Occurrence of Brachyspira hyodysenteriae in multiplier pig herds in Switzerland.

OBJECTIVE: This research was aimed to determine the occurrence of Brachyspira (B.) hyodysenteriae in Swiss multiplier pig herds. MATERIALS AND METHODS: In a pilot study a direct real-time polymerase chain reaction (PCR) method for B. hyodysenteriae was compared to culture followed by PCR on 106 samples from three herds. Subsequently 40 multiplier herds were epidemiologically characterized and analysed for the presence of B. hyodysenteriae using direct PCR on 1412 rectal swabs. For external validation 20 swabs obtained from two positive conventional herds were analysed. RESULTS: The comparison of direct PCR with culture followed by PCR resulted in a moderate agreement (kappa index: 0.58). In the two conventional herds, 35% of the samples (7/20) tested positive. Samples from 39 multipliers tested negative. In one multiplier herd, 25% (9/36) of the samples tested PCR positive. Risk factors in the multiplier herd may have been rodents or birds, but not pig purchase. CONCLUSION AND CLINICAL RELEVANCE: B. hyodysenteriae have been detected in a Swiss multiplier herd, which underlines the threat of potential spread by replacement pigs. Consequently, a Brachyspira monitoring programme was established for Swiss multiplier herds.

Physiochemical aspects of tubulin-interacting antimitotic drugs.

A diverse group of natural biological compounds bind to microtubules and suppress microtubule dynamics. Here we review the mechanism of microtubule assembly and dynamics as well as structural features that are important for nucleotide binding, GTP hydrolysis and stabilization of longitudinal and lateral protofilament contacts. Specific emphasis is placed upon the polar structure of the microtubule, the exposure of the nucleotide hydrolysis site at the + end and the conformational and configurational plasticity of the microtubule lattice. These features have important implications for the mechanism of dynamic instability and the disruptive action of antimitotic drugs. We then discuss the various classes of tubulin binding drugs emphasizing their site and mode of binding as well as the structural and energetic basis for their effects on microtubule assembly and dynamics. A common feature of tubulin-interacting compounds is a linkage to assembly, either the stabilization of a microtubule lattice by compounds like taxol or epothilone A, or the preferential formation of alternate lattice contacts and polymers at microtubule ends by compounds like colchicine, vinca alkaloids and cryptophycin-52. Finally, we explore the likely possibility that these drugs also disrupt the regulation of microtubule dynamics. Future generations of these compounds may be selectively developed to directly target the proteins that regulate mitotic spindle dynamics.

Vinca alkaloid-induced tubulin spiral formation correlates with cytotoxicity in the leukemic L1210 cell line.

The ability of a class of C-20' modified vinca alkaloid congeners to induce tubulin spiral formation was investigated relative to their ability to inhibit microtubule assembly, their cytotoxicity against a leukemic cell line, L1210, and their measured and calculated partition coefficients. These studies were prompted by the observation that the energetics of vinca alkaloid-induced tubulin spiral polymers, or spiraling potential, is inversely related to their clinical dosage and are aimed at the long-term goal of developing the ability to predict the cytotoxic and antineoplastic properties of antimitotic drugs. We demonstrate here that vinca-induced tubulin-spiraling potential is significantly correlated with cytotoxicity against L1210 cells. This is consistent with the size of spirals formed being proportional to the relaxation time for polymer redistribution, the lifetime of cell retention, and effects on microtubule ends and dynamics. Spiraling potential also correlates with calculated but not measured partition coefficients. Surprisingly, spiraling potential does not correlate with the ability to inhibit microtubule formation with purified tubulin or microtubule protein. For the set of C-20' modified compounds studied, the largest inhibitory effects on spiraling potential and cytotoxicity are caused by multiple sites of halogen (-F, -Cl) substitution with the introduction of increased rigidity in the ring. This suggests the C-20' position interacts with a hydrogen bond acceptor or an electrophilic region on the protein that electrostatically disfavors halogen substitutions. These studies are discussed in terms of the cellular mode of action of antimitotic drugs, particularly the importance of microtubule dynamics during mitosis and the factors that regulate those dynamics.

Ethanol, isopropanol, methanol, and ethylene glycol poisoning.

Alcohol intoxication, commonly encountered in emergency department and clinic settings, is by no means a benign condition. Ethanol ingested alone or in combination with other CNS depressants (eg, isopropanol, methanol, ethylene glycol, sedatives, opioids) can be fatal. Obtaining the patient's history and careful observation for clinical signs and symptoms, along with appropriate analysis of results of laboratory tests, are the key to determining and differentiating the agent ingested. It is critical that poisoning due to ethanol and/or other related alcohols should be recognized early in order to initiate appropriate treatments and prevent fatalities. Emergency department nurses may be the first persons to collect the essential data, and it is incumbent upon them to plan and initiate appropriate care. In continuing management for these patients, critical care nurses must understand the factors contributing to the observed signs and symptoms in order to initiate and monitor ongoing care and prevent serious complications.

Additivity of dilantin and vinblastine inhibitory effects on microtubule assembly.

Dilantin (phenytoin) is a commonly used antiepileptic agent that is known to decrease conductance of sodium and calcium ions and delay outward potassium currents. Separate from its antiseizure activity, dilantin interferes with microtubule protein polymerization. It induces metaphase arrest and potentiates the effects of the antimitotics vincristine and vinblastine in cell culture. We show here by fluorescence binding studies that dilantin interacts directly with tubulin at a low affinity site [Ka = 3.5 (+/- 2.5) x 10(3) M(-1); Kd = 286 microM]. We quantitatively examined the effect of dilantin on bulk microtubule formation and found that the drug raises the critical concentration for microtubule polymerization in 2 M glycerol identically in the presence or absence of vinblastine. The change in free energy for microtubule polymerization attributable to 400 microM dilantin [deltadelta G = 117 (+/- 28) cal/mol] is additive with vinblastine effects. Under the same conditions, mean microtubule lengths are 7.7 +/- 4.3 microm (n = 558) and 7.4 +/- 4.0 microm (n = 477) in the presence or absence of dilantin, respectively. Dilantin has no effect on vinblastine-induced tubulin spiral formation, as measured by sedimentation velocity. Our data suggest that the mechanism for the antimicrotubule effects of dilantin involves sequestration of tubulin heterodimers in 1:1 drug:tubulin complexes that do not participate in tubulin polymerization. The dilantin binding site is distinct from the Vinca binding site, and these independent binding modes account for the additive effects in vitro. The sequestration of tubulin heterodimers could explain the combined drug synergy in cell cultures if it disrupted interactions with proteins that regulate microtubule dynamics and/or cell cycle events.

A comparison of thermodynamic parameters for vinorelbine- and vinflunine-induced tubulin self-association by sedimentation velocity.

We present a comparison of the energetics of spiral formation for two vinca alkaloids: a novel difluorinated vinorelbine derivative 20',20'-difluoro-3',4'-dihydrovinorelbine (F12158, or vinflunine) and the parent compound, vinorelbine. Vinca alkaloids are antineoplastic agents that halt cell division at metaphase by inhibiting microtubule assembly and inducing tubulin self-association into spiral aggregates. The overall affinities for tubulin of vincristine, vinblastine, and vinorelbine seem to correlate with their clinical doses, where vincristine with the highest overall affinity is used at the lowest doses. Doses of chemotherapeutic agents, however, also are determined by toxicities. In the physicochemical study described here, we used sedimentation velocity to compare vinorelbine- and vinflunine-induced self-association of porcine brain tubulin in the presence of 50 micro M GDP or 50 micro M GTP. Vinflunine demonstrates 3-16-fold lower overall affinity for tubulin and induces smaller polymers compared with vinorelbine. Sedimentation velocity provides the only direct evidence to date that vinflunine is a tubulin-binding drug. Stopped-flow light scattering demonstrates the shortest relaxation times for polymer redistribution for vinflunine consistent with induction of the shortest spirals. Data collected at 5 degrees, 15 degrees, 25 degrees, and 37 degrees show increasing 20,w values with increasing temperature and are consistent with an entropically driven process. These data are entirely consistent with our hypothesis that vinflunine is likely to result in reduced clinical neurotoxicity relative to vinorelbine, vinblastine, and vincristine.

Energetics of vinca alkaloid interactions with tubulin isotypes: implications for drug efficacy and toxicity.

A number of vinca alkaloids, including vincristine, vinblastine, and vinorelbine, are currently used in cancer chemotherapy. These three vinca alkaloids interact differently with a range of solid and hematologic tumors. To test the possibility that the tubulin isotype composition is an important determinant in antineoplastic efficacy, we determined thermodynamic parameters for vinca alkaloid interactions with purified beta-tubulin isotypes, alphabetaII or alphabetaIII, as well as mixtures of alphabetaII and alphabetaIII, alphabetaII and alphabetaI&IV, or alphabetaIII and alphabetaI&IV (referred to as isotype-depleted tubulin) by quantitative sedimentation velocity. Vincristine-, vinblastine-, or vinorelbine-induced isotype self-association was studied at 25 degrees C in 10 mM Pipes, pH 6.9, 1 mM MgSO4, and 2 mM EGTA in the presence of 50 microM GTP or GDP. For all three drugs, we observed no significant differences in overall affinities, K1K2, or in GDP enhancement of purified isotypes compared to unfractionated tubulin, suggesting that differential antitumor efficacy observed clinically for these vinca alkaloids is not determined by tissue isotype composition. Small, but significant differences in the individual binding parameters, K1 and K2, are found in the vincristine data. In the presence of vincristine and GTP, K1, the affinity of drug for tubulin heterodimers, tends to be larger for purified alphabetaII- or alphabetaIII-tubulin compared to unfractionated tubulin. Furthermore, the apparent dimerization constant, K2app, at physiologically significant drug concentrations is larger for these purified isotypes. When alphabetaII- and alphabetaIII-tubulin are combined, the cooperativity between drug binding and spiral formation approaches that of unfractionated PC-tubulin. These differences are not observed in the presence of vinblastine or vinorelbine. The differences found with vincristine may be implicated in the dose-limiting neurotoxicity found with this drug, but not found with vinblastine or vinorelbine.

Role of guanine nucleotides in the vinblastine-induced self-association of tubulin: effects of guanosine alpha,beta-methylenetriphosphate and guanosine alpha,beta-methylenediphosphate.

It is now well established that guanine nucleotides are allosteric effectors of the vinca alkaloid-induced self-association of tubulin. GDP enhances self-association for vinblastine-, vincristine- and vinorelbine-induced spiral assembly relative to GTP by 0.90 +/- 0.17 kcal/mol [Lobert et al. (1996) Biochemistry 35, 6806-6814]. Since chemical modifications of the vinca alkaloid structure are known to modulate the overall affinity of drug binding, it is very likely that, by Wyman linkage, chemical modifications of guanine nucleotide allosteric effectors also modulate drug binding. Here we compare the effects of the GTP and GDP alpha,beta-methylene analogues GMPCPP and GMPCP on vinblastine-induced tubulin association in 10 and 100 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (Pipes), 1 mM MgSO4, and 2 mM [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA), pH 6. 9, at different temperatures. We found that GMPCPP perfectly mimics GTP in its effect on spiral assembly under all ionic strength and temperature conditions. However, GMPCP in 10 mM Pipes behaves not as a GDP analogue, but as a GTP analogue. In 100 mM Pipes, GMPCP has characteristics that are intermediate between GDP and GTP. These data suggest that the alpha,beta methylene group in GMPCP and GMPCPP is sufficient to produce a GTP-like effect on vinblastine-induced tubulin self-assembly. This is consistent with previous observations that GMPCP-tubulin will assemble into microtubules in a 2 M glycerol and 100 mM Pipes buffer [Vulevic & Correia (1997) Biophys. J. 72, 1357-1375]. Our results demonstrate that an alpha,beta methylene modification of the guanine nucleotide phosphate moiety can induce a salt-dependent conformational change in the tubulin heterodimer that favors the GTP-tubulin structure. This has important implications for understanding allosteric interactions that occur in the binding of guanine nucleotides to tubulin.

Divalent cation and ionic strength effects on Vinca alkaloid-induced tubulin self-association.

We present here a systematic study of ionic strength and divalent cation effects on Vinca alkaloid-induced tubulin spiral formation. We used sedimentation velocity experiments and quantitative fitting of weight-average sedimentation coefficients versus free drug concentrations to obtain thermodynamic parameters under various solution conditions. The addition of 50-150 mM NaCl to our standard buffer (10 mM piperazine-N,N'-bis(2-ethanesulfonic acid), 1 mM Mg, 50 microM GDP or GTP, pH 6.9) enhances overall vinblastine- or vincristine-induced tubulin self-association. As demonstrated in previous studies, GDP enhances overall self-association more than GTP, although in the presence of salt, GDP enhancement is reduced. For example, in 150 mM NaCl, GDP enhancement is 0.24 kcal/mol for vinblastine and 0.36 kcal/mol for vincristine versus an average enhancement of 0.87 (+/- 0.34) kcal/mol for the same drugs in the absence of salt. Wyman linkage analysis of experiments with vinblastine or vincristine over a range of NaCl concentrations showed a twofold increase in the change in NaCl bound to drug-induced spirals in the presence of GTP compared to GDP. These data indicate that GDP enhancement of Vinca alkaloid-induced tubulin self-association is due in part to electrostatic inhibition in the GTP state. In the absence of NaCl, we found that vinblastine and 1 mM Mn2+ or Ca2+ causes immediate condensation of tubulin. The predominant aggregates observed by electron microscopy are large sheets. This effect was not found with 1 mM Mg2+. At 100 microM cation concentrations (Mn2+, Mg2+, or Ca2+), GDP enhances vinblastine-induced spiral formation by 0.55 (+/- 0.26) kcal/mol. This effect is found only in K2, the association of liganded heterodimers at the ends of growing spirals. There is no GDP enhancement of K1, the binding of drug to heterodimer, although K1 is dependent upon the divalent cation concentration. NaCl diminishes tubulin condensation, probably by inhibiting lateral association, and allows an investigation of higher divalent cation concentrations. In the presence of 150 mM NaCl plus 1 mM divalent cations (Mn2+, Mg2+, or Ca2+) GDP enhances vinblastine-induced spiral formation by 0.35 (+/- 0.21) kcal/mol. Relaxation times determined by stopped-flow light scattering experiments in the presence of 150 mM NaCl and vincristine are severalfold longer than those in the presence of vinblastine, consistent with a mechanism involving the redistribution of longer polymers. Unlike previous results in the absence of NaCl, relaxation times in the presence of NaCl are only weekly protein concentration dependent, suggesting the absence of annealing or an additional rate-limiting step in the mechanism.

Interaction of vinca alkaloids with tubulin: a comparison of vinblastine, vincristine, and vinorelbine.

Vinca alkaloids are antimitotic drugs that inhibit microtubule assembly and induce tubulin self-association into coiled spiral aggregates. Previous sedimentation velocity results with vinblastine have been interpreted by a mechanism involving isodesmic ligand-mediated or ligand-mediated plus ligand-facilitated self-association [Lobert et al. (1995) Biochemistry 34, 8050-8060]. In this study, we compare the vincristine- or vinorelbine-induced self-association of porcine brain tubulin with our prior vinblastine studies in the presence of 50 microM GDP or 50 microM GTP. Vincristine demonstrates the highest overall affinity for tubulin, K1K2, and vinorelbine the lowest (vincristine > vinblastine > vinorelbine). These and the first quantitative studies comparing the interaction of a new vinca alkaloid derivative, vinorelbine (Navelbine), with other vinca alkaloids. The relative binding affinities reported here correlate with the weekly drug doses used clinically in cancer chemotherapy, where vincristine is used at the lowest dosages and vinorelbine at the highest. Surprisingly, K1, the affinity of drug for tubulin heterodimers, is identical for all three drugs. When data are fit with the ligand-mediated model, the differences in overall affinity are due to effects on K2, the affinity of liganded heterodimers for spiral polymers. When data are fit with the ligand-mediated plus-facilitated model, affinity differences are also reflected in K3, the binding of the drug to unliganded polymers. We find that GDP enhances self-association in the presence of all three drugs 3-5-fold over GTP. The enhancement is manifested in K2 and K3 and amounts to an average of 0.90 +/= 0.17 kcal/mol. Thus, nucleotide enhancement is linked to the self-association step. Data collected at 5, 25, and 36 degrees C for all three drugs show increased maximum s-20,w values with increasing temperature and are consistent with an entropically driven reaction for the overall process. To investigate these results further, stopped-flow light scattering experiments have been conducted. Relaxation times are longest for the largest vincristine polymers and shortest for the smallest vinorelbine polymers, consistent with a cascade of events corresponding to successive dissociation events from spiral polymers, the larger the polymers the longer the relaxation time. Relaxation times for any single drug decrease with increasing tubulin concentration, consistent with the occurrence of oligomer annealing in addition to the association of liganded heterodimers to the ends of the growing spirals. Relaxation times were used to estimate on and off rates for liganded heterodimer association with spirals, and their ratio gives affinity constants (Kapp) that are independently consistent with K2 estimates from sedimentation velocity results for vinblastine and vinorelbine. For vincristine-induced tubulin polymers, a two-step process is observed with a second relaxation time more than 20-fold longer than times observed for vinblastine or vinorelbine. Sedimentation velocity experiments at low speeds and electron microscopy are consistent with the presence of a small amount of larger polymers (> or = 40S) in the vincristine samples, possibly involving alignment of spirals. Under our experimental conditions, these larger polymers appear to have a minimal effect on the estimated energetics of the vincristine-induced self-association of tubulin.

Binding of vinblastine to phosphocellulose-purified and alpha beta-class III tubulin: the role of nucleotides and beta-tubulin isotypes.

Vinblastine is an antimitotic drug that inhibits microtubule assembly and induces the self-association of tubulin into coiled spiral aggregates. Previous quantitative binding and sedimentation velocity results have been interpreted by a mechanism involving isodesmic ligand-mediated plus ligand-facilitated self-association [Na, G., & Timasheff, S. N. (1986) Biochemistry 25, 6214-6222, 6222-6228]. In this study, the vinblastine-induced self-association of porcine brain tubulin has been compared in the presence of 50 microM GDP or 50 microM GTP to investigate the role of nucleotides. Experiments at 1-4 microM tubulin in 10 mM Pipes, 1 mM MgSO4, 2 mM EGTA (pH 6.9), and varying concentrations of vinblastine (0.05-70 microM) demonstrate that GDP enhances self-association by 2-4-fold over GTP. In the presence of GDP or GTP, sedimentation velocity data can be best fit by either an indefinite ligand-mediated model or an indefinite ligand-mediated plus ligand-facilitated model. The association constant, K2, for the vinblastine-tubulin complex binding to a polymer is larger when GDP is present, while the association constant, K1, for the binding of vinblastine to tubulin heterodimers is identical in the presence of either nucleotide. The enhancement of K2 by GDP is confirmed by micropartition binding experiments with [3H]vinblastine. The fitting of sedimentation velocity and binding studies gives parameters for the interaction of vinblastine with GTP-tubulin that are identical, within error, to the previous results of Na and Timasheff. van't Hoff analysis of multiple temperature data reveals that this enhancement in the presence of GDP is due to a change in the enthalpy of self-association. Additional results suggest that the interaction of vinblastine with tubulin is identical for all beta-isotypes. Sedimentation velocity experiments in the presence of GDP or GTP show that the vinblastine-induced association of affinity-purified alpha,beta-class III tubulin is identical to that of unfractionated tubulin, although there is a difference in the abilities of unfractionated tubulin and alpha,beta III-tubulin to associate into taxol-stabilized microtubules.

Method for rapid electrophoretic transfer of isoelectric focusing gels to polyvinylidene difluoride.

We present here a protocol for unidirectional wet electrotransfer of isoelectric focusing (IEF) gels to polyvinylidene difluoride (PVDF) that can be carried out in < 2 h, including preequilibration steps. In developing this protocol, we systematically evaluated sodium dodecyl sulfate and methanol concentrations in the preequilibration washes and length of electrotransfer. We demonstrated that the method is useful for tubulin, a protein that resists usual semi-dry transfer procedures due to its limited solubility under IEF conditions. We also successfully used our method for transferring the microtubule-associated protein, tau, as well as carbamylyte standards, creatine phosphokinase and carbonic anhydrase.

Interaction of tubulin and microtubule proteins with vanadate oligomers.

Microtubule assembly is known to be regulated by the phosphorylation of microtubule-associated proteins (MAPs), and is thus sensitive to phosphatase inhibitors. We have investigated the direct interaction between phosphatase inhibitors (vanadate, sodium fluoride, and okadaic acid) and microtubule proteins. Vanadate self-assembles into oligomers, primarily dimer, tetramer, and decamer in 0.1 M Pipes, pH 6.9. Oligomer concentrations and their direct binding to tubulin and MAPs were determined by 51V NMR. The assembly of microtubule protein (MTP) is strongly inhibited by decavanadate binding to MAPs and only weakly inhibited by tetravanadate binding to MAPs. Decavanadate will inhibit both MAP2 and tau-induced assembly. Decavanadate binds to MAP2 at 26 sites [Ka > or = (1.0-1.3) x 10(5) M-1]. The mechanism appears to involve competitive binding to MAPs, presumably at or near the microtubule binding domains, and reduced affinity for microtubules. The assembly of MAP-free, phosphocellulose-purified tubulin (PC-tubulin) is only weakly inhibited by decavanadate, although decavanadate binds to tubulin at four independent sites (Ka > or = 1.0 x 10(5) M-1). Monomeric vanadate, a strong phosphatase inhibitor, does not interact with tubulin or MAPs, and thus does not bind to the exchangeable nucleotide binding site on tubulin. Sodium fluoride stimulates both PC-tubulin and MTP assembly by a nonspecific effect, probably involving water structure formation. Wyman analysis suggests an absence of direct or specific binding to tubulin (d ln K/d ln [NaF] = 0.214). NaCl is nearly as effective in promoting assembly of PC-tubulin, but inhibits MTP assembly.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple sites for subtilisin cleavage of tubulin: effects of divalent cations.

Limited digestion of pig brain GDP-tubulin by subtilisin was carried out in the presence of Mg2+, Mn2+, Ca2+, Zn2+, or Be2+. Isoelectric focusing, followed by SDS-PAGE, revealed characteristic divalent cation-dependent changes in the alpha- and beta-tubulin cleavage patterns. Previous studies revealed that the beta-cleavage pattern is different for heterodimers and microtubules [Lobert and Correia, 1992: Arch. Biochem. Biophys. 296: 152-160]. Divalent cation effects on subtilisin digestion of tubulin indicate different classes of divalent cation binding sites. Western blot analysis locates the proteolytic zone at residue 430 or higher in both subunits for all conditions. Turbidity and electron microscopy reveal that GDP-tubulin cleaved by subtilisin in the presence of Mg2+, Ca2+, or Mn2+ forms sheets of rings. Mn2+ induces ring formation in uncleaved GDP-tubulin. Isotype-depleted tubulin was generated by the removal of class III beta-tubulin using immunoaffinity chromatography. Subtilisin digestion of the depleted fraction and the purified class III beta-tubulin demonstrates that cleavage occurs at three to four distinct sites. Thus, subtilisin-digested tubulin is more heterogeneous than was previously reported and the cleavage sites depend on solution conditions, divalent cations, and the state of assembly. This has important implications for experiments that utilize subtilisin-digested tubulin for studying microtubule-associated protein binding.

Nondisulfide crosslinking and chemical cleavage of tubulin subunits: pH and temperature dependence.

Tubulin is known to be extremely unstable. The denaturation process partially involves irreversible aggregation, mediated by disulfide crosslinking. In addition, tubulin is known to undergo chemical cleavage during boiling in sodium dodecyl sulfate (SDS), a process that generates small peptides that have been mistaken for low molecular weight MAPs. Similar peptide cleavage has now been observed during two-dimensional denaturing isoelectric focusing-SDS-polyacrylamide gel electrophoresis. This phenomenon has complicated interpretation of limited proteolysis studies of tubulin by subtilisin. In an effort to avoid this problem we have undertaken a detailed study of the solution conditions that promote chemical cleavage of tubulin. The cleavage reaction is found to be strongly pH, time, and temperature dependent. Nondenatured and denatured tubulin is susceptible to peptide cleavage, suggesting that primary structure is more important than secondary structure in selection of susceptible bonds. After transfer of cleavage products from an SDS gel to a polyvinylidene difluoride membrane, amino acid sequencing has confirmed cleavage at Asp-Pro bonds, at position 306 in alpha-tubulin and at position 304 in beta-tubulin. We also infer cleavage at the only additional Asp-Pro peptide bond located at position 31 in beta-tubulin. Heat-induced cleavage at Asp-Pro accounts for 5 of the 13 bands observed on SDS gels. In addition, a minor alpha-tubulin band has been sequenced from a two-dimensional gel, corresponds to cleavage at Asp-Cys located at alpha-tubulin position 200, and accounts for two additional bands observed on SDS gels. Under nondenaturing and nonpolymerizing conditions tubulin undergoes extensive intermolecular, disulfide crosslinking. At elevated temperatures and high pH, a small fraction of the crosslinking is not reduced by beta-mercaptoethanol. Disulfide-crosslinked aggregates are not suspected because carboxymethylation of tubulin does not prevent their formation. Lysinoalanine has been found by amino acid analysis and thus covalent lysine-dehydroalanine crosslinks are suspected. Dehydroalanine is formed by beta-elimination at serine and thus the presence of lysinoalanine is consistent with cleavage at Gly-Ser peptides, the most unstable serine peptide bond, and accounts for most of the remaining cleavage data.

Subtilisin cleavage of tubulin heterodimers and polymers.

Native pig brain tubulin in heterodimer or polymer form was subjected to limited proteolysis by subtilisin, which is known to cleave at accessible sites within the last 50 amino acids of the highly variable carboxyl-termini of the alpha and beta subunits. Heterodimeric tubulin or tubulin polymerized in the presence of 4 M glycerol or taxol was used in these experiments. Digested tubulin was purified by cycles of polymerization and depolymerization, ammonium sulfate precipitation, or ion-exchange chromatography in the absence or presence of nonionic detergent; however, smaller cleaved products of about 34,000 to 40,000 MW remained associated with the major cleaved subunits, alpha' and beta', under all purification conditions. In order to determine the effect of subtilisin cleavage on tubulin heterogeneity, purified native or subtilisin-cleaved tubulin was subjected to isoelectric focusing, followed by SDS-PAGE. The total number of isotypes was reduced from 17-22 for native alpha,beta tubulin to 7-9 for subtilisin-cleaved alpha',beta' tubulin. When tubulin heterodimers were cleaved, a single major beta' isotype was evident; however, when tubulin polymerized in 4 M glycerol was cleaved, two major beta' isotypes were found. Monoclonal antibodies that recognize a beta carboxyl-terminal peptide, residues 410-430, reacted with both major beta' isotypes, indicating that subtilisin cleavage occurred within the last 20 of the 450 amino acids. In order to establish whether this difference was in fact associated with polymer or heterodimer forms of tubulin, digestion was carried out in the presence of taxol, which stabilizes tubulin polymers. A single major beta' isotype different from the cleaved heterodimer, but coincident with one of the bands of the cleaved glycerol-induced polymers, was found when taxol-treated tubulin was digested. This result suggests the presence of more than one subtilisin site in the beta subunit, near residues 430-435, with different accessibility to the enzyme in the heterodimer and polymer form.

Antimitotics in cancer chemotherapy.

Many agents used in cancer chemotherapy act on a specific phase of the cell cycle and are classified as cell cycle-specific. Some of these cell cycle-specific drugs, the antimitotics, interact with tubulin, the major protein of mitotic spindles, and cause metaphase arrest, thus halting mitosis. Understanding the dynamic processes involved in tubulin polymerization/depolymerization and how chemotherapeutic agents disrupt these processes is fundamental to understanding and anticipating common adverse effects and planning nursing interventions.

Studies of crystallization conditions for native and subtilisin-cleaved pig brain tubulin.

A survey of crystallization conditions for pig brain tubulin, using standard vapor diffusion techniques in sitting drops or capillaries, has resulted in irregular, fragile needles or plates with a largest dimension of 0.5 mm. These occurred in 2.5% PEG (MW 3350), 0.1 M Pipes, pH 6.2 and 6.4, 2-16 mM MgSO4, 10-15 mM DTE, and 0.1 mM GDP at 8 degrees C. When GTP replaced GDP these aggregates did not form under any of the conditions surveyed (temperature: 8-10 degrees C; MgSO4: 2-16 mM; pH 6-7; PEG, MW 3350: 1.25-12.5%). EM observations demonstrated that sheets of rings appear in crystal solutions in the presence of GDP or GTP. These results are consistent with the results of Howard and Timasheff (1986, Biochemistry 25, 8292-8300) that tubulin rings form in the presence of GDP or GTP but more readily in GDP. Tubulin crystallization experiments are hampered by tubulin's high degree of heterogeneity. Much of the variability lies in the carboxyl terminal region. Conditions for limited digestion of the heterodimer by subtilisin, removing only the carboxyl terminus, were determined. Reduction of heterogeneity was demonstrated by isoelectric focusing. The solubilities of native and subtilisin-cleaved tubulin in MgSO4, (NH4)2SO4, PEG (MW 1450, 3350, 10,000), DMSO, and MPD were compared. Subtilisin-cleaved tubulin precipitated more readily than native tubulin under all conditions surveyed, consistent with the removal of the highly acidic carboxyl terminus. Vapor diffusion experiments using subtilisin-cleaved tubulin under conditions where native tubulin forms needles or plates resulted in similar aggregates.