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pH-sensitive unimolecular polymeric micelles: synthesis of a novel drug carrier.

Novel amphiphilic star-shaped polymers showing pH-sensitivity were synthesized by atom transfer radical polymerization. These new polymers present a core-shell structure similar to polymeric micelles, but are inherently stable to dilution and are referred to as unimolecular polymeric micelles. A four-armed multifunctional initiator was used for the sequential polymerization of hydrophobic ethyl methacrylate and tert-butyl methacrylate and hydrophilic poly(ethylene glycol)methacrylate. Polymers of molecular weight ranging from 9000 to 20,000 were obtained. Results of dynamic light scattering showed micelle size ranging from 11 to 40 nm. Unimolecular micelles were also analyzed by static light scattering in aqueous environment. Star-shaped polymers which presented the highest molar ratio of hydrophobic monomers tended to form high molecular weight aggregates in water. Hydrolysis of the tert-butyl methacrylate units permitted the introduction of ionizable methacrylic acid functions. Size distributions were bimodal at both acidic and basic pH. Since, the polymers were designed as potential delivery systems for the oral administration of hydrophobic drugs, they were titrated to evaluate the degree of ionization as a function of pH. In the stomach, the carboxylic functions are expected to be fully protonated. However, in the intestine, the micelles will be more than 40% ionized. Fluorescence studies were conducted in order to evaluate the polarity of the micellar core. Results showed an increase in polarity with pH due to the ionization of the acid functions present along the polymer chains. The pH rise was associated with an increase in the in vitro release rate of progesterone, which was used as hydrophobic drug model.

Drug Carriers↗
PubMed1995

Kinetic model for the inhibition of actin polymerization by actobindin.

Although Acanthamoeba actobindin binds actin monomers, its inhibition of actin polymerization differs from that of a simple monomer-sequestering protein in that actobindin inhibits nucleation very much more than elongation [Lambooy, P. K., & Korn, E. D. (1988) J. Biol. Chem. 263, 12836-12843] and can induce the accumulation of actin dimers in stoichiometric excess of the actobindin concentration [Bubb, M. R., Knutson, J. R., Porter, D. K., & Korn, E. D. (1994) J. Biol. Chem. 269, 25592-25597]. We now describe a "catalytic" model for the interaction of actobindin with actin monomer that quantitatively accounts for the effects of actobindin on the kinetics of actin polymerization de novo and the elongation of actin filaments. We propose that, in a polymerizing buffer, actobindin binds to two actin subunits forming an heterotrimeric complex that is incompetent for nucleation, self-association, and elongation. Actobindin can, however, dissociate from this complex, leaving a novel actin dimer that can participate in elongation but remains incompetent for nucleation and self-association. Under appropriate conditions, the concentration of this novel actin dimer can exceed the actobindin concentration; thus, the model is catalytic rather than stoichiometric. The experimentally observed time course of actin polymerization de novo, the rate of elongation of filaments, and the amount of actin dimer formed as a function of actobindin concentration are all consistent with the catalytic model and inconsistent with the stoichiometric model. The rate of actobindin-induced actin dimer formation is consistent with the hypothesis that the rate-limiting step is this pathway is the formation of a precursor heterotrimeric complex.

Acanthamoeba↗
PubMed2000

In vitro polymerization of tau protein monitored by laser light scattering: method and application to the study of FTDP-17 mutants.

Tau polymerization into the filaments that compose neurofibrillary tangles is seminal to the development of many neurodegenerative diseases. It is therefore important to understand the mechanisms involved in this process. However, a consensus method for monitoring tau polymerization in vitro has been lacking. Here we demonstrate that illuminating tau polymerization reactions with laser light and measuring the increased scattering at 90 degrees to the incident beam with a digital camera results in data that closely approximate the mass of tau polymer formation in vitro. The validity of the technique was demonstrated over a range of tau concentrations and through multiple angle scattering measurements. In addition, laser light scattering data closely correlated with quantitative electron microscopy measurements of the mass of tau filaments. Laser light scattering was then used to measure the efficiency with which the mutant tau proteins found in frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17) form filamentous structures. Several of these mutant proteins display enhanced polymerization in the presence of arachidonic acid, suggesting a direct role for these mutations in tau the filament formation that characterizes FTDP-17.

Chromosomes, Human, Pair 17↗
PubMed1982

Chemical modification of actin. Acceleration of polymerization and reduction of network formation by reaction with N-ethylmaleimide, (iodoacetamido)tetramethylrhodamine, or 7-chloro-4-nitro-2,1,3-benzoxadiazole.

We examined the properties of rabbit skeletal muscle actin labeled at Cys-373 with N-ethylmaleimide or with (iodoacetamido)tetramethylrhodamine, and of N-ethylmaleimide-actin further modified with 7-chloro-4-nitro-2,1,3-benzoxadiazole (which primarily labels Lys-372). All three derivatives polymerize more rapidly than unlabeled actin. As measured by fluorescence photobleaching recovery and low-shear viscometry, all three also show a lower extent of network formation relative to native actin. N-Ethylmaleimide has a much smaller effect on the rate of polymerization and on network formation than do the other two derivatives. We suggest that chemical modification of actin with these compounds may stabilize nuclei, accounting for the acceleration of polymerization. Stabilization of nuclei also reduces the average filament length at equilibrium, thereby reducing the extent of network formation. We note a parallel between these results and the effects that cytochalasin and capping proteins have on the polymerization of actin.

4-Chloro-7-nitrobenzofurazan↗
PubMed1984

Bound-cation exchange affects the lag phase in actin polymerization.

The delay or lag phase at the onset of polymerization of actin by neutral salt is generally attributed to an actin nucleation reaction. However, when nucleation is circumvented by the use of phalloidin-stabilized nuclei, a lag phase persists when Ca2+-containing actin is polymerized with MgCl2. Pretreatment of actin with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and/or Mg2+ shortens or eliminates this lag phase, suggesting that exchange of the actin-bound divalent cation occurs during this nucleation-independent lag phase. Measurement of the actin-bound cation initially and after brief incubation with EGTA/Mg2+ directly verifies that Mg2+ has replaced Ca2+ as the actin-bound cation, producing a highly polymerizable Mg2+-actin species. Bound-cation exchange prolongs the lag phase in actin polymerization and probably explains what has been termed the monomer activation step in actin polymerization.

Actins↗
PubMed1984

Complement protein C9 labeled with fluorescein isothiocyanate can be used to monitor C9 polymerization and formation of the cytolytic membrane lesion.

Human complement protein C9 was covalently labeled with the fluorescent chromophore fluorescein isothiocyanate (FITC) with only a small reduction in the cytolytic activity of the protein. Polymerization of the labeled protein--either by incubating with lipid vesicles treated with complement proteins C5b-8 (activating the C5b-9 membrane lesion) or by heating the protein [Tschopp, J., Muller-Eberhard, H.J., & Podack, E.R. (1982) Nature (London) 298, 534]--resulted in a 40-60% decrease in the fluorescence emission from FITC. The decrease in total fluorescence was accompanied by an increase in the steady-state anisotropy following activation and polymerization of FITC-C9 by C5b-8 membranes, while heat-induced aggregation of the protein resulted in a dramatic depolarization of fluorescence. Only small changes in either the absorbance spectrum or fluorescence lifetime of the chromophore were detected upon FITC-C9 polymerization. Evidence is presented that the measured changes in FITC fluorescence upon C9 activation are due to self energy transfer between closely apposed fluorescein chromophores which occur in the polymerized form of the protein. The significance of these observations to the molecular structure of the assembled C5b-9 complex is discussed, as are the potential applications of this fluorescent derivative of C9.

Animals↗
PubMed1986

Localization of segments essential for polymerization and for calcium binding in the gamma-chain of human fibrinogen.

We have isolated an intermediate plasmic degradation product, D2, of fibrinogen that does not inhibit the polymerization of fibrin monomer but does bind Ca2+. Fibrinogen was digested to a limited extent with plasmin in the presence of Ca2+, and a "large" fragment D (fragment D1A) was isolated with a gamma-chain remnant consisting of residues 63-411. Fragment D1A was digested further in the presence of Ca2+, yielding fragment D1 (with its gamma-chain containing residues 86-411). The digestion of fragment D1 [in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) to complex Ca2+] led to a gradual shortening of the carboxyl-terminal portion of the gamma-chain. Fragment D2 (with its gamma-chain containing residues 86-335/356) was isolated from an intermediate digest in the presence of EGTA. The Lys-338-Cys-339 peptide bond of the gamma-chain is intact in this preparation of D2, even though it is split in the isolated peptide gamma303-355 (with an intact disulfide bond at Cys-326-Cys-339). Fragment D2 does not interfere with the polymerization of fibrin monomer, whereas fragment D1 is a potent inhibitor of this polymerization. We conclude that the gamma-chain segment 356/357-411, present in fragment D1 but absent from fragment D2, is essential for maintenance of a polymerization site located in the outer (D) nodule of fibrinogen. This segment (356/357-411) is longer than two shorter ones reported earlier [Olexa, S.A., & Budzynski, A. Z. (1981) J. Biol. Chem. 256, 3544-3549; Horwitz, B.H., Váradi, A., & Scheraga, H.A. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5980-5984]; the data for the earlier reports are reinterpreted here. Finally, fragment D2 possesses a single Ca2+ binding site, as revealed by equilibrium dialysis binding studies. Since fragment D3 (with its gamma-chain containing residues 86-302) fails to bind Ca2+, we conclude that segment gamma 303-355/356 plays a crucial role in Ca2+ binding.

Amino Acids↗
PubMed1988

Effect of pH on the mechanism of actin polymerization.

The effect of pH on the Mg2+-induced polymerization of rabbit skeletal muscle G-actin at 20 degrees C was examined. Polymerization data were obtained at various initial concentrations of Mg2+, Ca2+, and G-actin between pH 6 and 7.5. The data were found to fit a kinetic mechanism for actin polymerization previously proposed at pH 8 in which Mg2+ binding at a moderate-affinity site on actin induces an isomerization of the protein enabling more favorable nucleation [Frieden, C. (1982) J. Biol. Chem. 257, 2882-2886]. The data also suggest the formation of actin dimers induced by Mg2+ binding is over 2 orders of magnitude more favorable at pH 6 than at pH 8. Little effect on trimer formation is found over this pH range. In addition, the conformation induced by nonspecific binding of metal to low-affinity sites becomes more favorable as the pH is lowered. The critical concentration for filament formation is also decreased at lower pH. The kinetic data do not support fragmentation occurring under any of the conditions examined. Furthermore, as Mg2+ exchange for Ca2+ at a high-affinity site (Kd less than 10(-9) M) fails to alter significantly the polymerization kinetics, Ca2+ release from this site appears unnecessary for either the nucleation or the elongation of actin filaments.

Actins↗
PubMed1990

Immunochemical probing of the N-terminal segment on actin: the polymerization reaction.

The N-terminal segment of actin contains a cluster of acidic residues which are implicated in macromolecular interactions of this protein. In this work, the interrelationship between the N-terminal segment and the polymerization of actin was studied by using affinity-purified antibodies directed against the first seven N-terminal residues on alpha-skeletal actin (S alpha N). The Fab fragments of these antibodies showed equal affinities for G- and F-actin while the bivalent IgG bound preferentially to the polymerized actin. As monitored by pyrene fluorescence measurements, the binding of Fab to G-actin did not alter the kinetics of the MgCl2-induced polymerization; IgG accelerated this reaction considerably. Consistent with these observations, the binding of Fab to F-actin did not change its morphological appearance in electron micrographs and had no effect on the stability and the rate of dissociation of actin filaments. These results are discussed in terms of their implications to the spatial relationship between the N-terminal segment and the rest of the molecule and the context of the polymerization reaction of actin in vitro and in vivo.

Actins↗
PubMed1982

Polymerization of the tubulin-colchicine complex and guanosine 5'-triphosphate hydrolysis.

The tubulin-colchicine (1:1) complex was shown to be able to polymerize in vitro under the buffer conditions of microtubule assembly from pure native tubulin. The physical characteristics of this peculiar polymer have been investigated under a variety of conditions and compared with those of microtubules. Polymerization consisted of the nucleation followed by the growth process, was characterized by a critical concentration, and exhibited divalent ion, temperature, and pH dependences very similar to those of microtubules, Guanosine 5'-triphosphate (GTP) or 5' -guanylyl methylene-diphosphate (GMPPCP) was required for polymerization, and guanosine 5'-diphosphate (GDP) was a potent inhibitor. GTP hydrolysis was totally disconnected from the polymerization process and occurred as well under nonpolymerizing conditions. The results are discussed in view of the different types of protein-protein interactions exhibited by tubulin and of the possible relationship between the conformation of the GTP site and the interaction areas.

Animals↗
PubMed1980

Mechanism of action of polymeric aurintricarboxylic acid, a potent inhibitor of protein--nucleic acid interactions.

The mechanism of inhibition of protein--nucleic acid complex formation by polymeric aurintricarboxylic acid (ATA) was investigated by proton magnetic resonance spectroscopy. The approach was the synthesis of totally deuterated ATA, followed by a 100-MHz proton magnetic resonance study of its interaction with bovine pancreatic ribonuclease A (RNase), a model nucleic acid binding protein. The binding of ATA to RNase elicited chemical shift changes and line broadening in the C(2)--H resonances of histidyl residues 12 and 119, both of which are located in the active site, whereas that of histidyl residue 105, which resides on the exterior of the protein structure, is unaffected. (Histidyl residue 48 is not observed under our conditions except at high pH.) The epsilon-methylene protons of the lysyl side chains were also broadened upon the binding of ATA. Polymeric ATA displaces cytidine 2'-monophosphate and cytidine 3'-monophosphate from the active site of the enzyme as revealed by nuclear magnetic resonance spectroscopy. These observations suggest that the mechanism of action of ATA involves competition between the nucleic acid and the polymeric ATA for binding in the active site of the protein. Electron spin resonance spectroscopy reveals that polymeric ATA is a stable free radical, thus accounting for the major line broadening effect upon binding to protein. This finding may provide a powerful means of probing the nucleic acid binding site of proteins by proton magnetic resonance spectroscopy.

Animals↗
PubMed2002

Structural requirements for the intracellular subunit polymerization of the complement inhibitor C4b-binding protein.

C4b-binding protein (C4BP), an important inhibitor of complement activation, has a unique spider-like shape. It is composed of six to seven identical alpha-chains with or without a single beta-chain, the chains being linked by disulfide bridges in their C-terminal parts. To elucidate the structural requirements for the assembly of the alpha-chains, recombinant C4BP was expressed in HEK 293 cells. The expressed C4BP was found to contain six disulfide-linked alpha-chains. Pulse-chase analysis demonstrated that the recombinant C4BP was rapidly synthesized in the cells and the polymerized C4BP appeared in the medium after 40 min. The alpha-chains were polymerized in the endoplasmic reticulum (ER) already after 5 min chase. The polymerization process was unaffected by blockage of the transport from the ER to the Golgi mediated by brefeldin A or low temperature (10 degrees C). The C-terminal part of the alpha-chain (57 amino acids), containing 2 cysteine residues and an amphiphatic alpha-helix region, was required for the polymerization. We constructed and expressed several mutants of C4BP that lacked the cysteine residues and/or were truncated at various positions in the C-terminal region. Gel filtration analysis of these variants demonstrated the whole alpha-helix region to be required for the formation of stable polymers of C4BP, which were further stabilized by the formation of disulfide bonds.

Base Sequence↗
PubMed2003

Facile polymerization of dNTPs bearing unnatural base analogues by DNA polymerase alpha and Klenow fragment (DNA polymerase I).

The high fidelity of DNA replication is largely dependent upon accurate incorporation of dNTPs by DNA polymerases. To study the mechanism underlying nucleotide selection, we synthesized four nucleotide analogues bearing the unnatural bases benzimidazole, 5-nitrobenzimidazole, 6-nitrobenzimidazole, and 5-nitroindole and analyzed their incorporation by three DNA polymerases. We have found that human DNA polymerase alpha (pol alpha) and the Klenow fragment of Escherichia coli DNA polymerase I (KF) incorporate all four nucleotide analogues opposite all four canonical bases up to 4000-fold more efficiently than an incorrect natural dNTP (i.e., rates that approach those of a correct, natural dNTP), even though the shape of any base pair formed between the analogue and the template likely does not resemble a normal base pair. While pol alpha preferentially incorporated the analogues opposite template pyrimidines, KF surprisingly preferred to polymerize them opposite template purines. Although neither pol alpha nor KF readily polymerized a natural dNTP opposite either 5- or 6-nitrobenzimidazole in the template strand, the enzymes did incorporate the analogues to generate novel base pairs. Both pol alpha and KF polymerized the analogues up to 140-fold more efficiently than dATP both across from abasic sites and as 3'-overhangs on blunt-ended templates. Although Maloney murine leukemia virus reverse transcriptase did not measurably incorporate the analogues, this enzyme bound the analogues with K(I)'s only slightly higher than the K(m) for polymerization of the normal dNTP. The implications of these results with respect to how polymerases discriminate between correct and incorrect dNTPs are discussed.

Base Pairing↗
PubMed2005

GTP analogue inhibits polymerization and GTPase activity of the bacterial protein FtsZ without affecting its eukaryotic homologue tubulin.

The prokaryotic tubulin homologue FtsZ plays a key role in bacterial cell division. Selective inhibitors of the GTP-dependent polymerization of FtsZ are expected to result in a new class of antibacterial agents. One of the challenges is to identify compounds which do not affect the function of tubulin and various other GTPases in eukaryotic cells. We have designed a novel inhibitor of FtsZ polymerization based on the structure of the natural substrate GTP. The inhibitory activity of 8-bromoguanosine 5'-triphosphate (BrGTP) was characterized by a coupled assay, which allows simultaneous detection of the extent of polymerization (via light scattering) and GTPase activity (via release of inorganic phosphate). We found that BrGTP acts as a competitive inhibitor of both FtsZ polymerization and GTPase activity with a Ki for GTPase activity of 31.8 +/- 4.1 microM. The observation that BrGTP seems not to inhibit tubulin assembly suggests a structural difference of the GTP-binding pockets of FtsZ and tubulin.

Anti-Bacterial Agents↗
PubMed1996

Polymerization and in vitro motility properties of yeast actin: a comparison with rabbit skeletal alpha-actin.

Actin purified from the yeast (Saccharomyces cerevisae) was polymerized faster than rabbit skeletal alpha-actin by MgCl2. The two actins polymerized at similar rates in the presence of CaCl2. Yeast actin, up to 25 microM, was not polymerized by KCl (100-300 mM); the monovalent salt also inhibited the MgCl2-induced polymerization of actin. The local structure of the subdomain-2 region in yeast actin filaments was probed by subtilisin and trypsin digestions. Loop 38-52 appeared more flexible and accessible to subtilisin in yeast than in rabbit actin. In contrast, tryptic digestions at Lys-61 and -68 occurred at the same rate for yeast and alpha-actin filaments. Modification of yeast actin by a sulfhydryl reagent CPM [7-(diethylamino)-3-(4'-maleimidophenyl)-4-methylcoumain] was specific to the Cys-374 residue; no labeling of a yeast actin mutant containing an alanine substitution for cysteine 374 was observed. The rates of Cys-374 labeling by CPM were similar for yeast and muscle actin, suggesting a similar environment for the C terminus in both polymers. In the in vitro motility assays, yeast actin required higher concentrations of heavy meromyosin (HMM) for its sliding than did the rabbit actin. At saturating concentrations of HMM, the sliding velocities of both actins were the same (3.0 microns/s). Relative forces generated by HMM with yeast and muscle actin were assessed by monitoring their in vitro motility in the presence of NEM-HMM load. The sliding of yeast actin was stopped at a level of external load (molar ratio NEM-HMM/HMM = 0.25) lower than that of muscle actin (NEM-HMM/HMM = 0.43), suggesting lower force production with yeast actin. These results are discussed in terms of the myosin cross-bridge cycle and actomyosin interactions.

Actins↗
PubMed1998

The role of profilin in actin polymerization and nucleotide exchange.

Properties of human profilin I mutated in the major actin-binding site were studied and compared with wild-type profilin using beta/gamma-actin as interaction partner. The mutants ranged in affinity, from those that only weakly affected polymerization of actin to one that bound actin more strongly than wild-type profilin. With profilins, whose sequestering activity was low, the concentration of free actin monomers observed at steady-state of polymerization [Afree], was close to that seen with actin alone ([Acc], critical concentration of polymerization). Profilin mutants binding actin with an intermediate affinity like wild-type profilin caused a lowering of [Afree] as compared to [Acc], indicating that actin monomers and profilin:actin complexes participate in polymer formation. With a mutant profilin, which bound actin more strongly than the wild-type protein, an efficient sequestration of actin was observed, and in this case, the [Afree] at steady state was again close to [Acc], suggesting that the mutant profilin:actin had a greatly lowered ability to incorporate actin subunits at the (+)-end. The results from the kinetic and steady-state experiments presented are consonant with the idea that profilin:actin complexes are directly incorporated at the (+)-end of actively polymerizing actin filaments, while they do not support the view that profilin facilitates polymer formation.

Actins↗
PubMed1998

Kinetic studies on the effect of yeast cofilin on yeast actin polymerization.

The effect of yeast cofilin on the kinetics of polymerization of yeast actin has been examined at 20 degrees C at both pH 8.0 and 6.6. In the absence of cofilin, the kinetic data may be described by a simple nucleation-elongation mechanism. Kinetic data in the presence of cofilin suggests a complex dependence on the cofilin concentration. At low cofilin-to-actin ratios, cofilin increases the rate of polymerization in a way best fit by assuming filament fragmentation. The apparent fragmentation rate constants increase with increasing cofilin concentration leveling off above a cofilin-to-actin ratio of 1:8 and are independent of pH. At higher cofilin-to-actin ratios, a nonpolymerizable cofilin-G-actin complex forms resulting in a decreased rate of polymerization. The data from fluorescence photobleaching recovery experiments at low cofilin-to-actin ratios are consistent with the presence of severed filaments at both pH 8 and 6.6. However, at pH 8 and a cofilin-to-actin ratio of 1:16, about 40-50% of the total actin is present as G-actin after polymerization while at pH 6.6 little or no G-actin is present at the same cofilin-to-actin ratio. The results suggest some cooperativity with respect to cofilin binding to filamentous actin which may be pH dependent.

Actin Depolymerizing Factors↗
PubMed1998

Analysis of A alpha 251 fibrinogen: the alpha C domain has a role in polymerization, albeit more subtle than anticipated from the analogous proteolytic fragment X.

Numerous experiments have demonstrated that the C-terminal domain of the fibrinogen Aalpha-chain, the alphaC domain, has a role in polymerization. To further examine the role of this domain, we synthesized a recombinant fibrinogen, Aalpha251 fibrinogen, that lacks the alphaC domain. We examined thrombin-catalyzed fibrinopeptide release and found that the rate of FpB release from Aalpha251 fibrinogen was 2.5-fold slower than FpB release from normal fibrinogen, while the rate of FpA release was the same for both proteins. We examined thrombin-catalyzed polymerization and found that the rates of protofibril formation and lateral aggregation were similar for both proteins, although discernible differences in lateral aggregation were clear. The rate of protofibril formation for Aalpha251 fibrinogen was never less than 85% of normal fibrinogen, while the rate of lateral aggregation for Aalpha251 fibrinogen varied from 64 to 74% of normal. We examined polymerization of fibrin monomers and found that polymerization of Aalpha251 fibrin was similar to normal fibrin at 0.4 M NaCl, but clearly different from normal at 0.05 M NaCl. These results indicate that the alphaC domain has a role in lateral aggregation, but this role is more subtle than anticipated from previous experiments, particularly those with fibrinogen fragment X. We interpret this unanticipated finding as indicative of an important contribution from the N-terminus of the beta-chain, such that protein heterogeneity that includes small amounts of fibrin lacking that N-terminus of the beta-chain leads to markedly altered lateral aggregation.

Animals↗