RESUMEN

A new reliable method to assay the activity of cytidine monophosphate sialic acid (CMP-Sia) synthetase (CSS) has been developed. The activation of sialic acids (Sia) to CMP-Sia is a prerequisite for the de novo synthesis of sialoglycoconjugates. In vertebrates, CSS has been cloned from human, mouse, and rainbow trout, and the crystal structure has been resolved for the mouse enzyme. The mouse and rainbow trout enzyme have been compared with respect to substrate specificity, demonstrating that the mouse enzyme exhibits a pronounced specificity for N-acetylneuraminic acid (Neu5Ac), while the rainbow trout CSS is equally active with either of three Sia species, Neu5Ac, N-glycolylneuraminic acid (Neu5Gc), and deaminoneuraminic acid (KDN). However, molecular details that explain the pronounced substrate specificities are unknown. Understanding the catalytic mechanisms of these enzymes is of major importance, since CSSs play crucial roles in cellular sialylation patterns and thus are potential drug targets in a number of pathophysiological situations. The availability of the cDNAs and the obtained structural data enable rational approaches; however, these efforts are limited by the lack of a reliable high-throughput assay system. Here we describe a new assay system that allows product quantification in a reduced nicotinamide adenine dinucleotide (NADH)-dependent color reaction. The activation reaction catalyzed by CSS, CTP+Sia-->CMP-Sia+pyrophosphate, was evaluated by a consumption of Sia, which corresponds to that of NADH on the following two successive reactions: (i) Sia-->pyruvate+ManNAc (or Man), catalyzed by a sialic acid lyase (SAL), and (ii) pyruvate+NADH-->lactate+oxidized nicotinamide adenine dinucleotide (NAD+), catalyzed by a lactate dehydrogenase (LDH). Consumption of NADH can be photometrically monitored on a microtiter plate reader for a number of test samples at the same time. Furthermore, based on the quantification of CSS used in the SAL/LDH assay, relative activities toward Sia derivatives have been obtained. The preference of mouse CSS toward Neu5Ac and the ability of the rainbow trout enzyme to activate both KDN and Neu5Ac were confirmed. Thus, this simple and time-saving method is suitable for a systematic comparison of enzyme activity of structurally mutated enzymes based on the relative specific activity.

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RESUMEN

The CMP-sialic acid synthetase (CMP-Neu5Ac, synthetase) is responsible for the synthesis of CMP-Neu5Ac, which is the donor used by sialyltransferases to attach sialic acid to acceptor hydroxyl groups in various polysaccharides, glycolipids, and glycoproteins. Since CMP-Neu5Ac is unstable and relatively expensive, the CMP-Neu5Ac synthetase is valuable for the preparative enzymatic synthesis of sialylated oligosaccharides. We made a construct to over-express the Neisseria meningitidis CMP-Neu5Ac synthetase in Escherichia coli. The recombinant enzyme was expressed at very high level (over 70,000 U/L) in a soluble form. It was purified by a sequence of anion-exchange chromatography and gel filtration with an overall yield of 23% (specific activity 220 U/mg). The purified CMP-Neu5Ac synthetase was used in the gram-scale synthesis of CMP-Neu5Ac.

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Trout liver is a rich source of sialate cytidylyltransferase activity. Three procedures are described by which the enzyme was enriched between 67- and 647-fold with high specific activities varying between 0.67 and 1.88 U/mg protein. In the simplest procedure studied, 100,000 x g supernatant of liver homogenate was chromatographed on Q-Sepharose and beta-[3-(2-aminoethylthio)propyl]-N- acetylneuraminic acid as affinity matrix, leading to an enzyme preparation (0.67 U/mg protein) well suited for the synthesis of CMP-N-acetylneuraminic acid. The synthase has a molecular mass of 160 kDa, a temperature optimum of 28 degrees C, a pH-optimum of 9.3 and exhibits Km-values for CTP, N-acetylneuraminic acid and N-glycoloylneuraminic acid of 1.7 mM, 2.1 mM and 2.9 mM, respectively. It is inactive with N-acetyl-9-O-acetylneuraminic acid. The enzyme is inhibited by CMP, CDP and 2'-deoxy-CTP. The sialic acid fraction of trout liver after hydrolysis is composed by N-acetylneuraminic acid (86%), N-acetyl-9-O-acetylneuraminic acid (12%) and N-acetyl-9-O-lactoylneuraminic acid (2%).

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A significant positive correlation was found between the liquefaction time of human seminal coagula and bound sialic acid. There was also a similar relationship between bound sialic acid and the enzyme sialyl-transferase. This suggests that the degree of sialylation of the components of seminal coagulum are important in determining the liquefaction time of the coagulum. These results support previous findings. The coagulum is considered to be composed of glycoprotein-metal ion complexes, and the initial stage of liquefaction results from the reduction of these metal ions by L-ascorbic acid. The removal of hydrogen peroxide, generated by the oxidation of L-ascorbic acid, requires the presence of glutathione peroxidase and glutathione reductase. These enzymes have been identified in human seminal plasma and their possible physiological importance is discussed.

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An HPLC method has been developed for the assay of cytidine monophosphate-sialic acid synthetase (EC 2.7.7.43) using ion-pair chromatography and gradient elution. This procedure permits the assay of alternative substrates and inhibitors of the enzyme and is not subject to the limitation of the colorimetric method. The newly synthesized N-acetyl-9-deoxy-9-fluoro-D-neuraminic acid was found to be a good substrate of the enzyme with a Km of 6.35 mM as compared to 1.84 mM for N-acetylneuraminic acid.

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