Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Galactose-1-phosphate uridyltransferase (GALT) is expressed in most tissues, but the near total absence of catalytic activity in humans with the disease galactosemia leads to specific organ dysfunction, the pathophysiology of which remains an enigma. To characterize the transcriptional regulation of the mouse GALT gene, we isolated and sequenced over 3 kb of a 5'-flanking sequence and functionally characterized the region using in vitro transient transfection and in transgenic mice. A minimal promoter of 145 bp was found to function in both HepG2 cells and NS20Y mouse neuroblastoma cells. The minimal promoter contains regions of homology to the corresponding rat and human GALT genes. In transgenic mice expressing a luciferase transgene under control of a 1.9-kb fragment of the mGALT promoter region, reporter activity was found in most tissues, with higher than expected reporter levels in neonatal brain. To determine if high galactose levels in tissues could induce promoter activity, we bred the mGALT:luciferase transgene into a line of mice in which the GALT gene function has been eliminated by homologous recombination. High tissue levels of galactose and metabolites did not induce reporter activity above background. The studies show that GALT transcriptional regulation is complex and not directly induced by substrate levels.
Mol Genet Metab 2001 Jan
PMID:Functional analysis of the mouse galactose-1-phosphate uridyl transferase (GALT)promoter. 1116 26

Human galactose-1-phosphate uridyltransferase (hGALT) is an evolutionarily conserved enzyme central to D-galactose metabolism. The impairment of hGALT causes galactosemia. One missense mutation, an aspartate to asparagine substitution at amino acid 314 (N314D), impairs 50% activity in the homozygous state in some patients but gives near normal activity in others. The former condition is called Duarte (D) and the latter, Los Angeles (LA). The D allele is linked to hGALT polymorphisms including a deletion 5'to the translation start site (-119 to -116delGTCA), g1391G --> A and g1105G --> C. The LA allele is linked to a g1721C --> T transition. To investigate possible mechanisms for differences in hGALT activity between the D and LA alleles, we sequenced 3951 nucleotides of genomic DNA 5' to the hGALT translation start site. Using a dual-luciferase reporter system to express deletion constructs of the hGALT promoter, we noted both positive and negative regulatory regions. Two putative positive regulatory domains overlap with the naturally occurring -119 to -116delGTCA linked to Duarte. One is an E-box motif (CACGTG) at -117 to -112 bp. The second is an AP-1 motif (TCAGTCAG) at -124 to -119 bp. The delGTCA mutation confers reduced luciferase activity to transfected cell lines derived from human ovarian and liver neoplasms. Additionally, human lymphoblasts derived from patients with the Duarte allele have reduced GALT mRNA. We conclude that the human GALT gene is regulated in the first -165 bp of its promoter region by positive regulators of GALT gene expression. The -119 to -116delGTCA reduces hGALT transcription resulting in reduced GALT activity in the Duarte allele.
Mol Genet Metab 2001 Apr
PMID:Functional analysis of the human galactose-1-phosphate uridyltransferase promoter in Duarte and LA variant galactosemia. 1128 3

Mice deficient in galactose-1-phosphate uridyltransferase (GALT) demonstrate abnormal galactose metabolism but no obvious clinical phenotype. To further dissect the pathways of galactose metabolism in these animals, galactose oxidation and metabolite levels were studied in 16-day-old sucklings and the effect of a 4 week prior exposure to a 40% glucose or 40% galactose diet was determined in 7-week-old mice. Suckling GALT-deficient (G/G) mice slowly oxidized [1-14C]galactose to 14CO2, 4.0% of the dose when fed and 7.9% when fasted compared to normal animals 38.3 and 36.4% in 4 h, respectively. Plasma of G/G sucklings contained 11.1 mM galactose and erythrocyte galactose 1-phosphate levels were 28.2 and 31.9 mg/dl packed cells. Galactose, galactitol, galactonate, and galactose 1-phosphate were found in G/G suckling mouse tissues. The tissue galactose concentrations were 10% or less of that in plasma, suggesting that there was limited cellular entry of galactose. In 7-week-old fasted mice with 4 weeks prior exposure to glucose or galactose-containing diet, 4-h oxidation was 12.9 and 15.0% of the administered radiolabeled galactose, respectively. Normal animals oxidized 33.9 and 37.9% of the dose when fed the same diets, respectively. The ability of G/G mice to oxidize galactose in the absence of GALT activity suggests the presence of alternate metabolic pathways for galactose disposition. G/G mice fed the galactose-free 40% glucose diet had erythrocyte galactose 1-phosphate levels ranging from 6.4 to 17.7 mg/dl packed cells and detectable galactose and galactose metabolites in tissues, suggesting that these animals endogenously produced galactose. The plasma of 40% galactose-fed G/G mice contained 9.1 mM galactose with red blood cell galactose 1-phosphate averaging 43.6 mg/dl. Tissues of these animals also contained high levels of galactose and galactose 1-phosphate. Liver contained over 4 micromol/g galactonate but little galactitol. Despite the elevated galactose and galactose 1-phosphate, the animals tolerated the high-galactose diet and were indistinguishable from normal animals, exhibiting no manifestations of galactose toxicity seen in human GALT-deficient galactosemia. The data suggest that high galactose 1-phosphate levels do not cause galactose toxicity and that high galactitol in combination with galactose 1-phosphate may be a prerequisite. Absence of GALT appears necessary but insufficient to produce human galactosemic phenotype.
Mol Genet Metab 2001 Apr
PMID:Galactose metabolism in mice with galactose-1-phosphate uridyltransferase deficiency: sucklings and 7-week-old animals fed a high-galactose diet. 1128 4

The persistent, dietary-independent elevation of galactose metabolites in patients with galactose-1-phosphate uridyltransferase (GALT) deficiency is probably secondary to de novo synthesis of galactose. Relatively constant steady-state levels of galactose metabolites in patients also suggest that non-GALT metabolic pathways must function to dispose of the galactose synthesized each day. The discovery of a patient with a rare deletion of the GALT gene provided a unique opportunity to examine the availability of any alternate galactose oxidative capacity both in vivo and in vitro. Utilizing genomic DNA from the patient, Southern blot data demonstrated that 10 of the 11 GALT exons were homozygously deleted. By measurement of 13CO2 in expired air for up to 24 h after an oral bolus of [1-13C]galactose, it was demonstrated that 17% of the galactose was metabolized, a value comparable to the 3-h elimination rate in a control subject. Furthermore, lymphoblasts prepared from the patient could also convert [1-14C]galactose to 14CO2. This unique study provides the first unambiguous evidence that another pathway exists in man that can be responsible for galactose disposal. Further knowledge of this alternate galactose oxidative route and its regulation may aid in formulating new strategies for the treatment of galactosemia.
Mol Genet Metab 2001 Apr
PMID:Evidence for alternate galactose oxidation in a patient with deletion of the galactose-1-phosphate uridyltransferase gene. 1128 5

We previously identified a missense mutation at amino acid 135 of human galactose 1-phosphate uridyltransferase (hGALT) in which a leucine (TTG) was substituted for a serine (TCG), S135L. This mutation was common in black patients with galactosemia and homozygotes (S135L/S135L) had no GALT activity or protein in their erythrocytes or lymphoblasts. However, there was residual GALT activity and protein in their leukocytes, and they had near normal total body [13C]galactose oxidation to 13CO2 in breath. To evaluate the biochemical mechanism(s) producing these effects, we overexpressed hGALT proteins with site-directed mutations in this nonconserved amino acid in a GALT-minus Escherichia coli. Enzyme activities detected in bacterial lysates overexpressing either S135 (wild type), A135, C135, H135, L135, S132-H135, T135, or Y135 were 100, 4.7, 3.0, 4.0, 2.7, 0.7, 35.4, and 1.4%, respectively. Only the threonine substitution (S135T) had significant enzyme activity in these lysates. There was also decreased abundance of all mutant proteins in the lysates exposed to bacterial proteolysis during preparation and analysis. This added the variable of bio-instability to analysis of enzyme activities in lysates. To further characterize the catalytic role of serine at amino acid 135 and to differentiate bio-instability from impaired catalysis by the leucine substitution, we purified wild-type and L135-hGALT proteins to homogeneity and analyzed identical amounts of enzyme protein. We found that the apparent Vmax of the purified L135-hGALT protein was significantly reduced from 80 +/- 5.9 to 5.8 +/- 1.8 micromol glucose 1-phosphate released/min/mg hGALT protein with no increase in KM for galactose 1-phosphate for the second displacement. The first displacement reaction, although three orders of magnitude slower, was similar between the wild type and L135-hGALT. We conclude that a hydroxyl group on amino acid 135 is required for the catalysis of uridyl transfer from UDP-glucose to UDP-galactose in the presence of galactose 1-phosphate, and plays a role in the bio-stability of hGALT.
Mol Genet Metab
PMID:Structure-function analyses of a common mutation in blacks with transferase-deficiency galactosemia. 1159 23

Impairment of the human enzyme galactose-1-P uridylyltransferase (hGALT) results in the potentially lethal disorder, galactosemia. One of the fundamental questions with regard to this dimeric enzyme involves the possible influence of patient mutations on heterodimer formation and activity. Indeed, considering that many if not most galactosemia patients are compound heterozygotes, this is an issue of clinical as well as basic science interest. We have utilized a yeast expression system for the human enzyme to test whether each of a small number of mutations in hGALT (S135L, F171S, F171W, Q188R, N314D, and R333W) impact either heterodimer formation or function. Our results clearly demonstrate that while a majority of the alleles tested show precisely random patterns of subunit assortment, two deviate slightly but significantly from this pattern. Similarly, while some heterodimers exhibit apparent independence of subunit activity, others do not. These data not only demonstrate that common patient mutations in hGALT can influence both heterodimer formation and function in heterozygotes, they further raise the question of whether such interactions may also occur between different mutant alleles in compound heterozygotes (i.e., patients). Indeed, such influences may underlie some of the biochemical and clinical heterogeneity observed in the galactosemia patient population.
Mol Genet Metab 2002 Aug
PMID:Impact of patient mutations on heterodimer formation and function in human galactose-1-P uridylyltransferase. 1220 37

Classical galactosemia is a genetic disease caused by mutations in the galactose-1-phosphate uridyl transferase (GALT) gene. Prospective newborn screening for galactosemia is routine and utilizes the universally collected newborn dried blood specimen on filter paper. Screening for galactosemia is achieved through analysis of total galactose (galactose and galactose-1-phosphate) and/or determining the activity of the GALT enzyme. While this approach is effective, environmental factors and the high frequency of the Duarte D2 mutation (N314D) does lead to false positive results. Using DNA derived from the original newborn dried blood specimen and Light Cycler technology a panel of five assays able to detect the four most frequently encountered classical galactosemia alleles (Q188R, S135L, K285N, and L195P) and the N314D Duarte variant mutation are described. The five assays are performed simultaneously using common conditions. Including DNA preparation, set-up, amplification, and analysis the genotype data for all five loci is obtained in less than 2 hours. The assays are easily interpreted and amenable to high-throughput newborn screening. Mutational analysis is useful to reduce false positive results, differentiate D/G mixed heterozygotes from classical galactosemia, and to clearly identify a very high percentage of those affected by classical galactosemia.
J Mol Diagn 2003 Feb
PMID:Analysis of common mutations in the galactose-1-phosphate uridyl transferase gene: new assays to increase the sensitivity and specificity of newborn screening for galactosemia. 1255 79

The red blood cell (RBC) concentration of galactitol and galactonate was measured in 27 patients with galactose-1-phosphate uridyltransferase (GALT) deficiency galactosemia and 19 non-galactosemic subjects by a newly devised isotope dilution gas chromatography/mass spectrometry (GC/MS) method. The method utilizing UL[13C]galactitol and UL[13C]galactonate was reproducible with excellent precision and recovery of 99%. The RBC galactitol in galactosemic patients on galactose-restricted diets averaged 5.98+/-1.2 microM (M+/-SD) with a range of 3.54-8.81 microM. The mean in non-galactosemic patients was 0.73+/-0.31 microM with a range of 0.29-1.29 microM. The mean of RBC galactonate in the same galactosemic patients was 4.16+/-1.32 microM (M+/-SD) with a range of 0.68-6.47, while the mean in non-galactosemic subjects was 1.94+/-0.96 (M+/-SD) with a range of 0.69-3.84. In galactosemic RBC the galactitol was higher than galactonate while this was reversed in non-galactosemic cells. RBC galactose-1-phosphate (Gal-1-P) measured at the same time as galactitol and galactonate was 30 times the level of the other two metabolites. There was no relationship between RBC Gal-1-P and galactitol or galactonate. The ability to measure all three galactose metabolites in the same procedure offers the possibility of augmented monitoring of the galactose metabolic status of patients. The measurement of RBC galactitol and galactonate presents a new means of characterizing galactosemic patients and their levels monitored over time may provide new insight in the development of long-term complications observed in afflicted patients.
Mol Genet Metab 2003 Nov
PMID:Galactitol and galactonate in red blood cells of galactosemic patients. 1468 Sep 73

Using both a continuous infusion of isotopically labeled [1-13C]galactose with a steady-state analysis and a single injection kinetic approach, we have calculated the apparent galactose appearance rate (GAR) in patients with galactose-1-phosphate uridyltransferase deficiency and control subjects. With the steady-state protocol, the GAR in 18 patients less than 18 years of age was 1.34+/-0.53 mg/kg/h (mean+/-SD) and was significantly greater than the mean of 0.56+/-0.01 mg/kg/h (p=0.004) in five patients above 18 years of age. Patients who were given a priming dose of [1-13C]galactose had a reduced GAR compared to those without a priming dose, 0.73+/-0.05 (n=9) vs 1.46+/-0.62 (n=14)mg/kg/h (p=0.005). The GAR in controls was lower than in patients ranging from 0.58 to 0.68 mg/kg/h in children and 0.07-0.09 mg/kg/h in adults. In the single bolus studies the plasma [13C]galactose enrichment decreased in a biexponential pattern suggesting at least a two-compartment system. The calculated GAR in three adult patients was similar to that found in them by the continuous infusion technique. The GAR in patients suggests the source of galactose for the continued elevation of galactose metabolites as well as the basis for the long-term complications in galactosemia despite restricted dietary galactose intake.
Mol Genet Metab 2004 Jan
PMID:The rate of de novo galactose synthesis in patients with galactose-1-phosphate uridyltransferase deficiency. 1472 88

Galactokinase (GalK) catalyses the first step of the Leloir pathway of galactose metabolism, the ATP-dependent phosphorylation of galactose to galactose-1-phosphate. In man, defects in galactose metabolism can result in disorders with severe clinical consequences, and deficiencies in galactokinase have been linked with the development of cataracts within the first few months of life. The crystal structure of GalK from Pyrococcus furiosus in complex with MgADP and galactose has been determined to 2.9 A resolution to provide insights into the substrate specificity and catalytic mechanism of the enzyme. The structure consists of two domains with the active site in a cleft at the domain interface. Inspection of the substrate binding pocket identifies the amino acid residues involved in galactose and nucleotide binding and points to both structural and mechanistic similarities with other enzymes of the GHMP kinase superfamily to which GalK belongs. Comparison of the sequence of the Gal3p inducer protein, which is related to GalK and which forms part of the transcriptional activation of the GAL gene cluster in the yeast Saccharomyces cerevisiae, has led to an understanding of the molecular basis of galactose and nucleotide recognition. Finally, the structure has enabled us to further our understanding on the functional consequences of mutations in human GalK which cause galactosemia.
J Mol Biol 2004 Mar 19
PMID:Substrate specificity and mechanism from the structure of Pyrococcus furiosus galactokinase. 1500 54


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