We synthesized five peptides homologous to the potentially antigenic positions alpha(214-226), alpha(430-443), alpha(415-443), beta(241-256), and beta(412-431) of the porcine brain tubulin sequences. ...These peptides were successfully employed to raise tubulin-cross-reactive antibodies. The antibodies are specific of the regions of tubulin spanned by the peptides. They react specifically with the tubulin bands in immunoblots and with microtubules in immunofluorescence assays of cytoskeletons. The peptides of the C-terminal regions have also been employed to localize determinants recognized by two available monoclonal antibodies to tubulin in the positions alpha(415-430) and beta(412-431), respectively. In a first application of the anti-peptide antibodies, we have mapped the fragments of limited proteolysis of purified calf brain tubulin by trypsin, chymotrypsin, papain, thermolysin, subtilisin, and protease V8 from Staphylococcus aureus. Thirty-seven peptides have been identified, of which 32 have been unequivocally aligned into the tubulin sequences on the basis of their antigenic reactivity. There are three major, well-defined zones of preferential cleavage by the proteases: the C-termini and two internal zones in each chain. C-Terminal cleavages of both chains by subtilisin do not remove the antigenic reactivity of the zones alpha(415-430) and beta(412-431). C-Terminal cleavages by protease V8 are preferential of beta-tubulin. All six proteases tested cleave alpha- and/or beta-tubulin at one or both of the internal zones. These zones are located roughly at one-third and two-thirds of the chain length in both subunits. Therefore, a model of the tubulin monomers is proposed which consists of three major, proteolytically defined, compact regions (N-terminal, middle, and C-terminal thirds) and the cleavable zones. This model is discussed with the tubulin structural information presently available.
Thioredoxin reductase 1 (Trr1) from Saccharomyces cerevisiae is a member of the family of pyridine nucleotide‐disulfide oxidoreductases capable of reducing the redox‐active disulfide bond of the ...cytosolic thioredoxin 1 (Trx1) and thioredoxin 2 (Trx2). NADPH, Trr1 and Trx1 (or Trx2) comprise the thioredoxin system, which is involved in several biological processes, including the reduction of disulfide bonds and response to oxidative stress. Recombinant Trr1 was expressed in Escherichia coli as a His6‐tagged fusion protein and purified by nickel‐affinity chromatography. The protein was crystallized using the hanging‐drop vapour‐diffusion method in the presence of PEG 3000 as precipitant after treatment with hydrogen peroxide. X‐ray diffraction data were collected to a maximum resolution of 2.4 Å using a synchrotron‐radiation source. The crystal belongs to the centred monoclinic space group C2, with unit‐cell parameters a = 127.97, b = 135.41, c = 75.81 Å, β = 89.95°. The crystal structure was solved by molecular‐replacement methods and structure refinement is in progress.
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Gossypol is a polyphenolic pigment, which is employed as a male antifertility drug. It inhibits, among other reported effects, the growth of cultured mammalian cells, spermiogenesis, flagellar ...motility in Trypanosoma and sperm, dynein ATPase and the lactate dehydrogenase X (LDH‐X) isozyme.
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We have characterized the non‐covalent binding of gossypol to purified calf brain tubulin in 10 mM phosphate buffer, 0.1 mM GTP pH 7.0 at 25°C. Equilibrium measurements were performed by difference spectroscopy. A peak at 435 nm was produced by the perturbation of gossypol light absorption upon binding to tubulin. The experimental isotherm was fitted by 1.96 ± 0.06 gossypol binding sites per tubulin molecule, with identical apparent equilibrium binding constants of (7.5 ± 1.1) × 104 M−1. The complex formed could be separated from free gossypol by gel chromatography.
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Binding of gossypol was independent of the presence of 0.1 mM GTP in the buffer. Gossypol did not affect the binding of ligands to the colchicine site. Gossypol interacted with vinblastine but apparently did not bind to the vinblastine sites of tubulin. Gossypol did not displace anilinonaphthalene sulphonate (ANS) bound to tubulin, but caused a strong (fivefold) quenching of its fluorescence. This indicated that gossypol probably binds in the vicinity of the ANS site of tubulin.
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Gossypol inhibited in vitro microtubule assembly at the same concentration range employed in the binding studies. An increase in the critical protein concentration required for polymerisation was observed, most simply interpreted by a stoichiometric mechanism. Gossypol did not induce any noticeable distortion of the microtubules observed under the electron microscope. This compound constitutes a new tubulin ligand and an inhibitor of microtubule assembly in vitro.
In this paper, we review electronic design free software tools. We have searched open source programs that help with several tasks of the electronic design flow: analog and digital simulation, ...schematic capture, printed circuit board design and hardware description language compilation and simulation. Using some rapid criteria for verifying their availability, we have selected some of them which are worth working with. This work intends to perform a deeper analysis of free software tools and select some of them to use in education or in professional electronic design.
La Sierra Monte Grande tiene una extensión de 41.8 km2. Se localiza en la zona norte del altiplano potosino; su flora vascular está compuesta por 74 familias, 242 géneros y 397 especies que se ...encuentran en los siguientes tipos de vegetación: matorrales crasicaule, desértico micrófilo y desértico rosetófilo, así como encinar arbustivo y piñonar. Se consignan 45 registros inéditos para la región del altiplano potosino.
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