RESUMEN
The manner by which the bacterial chemotaxis system responds to a wide range of attractant concentrations remains incompletely understood. In principle, positive cooperativity between chemotaxis receptors could explain the ability of bacteria to respond to extremely low attractant concentrations. By utilizing an in vitro receptor-coupled kinase assay, the attractant-dependent response curve has been measured for the Salmonella typhimurium aspartate chemoreceptor. The attractant chosen, alpha-methyl aspartate, was originally used to quantitate high receptor sensitivity at low attractant concentrations by Segall, Block, and Berg [(1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8987-8991]. The attractant response curve exhibits limited positive cooperativity, yielding a Hill coefficient of 1.7-2.4, and this Hill coefficient is relatively independent of both the receptor modification state and the mole ratio of CheA to receptor. These results disfavor models in which there are strong cooperative interactions between large numbers of receptor dimers in an extensive receptor array. Instead, the results are consistent with cooperative interactions between a small number of coupled receptor dimers. Because the in vitro receptor-coupled kinase assay utilizes higher than native receptor densities arising from overexpression, the observed positive cooperativity may overestimate that present in native receptor populations. Such positive cooperativity between dimers is fully compatible with the negative cooperativity previously observed between the two symmetric ligand binding sites within a single dimer. The attractant affinity of the aspartate receptor is found to depend on the modification state of its covalent adaptation sites. Increasing the the level of modification decreases the apparent attractant affinity at least 10-fold in the in vitro receptor-coupled kinase assay. This observation helps explain the ability of the chemotaxis pathway to respond to a broad range of attractant concentrations in vivo.
Asunto(s)
Aspartato Quinasa/metabolismo , Quimiotaxis/fisiología , Proteínas de Escherichia coli , Proteínas Quinasas/metabolismo , Receptores de Aminoácidos/metabolismo , Adaptación Fisiológica , Aspartato Quinasa/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Histidina Quinasa , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas Quimiotácticas Aceptoras de Metilo , Modelos Biológicos , Modelos Químicos , Mutagénesis Sitio-Dirigida , Proteínas Quinasas/fisiología , Receptores de Aminoácidos/fisiología , Salmonella typhimurium/enzimología , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Salmonella typhimurium/fisiología , Transducción de SeñalRESUMEN
A 2.9-kb SacI fragment containing the ask-asd operon, encoding aspartokinase and aspartatesemialdehyde dehydrogenase, was cloned from an aminoethylcysteine-resistant, lysine-producing Corynebacterium lactofermentum strain. Enzymatic analysis showed that the aspartokinase (ASK) activity was completely resistant to inhibition by mixtures of lysine and threonine. Comparison of the deduced amino acid sequence of the beta submit of the ask gene showed three amino acid residue changes with ask gene encoding wild-type, feedback-sensitive enzymes. Three C. lactofermentum strains, one being aspartokinase-negative, one carrying two ask genes on the chromosome and one having a sixfold higher specific ASK activity than the parental strain, were constructed by transconjugation and electroporation, and used to analyse the role of ASK in the lysine production by C. lactofermentum. The results indicate that, in this study, feed-back-resistant ASK is necessary for high-level lysine production, but dispensable for lysine and diaminopimelate synthesis required for cell growth.
Asunto(s)
Aspartato Quinasa/fisiología , Corynebacterium/metabolismo , Lisina/biosíntesis , Secuencia de Aminoácidos , Aspartato Quinasa/química , Aspartato Quinasa/genética , Clonación Molecular , Retroalimentación , Fermentación , Datos de Secuencia MolecularRESUMEN
Aspartate kinase activity in cells of A. variabilis has been studied in the dynamics of development of virus infection. An early period of reproduction of cyanophage A-1 has been determined to be conjugated with the increase of biosynthesis of amino acids from aspartate family. Five isoenzymes of aspartate kinase were isolated and purified from A. variabilis cells during early development period of cyanophage A-1. Physicochemical properties and influence of amino acids of aspartate family on the activity of homogeneous isoenzymes have been studied. Retroinhibition effect was not observed in infected cyanobacteria cells, which probably enables one to increase 2-7 times the concentration of amino acids in a cell. Such an increase of the amino acids pool is apparently necessary for realization of viral genome strategy.
Asunto(s)
Aspartato Quinasa/fisiología , Bacteriófagos/fisiología , Cianobacterias/virología , Isoenzimas/fisiología , Replicación Viral/fisiología , Aspartato Quinasa/química , Fenómenos Químicos , Química Física , Isoenzimas/químicaRESUMEN
La barrera hematoencefálica formada principalmente por los microvasos cerebrales, limita y controla el movimiento de iones y solutos entre la sangre y el cerebro. La enzima Na+K+ATSasa constituye una de las más importantes bombas de membrana, dispuesta para mantener las composiciones iónicas intra y extracelulares. esta bomba ha sido localizada en la membrana abluminal de distintos epitelios y se ha determinado que su funcionalidad depende de la fosforilación en residuos de aspartato. Por otra parte, la enzima aspartato quinasa (AK), desempeña el papel de fosforilar ácido aspártico, formando un compuesto altamente inestable. En trabajos anteriores, hemos inmunodetectado esa quinasa, asociada tanto a membranas como al citoplasma, en células de diferentes tejidos. En este estudio, basado en la inmunodetección de las enzimas por anticuerpos policlonales específicos, en secciones ultrafinas de cerebro de rata, hemos notado una asociación en la ubicación de Na+/K+APTasa y AK, en la membrana luminal de los endotelios de los capilares cerebrales. También, hemos observado vesículas en el citoplasma de los vasos, que tienen una reacción positiva al anticuerpo de AK marcado con peroxidasa. La presencia de Na+/K+APTasa en la membrana luminal y abluminal del endotelio de los microvasos cerebrales, indica una falta de polaridad de esta enzima. El análisis de las observaciones sugiere que ambas enzimas podrían estar funcionalmente relacionadas