Improved technique for electrophoresis of human galactose-1-P uridyl transferase (EC 2.7.7.12)

Summary A newly developed electrophoretic technique for human galactose-1-phosphate uridyl transferase confirms the multiple band patterns for the Duarte and Los Angeles variants. This represents the first confirmation for the Los Angeles variant. The observed frequencies of N, D, and LA types are similar to earlier reports for these variants.     

Probable linkage between the human galactose-1-P uridyl transferase locus and 9qh.

Evaluation of a family in which electrophoretic variants of the eznyme galactose-1-phosphate uridyl transferase (GALT) and 9qh variants occur demonstrates close linkage between these two traits: lod score of 3.67 at theta = 0. Taken with information indicating GALT is on the short arm of chromosome 9, these linkage data suggest that this locus is close to the centromere on the short arm of chromosome 9.     

Galactose metabolism in relation to cataract formation in marsupials.

Erythrocytic galactokinase and/or galactose-1-phosphate uridyl transferase activity were low in many species of marsupials. However, cataract formation was observed only in pouch-young members of these species when reared on cow's milk. The galactose tolerance of young kangaroos was found to be greatly impaired, but improved rapidly and markedly at the stage of which the definitive structure of the ruminant type of stomach as in adults is formed. The combination of high absorption of galactose and low levels of galactokinase and/or transferase thus appears to determine the predisposition of pouch-young marsupials to galactose cataractogenesis.     

Galactose-1 phosphate uridyl transferase deficiency in the western grey kangaroo (Macropus fuliginosus; marsupialia): a model system for gene therapy studies.

The western grey kangaroo (Macropus fuliginosus) was found to be deficient for galactose-1 phosphate uridyl transferase (GPUT). This species could be used therefore for studies of gene therapy techniques. An improved GPUT assay procedure was developed. It was found that phage particles injected intravenously remain in the blood of kangaroos until the particles are removed by the reticuloendothelial system or, if this system is overloaded, the particles are inactivated by the immune system four days later. No effective transgenosis was detected of the bacterial gal operon contained in the phage used.     

Polymorphism of human red cell galactose-1-phosphate uridyl transferase in Serbia, Yugoslavia

Phenotypes of human red cell galactose-1-phosphate uridyl transferase (GALT) were determined in 283 unrelated adults from Serbia (Yugoslavia). The gene frequencies were 0.959 for GALT N, 0.018 for GALT D and 0.023 for GALT N.     

The occurrence of the Leloir pathway in non-pathogenic mycobacteria

It has been found that saprophytic strains of mycobacteria can utilize D-galactose via the Leloir pathway which involves galactokinase, galactose-1-phosphate uridyl transferase and UDP-galactose-4-epimerase. The resulted glucose-1-phosphate is further converted by phosphoglucomutase to glucose-6-phosphate and the latter catabolized in glycolitic cycle to pyruvate. The particular enzymes of the galactose pathway have been fully separated by chromatography on a DEAE-cellulose column and some of them partially characterized.     

Molecular analysis of 11 galactosemia patients.

Galactosemia is a human inborn error of galactose metabolism due to deficiency of galactose-1-phosphate uridyl transferase. In this paper, I describe the molecular analysis of genomic DNA, mRNA and protein from 11 different galactosemic patients by Southern, Northern and Western blotting. The results of these experiments lead me to conclude that galactosemia is caused mostly by missense mutations. The unusual preponderance of missense mutations in galactosemia led me to investigate its cause. I demonstrate that all 9 patients I investigated have detectable residual enzyme activity (ranging from 0.7-6.9% of normal). This finding is of potential importance in addressing the long-term complications of galactosemia.     

Biochemistry and Genetics of Galactose Metabolism in Group H Streptococcus Strain Challis

Galactose-negative mutants of the group H Streptococcus strain Challis were obtained by treatment with nitrosoguanidine. Enzyme assays of extracts of these mutants revealed that 12 of the mutants were lacking one of the enzymes of the Leloir pathway. Thus, the Leloir pathway is the major means of galactose metabolism in strain Challis. In addition, uridyl diphosphate galactose pyrophosphorylase, a permease function, and at least one other function are required for the utilization of galactose. The enzymes of the Leloir pathway are induced by galactose and fucose; no compounds which act as repressors of these enzymes have been found, although the system appears to be sensitive to catabolite repression. Transformation was used to map the mutants. The genes for galactose-1-phosphate uridyl transferase and glucose-4-epimerase appear to be closely linked. Within the transferase gene, six mutations have been mapped. The permease function and the undetermined functions are not linked to the Leloir pathway.     

On the molecular nature of the Duarte variant of galactose-1-phosphate uridyl transferase (GALT)

Galactosemia is an inborn error of galactose metabolism secondary to deficiency of galactose-1-phosphate uridyl transferase (GALT). GALT is a polymorphic enzyme and Duarte (D) is the most common enzyme variant. This variant is characterized by faster electrophoretic mobility and reduced activity. Duarte/galactosemia compound heterozygotes (D/G) are commonly identified in galactosemia newborn screening programs. However, these patients do not generally require treatment. By using a candidate mutation approach to define the molecular basis of the Duarte variant of GALT, a close association between the previously reported N314D polymorphism and the Duarte variant of GALT was found. We suggest that N314D encodes the D variant of GALT and that molecular testing for N314D might be useful to confirm a biochemical diagnosis of Duarte variant of GALT.     

Effect of uridine on hepatic galactose-1-phosphate uridyltransferase

Uridine sugar nucleotides are important intermediates in galactose metabolism and may play a role in the long-term galactose toxicity in human galactose-1-phosphate uridyltransferase deficiency galactosemia. Since administration of uridine, a precursor of uridine nucleotides, has been considered as a therapeutic measure, we have investigated the effects of this compound on the activity of rat hepatic transferase. Uridine has been found to be an inhibitor of the enzyme in in vitro studies and to cause an increase in galactose-1-phosphate in liver perfused with galactose which is consistent with physiologic inhibition of the enzyme. Uridine is a partial linear competitive inhibitor of UDPglucose and an uncompetitive inhibitor of galactose-1-phosphate. These findings suggest caution should be applied in giving the compound to subjects with genetically limited transferase activity because of the possibility of inhibiting the small amount of residual enzyme.     

Molecular characterization of two galactosemia mutations: correlation of mutations with highly conserved domains in galactose-1-phosphate uridyl transferase.

Galactosemia is an autosomal recessive disorder of human galactose metabolism caused by deficiency of the enzyme galactose-1-phosphate uridyl transferase (GALT). The molecular basis of this disorder is at present not well understood. We report here two missense mutations which result in low or undetectable enzymatic activity. First, we identified at nucleotide 591 a transition which substitutes glutamine 188 by arginine. The mutated glutamine is not only highly conserved in evolution (conserved also in Escherichia coli and Saccharomyces cerevisiae), but is also two amino acid residues downstream from the active site histidine-proline-histidine triad and results in about 10% of normal enzymatic activity. The arginine 188 mutation is the most common galactosemia mutation characterized to date. It accounts for one-fourth of the galactosemia alleles studied. Second, we report the substitution of arginine 333 by tryptophan, caused by a transition at nucleotide 1025. The area surrounding this missense mutation is the most highly conserved domain in the homologous enzymes from E. coli, yeast, and humans, and this mutation results in undetectable enzymatic activity, suggesting that this is a severe mutation. This second mutation appears to be rare, since it was found only in the patient we sequenced. Our data provide further evidence for the heterogeneity of galactosemia at the molecular level, heterogeneity which might be related to the variable clinical outcome observed in this disorder.     

Galactosemia: a strategy to identify new biochemical phenotypes and molecular genotypes.

We describe a stratagem for identifying new mutations in the galactose-1-phosphate uridyl transferase (GALT) gene. GALT enzyme activity and isoforms were defined in erythrocytes from probands and their first-degree relatives. If the biochemical phenotypes segregated in an autosomal recessive pattern, we screened for common mutations by using multiplex PCR and restriction endonuclease digestions. If common mutant alleles were not present, the 11 exons of the GALT gene were amplified by PCR, and variations from the normal nucleotide sequences were identified by SSCP. The suspected region(s) was then analyzed by direct DNA sequencing. We identified 86 mutant GALT alleles that reduced erythrocyte GALT activity. Seventy-five of these GALT genomes had abnormal SSCP patterns, of which 41 were sequenced, yielding 12 new and 21 previously reported, rare mutations. Among the novel group of 12 new mutations, an unusual biochemical phenotype was found in a family whose newborn proband has classical galactosemia. He had inherited two mutations in cis (N314D-E203K) from his father, whose GALT activity was near normal, and an additional GALT mutation in the splice-acceptor site of intron C (IVSC) from his mother. The substitution of a positively charged E203K mutation created a unique isoform-banding pattern. An asymptomatic sister''s GALT genes carries three mutations (E203K-N314D/N314D) with eight distinct isoform bands. Surprisingly, her erythrocytes have normal GALT activity. We conclude that the synergism of pedigree, biochemical, SSCP, and direct GALT gene analyses is an efficient protocol for identifying new mutations and speculate that E203K and N314D codon changes produce intraallelic complementation when in cis.     

E. coli galactose-1-phosphate uridyl transferase: N-terminal and C-terminal sequences

Summary A modified procedure for the purification of E. coli galactose-1-phosphate uridyl transferase (E.C. 2.7.6.12) was developed which reproducibly gives pure enzyme. The purified enzyme was shown to be a dimeric protein with a subunit molecular weight of 41,000 and its amino acid composition and content of free sulfhydryl groups were determined. The N-terminal and C-terminal amino acid sequences were found to be NH₂-thr-gln-phe-asn-pro-val-asp and -ser(val leu)-ala-COOH respectively. This N-terminal sequence allowed the identification of the start of the transferase gene in the DNA sequence determined by GRINDLEY. Furthermore it appears to define a nine base intercistronic region between the epimerase and transferase genes.Abbreviations Cyclic AMP Cyclic adenosine 2¹5¹ monophosphate - DPN Diphosphopyridine nucleotide - UDP Uridine diphosphate - EDTA Ethylene diamine tetra acetic acid - SDS sodium dodecyl sulfate - NEM N-ethylmaleimide     

Osmotic Remedial Response in a Galactose-negative Mutant of Saccharomyces cerevisiae

We investigated an osmotic remedial mutant of Saccharomyces which was deficient in galactose-1-phosphate uridyl transferase. Both the rate of growth and the transferase activity of the mutant culture were dependent on the osmotic activity of the growth medium. Organic and ionic solutes proved to be equally effective in inducing the osmotic remedial response. The galactose pathway enzymes were separable by ultracentrifugation, indicating that a stable interenzyme complex was not formed. The results were consistent with the hypothesis that the corrective effect occurs at the tertiary or quaternary level of organization in an environmentally sensitive protein. The possibility that the osmotic remedial response represents an effect of translation is discussed.     

A common mutation associated with the Duarte galactosemia allele.

The human cDNA and gene for galactose-1-phosphate uridyl transferase (GALT) have been cloned and sequenced. A prevalent mutation (Q188R) is known to cause classic galactosemia (G/G). G/G galactosemia has an incidence of 1/38,886 in 1,396,766 Georgia live-born infants, but a more common variant of galactosemia, Duarte, has an unknown incidence. The proposed Duarte biochemical phenotypes of GALT are as follows: D/N, D/D, and D/G, which have approximately 75%, 50%, and 25% of normal GALT activity respectively. In addition, the D allele has isoforms of its enzyme that have more acidic pI than normal. Here we systematically determine (a) the prevalence of an A-to-G transition at base pair 2744 of exon 10 in the GALT gene, transition that produces a codon change converting asparagine to aspartic acid at position 314 (N314D), and (b) the association of this mutation with the Duarte biochemical phenotype. The 2744G nucleotide change adds an AvaII (SinI) cut site, which was identified in PCR-amplified DNA. In 111 biochemically unphenotyped controls with no history of galactosemia, 13 N314D alleles were identified (prevalence 5.9%). In a prospective study, 40 D alleles were biochemically phenotyped, and 40 N314D alleles were found. By contrast, in 36 individuals known not to have the Duarte biochemical phenotype, no N314D alleles were found. We conclude that the N314D mutation is a common allele that probably causes the Duarte GALT biochemical phenotype and occurs in a predominantly Caucasian, nongalactosemic population, with a prevalence of 5.9%.     

Enzyme kinetics in mammalian cells. 3. Regulation of activities of galactokinase, galactose-1-phosphate uridyl transferase and uridine diphosphogalactose-4-epimerase in human erythrocytes

The sequential enzyme assay as previously described has been used to study various effects on the three enzymes in human red cells involved in the phosphorylation of galactose: galactokinase, galactose-1-phosphate uridyl transferase and uridine diphospho-galactose-4-epimerase.

• 1 Enzyme activities in undiluted lysates appear to reflect the respective activities in whole cells.
• 2 Added extracellular Gal-1-P, G-1-P, UDPGal and UPDG do not affect enzyme activities in whole cells.
• 3 The kinase and transferase enzymes do not appear to be associated with the membrane fraction of the red cells.
• 4 Galactokinase activity is inhibited by G-6-P and Gal-1-P, but not by glucose, G-1-P, UDPG, UDPGal, UTP or NAD⁺. It is inhibited by ATP and ADP in high concentration.
• 5 Galactose-1-phosphate uridyl transferase activity is inhibited by G-1-P, G-6-P, UDPG, UDPGal, ATP, and ADP. It is not affected by UTP, NAD⁺, or galactose.
• 6 Uridine diphospho-galactose-4-epimerase activity is inhibited by UDPG, ATP, ADP, UTP and NADH. It is stimulated by NAD⁺ and possibly by Gal-1-P. It is unaffected by G-1-P, G-6-P.
• 7 The rates of the three reactions decrease with decreasing temperature. The activities of transferase and epimerase are inactivated at the same rate, the kinase activity is inactivated more slowly.
• 8 Dilution experiments indicate the presence in lysates of a pool of UDPG (or, possibly UDPGal) which regulates the activities transferase and the epimerase enzymes.
• 9 Results of dilution experiments suggest that the radioactive product of the transferase enzyme is different from commercially available UDPGal-u-¹⁴C.
• 10 ATP, UTP and UDPG interact with some substance(s) in the red cell lysate to cause a time dependent inactivation of the epimerase. These interactions are the result of glucose metabolism.

     

Demonstration of gene dosage effects for AK3 and GALT in fibroblasts from a fetus with 9p trisomy

After prenatal detection of a de novo unbalanced translocation 46,XX,18p+, fetal fibroblasts were obtained for further studies. Chromosome banding suggested that the fetus might have trisomy of the short arm of chromosome 9. This tentative diagnosis could be confirmed by demonstration of gene dosage effects for galactose-1-phosphate uridyl transferase (GALT, EC 2.7.7.12) and nucleoside triphosphate adenylate kinase (AK3, EC 2.7.4.10) both known to be controlled by genes assigned to 9p. These findings demonstrate for the first time a gene dosage effect for AK3 in a case of 9p trisomy and show again that gene mapping information can be used to define partial aneuploidies in fetal fibroblasts.