable of targeting for the treatment of this lethal disease particularly neuroendocrine CaP (NE-CaP). Using a genetically engineered transgenic mouse model of NE-CaP, we show that knocking down of S100A4 significantly inhibited growth of prostate tumorigenesis and metastasis. Our noticeable finding is that S100A4 is secreted by prostatic tumors, and extracellular/soluble S100A4 acts as a growth factor that has the ability to confer aggressive potential to less aggressive or indolent tumor cells. We show that serum-S100A4 level is highly elevated in human CaP patients with aggressive disease regardless of their serum-PSA levels. We next asked if small molecule inhibitors could be developed to inhibit the activity if intracellular as well as extracellular S100A4. Using a highly robust Blue/Gene supercomputer-based in silico method, we screened a library of 5000 molecules and based on their binding efficacy to S100A4, identified potential inhibitors (SMI1 and SMI2). We next tested if SMI1 and SMI2 bind to the S100A4 protein in biological solution. We generated recombinant S100A4 protein and standardized an isothermal titration Calorimetry (ITC) assay for S100A4 binding. The ITC analysis (Kcal vs time) shows that both SMI1 and SMI2 inhibitors significantly bind to the S100A4 protein nevertheless SMI2 exhibited higher binding affinity to S100A4 than SMI1. Next, we used Surface Plasmon Resonance (SPR) method (accurate and sensitive technique) for detecting binding of inhibitors to S100A4. The SPR data (sensogram) shows that SMI1 and SMI2 bind to the S100A4 protein. The binding of S100A4 to Myosin IIA (MIIA) is known to disrupt the latter’s monomer-polymer equilibrium. This phenomenon is captured in solution (in terms of disassembly of MIIA filaments and change in turbidity). Using disassembly/ or turbidity assays as an index of S100A4 activity (where recombinant S100A4 and MIIA are incubated +/- inhibitors), we show that SMI1 and SMI2 inhibit the activity of S100A4 protein. Next, we tested efficacy of inhibitors in vitro and show that SMI1 and SMI2 therapies inhibit the growth, proliferation, migration and invasiveness of NE-CaP (TRAMPC2) and AI-CaP (DU145, PC3) cells. Notably, SMI1 and SMI2 therapies inhibited the growth-promoting effects of extracellular S100A4 and decreased activities of S100A4 downstream target proteins (MMP9 and NFκB). These data suggest that S100A4 inhibitors (SMI1 and SMI2) exhibit high anti-metastatic efficacy and are the potential candidates for treating NE-CaP and metastatic AI-CaP. The validation of SMI1 and SMI2 under in vivo models is underway in our laboratory. Citation Format: Mohammad Saleem, Arsheed A. Ganaie, Reihana Maqbool, Firdous A. Beigh, Syed Umbreen, Natalya G. Dulyaninova, Badrinath R. Konety. Developing novel inhibitors of S100A4 for neuroendocrine (NE) and metastatic prostate cancer: systematic testing using relevant models and drug development techniques [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1586. doi:10.1158/1538-7445.AM2017-1586

Two Functional S100A4 Monomers Are Necessary for Regulating Nonmuscle Myosin-IIA and HCT116 Cell Invasion

S100A4, a member of the Ca(2+)-activated S100 protein family, regulates the motility and invasiveness of cancer cells. Moreover, high S100A4 expression levels correlate with poor patient survival in several cancers. Although biochemical, biophysical, and structural data indicate that S100A4 is a noncovalent dimer, it is unknown if two functional S100A4 monomers are required for the productive recognition of protein targets and the promotion of cell invasion. To address this question, we created covalently linked S100A4 dimers using a glycine rich flexible linker. The single-chain S100A4 (sc-S100A4) proteins exhibited wild-type affinities for calcium and nonmuscle myosin-IIA, retained the ability to regulate nonmuscle myosin-IIA assembly, and promoted tumor cell invasion when expressed in S100A4-deficient colon carcinoma cells. Mutation of the two calcium-binding EF-hands in one monomer, while leaving the other monomer intact, caused a 30-60-fold reduction in binding affinity for nonmuscle myosin-IIA concomitant with a weakened ability to regulate the monomer-polymer equilibrium of nonmuscle myosin-IIA. Moreover, sc-S100A4 proteins with one monomer deficient in calcium responsiveness did not support S100A4-mediated colon carcinoma cell invasion. Cross-linking and titration data indicate that the S100A4 dimer binds a single myosin-IIA target peptide. These data are consistent with a model in which a single peptide forms interactions in the vicinity of the canonical target binding cleft of each monomer in such a manner that both target binding sites are required for the efficient interaction with myosin-IIA.

Methyl methacrylate monomer–polymer equilibrium in solid polymer

Objective Poly(methyl methacrylate) (PMMA) is commonly processed in dentistry by thermally initiating the free-radical polymerization of methyl methacrylate (MMA). Residual MMA, a tissue irritant, is a concern. The concentration of MMA ([MMA]) versus time and temperature was studied to identify optimum processing conditions. Materials and methods One hundred milligram portions of plain and dental PMMA powders were incubated (10–170 °C, 1–384 h), with and without 6.0 μL MMA added. After incubation, [MMA] was determined by GC. Results For plain PMMA alone, equilibrium was attained in about 100 h. The equilibrium data for log[MMA] versus 1/ T was better fitted by a quadratic than a straight line, and formed an upper bound to the values of [MMA] when PMMA was incubated with MMA at temperatures >∼120 °C. The response surface for [MMA] versus log(time) and reciprocal temperature was fitted. An ‘overshoot’ in the equilibration process was identified, and postulated to be due to a rapidly formed intermediate of unknown chemistry. Significance Minimization of the residual MMA in acrylic denture bases prepared by processing a mixture of PMMA and MMA is important for reasons of mechanical properties and irritancy. The response surface mapped here allows direct identification of the optimum processing conditions.

Hyperbranched polymers from divinylbenzene and 1,3-diisopropenylbenzene through anionic self-condensing vinyl polymerization

Hyperbranched polymers were synthesized using anionic self-condensing vinyl polymerization (ASCVP) by forming ‘inimer’ (initiator within a monomer) in situ from divinylbenzene (DVB) and 1,3-diisopropenylbenzene (DIPB) using anionic initiators in THF at −40 °C. The reaction of equimolar amounts of DVB and nBuLi results in the formation of hyperbranched poly(divinylbenzene) through self-condensing vinyl polymerization (SCVP). The hyperbranched polymers were invariably contaminated with small amount of gel (<15%). No gelation was observed when using DIBP with anionic initiators. The presence of monomer–polymer equilibrium in the SCVP of DIPB restricts the growth of hyperbranched poly(DIPB). The inimer synthesized from DIPB at 35 °C undergoes intermolecular self-condensation to different extent depending on the nature of anionic initiator at −40 °C. The molecular weight of the hyperbranched polymers was higher when DPHLi was used as initiator. A small amount of styrene ([styrene]/[Li +]=1) was used to promote the chain growth by inducing cross-over reaction with styrene, and subsequent reaction of styryl anion with isopropenyl groups of inimer/hyperbranched oligomer. The hyperbranched polymers were soluble in organic solvents and exhibited broad molecular weight distribution (2< M w/ M n<17).

Quantitative Analysis of Tropomyosin Linear Polymerization Equilibrium as a Function of Ionic Strength

Tropomyosin is a coiled-coil protein that polymerizes by head-to-tail interactions in an ionic strength-dependent manner. We produced a recombinant full-length chicken alpha-tropomyosin containing a 5-hydroxytryptophan residue at position 269 (formerly an alanine), 15 residues from the C terminus, and show that its fluorescence intensity specifically reports tropomyosin head-to-tail interactions. We used this property to quantitatively study the monomer-polymer equilibrium in tropomyosin and to calculate the equilibrium constant of the head-to-tail interaction as a function of ionic strength. Our results show that the affinity constant changes by almost 2 orders of magnitude over an ionic strength range of 50 mm (between I = 0.045 and 0.095). We were also able to calculate the average polymer length as a function of concentration and ionic strength, which is an important parameter in the interpretation of binding isotherms of tropomyosin with other thin filament proteins such as actin and troponin.

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.

Thermodynamic Recycling—On Ring-Opening Polymerization and Ring-Closing Depolymerization

Abstract The ring-opening polymerization of various cyclic monomers has been extensively investigated in the past, e.g., the cationic polymerization of tetrahydrofuran, the anionic polymerization of ∊-caprolactone, the metathesis polymerization of cycloalkenes, and the anionic or cationic polymerization of cyclohexamethyltrisiloxane and cyclooctamethyltetrasiloxane. In conjunction with ring–opening polymerization, monomer–polymer equilibrium (ceiling temperature) and/or the formation of a ring–chain equilibrium have also been studied. In the last few years we have studied extensively the anionic ring-opening polymerization and copolymerization of cyclic carbonates, the monomers mostly being provided by depolymerization of the polymers which were obtained by polycondensation polymerization. This fact has led to the development of a new polymer recycling concept by depolymerization and repolymerization, which we call thermodynamic recycling.

A lower proportion of filamentous to monomeric actin in the developing cerebellum of thyroid-deficient rats

Using a DNase I inhibition assay, in the presence or the absence of guanidine hydrochloride (which depolymerizes the actin filaments), developmental changes in total and filamentous actin were determined in the cerebellum of normal and hypothyroid rats. The total actin content per mg protein was not modified by hypothyroidism. As in normal animals, it reached a maximum around the age of 8 days and then decreased until adulthood. In contrast, the proportion of filamentous actin, which increased after the first postnatal week during normal development, was significantly reduced in the thyroid-deficient rats, only reaching normal values at 35 days. Thyroxine treatment for at least 4 days returned the filamentous actin content to normal at 14 days. The present study shows that the morphogenetic action of thyroid hormone is exerted not only on the microtubular apparatus, as previously described, but also in part through a control of actin monomer-polymer equilibrium.

Polymerization of vertebrate non-muscle and smooth muscle myosins

We investigated how light chain phosphorylation controls the stability of filaments of vertebrate non-muscle myosins (from bovine thymocytes and chicken intestine epithelial brush border cells) and smooth muscle myosin (from chicken gizzard) in vitro. Using a sedimentation assay, the solubilities of the myosins were determined by measuring the amounts of myosin monomers ( C m) and filaments ( C p) present under a given set of conditions as a function of the total myosin concentration ( C t). Below 200 m m-NaCl, each myosin displayed distinct “critical monomer concentrations” ( C c) for polymerization, which were dependent on the salt concentration, the state of light chain phosphorylation and the presence of MgATP. At 150 m m-NaCl, MgATP increased the C c of non-phosphorylated brush border myosin approximately five to tenfold, thymus myosin approximately 10 to 15-fold, and gizzard myosin approximately 25 to 50-fold. When these myosins were phosphorylated, MgATP had little effect on their solubilities, and their C c values remained low. Analytical ultracentrifugation and electron microscopy demonstrated that the myosins were present in three different conformational states under the conditions used in the sedimentation assays, i.e. filaments, extended monomer (6 S) and folded monomer (10 S). Since at equilibrium only filaments and monomers were observed, we suggest that the polymerization pathway for these myosins can be analysed in terms of a dynamic monomer-polymer equilibrium (polymer ⇔ 6 S monomer ⇔ 10 S monomer). At roughly physiological ionic strength, light chain dephosphorylation (in the presence of MgATP) promotes the folded state (10 S), whereas phosphorylation promotes the extended state (6 S), and thereby favours filament assembly. The relevance of the monomer-polymer equilibrium to the state of organization of the myosin in vivo is discussed.

Modulation of monomer-polymer equilibrium of phosphorylated smooth muscle myosin: effects on actin activation.

Actin activation of the adenosinetriphosphatase (ATPase) of phosphorylated gizzard myosin at low (2 mM) free Mg2+ concentration and 50 mM total ionic strength continues to increase on raising the free Ca2+ concentration near pCa 3. Similar levels of activity can be obtained by increasing the free Mg2+ concentration to a higher (in excess of 4 mM free) concentration. In the presence of micromolar concentrations of free Ca2+ and low free Mg2+ concentration, the actin-activated adenosine 5'-triphosphate (ATP) hydrolysis exhibits an initial rapid rate which progressively slows to a final, lower but more linear rate. In the presence of high divalent cation concentrations, the fast rate of ATP hydrolysis is maintained during the entire ATPase assay. The ionic conditions which favor the slow rate of ATP hydrolysis are correlated with increased proportions of folded myosin monomers while higher rates of ATP hydrolysis are correlated with increased levels of aggregated myosin. Elevating the thin filament proteins to saturating concentrations does not abolish the change in ATPase rate or the final distribution of myosin aggregates and monomers; however, the stability of the myosin aggregates is enhanced by the presence of thin filament proteins in low divalent cation conditions. The nonlinear profile of the actin-activated ATP hydrolysis in low divalent cation concentrations is eliminated by utilizing nonfilamentous, phosphorylated heavy meromyosin. The data presented indicate that Ca2+ and Mg2+ alter monomer-polymer equilibrium of stably phosphorylated myosin. The alteration of monomer-polymer equilibrium by Ca2+ at low Mg2+ concentration modulates ATPase rates.

Differential association of the different brain microtubule proteins in different in vitro assembly conditions

Microtubule protein was assembled in vitro for different time periods and at different protein concentrations, near and far from the critical concentration needed for assembly. When the isolated polymers were characterized by electrophoresis, a higher association of high-molecular-weight microtubule-associated proteins, particularly of the larger microtubule-associated proteins, was found in the polymers assembled close to the critical concentration, while tau factor polypeptides are predominantly present in those polymers assembled at higher protein concentration. Also, a higher proportion of high-molecular-weight microtubule-associated proteins MAP 1 and MAP 2 was observed in microtubules assembled for short time periods, compared with those obtained after the monomer-polymer equilibrium was reached. When the assembled protein was further characterized by isoelectric focusing, no differences were found in the proportion of the different isotubulins present in the polymers assembled under different conditions tested.

Monomer-polymer equilibria in the axon: direct measurement of tubulin and actin as polymer and monomer in axoplasm.

The monomer-polymer equilibria for tubulin and actin were analyzed for the cytoskeleton of the squid giant axon. Two methods were evaluated for measuring the concentrations of monomer, soluble (equilibrium) polymer, and stable polymer in extruded axoplasm. One method, the Kinetic Equilibration Paradigm ( KEP ), employs the basic principles of diffusion to distinguish freely diffusible monomer from proteins that are present in the form of polymer. The other method is pharmacological and employs either taxol or phalloidin to stabilize the microtubules and microfilaments, respectively. The results of the two methods agree and demonstrate that 22-36% of the tubulin and 41-47% of the actin are monomeric. The in vivo concentration of monomeric actin and tubulin were two to three times higher than the concentration required to polymerize these proteins in vitro, suggesting that assembly of these proteins is regulated by additional mechanisms in the axon. A significant fraction of the polymerized actin and tubulin in the axoplasm was stable microtubules and microfilaments, which suggests that the dissociation reaction is blocked at both ends of these polymers. These results are discussed in relationship to the axonal transport of the cytoskeleton and with regard to the ability of axons to change their shape in response to environmental stimuli.