Ácidos siálicos: distribución, metabolismo y función biológica / Sialic Acids: ocurrence, metabolism and biological function

Los ácidos siálicos están entre las moléculas de mayor importancia del reinoanimal encontrándose también en algunos microorganismos. Son cetoácidos con unesqueleto glucídico de nueve carbonos, están cargados negativamente y se descubrieronen mamíferos aunque se encuentran en la mayor parte de los celomados, enprotostomados (por ejemplo, artrópodos), y deutorostomados (por ejemplo, cordadosy equinodermos). La ruta biosintética del Neu5Ac tiene lugar a través de las siguientesreacciones: a) síntesis de ManAc-6-P, b) síntesis de ManNAc, c) síntesis deNeu5Ac, d) activación del monómero to CMP-Beta-Neu5Ac, y e) transferencia delNeu5Ac a un aceptor. En el catabolismo de estos compuestos las enzimas neuraminidasay N-acetil neuraminato liasa tienen un papel importante. En animales losácidos siálicos están involucrados en interacciones célula-célula e intervienen en laregulación de procesos de reconocimiento celular. En microorganismos están presentesen un número escaso de bacterias y hongos. Estos microorganismos los utilizanpara establecer relaciones simbióticas o parasitarias con animales, para utilizarloscomo fuente de nitrógeno o carbono o para sialilar su propia superficie

Sialic acids are among the most important molecules in the animal kingdomand also occur in some microorganisms. They are alpha-ketoacids with a nine-carbonglycid backbone. They are negatively charged and they were first discovered inmammals, although it appears that they are present in most Coelomata in bothprotostomes (e.g. Arthropoda) and deutorostomes (e.g. Chordata or Echinodermata).The biosynthetic pathway of Neu5Ac proceeds through the following reactions:a) synthesis of ManAc-6-P, b) synthesis of ManNAc, c) synthesis of Neu5Ac, d)activation of the monomer to CMP-Beta-Neu5Ac, and e) transfer of Neu5Ac to theacceptor structure. In the catabolism of this compound, the neuraminidase and Nacetyl-neuraminidate-lyase activities play important roles. In animals, sialic acidsare involved in cell-to-cell interactions and mediate the regulation of recognitionprocess. In microorganisms they are present in a few taxonomically scatteredbacterial and fungal species. These microorganisms establish either symbiotic orparasitic relationships with animals and use host sialic acids either as a carbonnitrogensource or to sialylate their own cell surface

Spectrophotometric characterisation of the Cu(II):PPi system: implementation as a method for measuring pyrophosphate (PPi) in solution

El pirofosfato (PPi) tiene una importante función en numerosos procesos biológicos. Participa en muchas reacciones enzimáticas catalizadas por transferasas, hidrolasas, ligasas o sintetasas. La interacción en solución del Cu(II) con pirofosfato (PPi) modifica el espectro de absorción del catión. Se describen las interacciones químicas involucradas en el cambio de absorbancia, se identifican los coeficientes de absorción de las diferentes especies en solución y proponemos un modelo del sistema químico Cu(II)-PPi. Ya que los cambios en el espectro de absorción son dependientes de la concentración, nosotros proponemos un método para cuantificar PPi en soluciones acuosas basado en la modificación del espectro de absorción de Cu(II) en presencia PPi. Los cambios en la absorción pueden ser monitorizados y utilizados para cuantificar PPi en solución. La determinación es simple, rápida y barata. El límite de detección del método es 0,1 ìmol de PPi en las condiciones del ensayo. Además es posible usar este método en sistema biológicos que contienen proteínas, nucleótidos, EDTA o magnesio

The interaction of Cu(II) with pyrophosphate (PPi) in solution modifies the absorption spectrum of the cation. We provide here a proper description of the chemical interactions involved in the absorbance shift, identify the absorption coefficients of the species in solution and afford a model of the Cu(II)-PPi chemical system. Since the changes in the absorption spectrum are concentration dependent, we describe a new method for quantifying PPi in aqueous solutions, based on the modification of the Cu(II) absorption spectrum in the presence of PPi. Changes in absorptivity can be monitored and used to quantify PPi in solution. The determination is simple, fast and cheap. The detection limit of the method is 0.1 ìmol of PPi in the assay conditions. The presence of orthophosphate does not interfere in the determination of PPi. Furthermore, it is possible to use this method in biological systems containing proteins, nucleotides, EDTA or magnesium

Application of a normalised plot to the study of ter ter enzyme systems.

The kinetic characterisation of multisubstrate systems is not a trivial task. Common approaches simplify the experimental procedures by sequentially fixing saturating concentrations of different substrates/products, thereby attempting to isolate the influence of the varying molecule. Even after such tedious work, only apparent Km values can be determined, preventing serious comparison among differential substrate behaviours. Moreover, the choice among rival kinetic models is not statistically guaranteed; instead, classical tools such as re-plots continue to be used. Here, we report the application of a normalisation of kinetic data, formerly applied to simpler systems, to the description of ter ter systems. This data treatment is able to provide true Km values and a reliable description of the system, at the same time reducing the experimental work and statistically supporting the choice of kinetic schemes.

Application of a normalised plot to the study of uni-uni enzyme-inhibitor systems.

Normalisation of kinetic data is a useful tool in the study of complex enzyme systems. In the present paper, we have applied the premises of the normalised plot to the description of uni-uni enzyme inhibition. Guidelines to the design of the experiments and to data managing using the freeware program SIMFIT (http:\\www.simfit.man.ac.uk) are offered. The treatment has a lessened demand in experimental data while ensuring biological consistence of the results. Moreover, the results are obtained without resorting to secondary plots, and the election between rival mechanisms is statistically granted. Hyperbolic mixed-type inhibition is studied as a general model for enzyme-inhibitor/activator interaction, and equations describing classical cases of linear inhibition are also considered.

Transport of N-acetyl-D-mannosamine and N-acetyl-D-glucosamine in Escherichia coli K1: effect on capsular polysialic acid production.

N-Acetyl-D-mannosamine (ManNAc) and N-acetyl-D-glucosamine (GlcNAc) are the essential precursors of N-acetylneuraminic acid (NeuAc), the specific monomer of polysialic acid (PA), a bacterial pathogenic determinant. Escherichia coli K1 uses both amino sugars as carbon sources and uptake takes place through the mannose phosphotransferase system transporter, a phosphoenolpyruvate-dependent phosphotransferase system that shows a broad range of specificity. Glucose, mannose, fructose, and glucosamine strongly inhibited the transport of these amino-acetylated sugars and GlcNAc and ManNAc strongly affected ManNAc and GlcNAc uptake, respectively. The ManNAc and the GlcNAc phosphorylation that occurs during uptake affected NeuAc synthesis in vitro. These findings account for the low in vivo PA production observed when E. coli K1 uses ManNAc or GlcNAc as a carbon source for growth.

Catabolism of D-glucose by Pseudomonas putida U occurs via extracellular transformation into D-gluconic acid and induction of a specific gluconate transport system.

Pseudomonas putida U does not degrade D-glucose through the glycolytic pathway but requires (i) its oxidation to D-gluconic acid by a peripherally located constitutive glucose dehydrogenase (insensitive to osmotic shock), (ii) accumulation of D-gluconic acid in the extracellular medium, and (iii) the induction of a specific energy-dependent transport system responsible for the uptake of D-gluconic acid. This uptake system showed maximal rates of transport at 30 degrees C in 50 mM potassium phosphate buffer, pH 7.0. Under these conditions the K(m) calculated for D-gluconic acid was 6.7 microM. Furthermore, a different transport system, specific for the uptake of glucose, was also identified. It is active and shows maximal uptake rates at 35 degrees C in 50 mM potassium phosphate buffer, pH 6.0, with a K(m) value of 8.3 microM.