Galactose-metabolizing Enzymes Research Articles

Consumption of Galactose by Trypanosoma cruzi Epimastigotes Generates Resistance against Oxidative Stress.

In this study, we demonstrate that Trypanosoma cruzi epimastigotes previously grown in LIT medium supplemented with 20 mM galactose and exposed to sub-lethal concentrations of hydrogen peroxide (100 μM) showed two-fold and five-fold viability when compared to epimastigotes grown in LIT medium supplemented with two different glucose concentrations (20 mM and 1.5 mM), respectively. Similar results were obtained when exposing epimastigotes from all treatments to methylene blue 30 μM. Additionally, through differential centrifugation and the selective permeabilization of cellular membranes with digitonin, we found that phosphoglucomutase activity (a key enzyme in galactose metabolism) occurs predominantly within the cytosolic compartment. Furthermore, after partially permeabilizing epimastigotes with digitonin (0.025 mg × mg−1 of protein), intact glycosomes treated with 20 mM galactose released a higher hexose phosphate concentration to the cytosol in the form of glucose-1-phosphate, when compared to intact glycosomes treated with 20 mM glucose, which predominantly released glucose-6-phosphate. These results shine a light on T. cruzi’s galactose metabolism and its interplay with mechanisms that enable resistance to oxidative stress.

Galactosemia.

Galactosemia.

Functional and structural impact of the most prevalent missense mutations in classic galactosemia.

Galactose-1-phosphate uridylyltransferase (GALT) is a key enzyme in galactose metabolism, particularly important in the neonatal period due to ingestion of galactose-containing milk. GALT deficiency results in the genetic disorder classic galactosemia, whose pathophysiology is still not fully elucidated. Whereas classic galactosemia has been hypothesized to result from GALT misfolding, a thorough functional–structural characterization of GALT most prevalent variants was still lacking, hampering the development of alternative therapeutic approaches. The aim of this study was to investigate the structural–functional effects of nine GALT mutations, four of which account for the vast majority of the mutations identified in galactosemic patients. Several methodologies were employed to evaluate the mutations' impact on GALT function, on the protein secondary and tertiary structures, and on the aggregation propensity. The major structural effect concerns disturbed propensity for aggregation, particularly striking for the p.Q188R variant, resulting from the most frequent (∼60%) allele at a worldwide scale. The absence of major effects at the secondary and tertiary structure levels suggests that the disturbed aggregation results from subtle perturbations causing a higher and/or longer exposure of hydrophobic residues in the variants as compared to WT GALT. The results herein described indicate a possible benefit from introducing proteostasis regulators and/or chemical/pharmacological chaperones to prevent the accumulation of protein aggregates, in new avenues of therapeutic research for classic galactosemia.

Deletion of the HXK2 gene in Saccharomyces cerevisiae enables mixed sugar fermentation of glucose and galactose in oxygen-limited conditions

Galactose is one of the major sugar components of red seaweeds along with glucose. In Saccharomyces cerevisiae, synthesis of galactose-metabolizing enzymes (the Leloir proteins) is under tight and complex regulation in the presence of glucose by a mechanism called catabolite repression. As a result, when both sugars are concurrently present in the medium under oxygen-limited conditions, the yeast cannot utilize galactose even after glucose consumption. Research efforts on mixed sugar fermentation of glucose/galactose, therefore, were made in the presence of oxygen, thereby resulting in sequential substrate consumption, and low ethanol yield and productivity. In this study, mixed sugar fermentation of glucose and galactose in oxygen-limited conditions was achieved by deleting the HXK2 gene, a moonlighting protein acting as hexokinase or repressor involved in catabolite repression of S. cerevisiae. Remarkably, the S. cerevisiae D452-2Δhxk2 strain utilized galactose after glucose depletion without a diauxic lag period, whereas the parental strain could not use galactose at all under oxygen-limited conditions. The Δhxk2 strain fermented galactose at the consumption rate of 3.02±0.10g/L/h to produce ethanol with a yield of 0.44±0.01g ethanol/g galactose. Complementation of the Δhxk2 strain with the plasmid-harbored HXK2 gene under the control of various promoters clearly showed that the galactose consumption rates were inversely related to the mRNA levels of the HXK2 gene. The transcription of the GAL genes was dramatically elevated by deleting the HXK2 gene, whereas the expressions of hexokinase genes were not significantly affected. We concluded that the HXK2-deleted strain is able to efficiently utilize both glucose and galactose in the oxygen-limited conditions by alleviating catabolite-repression.

Proteomic Analysis of Bifidobacterium longum subsp. infantis Reveals the Metabolic Insight on Consumption of Prebiotics and Host Glycans

Bifidobacterium longum subsp. infantis is a common member of the intestinal microbiota in breast-fed infants and capable of metabolizing human milk oligosaccharides (HMO). To investigate the bacterial response to different prebiotics, we analyzed both cell wall associated and whole cell proteins in B. infantis. Proteins were identified by LC-MS/MS followed by comparative proteomics to deduce the protein localization within the cell. Enzymes involved in the metabolism of lactose, glucose, galactooligosaccharides, fructooligosaccharides and HMO were constitutively expressed exhibiting less than two-fold change regardless of the sugar used. In contrast, enzymes in N-Acetylglucosamine and sucrose catabolism were induced by HMO and fructans, respectively. Galactose-metabolizing enzymes phosphoglucomutase, UDP-glucose 4-epimerase and UTP glucose-1-P uridylytransferase were expressed constitutively, while galactokinase and galactose-1-phosphate uridylyltransferase, increased their expression three fold when HMO and lactose were used as substrates for cell growth. Cell wall-associated proteomics also revealed ATP-dependent sugar transport systems associated with consumption of different prebiotics. In addition, the expression of 16 glycosyl hydrolases revealed the complete metabolic route for each substrate. Mucin, which possesses O-glycans that are structurally similar to HMO did not induced the expression of transport proteins, hydrolysis or sugar metabolic pathway indicating B. infantis do not utilize these glycoconjugates.

Newborn screening for galactosemia by a second‐tier multiplex enzyme assay using UPLC‐MS/MS in dried blood spots

Galactosemia is one of the most important inherited metabolic disorders detected by newborn screening tests. Abnormal results during screening should be confirmed by enzyme activity assays. Recently, we developed a multiplex enzyme assay for galactosemia in erythrocytes using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). In this study, we proposed a second-tier multiplex enzyme assay for galactosemia that can be directly applied to dried blood spots (DBSs). Supernatants from two rehydrated-punched 3.2-mm DBSs were incubated with a reaction mixture containing [¹³C6]galactose, [¹³C2]galactose-1-phosphate, and UDP-glucose as substrates for three galactose-metabolizing enzymes. After a 4-hour incubation, the end products from the combined reaction mixture, [¹³C6]galactose-1-phosphate, UDP-[¹³C2]galactose, and UDP-galactose, were simultaneously measured using UPLC-MS/MS. Substrates, products, and internal standards from the mixture of the three enzyme reactions were clearly separated in the UPLC-MS/MS system, with an injection cycle time of 10 min. Intra- and inter-assay imprecisions of the UPLC-MS/MS were 8.4-14.8% and 13.2-15.7% CV, respectively. Enzyme activities in DBSs from 37 normal individuals and 10 patients with enzyme deficiencies were analyzed. DBSs from galactosemia patients showed consistently lower enzyme activities as compared to those of normal individuals. In conclusion, multiplex enzyme assays using UPLC-MS/MS can be successfully applied to DBS analysis. This method allows a fast and effective second-tier test for newborns showing abnormal screening results.

Multiplex Enzyme Assay for Galactosemia Using Ultraperformance Liquid Chromatography–Tandem Mass Spectrometry

Galactosemia is one of the most important inherited disorders detected by newborn screening tests. Abnormal results in screening tests should be confirmed by enzyme activity assays, but existing methods are time and labor intensive. We developed a novel multiplex enzyme assay for galactosemia using ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). [(13)C6]-galactose, [(13)C2]-galactose-1-phosphate, and UDP-glucose were used as substrates for 3 galactose-metabolizing enzymes. The end products from the combined reaction mixtures, [(13)C6]-galactose-1-phosphate, UDP-[(13)C2]-galactose, and UDP-galactose, were simultaneously measured using UPLC-MS/MS. Linearity, imprecision, ion suppression, and the effects of substrate were evaluated to determine assay performance. Enzyme activities from 35 healthy individuals, 8 patients with enzyme deficiency, and 18 mutant cells were analyzed. Substrates, products, and internal standards from the mixture of 3 enzyme reactions were clearly separated by using UPLC-MS/MS, with an injection cycle time of 10 min. Ion suppression was 0.1%-2.5%, the interassay imprecision of UPLC-MS/MS was 3.3%-10.6% CV, and the linearity of each system was good (R(2) = 0.994-0.999). Patient samples and mutated cells showed consistently low enzyme activities compared with those of normal individuals and wild-type cells. This method allows for a high-throughput and reproducible multiplex enzyme assay for galactosemia in erythrocytes.

Systems biology of GAL regulon in Saccharomyces cerevisiae

Evolutionary success of an organism depends on its ability to express or adapt to constantly changing environmental conditions. Saccharomyces cerevisiae has evolved an elaborate genetic circuit to regulate the expression of galactose-metabolizing enzymes in the presence of galactose but in the absence of glucose. The circuit possesses molecular mechanisms such as multiple binding sites, cooperativity, autoregulation, nucleocytoplasmic shuttling, and substrate sensing mechanism. Furthermore, the GAL system consists of two positive (activating) feedback and one negative (repressing) feedback loops. These individual mechanisms, elucidated through experimental approach, can be integrated to obtain a system-wide behavior. Mathematical models in conjunction with guided experiments have demonstrated system-level properties such as ultrasensitivity, memory, noise attenuation, rapid response, and sensitive response arising out of the molecular interactions. These system-level properties allow S. cerevisiae to adapt and grow in a galactose medium under noisy and changing environments. This review focuses on system-level models and properties of the GAL regulon.

Overexpression of the aldose reductaseGRE3 suppresses lithium-induced galactose toxicity inSaccharomyces cerevisiae

In Saccharomyces cerevisiae, lithium induces a 'galactosemia-like' phenotype as a consequence of inhibition of phosphoglucomutase, a key enzyme in galactose metabolism. Induced galactose toxicity is prevented by deletion of GAL4, which inhibits the transcriptional activation of genes involved in galactose metabolism and by deletion of the galactokinase (GAL1), indicating that galactose-1-phosphate, a phosphorylated intermediate of the Leloir pathway, is the toxic compound. As an alternative to inhibiting entry and metabolism of galactose, we investigated whether deviation of galactose metabolism from the Leloir pathway would also overcome the galactosemic effect of lithium. We show that cells overexpressing the aldose reductase GRE3, which converts galactose to galactitol, are more tolerant to lithium than wild-type cells when grown in galactose medium and they accumulate more galactitol and less galactose-1-phosphate. Overexpression of GRE3 also suppressed the galactose growth defect of the 'galactosemic'gal7- and gal10-deleted strains, which lack galactose-1-P-uridyltransferase or UDP-galactose-4-epimerase activities, respectively. Furthermore, the effect of GRE3 was independent of the inositol monophosphatases INM1 and INM2. We propose that lithium induces a galactosemic state in yeast and that inhibition of the Leloir pathway before the phosphorylation step or stimulation of galactitol production suppresses lithium-induced galactose toxicity.

Regulating the Regulon

In the presence of galactose, the yeast Saccharomyces cerevisiae transports the sugar into its cytoplasm, where the galactose binds the Gal3 protein, which in turn sequesters the Gal80 repressor away from the transcriptional activator Gal4, resulting in the induction of galactose-metabolizing enzymes. The induction is so rapid that it has been suggested that another unknown factor must somehow be involved in this "galactose regulon." Kumar et al. have identified this factor as nicotinamide adenine dinucleotide (NAD) and its phosphate (NADP). In the Gal4:Gal80 complex, NAD is sandwiched between the two proteins. NADP, on the other hand, inhibits the interaction between Gal4 and Gal80. Mutations that have the potential to affect the NAD-binding site in Gal80 result in even faster induction, which suggests that Gal80 is sensing the balance between NAD and NADP and thus the metabolic state of the cell. P. R. Kumar, Y. Yu, R. Sternglanz, S. A. Johnston, L. Joshua-Tor, NADP regulates the yeast GAL induction system. Science 319 , 1090-1092 (2008). [Abstract] [Full Text]

The key enzyme in galactose metabolism, UDP-galactose-4-epimerase, affects cell-wall integrity and morphology in Candida albicans even in the absence of galactose

The enzyme UDP-galactose-4-epimerase ( GAL10) catalyzes a key step in galactose metabolism converting UDP-galactose to UDP-glucose which then can get metabolized through glycolysis and TCA cycle thus allowing the cell to use galactose as a carbon and energy source. As in many fungi, a functional homolog of GAL10 exists in Candida albicans. The domainal organization of the homologs from Saccharomyces cerevisiae and C. albicans show high degree of homology having both mutarotase and an epimerase domain. The former is responsible for the conversion of β- d-galactose to α- d-galactose and the latter for epimerization of UDP-galactose to UDP-glucose. Absence of C. albicans GAL10 (Ca GAL10) affects cell-wall organization, oxidative stress response, biofilm formation and filamentation. Cagal10 mutant cells tend to flocculate extensively as compared to the wild-type cells. The excessive filamentation in this mutant is reflected in its irregular and wrinkled colony morphology. Cagal10 strain is more susceptible to oxidative stress when tested in presence of H 2O 2. While the S. cerevisiae GAL10 (Sc GAL10), essential for survival in the presence of galactose, has not been reported to have defects in the absence of galactose, the C. albicans homolog shows these phenotypes during growth in the absence of galactose. Thus a functional CaGal10 is required not only for galactose metabolism but also for normal hyphal morphogenesis, colony morphology, maintenance of cell-wall integrity and for resistance to oxidative stress even in the absence of galactose.

Two familial cases of high blood galactose of unknown aetiology

We report two male siblings presenting as newborns with increased blood galactose, urinary excretion of galactitol, and normal galactose 1-phosphate on a breast milk diet. A lactose-free diet led to normalization of all metabolites, while reintroduction of galactose in the diet resulted in an accumulation of metabolites. Potential causes of galactosaemia include: (1) activities of three enzymes of galactose metabolism: galactokinase (GALK), galactose-1-phosphate uridyltransferase (GALT), and uridine diphosphate galactose 4'-epimerase (GALE), (2) portosystemic shunting, (3) Fanconi-Bickel syndrome, (4) tyrosinaemia. Each was excluded with appropriate tests. These two familial cases may represent a novel autosomal or X-linked recessive disorder of galactose metabolism, possibly due to a novel defect in the transport of galactose across the plasma membrane.

Galactosyl‐sucrose metabolism and UDP‐galactose pyrophosphorylase from Cucumis melo L. fruit

In muskmelon (Cucumis melo L.), sink tissues receive stachyose, raffinose and sucrose through phloem translocation of carbohydrates that are formed as products of leaf photosynthesis. Melon fruits accumulate sucrose massively during the final stages of maturation. This sucrose is derived partially from the catabolism of raffinose saccharides. Rapid galactose metabolism is required, because liberation of free galactose is the first step in the metabolic utilization of the raffinose sugars. The current study demonstrates that the enzyme UDP‐glucose‐hexose‐1‐P uridylyltransferase (EC 2.7.7.12), the central enzyme in the classical Lelior pathway, is not the central enzyme in galactose metabolism in muskmelon fruit. Rather, a broad substrate specificity UDP‐galactose pyrophosphorylase (PPase) serves the same functional role. This enzyme accepts either UDP‐galactose or UDP‐glucose as a substrate and is different from a UDP‐glucose PPase with more strict substrate specificity for UDP‐glucose that is also present in melon tissue. UDP‐galactose PPase was purified 113‐fold from melon tissue and was shown to be a 54 kDa (size exclusion chromatography) to 68 kDa (SDS‐PAGE) protein that is enzymatically active as a monomer. We also present evidence that the enzyme likely accepts UDP‐galactose and UDP‐glucose at the same catalytic site. Polyclonal antibodies prepared against this protein reacted with numerous other antigens in melon extracts, apparently as a result of the presence of common antigenic epitopes.

Galactose-dependent reversible interaction of Gal3p with Gal80p in the induction pathway of Gal4p-activated genes of Saccharomyces cerevisiae.

When galactose is added to logarithmically growing culture of the yeast Saccharomyces cerevisiae, a set of genes encoding galactose-metabolizing enzymes (GAL genes) starts to be transcribed within a few minutes. This rapid induction involves a serial interplay of Gal3p, Gal80p, and Gal4p. Recent experiments have indicated that a direct interaction between Gal3p and Gal80p plays a pivotal role in an early step of GAL induction. Here we demonstrate that complex of Gal3p and Gal80p, otherwise unstable, is stabilized in the presence of 0.1 mM galactose and 0.5 mM ATP. The requirement for galactose and ATP for stable complex formation is also observed by using highly purified Gal3p and Gal80p from yeast. We further show that thus formed Gal3p/Gal80p complex can easily be dissociated when it is washed with buffer lacking galactose. Finally, we show that mutant proteins encoded by GAL80S or GAL80DE21, which confer galactose-uninducible phenotype, fail to interact with Gal3p. These results strongly suggest that Gal3p functions as the sensor and transducer of galactose signal in the induction pathway of Gal4p-activated genes.

Metabolic effects of galactose on human HepG2 hepatoblastoma cells

HepG2 cells were used as a model system to study the effects of galactose overload on the liver, a target organ of galactose toxicity in patients suffering from transferase-deficient galactosemia. In the presence of galactose, HepG2 cell growth was slow and the pattern of gene expression remained characteristic of liver cells (secretion of α-fetoprotein [AFP] albumin, and transferrin). Galactose-1-phosphate (Gal-1-P) accumulated, as it does in galactosemic cells, but did not affect the energetic status of the cells (no adenosine triphosphate [ATP] depletion). However, the substitution of galactose for glucose as the sole hexose in the medium affected the specific activities of the galactose-metabolizing enzymes. Galactokinase (GALK) activity was decreased, and those of galactose-1-phosphate uridyltransferase (GALT), phosphoglucomutase, and glucose-6-phosphate dehydrogenase (G6PDH) were increased. The conversion of radiolabeled galactose to glucose (CO 2 production and glycogen level) was greater in galactose medium than in glucose medium after a 7-day culture. Therefore, the culture of HepG2 cells in galactose medium indicates that the enhanced utilization of this hexose is due to the increased enzyme activities regulating its own metabolism. Hence, HepG2 cells constitute a good model for the study of modulation of galactose-metabolizing enzymes by galactose.

Studies of DNA in galactose-1-phosphate uridyltransferase deficiency and the Duarte variant in Germany.

Classical galactosaemia is caused by deficiency of galactose-l-phosphate uridyltransferase (GALT; McKusick 230400) with an incidence of 1 : 40 000 to 1 : 60 000 in Caucasian populations. The clinical symptoms of untreated newborns include hepatomegaly, jaundice, vomiting, ataxia and cataracts. GALT is a key enzyme in galactose metabolism because it interconverts galactose 1-phosphate to UDPgalactose. This enzyme is polymorphic (Shin et al 1987). The most common variant, the Duarte variant was found to exist as two different types, the Duarte-1 (D1) with about 100-110% of the enzyme activity of the normal (N) allele and the Duarte-2 (D2) with about 35-45%. Compound heterozygotes D2-G, with about 17-23% of normal activity, therefore show increased galactose metabolites during the newborn period. We have recently observed that the other phenotypes such as D2-D2, D1 G and N-G can be detected through newborn screening programmes. Recently Leslie and coworkers (1992) have characterized the human GALT gene and various disease-causing mutations and polymorphisms have been identified (Reichhardt 1992). The most common galactosaemia mutation was reported to be Q188R (Reichardt and Woo 1991; Leslie et al 1992), which accounts for more than 60% among Caucasians. Recent studies (Lin et al, in press) have revealed a close association between the Duarte variant of GALT and the N314D polymorphism. We report here nucleic acid screening for Q188R, R333W and N314D in 54 galactosaemic patients and 29 Duarte variants in Germany.

Selection and Analysis of Galactose Metabolic Pathway Variants of a Mouse Liver Cell Line

To study the genetic expression and regulation of galactose-metabolizing enzymes, we mutagenized the mouse liver H2.35 cell line and selected for cell clones resistant to the toxic galactose analog, 2-deoxy-D-galactose (2-DOG). One cloned line, designated H12.10, was stably resistant to high levels of 2-DOG and was completely deficient in galactokinase activity. Galactokinase activity and growth sensitivity to 2-DOG could be restored by transfecting H12.10 cells with a plasmid containing the Escherichia coli galactokinase (galK) gene fused to a eucaryotic promoter; thus, the 2-DOG selection could be directed against transfected recombinant constructs in a liver cell line. We also found that H2.35 cells could not utilize galactose as a primary carbon source because of a deficiency in galactose-1-phosphate uridyltransferase; a variant line of H2.35 cells selected in galactose medium expressed higher levels of uridyltransferase activity. Finally, we found that in all mammalian cell lines tested, galactokinase expression was the same whether the medium contained glucose, galactose, or both sugars. These studies demonstrate differences between mammalian cells and yeast cells in the regulation of gal enzymes, and they define different schemes for obtaining altered expression of genes in the galactose metabolic pathway. The isogenic liver cell lines described here can also serve as model systems for studying galactosemias, which are inherited disorders of galactose metabolism in humans.

Selection and analysis of galactose metabolic pathway variants of a mouse liver cell line.

To study the genetic expression and regulation of galactose-metabolizing enzymes, we mutagenized the mouse liver H2.35 cell line and selected for cell clones resistant to the toxic galactose analog, 2-deoxy-D-galactose (2-DOG). One cloned line, designated H12.10, was stably resistant to high levels of 2-DOG and was completely deficient in galactokinase activity. Galactokinase activity and growth sensitivity to 2-DOG could be restored by transfecting H12.10 cells with a plasmid containing the Escherichia coli galactokinase (galK) gene fused to a eucaryotic promoter; thus, the 2-DOG selection could be directed against transfected recombinant constructs in a liver cell line. We also found that H2.35 cells could not utilize galactose as a primary carbon source because of a deficiency in galactose-1-phosphate uridyltransferase; a variant line of H2.35 cells selected in galactose medium expressed higher levels of uridyltransferase activity. Finally, we found that in all mammalian cell lines tested, galactokinase expression was the same whether the medium contained glucose, galactose, or both sugars. These studies demonstrate differences between mammalian cells and yeast cells in the regulation of gal enzymes, and they define different schemes for obtaining altered expression of genes in the galactose metabolic pathway. The isogenic liver cell lines described here can also serve as model systems for studying galactosemias, which are inherited disorders of galactose metabolism in humans.

GAL4 transcription factor is not a "zinc finger" but forms a Zn(II)2Cys6 binuclear cluster.

The DNA-binding domain of the transcription factor GAL4, consisting of the 62 N-terminal residues and denoted GAL4(62*), contains a Cys-Xaa2-Cys-Xaa6-Cys-Xaa6-Cys-Xaa2-Cys-Xaa6+ ++-Cys motif, which has been shown previously to bind two Zn(II) or Cd(II) ions. Binding of Zn(II) or Cd(II) is essential for the recognition by GAL4 of the specific palindromic DNA sequence to which it binds upstream of genes for galactose-metabolizing enzymes, the UASG sequence. On the basis of the 113Cd NMR chemical shifts of the two bound 113Cd(II) ions, we propose a binuclear cluster model for this Zn(II)-binding subdomain. 1H-113Cd heteronuclear multiple-quantum NMR spectroscopy and phase-sensitive double-quantum filtered 1H correlation spectroscopy of the 112Cd(II)- and 113Cd(II)-substituted GAL4(62*) derivatives provide direct evidence that the two bound 113Cd(II) ions are coordinated only by the six cysteine residues, two of which form bridging ligands between the two 113Cd(II) ions. The latter can be identified from the pattern of 1H-113Cd J coupling. Thus a binuclear metal ion cluster rather than a "zinc finger" is formed by the six cysteine residues of the GAL4 DNA-binding domain. This model can be directly applied to eight other fungal transcription factors which have been shown to contain similarly spaced Cys6 clusters. 1H NMR spectra of apo-GAL4(62*) suggest conformational fluctuation of the metal-binding subdomain upon removal of Zn(II) or Cd(II). Both Cd(II)2- and Zn(II)2-containing species of GAL4 can be formed, and the similar 1H NMR spectra suggest similar conformations.

Galactose as a regulatory factor of its own metabolism by rat liver

As part of a series of studies to assess the regulation of hepatic galactose-metabolizing enzymes, galactokinase, galactose-1-phosphate uridyltransferase, and UDPgalactose-4-epimerase, the effect of feeding a high galactose-containing diet to normal adult and pregnant female rats was examined. Sixteen days of galactose exposure of adult virgin females produced a different response in the specific activity of each of the enzymes, that of galactokinase being lower, transferase higher, and epimerase transiently elevated. Galactose feeding increased the specific activity of transferase in pregnant rat liver above the elevated level that already exists in the pregnant state but failed to influence the enzyme of the developing fetal liver. Galactose added to liver homogenates did not activate transferase. The increased activity in liver of adult, fed animals was not associated with a change in isoenzyme patterns examined by isoelectric gel electrophoresis but was characterized on kinetic analysis by an increase in Vmax for UDPglucose. The changes in enzyme specific activity in liver of animals fed galactose appear to have physiologic significance because hepatocytes isolated from galactose exposed livers take up more galactose and convert more to glucose and lactate than cells from control animals.