PubMed · 5833777
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J F Buri. 1965-10-07. [Chromatography].. https://pubmed.ncbi.nlm.nih.gov/5833777/
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Biosynthesis of the molybdenum cofactor, a chelate of molybdenum or tungsten with a novel pterin, occurs in virtually all organisms including humans. In the cofactor, the metal is complexed to the unique cis-dithiolene moiety located on the pyran ring of molybdopterin. Escherichia coli molybdopterin synthase, the protein responsible for adding the dithiolene to a desulfo precursor termed precursor Z, is a dimer of dimers containing the MoaD and MoaE proteins. The sulfur used for dithiolene formation is carried in the form of a thiocarboxylate at the MoaD C terminus. Using an intein expression system for preparation of thiocarboxylated MoaD, the mechanism of the molybdopterin synthase reaction was examined. A stoichiometry of 2 molecules of thiocarboxylated MoaD per conversion of a single precursor Z molecule to molybdopterin was observed. Examination of several synthase variants bearing mutations in the MoaE subunit identified Lys-119 as a residue essential for activity and Arg-39 and Lys-126 as other residues critical for the reaction. An intermediate of the synthase reaction was identified and characterized. This intermediate remains tightly associated with the protein and is the predominant product formed by synthase containing the K126A variant of MoaE. Mass spectral data obtained from protein-bound intermediate are consistent with a monosulfurated structure that contains a terminal phosphate group similar to that present in molybdopterin.
The ion chromatographic method for the quantification of phytate (InsP(6)) in foods was adapted for the analysis of roots and tubers. To maximize sensitivity, ultraviolet (UV) detection following postcolumn derivatization was compared with evaporative light-scattering detection (ELSD). Detection limits for phytate were 0.5 and 1 microg for UV and ELSD, respectively. Unidentified peaks eluting close to and after InsP(6) were removed by solid-phase extraction. Phytate was detected in 11 of 15 roots or tubers. The highest phytate levels were 0.169 and 0.133% of the fresh weight of taro (Colocasia esculenta) and yuca (Manihot esculenta), respectively. Potatoes (Solanum tuberosum) contained 0.035-0.073% phytate, whereas no phytate at a detection limit of 0.003% of fresh weight was observed in sweet potatoes (Ipomoea batatas).
A rapid procedure for the preparation of D-rhamnose from bacterial lipopolysaccharide (LPS) has been developed. It involves purification of LPS from Pseudomonas syringae pv. phaseolicola by phenol extraction and hydrophobic interaction chromatography (HIC), followed by mild hydrolysis and cleavage of the O-antigen into D-fucose and D-rhamnose. The monosaccharides were separated by column chromatography, and D-rhamnose recovered after filtration over Sephadex-LH 20.