Ethyl Ester Moiety Research Articles

Restoration of enzymatic activity of energy related proteins in traumatic brain injured rats following the administration of gamma‐glutamylcysteine ethyl ester

The brain serves as the control center for the body. Its function is primarily dependent upon sufficient supply of glucose for energy metabolism. The glycolytic pathway and Kreb's cycle are important in producing ATP for the brain. Brain dysfunction resulting from an external force is known as traumatic brain injury (TBI). Symptoms range from physical to psychological and effects can be mild, moderate, or severe depending on the extent of the injury. TBI is associated with oxidative damage resulting in reduced energy metabolism. Antioxidants are substances responsible for the inhibition of oxidation. Gamma‐glutamylcysteine ethyl ester (GCEE) is an ethyl ester moiety of gamma‐glutamylcysteine that exhibits antioxidant activity by increasing glutathione production. This study investigates the enzymatic activity of two energy related enzymes, malate dehydrogenase and glyceraldehyde‐3‐phosphate dehydrogenase that have been identified as being nitrated in moderate TBI. To test the hypothesis that the administration of GCEE will normalize enzymatic activity post‐TBI, adult male Wistar rats were divided equally into three groups: Sham, Saline, and GCEE treatment (150 mg/kg). Upon sacrifice, enzymatic activity was indirectly measured spectrophotometrically by assessing the absorbance of reduced β‐nicotinamide adenine dinucleotide at 340nm. Preliminary data demonstrates that the administration of GCEE post‐TBI robustly increases enzymatic activity bolstering the hypothesis. These results are promising and indicate potential therapeutic strategies to restore enzymatic activity in the brain post‐TBI.

Synthesis of Thiazole Derivatives and Evaluation of Their Antiamoebic Activity and Cytotoxicity

Five ethyl (5-alkyl-2-amino-1,3-thiazol-4-yl) acetates (designated compounds 4a-e) incorporating octyl, decyl, dodecyl, tetradecyl, and hexadecyl alkyl chains, respectively, were prepared by reacting 4-alkyl-4-bromo-3-oxobutyric acid ethyl esters (3a-e) with thiourea in dried acetonitrile. Compounds 3a-e were synthesized by reacting alkylated ethyl acetoacetates with bromine. The newly synthesized compounds were characterized by mass spectrometry, NMR, and elemental analysis. Compounds 4a-c demonstrated good in vitro antiamoebic activity against Acanthamoeba polyphaga exposed to 10 mg L(-1) for 6 h at 28 °C. Compound 4b showed the highest antiamoebic activity among the tested compounds, comparable to that of chlorhexidine dihydrochloride (CHX), decreasing the number of viable cells to below the detection limit of 1 cell mL(-1). The activity of compounds 4a and 4c was similar to that of the commercial antifungal agent fluconazole (Flu). The cytotoxic and hemolytic activity of the compounds was assayed against human neonate dermal fibroblasts and sheep erythrocytes, respectively. Compounds 4a-c were less cytotoxic than Flu and CHX. Our results suggest that compound 4b, which is composed of a 2-amino-thiazole attached to a decyl group and an ethyl ester moiety, is a particularly safe and effective alternative amoebicidal agent.

Synthesis of trans/cis‐3,4‐Disubstituted 1,2,3,4‐Tetrahydroisoquinolines by Nucleophilic Acyl Substitution of Homophthalic Anhydride with Aldimines

The reaction of the functionalized N‐protected imines 3 and 7 containing either the chloromethyl or ethyl ester moieties, respectively, attached to the imine carbon with homophthalic anhydride 2 afforded cis/tran‐3‐subsituted‐1‐oxo‐1,2,3,4‐tetrahydroisoquinoline‐4‐caboxylic acids appropriate for further synthetic elaboration.

Cyclometalated cinchophen ligands on iridium(iii): towards water-soluble complexes with visible luminescence

Eight cationic heteroleptic iridium(III) complexes, [Ir(epqc)2(N^N)](+), were prepared in high yield from a cyclometalated iridium bridged-chloride dimer bearing two ethyl-2-phenylquinoline-4-carboxylate (epqc) ligands. Two X-ray crystallographic studies were undertaken on selected complexes (where the ancillary ligand N^N = 4,4'-dimethyl-2,2'-bipyridine and 4,7-diphenyl-1,10-phenanthroline) each confirming the proposed formulations, showing an octahedral coordination at Ir(III). In general, the complexes are luminescent (620-630 nm) with moderately long lifetimes indicative of phosphorescence. Hydrolysis of the ethyl ester moieties of the epqc ligands gave the analogous cinchophen-based complexes, which were water-soluble and visibly luminescent (568-631 nm). The spectroscopic and redox characterisation of the complexes was complemented by DFT and TD-DFT calculations, supporting the assignment of dominant (3)MLCT to the emissive character.

Synthesis, labelling and evaluation of hydantoin‐substituted indole carboxylic acids as potential ligands for positron emission tomography imaging of the glycine binding site of the N‐methyl‐d‐aspartate receptor

The N‐methyl‐ d‐aspartate (NMDA) receptor as a type of ionotropic glutamatergic receptors is essential for physiological processes such as learning, memory and synaptic plasticity. A glutamate‐induced overactivation of these receptors, accompanied by increased intracellular calcium concentration, causes cell injury and leads to a large number of acute or chronic neurological disorders, such as stroke, trauma, Parkinson's disease and Alzheimer's disease. In an attempt to visualise the glutamatergic neurotransmission in vivo with positron emission tomography, novel fluoroethoxy‐ and methoxy‐substituted reference compounds based on the lead structure of a hydantoin‐substituted indole‐2‐carboxylic acid were synthesised. The affinities towards the glycine binding site of the NMDA receptor showed Ki values between 322 and 11 nM and the lipophilicities ranged from logD values of 1.51 to 2.53. On the basis of these results, precursor compounds were synthesised containing a phenolic hydroxy moiety to obtain the radiolabelled ligands through an alkylation reaction. Radiosynthesis was achieved by labelling the precursor ethyl 4,6‐dichloro‐3‐((3‐(4‐hydroxyphenyl)‐2,4‐dioxoimidazolidin‐1‐yl)methyl)‐indole‐2‐carboxylate with 2‐[18F]fluoroethyl tosylate or [11C]methyl iodide and subsequent cleavage of the ethyl ester moiety. This gave the final products in overall decay‐corrected radiochemical yields of 5–7% and 6–9% and specific activities of 24–67 GBq/µmol and 8–26 GBq/µmol, respectively.

Synthesis of Novel Photochromic Calix[4]arene Ligand Bearing a Spirobenzopyran and Three Ethyl-Ester Moieties and Photocontrol of Its Alkali Metal Ion Extractability

Abstract A novel photochromic calix[4]arene ligand bearing a spirobenzopyran and three ethyl-ester moieties (compound 1) was synthesized, and photocontrol of its alkali metal ion extractability was studied. In the dark, compound 1 showed the largest changes of absorption spectra for Na+ among alkali metal ions. When Na+ was gradually added to the solution of compound 1, its complexation behavior was evaluated by 1H NMR, which suggests that Na+ was taken into the cavity formed by four carbonyl oxygens and four ether oxygens, and that compound 1 formed a 1:1 complex with Na+. The proton signals originating from the spirobenzopyran moiety were slightly changed by metal-ion complexation. This means that the spirobenzopyran moiety was not very close to Na+ under dark conditions. Liquid–liquid extraction experiments with alkali metal ion using compound 1 made it clear that the extractability of Na+ was enhanced under UV-light irradiation, compared with that under visible-light irradiation. This suggests that the electrostatic interaction between a phenolate anion of the merocyanine moiety generated by UV-light irradiation and Na+ promotes the extractability to some extent.

Photopolymerization and Formation of a Stable Purple Langmuir−Blodgett Film Based on the Gemini-Type Amphiphilic Diacetylene Derivatives

In this paper, we designed two gemini-type amphiphiles containing diacetylene units which were covalently linked through L-lysine or L-lysine ethyl ester moiety (abbreviated as TCDA-LysAc or TCDA-LysEs) as the spacer. These amphiphiles can be easily fabricated into organized molecular films though the Langmuir-Blodgett technique and the films showed an excellent photopolymerization ability. Upon photopolymerization, TCDA-LysEs formed a purple LB film with an excellent stability, while TCDA-LysAc formed the film with mixed blue, purple, and red phases. Aligned nanofibers were observed for the TCDA-LysEs film, while a mixed domain with short fiber and rotted wood structure was obtained for TCDA-LysAc. Different supramolecular chirality was obtained for the transferred LB films although both the compounds have spacers with similar chirality. It was suggested that the subtle change in the spacer structure led to different noncovalent interactions, which subsequently affected the polymerization. Through such a molecular design, we have successfully obtained the stable purple poly(diacetylene) film.

Porphyrin on a Half-Shell! Synthesis and Characterization of Corannulenoporphyrins

An efficient synthetic method for preparing corannulenoporphyrins is described. Nitrocorannulene was reacted with ethyl isocyanoacetate in the presence of a phosphazene base to generate the pivotal corannulenopyrrole intermediate. Following cleavage of the ethyl ester moiety with KOH in ethylene glycol at 180-190 degrees C, the heptacyclic system was reacted with acetoxymethylpyrroles under mildly acidic conditions to afford tripyrranes. The proton NMR spectra for these tripyrrolic intermediates suggest that they can take on helical geometries, but the conformations are dependent on the nature of the terminal ester groupings and may be altered by solvent interactions. Treatment of a di-tert-butyl ester tripyrrane with TFA cleaved the protective groups, and subsequent condensation with diformylpyrroles in TFA-CH(2)Cl(2), followed by oxidation with DDQ or ferric chloride, gave excellent yields of corannulenoporphyrins. Nickel(II), copper(II), and zinc complexes of this system were also prepared, and the nickel derivative was also further characterized by X-ray crystallography. The same synthetic strategy was also used to prepare a porphyrin with fused acenaphthylene and corannulene subunits. The fused bowl-shaped corannulene provides these porphyrins with a unique structural component that increases solubility by reducing pi-pi stacking interactions.

Proteomic identification of nitrated brain proteins in traumatic brain‐injured rats treated postinjury with gamma‐glutamylcysteine ethyl ester: Insights into the role of elevation of glutathione as a potential therapeutic strategy for traumatic brain injury

Traumatic brain injury (TBI) occurs suddenly and has damaging effects to the brain that are dependent on the severity of insult. Symptoms can be mild, moderate, or severe. Oxidative damage is associated with traumatic brain injury through reactive oxygen/nitrogen species production. One such species, peroxynitrite, is elevated in TBI brain tissue (Orihara et al. [2001] Forensic Sci. Int. 123:142-149; Deng et al. [2007] Exp. Neurol. 205:154-165). Peroxynitrite can react with carbon dioxide and decompose to produce NO(2) and carbonate radicals, which in turn can lead to 3-nitrotyrosine, an index of protein nitration. Gamma-glutamylcysteine ethyl ester (GCEE) is an ethyl ester moiety of gamma-glutamylcysteine, an agent that up-regulates glutathione (GSH) production in brain (Drake et al. [2002] J. Neurosci. Res. 68:776-784). Many preclinical studies of TBI have employed pretreatment of animals with proposed beneficial agents prior to the injury itself. However, in the real world of TBI, treatment begins postinjury. Hence, insights into agents that improve outcome following injury are desperately needed. This study is one of the first to investigate a potential GSH-based therapy for TBI postinjury. Protein carbonyls, an index of protein oxidation, were significantly elevated in brain of animals subjected to TBI. However, if, after TBI, GCEE was administered i.p., protein carbonyl levels were significantly reduced. Similarly, 3-nitrotyrosine levels were elevated in brain following TBI but significantly decreased following TBI if GCEE was administered i.p. Redox proteomics analysis showed that several brain proteins were nitrated after TBI. However, if GCEE was given i.p. following TBI, many of these proteins were protected from nitration. The results are encouraging and are discussed with reference to potential therapeutic strategies for TBI involving elevated GSH.

Kinetics and thermodynamics of complex formation with iron of a new series of dicatecholspermidine siderophore-like ligands

This article deals with the kinetics and thermodynamics of complex formation between Fe3+ and a series of four synthetic chelators of the 1,2-dicatecholspermidine family (LA5, LA3, LE5 and LE3). LA5 and LA3 bear a carboxylic moiety linked to the central nitrogen by either a C5 or a C3 chain, whereas LE5 and LE3 bear an ethyl ester moiety. The following data concern LE5, LE3, LA5 and LA3, respectively. Each species undergoes four acid-base dissociations of the hydroxyls of the catechols with, for the two hydroxyls in position 1; average pK2a=7.30, 7.25, 7.45, 7.34 and, for the two hydroxyl in position 2; average pK3a=12.35, 12.65, 12.10, 12.60. The LA5 and LA3 species also undergo proton-dissociations of their carboxylic moieties; pK1a=5.20 and 5.10. The four species form one-to-one iron complexes with, for the 1-hydroxyl; an average pK22a=2.65, 2.25, 2.95, 2.80, for the 2-hydroxyl; pK33a=5.20, 5.40, 6.10, 5.40 and, for the carboxylic moieties; pK11a=3.90 and 4.45. In the vicinity of pH 5, Fe3+ is rapidly exchanged between FeNta and the four ligands. This occurs with direct rate constants: k1=(1.3±0.1)×104, (1.4±0.2)×104, (3.3±0.2)×104, (1.4±0.1)×104M−1s−1, and reverse rate constants: k−1=(7±0.5)×104, (9±1)×104, (1.15±0.15)×105, (7±0.5)×104M−1s−1. The kinetic data, the pKa values of the free ligands, those of the iron complexes and the β value of FeNta allow us to determine the affinity constants of the four ligands for iron: logβ1=33, 34, 33, 34, and pFe=23.3, 24.6, 22.2, 24.3. This implies that these ligands of the dicatecholspermidine family may act as siderophores. They may also be used as drug carriers which can utilize the bacterial iron-acquisition paths.

Synthesis and cytotoxic evaluation of new (4,5,6,7-tetrahydro-indol-1-yl)-3- R-propionic acids and propionic acid ethyl esters generated by molecular mimicry

Indolones 4 and 5, and indolyl-aminoacids 6a– e, 7a– e, and 8a and 8b were designed by structural modification of lead compound 3. These compounds were tested on six tumor cell lines to determine the role of the azepinone ring and the N-phenyl substituent in the cytotoxicity of 3. Our results show that 4 and 5 have dramatically reduced cytotoxicity, due to the loss of the azepinone moiety of lead compound 3. In contrast, indolyl-aminoacids 6a, 7a, and 8a ( N-( l)-cysteine ethyl ester derivatives) inhibited the proliferation of almost all cancer cell lines tested, even though they lack the azepinone ring. In addition, derivative 6c ( N-( d)-alanine methyl ester group) was selectively cytotoxic to HCT-15 cells. Preliminary structure–activity relationship (SAR) studies with these compounds revealed the importance of the ethyl ester moiety on the amino acid moiety. Compounds 6a– e, 7a– e, and 8a and 8b were obtained in good yields by a catalytic Paal–Knorr reaction carried out under microwave irradiation using commercially available chiral amino esters or amino acids and 1,4-dicarbonyl compounds.

Novel Functionally Grafted Pseudo-Semi-interpenetrating Networks Constructed by Reactive Linear−Dendritic Copolymers1

This paper describes the synthesis of amphiphilic pseudo-semi-interpenetrating polymer networks (pseudo-semi-IPNs) containing linear poly(styrene) and poly(ethylene glycol) (PEG) cross-linked through monodendritic fragments. A unique feature of the synthetic strategy is the permanent attachment of the linear segment to the PEG network by a transesterification reaction between the hydroxyl groups at both ends of the PEG and peripheral ethyl ester moieties in the monodendron portion of a linear poly(styrene)-dendritic poly(benzyl ether) AB block copolymer. The proceeding of the reaction is monitored by (1)H NMR and size exclusion chromatography (SEC). The formation of an interlock structure between the linear block and the network matrix in the pseudo-semi-IPN is evidenced by the results from spectroscopic analyses and differential scanning calorimetry measurements. The accessibility of functional centers in the grafted semi-IPN is confirmed by model reactions with fluorescent markers, fluorescence spectroscopy, and NMR techniques and shows the potential of these novel materials as sequestering reagents for resin capture-release applications in parallel synthesis, combinatorial chemistry, and advanced drug design.

Complexation Dynamics of Xanthone and Thioxanthone to β-Cyclodextrin Derivatives

The complexation dynamics of xanthone to substituted β-cyclodextrins (CDs) and of thioxanthone to β-CD was studied by directly following the relocation of the excited triplet-state ketones from the CD cavity to the aqueous phase. Equilibrium constants for the ground-state complexation of both ketones with CDs were determined from fluorescence studies. The complexation efficiency and dynamics of thioxanthone with β-CD were the same as previously observed for the xanthone/β-CD system. Substitution of some of the hydrogens on the alcohol groups by methyl moieties at both entrances to the β-CD cavity increased the equilibrium constant for ground-state xanthone, but decreased that for the excited triplet state, leading to a larger difference for the binding behavior of these two electronic states of the guest. Addition of one pendant arm containing a carboxyl moiety to the rim where xanthone is bound does not change the binding efficiency of the ground state or the binding dynamics of triplet xanthone. In contrast, when this arm contains an ethyl ester moiety, the binding efficiencies of both the ground and excited triplet states are increased. The higher binding constant for triplet xanthone is due to an increase of the association rate constant and a decrease for the dissociation rate constant.

Forward recoil spectrometry study of the diffusion of PMDA/ODA-based poly(amic ethyl esters)

The poly(amic ethyl esters) (PAEs) of pyromellitic dianhydride/4,4′-oxydianiline (PMDA/ODA) were prepared in three isomeric forms, with meta-linkages, para-linkages and randomly alternating meta- and para-linkages (referred to as mixed PAEs). In addition to the normal PAEs (which contained only hydrogen and which were cast as base layers for diffusion studies), each of these isomeric PAE was synthesized in two other forms: (1) labelled with deuterium on the ODA segments, and (2) labelled with deuterium on the ethyl ester moiety to allow determination of the imide fraction, f. Forward recoil spectrometry (FRES) was utilized to provide information about the polyimide—polyimide interface by measuring the depth profile of hydrogen and deuterium for bilayer samples consisting of a thick non-deuterated base layer, and a thin deuterated tracer layer. FRES measurements indicated that the interdiffusion distance, w, decreased markedly with increasing imidization temperature, T i. For base layers prepared from N-methylpyrrolidone, w for the para- and mixed PAE base layers decreased more rapidly with T i than with w for the meta-PAE base layers. However, these differences were found to result from the differences in imidization kinetics of the various isomers with f increasing more rapidly with T i, for para- and mixed PAE than for the meta-PAE. Nevertheless, when w was plotted as a function of f, all of the isomers exhibited similar behaviour, i.e. w decreased steadily as the imide fraction f approached 1. Base layers cast from dimethyl sulfoxide did not show differences in either f or w after imidization at a given T i.

Synthesis and inhibitory effect of a trisubstrate transition state analogue for UDP glucuronosyltransferases

Trisubstrate UGT transition state analogue 2 is readily accessible by nucleophilic ring-opening of 1,2-anhydroglucose precursor 5 with diethylmalonate anion followed by reduction of the ethyl ester moieties ( 6→7). Subsequent C 6 oxidation ( 8→9), NIS/ cat. TfOH-mediated introduction of the androsterylmethylene unit ( 12→15) and phosphitylation with 5′-uridine phosphoramidite 16 furnished, after oxidation and deprotection, target derivative 2, the two individual diastereomers of which ( 2a and 2b) were separated by HPLC. Trisubstrate analogues 2a,b show a different inhibition pattern for several UGT isoforms, indicating isoenzyme selectivity. Moreover, C 7″-epimers 2a and 2b exert a different inhibitory effect on UGT2B15.

Characteristics of cyanopindolol analogues active at the beta 3-adrenoceptor in rat ileum.

1. Cyanopindolol (CYP) is a potent antagonist at the beta 3-adrenoceptor in rat ileum. Several analogues of CYP and pindolol were synthesized that also produced antagonist effects at the beta 3-adrenoceptor. However, at high concentrations, these compounds appear to act as "partial agonists'. This study was conducted to determine the structural requirements of CYP analogues necessary for antagonist activity and to examine the possibility that the agonist effects of CYP and its analogues may occur through a mechanism independent of beta-adrenoceptor activation. 2. Analogues of CYP and pindolol were tested for antagonist activity in rat ileum in which the beta 1- and beta 2-adrenoceptors were blocked. Fourteen compounds were tested against (-)-isoprenaline, and four of the more potent analogues were then tested against BRL 37344. The two most potent antagonists were CYP and iodocyanopindolol. The pKb values (negative log of equilibrium dissociation constant) obtained against (-)-isoprenaline were significantly higher than those obtained against BRL 37344, but the cause of this difference is not known. 3. Several structural requirements were determined for antagonist activity. Modification at the carbon atom alpha to the secondary amine caused the antagonist potency to fall as the level of saturation was reduced. Thus, a quaternary carbon group, such as t-butyl, produced the most potent antagonist. Substitution with a large moiety such as a cyclohexyl or benzyl group reduced antagonist activity, probably due to steric hindrance. Inclusion of an electron-withdrawing group, such as a cyano or ethylester moiety, alpha to the indole nitrogen, also increased the potency. Iodination of CYP and ethylesterpindolol at the 3-position of the indole ring did not increase antagonist potency. In contrast, iodination of the almost inactive analogues produced a significant increase in potency, suggesting that a beneficial electronic effect on the indole ring imparted by the iodo moiety may be able to offset partially the negative effects caused by either the steric hindrance, of lack of a quaternary carbon alpha to the secondary amine. 4. Values for pseudo-pD2 were also determined by conducting cumulative concentration-response studies up to the limit of drug solubility. For nine of the compounds tested, the pKb was significantly higher than the pseudo-pD2 value. 5. The discrepancy between the pKb and pseudo-pD2 values was examined further. The agonist effects of iodocyanopindolol, the agonist with the highest potency, were not antagonized by CYP which was the most potent antagonist of (-)-isoprenaline and BRL 37344 at the beta 3-adrenoceptor. This suggests that the agonist effects of iodoCYP were produced through a different mechanism: either via another receptor, another isoform of the rat beta 3-adrenoceptor, or through a non-receptor-mediated effect. Pseudo-pD2 values did not correlate with log P values for these compounds, indicating that their relaxant effects were not simply a function of their lipid solubility. 6. This study has highlighted several structural requirements for antagonist binding potency at the rat ileum beta 3-adrenoceptor and should assist in the development of potent selective antagonists for this receptor.

2',3'-Dideoxy-3'-C,2'-N-[(3R,5R)-5-ethoxy-carbonyl-2-methyl-1, 2-isoxazolidine]-ribothymidine.

The title compound, C15H21N3O7, is a thymidine derivative with a tetrahydroisoxazole (THI) ring fused to the sugar ring at the 2'- and 3'-C atoms. The thymine base is in an anti conformation [chi = -122.5 (3) degrees] while the ribose moiety has C2'-exo,C3'-endo (3T2) conformation with a highly flattened sugar ring [P = -5.8, tau m = 13.1 degrees]. The molecule contains a pseudo-twofold axis on the C2'-C3' bond with the ethyl ester group acting as the base and the N-methyl group acting as the 5'-hydroxyl group in a nucleoside. The THI ring fused to the furanose has a C3'-endo,C4'-exo (3T4) conformation [P = 4.18, tau m = 19.3 degrees]. The ethyl ester moiety is disordered with respect to two conformations, the population of the major form being 87.4 (8)% and that of the minor form 12.6 (8)%.

Macromolecular Prodrugs of 5-Fluorouracil. 1: Synthesis and Hydrolytic Stability

The hydroxyl groups of poly(ethylene glycol), dextran and poly-[N5-(2-hydroxy-ethyl-L-glutamineI were activated using the 4-nitrophenyl chloroformate method. The resulting phenyl carbonate groups reacted quantitatively with the primary amine groups of oligopeptides with 2-(5-fluorouracil-1-yl) glycine ethyl ester moiety as C-terminus. Following this procedure, 5-fluorouracil-polymer conjugates with peptide chains of different amino acid sequences and configurations were synthesized. The conjugates showed a good hydrolytic stability in buffer solutions of pH 7.4 and 5.5, as well as in calf serum, which indicated good stability of the polymer-drug linkage in the blood.

Polymerization of Antarctic fish tubulins at low temperatures: role of carboxy-terminal domains.

We have proposed previously that the efficient polymerization of tubulins from Antarctic fishes at low, physiological temperatures (-1.8 to +2 degrees C) may result in part from adaptations (e.g., reductions in acidic residues) located in their carboxy-terminal (C-terminal) tails [Detrich & Overton (1986) J. Biol. Chem. 261, 10922-10930]. To test this hypothesis, we have examined the polymerization of Antarctic fish neural tubulins modified at their C termini by proteolysis or by neutralization of carboxyl groups. Addition of subtilisin to low concentrations of Notothenia coriiceps tubulin induced a biphasic assembly reaction: stage I corresponded to the C-terminal cleavage of beta chains to produce alpha beta s dimers, and stage II coincided with the slower, C-terminal cleavage of alpha chains to yield alpha s beta s. Both stage I and stage II polymers consisted of protofilament sheets and microtubules with attached sheets. The critical concentration for assembly of the stage II polymer was at least 10-fold lower than that of untreated tubulin. Neutralization of Glu and Asp carboxyls in Gobionotothen gibberifrons microtubules by the carbodiimide-catalyzed incorporation of glycine ethyl ester (GEE) moieties produced a tubulin, modified largely in its C termini, that assembled more readily than did control tubulin. When 12 GEE groups were incorporated per dimer, the critical concentrations for assembly of modified tubulin at 5-10 degrees C were 2-3-fold smaller than those for the unmodified protein. Comparably modified bovine tubulin (10 GEE/dimer) assembled at 37 degrees C with a critical concentration 2.6-fold lower than that for the unmodified tubulin.(ABSTRACT TRUNCATED AT 250 WORDS)