Purification, stabilization, and properties of bovine mammary UDP-galactose 4-epimerase

Tsai, Cheng-Hsien; Holmberg, Nels; Ebner, Kurt E.

doi:10.1016/0003-9861(70)90347-4

Cited by 40 publications

(13 citation statements)

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“…Unlike enzymes isolated from E. coli and yeast, mammalian GALE enzymes can convert UDP‐ N ‐acetylgalactosamine (UDP‐galNAc) to UDP‐ N ‐acetylglucosamine (UDP‐glcNAc), and are known to require exogenous NAD + [9]. We did not test the GALE mutations for enzyme activity on UDP‐galNAc.…”

Section: Discussionmentioning

confidence: 99%

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Functional analysis of mutations in UDP‐galactose‐4‐epimerase (GALE) associated with galactosemia in Korean patients using mammalian GALE‐null cells

et al. 2009

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Galactosemia is caused by defects in the galactose metabolic pathway, which consists of three enzymes, including UDP‐galactose‐4‐epimerase (GALE). We previously reported nine mutations in Korean patients with epimerase‐deficiency galactosemia. In order to determine the functional consequences of these mutations, we expressed wild‐type and mutant GALE proteins in 293T cells. GALEE165K and GALEW336X proteins were unstable, had reduced half‐life, formed aggregates and were partly degraded by the proteasome complex. When expressed in GALE‐null ldlD cells GALEE165K, GALER239W, GALEG302D and GALEW336X had no detectable enzyme activity, although substantial amounts of protein were detected in western blots. The relative activities of other mutants were lower than that of wild‐type. In addition, unlike wild‐type, GALER239W and GALEG302D were not able to rescue galactose‐sensitive cell proliferation when stably expressed in ldlD cells. The four inactive mutant proteins did not show defects in dimerization or affect the activity of other mutant alleles identified in patients. Our observations show that altered protein stability is due to misfolding and that loss or reduction of enzyme activity is responsible for the molecular defects underlying GALE‐deficiency galactosemia.

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Section: Discussionmentioning

confidence: 99%

“…GALE is a member of the short‐chain dehydrogenase/reductase superfamily. It is widely distributed in nature and has been isolated and characterized in several species [7–9]. The 3D structure of Escherichia coli [10], Trypanosoma brucei [11], Saccharomyces cerevisiae [12] and human [13] GALE has been determined by high‐resolution X‐ray crystallography.…”

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confidence: 99%

Functional analysis of mutations in UDP‐galactose‐4‐epimerase (GALE) associated with galactosemia in Korean patients using mammalian GALE‐null cells

et al. 2009

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“…', Thomson et al 1984;-, Dey 1984;\ Dressier et al 1984;', Fong and Feingold 1969;' Fukasawa et a] ! '98O-' MasweU and de Robichon-Szulmajster 1960;', Maxwell 1957;', Langer and Glaser 1974;'. Tsai et al 1970;'°, Geren and Ebner 1977a;"[ Wilson and Hogness 1964…”

Section: Resultsunclassified

Purification and characterization of UDP‐D‐galactose 4‐epimerase from the red alga Galdieria sulphuraria

Prosselkov

Groß

Igamberdiev

et al. 1996

Physiologia Plantarum

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UDP‐D‐galactose 4‐epimerase of the unicellular red alga Galdieria sulphuraria has been purified to apparent electrophoretic homogeneity by chromatography on DEAE‐Fractogel, hydroxylapatite and by affinity chromatography on Dyematrex Orange. The holoenzyme is a homodimer with an apparent molecular mass of 83 and 76 kDa as determined by gelfiltration and by sucrose gradient centrifugation, respectively. The size of the subunits was 42 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 4‐epimerase from G. sulphuraria does not require external NAD for activity, unlike the enzyme from some other organisms, and inhibition by NADH was not observed. The apparent Km for UDP‐D‐galactose was 64 μM. The pH optimum was at 8 and the apparent equilibrium constant for UDP‐Glc/UDP‐Gal was 3.5. The enzyme in crude as well as in purified samples was unusually stable and was not inactivated even on incubation at 46°C for several hours.

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“…The enzyme was very labile in dilute solutions. However, addition of NAD+ was efficient for stabilization of the enzyme throughout the purification procedures (13).…”

Section: Purification Of the Enzymementioning

confidence: 99%

Appearance of Uridine 5′‐Diphospho‐N‐Acetylglucosamine‐4‐Epimerase during Sporulation of Bacillus megaterium

Nishikawa

Iwawaki

Takubo

et al. 1986

Microbiology and Immunology

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In a biosynthetic study of the spore coat of Bacillus megaterium ATCC 12872 spore with galactosamine phosphate as a major component of the outer coat, high-performance liquid chromatography (HPLC) and enzyme immunoassay were applied for the measurement of UDP-N-acetylglucosamine-4-epimerase [EC 5.1.3.7] activity and the enzyme protein concentration, respectively. The new HPLC system using an ion-pair (or anion-exchange) column allowed us to determine successfully the enzyme activity and its application, proving that the specific activity of the enzyme in the cells increased at the later stage of sporulation. This increase in activity was parallel to the induction of enzyme protein synthesis, which was detected by sandwich enzyme immunoassay using antiserum to the purified enzyme. These results suggested that the regulation of this enzyme is at the genetic level and it plays an important role in the outer coat synthesis in the later sporulation stage of B. megaterium.The spore of Bacillus megaterium ATCC 12872 has sugar components, composed almost entirely of polymerized galactosamine-6-phosphate, in the outer coat (9, 10), which has been called the "exosporium" by Gerhardt's group (7). One of the enzymes involved in the synthesis of galactosamine-6-phosphate polymer must be UDP-N-acetylglucosamine-4-epimerase [EC 5.1.3.7], which catalyzes the conversion of UDP-N-acetylglucosamine (UDP-GlcNAc) to UDP-N-acetylgalactosamine (UDPGalNAc). Because the outer coat is chemically and morphologically specific to spores and is synthesized at the later stage of spore formation, it is assumed that the biosynthesis of this structure is regulated during the cell cycle. As the first step in this direction, the activity and the amount of UDP-GlcNAc-4-epimerase had to be determined in sporulating cells.Previous assays for this enzyme were carried out by quantitative analysis of galactosamine or glucosamine by paper chromatography (2) and/or enzymatic analysis (6) after acid hydrolysis. Without acid hydrolysis, measurement of UDPGlcNAc or UDP-GalNAc produced was performed using UDP-glucose dehydrogenase (8) or lectin affinity chromatography (11). These methods were not sufficient for our experiments because of inaccuracy and the long time required by the complicated procedures involved.

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Purification, stabilization, and properties of bovine mammary UDP-galactose 4-epimerase

Cited by 40 publications

References 20 publications

Functional analysis of mutations in UDP‐galactose‐4‐epimerase (GALE) associated with galactosemia in Korean patients using mammalian GALE‐null cells

Functional analysis of mutations in UDP‐galactose‐4‐epimerase (GALE) associated with galactosemia in Korean patients using mammalian GALE‐null cells

Purification and characterization of UDP‐D‐galactose 4‐epimerase from the red alga Galdieria sulphuraria

Appearance of Uridine 5′‐Diphospho‐N‐Acetylglucosamine‐4‐Epimerase during Sporulation of Bacillus megaterium

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