pyrophosphate and malonic-acid

This study describes the simultaneous determination of phosphonate, phosphate, and diphosphate by CE with direct UV detection, based on in-capillary complexation with Mo(VI). When a mixture of phosphonate, phosphate, and diphosphate was injected into a capillary containing 3.0 mM Mo(VI), 0.05 M malonate buffer (pH 3.0) and 45% v/v CH3CN, three well-defined peaks, due to the migration of the corresponding polyoxomolybdate anions, were separated. The respective calibration graphs were linear in the concentration range of 2 x 10(-6)-2 x 10(-4) M for phosphonate, 1 x 10(-6)-5 x 10(-5) M for phosphate, and 1 x 10(-6)-2 x 10(-4) M for diphosphate; the correlation coefficients were better than 0.9990. The present CE method is successfully applied to the simultaneous determination of phosphonate, phosphate, and diphosphate in tap water.

Topics: Acetonitriles; Diphosphates; Electrophoresis, Capillary; Hydrogen-Ion Concentration; Malonates; Molybdenum; Organophosphonates; Phosphates; Sensitivity and Specificity; Spectrophotometry, Ultraviolet; Time Factors

L-Gulonate 3-dehydrogenase (GDH) catalyzes the NAD(+)-linked dehydrogenation of L-gulonate into dehydro-L-gulonate in the uronate cycle. In this study, we isolated the enzyme and its cDNA from rabbit liver, and found that the cDNA is identical to that for rabbit lens lambda-crystallin except for lacking a codon for Glu(309). The same cDNA species, but not the lambda-crystallin cDNA with the codon for Glu(309), was detected in the lens, which showed the highest GDH activity among rabbit tissues. In addition, recombinant human lambda-crystallin that lacks Glu(309) displays enzymatic properties similar to rabbit GDH. These data indicate that GDH is recruited as lambda-crystallin without gene duplication. An outstanding feature of GDH is modulation of its activity by low concentrations of P(i), which decreases the catalytic efficiency in a dose dependent manner. P(i) also protects the enzyme against both thermal and urea denaturation. Kinetic analysis suggests that P(i) binds to both the free enzyme and its NAD(H)-complex in the sequential ordered mechanism. Furthermore, we examined the roles of Asp(36), Ser(124), His(145), Glu(157 )and Asn(196) in the catalytic function of rabbit GDH by site-directed mutagenesis. The D36R mutation leads to a switch in favor of NADP(H) specificity, suggesting an important role of Asp(36) in the coenzyme specificity. The S124A mutation decreases the catalytic efficiency 500-fold, and the H145Q, N196Q and N195D mutations result in inactive enzyme forms, although the E157Q mutation produces no large kinetic alteration. Thus, Ser(124), His(145) and Asn(196) may be critical for the catalytic function of GDH.

Topics: 3-Hydroxyacyl CoA Dehydrogenases; Amino Acid Sequence; Animals; Carbohydrate Dehydrogenases; Crystallins; Diphosphates; DNA, Complementary; Enzyme Stability; Humans; Hydrogen-Ion Concentration; Kinetics; Liver; Malonates; Phosphates; Protein Denaturation; Rabbits; Recombinant Proteins; Sequence Alignment; Triazines

The design and synthesis of several beta-1,4-galactosyltransferase inhibitors are reported. Mimics of the pyrophosphate-Mn2+ complex were the focus of the design. Malonic, tartaric, and monosaccharide moieties were used as replacements of the pyrophosphate moiety, and galactose or azasugars with potent galactosidase inhibitory activity were used as the 'donor' component. Compound 6, in which glucose was used as the pyrophosphate-Mn2+ complex mimic and galactose as the 'donor' component, showed the best inhibitory activity towards the transferase with a Ki of 119.6 microM.

Topics: Aminoglycosides; Anti-Bacterial Agents; Antifungal Agents; Antiviral Agents; Binding Sites; Diphosphates; Enzyme Inhibitors; Fucosyltransferases; Malonates; Manganese; Monosaccharides; N-Acetyllactosamine Synthase; Pyrimidine Nucleosides; Substrate Specificity; Tartrates; Tunicamycin