UDP-galactose 4-epimerase from Escherichia coli: equilibrium unfolding studies.

UDP-galactose 4-epimerase from Escherichia coli is a homodimer of 39 kDa subunit with non-covalently bound NAD acting as cofactor. The enzyme can be reversibly reactivated after denaturation and dissociation using 8 M urea at pH 7.0. There is a strong affinity between the cofactor and the refolded molecule as no extraneous NAD is required for its reactivation. Results from equilibrium denaturation using parameters like catalytic activity, circular-dichroism, fluorescence emission (both intrinsic and with extraneous fluorophore 1-aniline 8-naphthalene sulphonic acid), 'reductive inhibition' (associated with orientation of NAD on the native enzyme surface), elution profile from size-exclusion HPLC and light scattering have been compiled here. These show that inactivation, integrity of secondary, tertiary and quaternary structures have different transition mid-points suggestive of non-cooperative transition. The unfolding process may be broadly resolved into three parts: an active dimeric holoenzyme with 50% of its original secondary structure at 2.5 M urea; an active monomeric holoenzyme at 3 M urea with only 40% of secondary structure and finally further denaturation by 6 M urea leads to an inactive equilibrium unfolded state with only 20% of residual secondary structure. Thermodynamical parameters associated with some transitions have been quantitated. The results have been discussed with the X-ray crystallographic structure of the enzyme.

Alkaline phosphatase activity in plasma of pregnant chagasic patients

The Kinetic properties of plasma placental alkaline phosphatase patients with Chagas' disease were studied. When Cl2 Mg was used as activator the same increase of activity (17-20 per cent) was found in the chagasic and non chagasic groups. The enzyme was not inhibited by F-ion in any of the groups. No significant differences were detected between the two groups (chagasic and non chagasic) when the enzyme was treated with inhibitors such as EDTA and L-phenylamine. However, when the CN- ion was used, the enzyme of the normal pregnant women followed a Michaelian curve, whereas in the chagasic group a sigmoideal plot was observed. Thus, the Hill coefficient was 1.1 for the normal group and over 1.5 for the chagasic.