Presence of bound substrate in lactate dehydrogenase from carp liver.

While attempting to purify UDP-galactose 4-epimerase from carp liver extract at pH 8.0, it was observed that the preparation even after dialysis could reduce NAD to NADH, interfering epimerase assay. The NAD reduction activity and the epimerase were co-eluted in a series of chromatographic steps. Mass spectrometric analysis of semi-purified fraction revealed that carp liver lactate dehydrogenase (LDH) contained bound lactate which was converted to pyruvate in the presence of NAD. The enzyme-bound lactate and the association with epimerase stabilized LDH from trypsin digestion and thermal inactivation at 45°C by factors of 2.7 and 4.2 respectively, as compared to substrate-free LDH. LDH and epimerase do not belong to any one pathway, but are the rate-limiting enzymes of two different pathways of carbohydrate metabolism. Typically, strongly associated enzymes work in combination, such as two enzymes of the same metabolic pathway. In that background, co-purification of LDH and epimerase as reloaded in this study was an unusual phenomenon.

Generalized epimerase deficiency galactosemia.

Galactosemia is caused by inherited deficiencies in one of three enzymes involved in the metabolism of galactose: galactose- 1-phosphate uridyltransferase (GALT), galactokinase (GALK), and uridine diphosphate galactose-4-epimerase (GALE). The rarest and most poorly understood form of galactosemia is due to epimerase deficiency. We are reporting such a rarest form of galactosemia presenting with progressively increasing cholestatic jaundice and failure to thrive at one month of age. After confirmation of decreased epimerase level in RBC hemolysate, the patient was put on galactose restricted diet and vitamins supplementation, which reversed the clinical signs as well as altered liver function. Patient is on regular follow-up and now at 15 months of age he has no marked developmental delay.

effect of diet on antioxidant status in patients with galactosemia

Galactosemia is an autosomal recessively inherited disorder of galactose metabolism. It has a good prognosis, if detected in neonatal period or early infancy. Treatment consists of life long dietary restriction of galactose. Our study included eight patients with galactosemia on dietary treatment, five of them had galactose-1-phosphate unidyltransferase deficiency known as classical galactosemia and three had uridine-diphosphate galactose-4' epimerase deficiency. Delayed milestones were present in all patients, jaundice at birth was present in 4 and low birth weight was present in 3 patients. Craniofacial dysmorphism was present in 5 patients. Hepatomegaly was present in 6 patients. MRI of the brain showed brain atrophy in 3 patients and demyelination in 2 patients. There was cataract in 7 patients. The aim of this study was to asses the antioxidant status in response to dietary-therapy. The levels of zinc, copper and Iron, calcium, phosphate, magnesium, selenium, manganese, beta-carotene and vitamin A were evaluated in the blood of galactosemic patients on galactose restricted diet. Also, a comparison between trace elements, beta-carotene and vitamin A in studied patients with galactosemia and controls was done. Copper, calcium, phosphate, manganese and beta-carotene levels in blood were significantly decreased in our patients [p<0.001] than in controls. These findings suggest that treated galactosemic patients are at risk of oxidative stress and abnormal bone mineralization. Therefore, therapeutic intervention in these cases should be more appropriately targeted. The data emphasise the importance of antioxidants and trace elements in minimizing the neurological deficits in galactosaemic patients

Comparative study on the role of parent Campylobacter jejuni and galE mutant in inducing experimental peripheral nerve damage / 中华儿科杂志

<p><b>OBJECTIVE</b>A comparative study on the role of Campylobacter jejuni (CJ) HB9313 and galE mutant in inducing experimental sciatic nerve damage was conducted in guinea pigs in order to explore whether CJ lipo-oligosaccharide (LOS) is critical component associated with peripheral nerve lesions and find experimental evidence for the presumption of molecular mimicry on the pathogenesis of Guillain-Barre syndromes (GBS) with CJ antecedent infection.</p><p><b>METHODS</b>A total of 32 guinea pigs were randomly divided into four groups: parental strain group (n = 10), galE mutant group (n = 10), control group (n = 6) and PBS group (n = 6), and immunized with the whole cell antigens of CJ HB9313 with Freund's adjuvant (FA), the whole cell antigens of galE mutant (without ganglioside-like structure) with FA, PBS with FA, and PBS alone, respectively. Enzyme-linked immunosorbent assay (ELISA) was employed to detect anti-LOS and anti-ganglioside GM1 antibodies in sera of these animals, and comparative morphologic studies of pathologic changes were carried out on the sciatic nerves, including examination of teasing fibers, examination of semithin sections made from epon-embedded tissue blocks under light microscope and transmission electron microscope.</p><p><b>RESULTS</b>ELISA results indicated that after immunization, the levels of anti-LOS IgG antibody were significantly elevated in animals from parental strain group and galE mutant group as compared with those before immunization (P < 0.01). No statistically significant difference was found between the two groups. However, the mean optical densities (ODs) of IgG antibody against GM1 at 14 and 28 day after immunization, in parental strain group, were 0.661 +/- 0.290 and 0.984 +/- 0.025, respectively, significantly higher than those of galE mutant group, which were 0.193 +/- 0.078 and 0.180 +/- 0.063 (P < 0.01). The results of morphologic examination on sciatic nerves showed that for teased-fiber study, incidence of pathologic abnormalities of teased fibers from animals of galE mutant group was 4.9% (98/2000), significantly lower than that from parental strain group, which was 16% (320/2000), characterized by predominantly axonal degeneration. The difference between them was highly significant statistically (P < 0.01). Examination of semithin sections of sciatic nerves also revealed that obvious pathological changes occurred in the animals from parental strain group, while only minimal abnormalities could be seen from galE mutant group, there was a significant differences between them (P < 0.01). In parental strains group, the predominant pathologicanl change was axonal degeneration with considerable variation in severity. These morphologic changes were confirmed by electron microscopy.</p><p><b>CONCLUSION</b>Compared with parental strain, galE mutant without ganglioside-like structure no longer could induce anti-GM1 antibodies, nor induce obvious immune damage of peripheral nerves in experimental guinea pigs. The results of this study provide a strong support to the hypothesis of molecular mimicry as a pathogenesis in patients with GBS following CJ antecedent infection.</p>

Analysis of the Cell Lysate Proteome of a Korean Mycobacterium tuberculosis Isolate K01 with H37Rv and H37Ra Strains

Despite recent economic prosperity, Korea still has high prevalence of tuberculosis. Molecular biologic characterization of Korean Mycobacterium tuberculosis strains might provide a deeper understanding of the forces contributing to the spread of tuberculosis in Korea. Therefore, we analyzed the cell lysate proteome of a representative Korean Mycobacterium tuberculosis isolate (K01) in comparison with laboratory reference strains H37Rv and H37Ra. Seven spots were strongly expressed only in K01 strain compared with M. tuberculosis H37Rv and H37Ra. Through continuous MALDI-MS analysis, these spots were identified as hypothetical protein Rv3849, secreted immunogenic protein Mpt64, Acetyl/propionyl-CoA Carbpxylase (AccD1), alkyl hydroperoxide reductase C (AhpC), N-acetylmuramyl-L-alanine amidase, a putative UDP glucose epimerase, and a transposase. A deeper study of these proteins may provide a clue in the development of effective new anti-tuberculosis vaccines against Korean M. tuberculosis isolates.

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.

UDP-galactose 4-epimerase from Kluyveromyces fragilis: equilibrium unfolding studies.

UDP-galactose 4-epimerase from yeast (Kluyveromyces fragilis) is a homodimer of total molecular mass 150 kDa having possibly one mole of NAD/dimer acting as a cofactor. The molecule could be dissociated and denatured by 8 M urea at pH 7.0 and could be functionally reconstituted after dilution with buffer having extraneous NAD. The unfolded and refolded equilibrium intermediates of the enzyme between 0-8 M urea have been characterized in terms of catalytic activity, NADH like characteristic coenzyme fluorescence, interaction with extrinsic fluorescence probe 1-anilino 8-naphthelene sulphonic acid (ANS), far UV circular dichroism spectra, fluorescence emission spectra of aromatic residues and subunit dissociation. While denaturation monitored by parameters associated with active site region e.g. inactivation and coenzyme fluorescence, were found to be cooperative having delta G between -8.8 to -4.4 kcals/mole, the overall denaturation process in terms of secondary and tertiary structure was however continuous without having a transition point. At 3 M urea a stable dimeric apoenzyme was formed having 65% of native secondary structure which was dissociated to monomer at 6 M urea with 12% of the said structure. The unfolding and refolding pathways involved identical structures except near the final stage of refolding where catalytic activity reappeared.

Microenvironment at the substrate binding subsite of the active site of UDPglucose 4-epimerase from Kluyveromyces fragilis using a fluorescent analog of UMP.

A chromophorics and fluorescent analog of uridine 5'-monophosphate (UMP), a known competitive inhibitor of UDPglucose 4-epimerase was synthesised. This analog, namely 2',3'-O-(2,4,6-trinitrocyclohexadienylidene) uridine 5'-monophosphate, was found to be a powerful reversible inhibitor of UDPglucose 4-epimerase indicating its interaction with the substrate binding site of the enzyme. The extreme sensitivity of the fluorescence emission spectrum of this analog to solvent polarity makes it an excellent probe for the study of the environment at the active site of the enzyme. We report here the effective use of this UMP analog to demonstrate that the hydroxyl groups of the ribose moiety of UMP and presumably the substrates (UDPgalactose and UDPglucose) do not reside in a hydrophobic milieu.