Expression and characterization of an N-truncated form of the NifA protein of Azospirillum brasilense

Azospirillum brasilense is a nitrogen-fixing bacterium associated with important agricultural crops such as rice, wheat and maize. The expression of genes responsible for nitrogen fixation (nif genes) in this bacterium is dependent on the transcriptional activator NifA. This protein contains three structural domains: the N-terminal domain is responsible for the negative control by fixed nitrogen; the central domain interacts with the RNA polymerase σ54 co-factor and the C-terminal domain is involved in DNA binding. The central and C-terminal domains are linked by the interdomain linker (IDL). A conserved four-cysteine motif encompassing the end of the central domain and the IDL is probably involved in the oxygen-sensitivity of NifA. In the present study, we have expressed, purified and characterized an N-truncated form of A. brasilense NifA. The protein expression was carried out in Escherichia coli and the N-truncated NifA protein was purified by chromatography using an affinity metal-chelating resin followed by a heparin-bound resin. Protein homogeneity was determined by densitometric analysis. The N-truncated protein activated in vivo nifH::lacZ transcription regardless of fixed nitrogen concentration (absence or presence of 20 mM NH4Cl) but only under low oxygen levels. On the other hand, the aerobically purified N-truncated NifA protein bound to the nifB promoter, as demonstrated by an electrophoretic mobility shift assay, implying that DNA-binding activity is not strictly controlled by oxygen levels. Our data show that, while the N-truncated NifA is inactive in vivo under aerobic conditions, it still retains DNA-binding activity, suggesting that the oxidized form of NifA bound to DNA is not competent to activate transcription.

Production of the refolded oligopeptide-binding protein (OppA) encoded by the citrus pathogen Xanthomonas axonopodis pv. citri

The oligopeptide-binding protein, OppA, binds and ushers oligopeptide substrates to the membrane-associated oligopeptide permease (Opp), a multi-component ABC-type transporter involved in the uptake of oligopeptides expressed by several bacterial species. In the present study, we report the cloning, purification, refolding and conformational analysis of a recombinant OppA protein derived from Xanthomonas axonopodis pv. citri (X. citri), the etiological agent of citrus canker. The oppA gene was expressed in Escherichia coli BL21 (DE3) strain under optimized inducing conditions and the recombinant protein remained largely insoluble. Solubilization was achieved following refolding of the denatured protein. Circular dichroism analysis indicated that the recombinant OppA protein preserved conformational features of orthologs expressed by other bacterial species. The refolded recombinant OppA represents a useful tool for structural and functional analyses of the X. citri protein.

Vicilins (7S storage globulins) of cowpea (Vigna unguiculata) seeds bind to chitinous structures of the midgut of Callosobruchus maculatus (Coleoptera: Bruchidae) larvae

The presence of chitin in midgut structures of Callosobruchus maculatus larvae was shown by chemical and immunocytochemical methods. Detection by Western blotting of cowpea (Vigna unguiculata) seed vicilins (7S storage proteins) bound to these structures suggested that C. maculatus-susceptible vicilins presented less staining when compared to C. maculatus-resistant vicilins. Storage proteins present in the microvilli in the larval midgut of the bruchid were recognized by immunolabeling of vicilins in the appropriate sections with immunogold conjugates. These labeling sites coincided with the sites labeled by an anti-chitin antibody. These results, taken together with those previously published showing that the lower rates of hydrolysis of variant vicilins from C. maculatus-resistant seeds by the insect's midgut proteinases and those showing that vicilins bind to chitin matrices, may explain the detrimental effects of variant vicilins on the development of C. maculatus larvae

Important amino acid residues of potato plant uncoupling protein (StUCP)

Chemical modifications were used to identify some of the functionally important amino acid residues of the potato plant uncoupling protein (StUCP). The proton-dependent swelling of potato mitochondria in K+-acetate in the presence of linoleic acid and valinomycin was inhibited by mersalyl (Ki = 5 æM) and other hydrophilic SH reagents such as Thiolyte MB, iodoacetate and 5,5'-dithio-bis-(2-nitrobenzoate), but not by hydrophobic N-ethylmaleimide. This pattern of inhibition by SH reagents was similar to that of brown adipose tissue uncoupling protein (UCP1). As with UCP1, the arginine reagent 2,3-butadione, but not N-ethylmaleimide or other hydrophobic SH reagents, prevented the inhibition of StUCP-mediated transport by ATP in isolated potato mitochondria or with reconstituted StUCP. The results indicate that the most reactive amino acid residues in UCP1 and StUCP are similar, with the exception of N-ethylmaleimide-reactive cysteines in the purine nucleotide-binding site

Aislamiento y caracterizacion de proteinas unidas a prolactina en el plasma de pacientes hiperprolactinemicos / Isolation and characterization of prolactin-binding proteins in plasma of hyperprolactinemic patients

Se analizaron los resultados de la purificación y caracterización de las proteínas que se encuentran unidas a la PRL en el plasma de pacientes hiperprolactinémicos. Se demostró la existencia del complejo proteico mediante incubación del plasma con PRL marcada con yodo radiactivo (PRL-I125), seguido por separación cromatográfica de gel filtración en Sephacryl S-300HR. Se comparó el patrón de elución con proteínas de pesos moleculares conocidos y la radiactividad incorporada se determinó por conteo de las radiaciones gamma. El complejo prolactina-proteína eluyó en un rango de pesos moleculares elevados. Se aislaron las proteínas unidas a la prolactina por cromatografía de afinidad en una columna de prolactina ovina (oPRL) unida a Sepharosa 4B activada con bromuro de cianógeno. La concentración de proteínas en 5 lotes analizados en forma sucesiva, fue de 180"74Fg/fraccionamiento. Las proteínas aisladas mostraron homología inmunológica con la IgG humana mediante el análisis por inmunodifusión radial en geles de agarosa. Según el patrón electroforético encontrado, las proteínas que se unen a la PRL en el plasma están formadas por 4 bandas de pesos moleculares, 2 de las cuales coinciden con las cadenas ligeras y pesadas de la IgG, mientras que las otras 2 presentan pesos moleculares aproximados de 68 y 80 kDa, respectivamente. Los resultados encontrados permiten identificar a las proteínas unida a la PRL como un complejo con una estructura molecular en la cual participa la IgG

Isolation of 75Se binding protein from hepatic tissues of chick embryos.

Distribution study using 75Se shows that maximum accumulation was in liver tissues after 24h of 75Se administration. Induction of selenium binding protein (Se-P) in hepatic tissues of chick embryo was observed. Chick embryo hepatic Se-P was isolated after 24h of 75Se treatment using Sephadex G-75 column chromatography. Fractions of induced protein shows the presence of maximum concentration of 75Se. This induced protein was found to have an approximate molecular weight of 56 KD on molecular sieve. It also showed an absorbance maxima at 254 nm, which indicates the presence of high concentrations of sulphydryl groups.

Purification and partial characterization of acyl carrier proteins from developing oil seeds of pisa (Actinodaphne hookeri) and ground nut (Arachis hypogaea).

Acyl carrier proteins (ACP) were purified to homogeneity in the active form from developing seeds of pisa (Actinodaphne hookeri) which synthesizes exclusively trilaurin and from ground nut (Arachis hypogaea) which synthesizes triacylglycerols containing long chain fatty acids. Two major isoforms of ACPs were purified from developing pisa seeds using DEAE-cellulose, Superose-6 FPLC and C4 reversed phase HPLC chromatographic methods. In contrast, only a single form of ACP was present in ground nut seeds which was purified by anion-exchange and activated thiol-Sepharose 4B affinity chromatography. The two isoforms of ACPs from pisa showed nearly the same specific activity of 6,706 and 7,175 pmol per min per mg protein while ground nut ACP showed a specific activity of 3,893 pmol per min per mg protein when assayed using E. coli acyl-ACP synthetase and [1-14C]palmitic acid. When compared with E. coli ACP, the purified ACPs from both the seeds showed considerable difference in their mobility in native PAGE, but showed similar mobility in SDS-PAGE under reducing conditions. In the absence of reducing agents formation of dimers was quite prominent. The ACPs from both the seed sources were acid- and heat-stable. The major isoform of pisa seed ACP and the ground nut ACP contain 91 amino acids with M(r) 11,616 and 1,228 respectively. However, there is significant variation in their amino acid composition. A comparison of the amino acid sequence in the N-terminal region of pisa and ground nut seed ACPs showed considerable homology between themselves and with other plant ACPs but not with E. coli ACP.

Trypanosoma cruzi binds to laminin in a carbohydrate-independent way

The bindings of 125I-laminin to trypomastigotes is specific and 2-5 x 10**3 laminin-binding sites were calculated to be presented on the surface of a live trypomastigote. Anti-laminin antibodies were able to inhibit the invasion of cultured cells by trypomastigotes (62-75 per cent), suggesting that laminin may be involved in the adhesion of the parasite to host cells. By affinity chromatography, an 85-KDa glycoprotein was isolated (laminin-bindign glycoprotein, LBG) from trypomastigote lysates, but not from epimastigote lysates. It is suggested that at least fragment E8 (but not E1) from laminin could be involbed in the reaction which is independent of the carbohydrate moieties from both ligand and recepto. It is also shown that LBG is member of the Tc-85 family, previously shown to be related to the invasion process of the parasite

Lipophorin as a lipid carrier in the hemolymph of the horseshoe crab, Limulus polyphemus

A high-density lipoprotein (HDL) was obtained from the hemolymph of Limulus polyphemus in yields generally less than 30 micrograms/ml by ultracentrifugal flotation. SDS-PAGE revealed two apolipoproteins with masses similar to those of apolipophorins (apoLp-I, 265 +/- 14 kDa; apoLp-II, 89 +/- 6 kDa). Lipid composition was different from both insect lipophorin and crustacean HDL, and showed less diacylglycerols than triacylglycerols (3.8 and 36.2 of total lipids, respectively). Since Limulus polyphemus is closely related to precambrian chelicerates, our results confirm that lipophorin was present early in the evolution of arthropods.

Biochemistry of the evolution of Triatoma infestans: XI. Hemolymph lipophorin

Se aisló y purificó la lipoforina del T.infestans a partir de hemolinfa de machos adultos por ultracentrifugación en gradiente de densidad en dos etapas. Esta lipoproteína tiene una densidad de 1.10 g/ml y está constituida por 53% de proteínas y 47% de lípidos. El radio de Stokes (Rs) de la molécula, determinado por cromatografía de filtración en gel, es aproximadamente de 73A y e;l peso molecular (Mr) calculado por el método de Margolis, es de 743 000 daltons. La lipoforina contiene dos apoproteínas: apolipoforina I (apoLp-I) con Mr = 255 000 y apolipoforina II (apoLp-II) con Mr = 78 000, siendo ambas glicosiladas. La composición de aminoácidos de la apoLp-I y de la apoLp-II presenta un elevado contenido de aspartato, glutamato y leucina y muy pequeña cantidad de metionina y cisteína. Los lípidos de la lipoforina comprenden: diacilgliceroles (41.4% de los lípidos totales), fosfolípidos (31.5%), hidrocarburos (12.2%), ácidos grasos libres (6.1%), colesterol (4.7%) y triacilgliceroles (4.1%). La fosfatidiletanolamina es el fosfolípido predominante con cantidades menores de fosfatidilcolina. Al tratar la lipoforina con tripsina, la apoLp-I sufre ruptura proteolítica, mientras que la apoLp-II es resistente. La anisotropía de fluorescencia del difenilhexatrieno (DPH) incluido en la lipoforina señala una fuerte interacción lípido-apoproteína, la cual no se modifica después de una extensa proteólisis de la apoLp-I con tripsina. Durante la tripsinización de la lipoproteína no se detectó...