A 1 K bit Associative Memory LSI

Nikaido, T.; Ogura, Takeshi; Hamaguchi, S.; Muramoto, Susumu

doi:10.7567/jjaps.22s1.51

Cited by 8 publications

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“…The activity was low compared to that of strain P9254, which contains cloned DNA for all dTDP-L-rhamnose-synthesizing enzymes. The enzyme outside the rj8 gene cluster with glucose-1-phosphate thymidylyltransferase activity may be the same as one of the four glucose-1-phosphate uridylyltransferases found in S. enterica, strain LT2 [7] : that enzyme has a low substrate specificity and also catalyzes the glucose-1-phosphate thymidylyltransferase reaction [7]. We also surmise that this second activity, together with the high velocity of dTTP breakdown, may explain how a strain carrying pPR1123 containing orf5.2 of the rjh gene cluster, was identified as having a rfb glucose-1 -phosphate thymidylyltransferase gene [5].…”

Section: Discussionmentioning

confidence: 89%

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Purification, characterization and HPLC assay of Salmonella glucose‐1‐phosphate thymidylyltransferase from the cloned rfbA gene

Lindquist

Kaiser

Reeves

et al. 1993

European Journal of Biochemistry

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We here report on the purification and characterization of glucose-1-phosphate thymidylyltransferase, the first of four enzymes commited to biosynthesis of dTDP-L-rhamnose from Salmonella enterica strain LT2. The purification was greatly facilitated by the cloning of the IjhA gene encoding this enzyme. Pure enzyme was obtained by 109-fold enrichment in three chromatography steps.The glucose-1 -phosphate thymidylyltransferase catalyzes a reversible bimolecular group transfer reaction and kinetic measurements indicate that it acts by a 'ping-pong' mechanism. The K , values for dTTP and a-D-glucose 1-phosphate in the forward reaction are 0.020 mM and 0.11 mM, respectively. In the reverse reaction the K , values for dTDP-D-glucose and diphosphate are 0.083 mM and 0.15 mM, respectively. The enzyme also accepts UTP and UDP-D-glucose and a-D-glucosamine 1-phosphate is accepted equally as well as a-D-glucose 1-phosphate.The NH,-terminal sequence of glucose-1 -phosphate thymidylyltransferase agrees with the sequence predicted from the nucleotide sequence of the 0lf6.l gene of the rj& gene cluster. The SDS/ PAGE estimated subunit mass of 31 kDa agrees well with that calculated from the amino acid composition deduced from the nucleotide sequence of the 0rf6.l gene (32453 Da).Saccharides are important surface structures in eukaryotic and prokaryotic cells and involved in cell recognition phenomena. They occur as free saccharides and as glycoconjugates linked to proteins and lipids. One of the problems in studies of their activities is the difficulty in obtaining them in pure form in sufficient quantities. In recent years advances have been made in the chemical synthesis of saccharides [l]. However, saccharide synthesis is still a relatively complicated procedure, in particular when larger saccharides or large quantities are desirable. In the last few years we have been investigating the feasibility of in vitro enzymatic synthesis of the Salmonella enterica 0-antigen-specific oligosaccharide and the nucleotide sugar precursors using enzymes overexpressed by cloned genes [Z].The 0-antigen part of S. enterica sero-group B strains is a repeating tetramer oligosaccharide composed of abequose, mannose, rhamnose and galactose. This saccharide is assembled from appropriate nucleoside &phosphate monosaccharides. The enzymes which participate in the biosynthesis of S. enterica 0-antigen polysaccharide are encoded by genes Abbreviations. Buffer A, 50mM Tris/HCl pH7.6, 10mM MgCl, and 1 mM EDTA; buffer B, 20 mM Tris/HC pH 8.0, 1 mM MgC1, and 22% glycerol; buffer C, 50mM sodium phosphate pH 7.0, 1 M ammonium sulphate and 22% glycerol.Enzymes. Glucose-I-phosphate thymidylyltransferase (EC 2.7.7.24), inorganic pyrophoshatase (EC 3.6.1.1).which are located mostly in the rj8 gene cluster [3]. The rj& gene cluster has been cloned [4-61, and Escherichia coli K12 strains harboring plasmids containing different parts of the rj8 gene cluster of S. enten'ca LT2 was the source of the enzymes used for in vitro enzymatic synthesis of dTDP-Lrhamnose, ...

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Section: Discussionmentioning

confidence: 89%

“…The enzyme which catalyzes the reaction is glucose 1phosphate thymidylyltransferase, which is encoded by the @A gene [7].…”

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confidence: 99%