Binding properties and esterase activity of monoclonal antibodies elicited against sucrose 6-heptylphosphonate.

Various sugar phosphonates were prepared by a Mitsunobu condensation between phosphonic diacids and properly protected carbohydrates. 6'-O-p-Aminophenylsucrose 6-heptylphosphonate was coupled to Bovine Serum Albumin (BSA) and Keyhole Limpet Hemocyanin (KLH) and the KLH conjugate was used for generation of monoclonal antibodies. Binding properties of these antibodies were screened by competitive enzyme-linked immunosorbent assay (ELISA) using the BSA conjugate. A monoclonal antibody with good binding properties showed a regioselective esterase activity toward 6-octanoylsucrose compared with 6'-octanoylsucrose.

Conversion of 5-aminoimidazole ribotide to the pyrimidine of thiamin in enterobacteria: study of the pathway with specifically labeled samples of riboside.

Samples of 5-amino-1-(beta-D-ribofuranosyl)imidazole labeled with 13C at position C-1 or C-2 of the ribose part or with 15N at position N-3 or amino of the imidazole part were prepared by chemical synthesis. The incorporation of label from these samples into the pyrimidine of thiamin biosynthesized by a mutant strain of Salmonella typhimurium was studied by GC-MS. The results show clearly that in enterobacteria the methyl carbon atom and the N-1 nitrogen atom of one molecule of thiamin pyrimidine derive from the same molecule of 5-aminoimidazole ribotide. More specifically, the methyl carbon atom comes from the carbon C-2' of the ribose part and the nitrogen N-1 from nitrogen N-3 of the imidazole; furthermore, the amino nitrogen of the aminoimidazole becomes the amino nitrogen of the pyrimidine.

Biosynthesis of thiamine: origin of the methyl carbon atom of the pyrimidine moiety in Salmonella typhimurium.

Two biosynthetic, unevenly labeled samples of 5-aminoimidazole riboside were prepared. The incorporation of radioactivity from these labeled samples was studied in a Salmonella typhimurium strain able to use this riboside as precursor of the pyrimidine moiety of thiamine. In one sample, the ribose part was labeled almost equally at C-1' and C-2' and threefold more at C-3' but unlabeled at C-4' and C-5'. The pyrimidine moiety of thiamine derived from it was found almost inactive at C-5, C-7; the specific activity of the methyl carbon atom was found close to that of C-1' or C-2' of ribose in the precursor. On the other hand, only a poor incorporation of radioactivity occurred from a sample in which the ribose part was labeled mainly at C-1'. We conclude that C-5, C-7 of the pyrimidine moiety of thiamine derive from C-4', C-5' and the methyl carbon atom of the pyrimidine derives from C-2' of the ribose part of 5-aminoimidazole ribotide.

[Biosynthesis of thiazole from thiamine in Escherichia coli]. / Biosynthèse du thiazole de la thiamine chez Escherichia coli.

The incorporation into the thiazole moiety of thiamine of several labeled compounds has been studied on short time incubations of washed-cells suspensions. No incorporation of radioactivity from [G-14C] methionine was found in a mutant auxotrophic for methionine. No radioactivity was incorporated from [U-14C] aspartate or from [U-14C] serine. The incorporation of 35S from sulphate was lowered by cysteine or glutathione but was unaffected by methionine or homocystine. Although the synthesis of thiazole is dependent on methionine, neither the sulphur atom nor the carbon chain of thiazole originate from methonine in E. coli. No carbon originates from cysteine which is the likely direct donor of sulphur.