A new genetic variant of galactose-1-phosphate uridyl transferase

Summary The enzyme galactose-1-phosphate uridyl transferase (E.C. 2.7.7.12), which has an important function in the metabolism of galactose, exists in multiple molecular forms. The different phenotypes are genetically determined. They can be distinguished according to their electrophoretic mobility. The enzymatic activity of the different gene products varies within certain limits. A new phenotype of the enzyme has been detected in the red cells of a healthy individual. The electrophoretic migration of this phenotype is slower compared to the wild type and its enzymatic activity is lower, but still sufficient as not to cause galactosemia. An extensive family study revealed that the rare gene is inherited according to mendelian law. Independently the same gene product has been detected in two other, nonrelated individuals out of a total of 1668 samples tested. The gene frequency can therefore be estimated to 0.0009 in the Swiss population. We suggest that the new type be called Berne variant of galactose-1-phosphate uridyl transferase.     

Molecular characterization of two galactosemia mutations and one polymorphism: implications for structure-function analysis of human galactose-1-phosphate uridyltransferase.

We report here the molecular characterization of two galactosemia mutations, L74P and F171S, and one polymorphism, S135L, in human galactose-1-phosphate uridyltransferase (GALT). Both galactosemia mutations result in reduced enzymatic activity when reconstructed in the cDNA and overexpressed. The polymorphism, in contrast, has near normal activity. Both mutations affect evolutionarily conserved residues, suggesting that they are functionally important, while the polymorphism occurs in a nonconserved domain which is presumably not critical for enzymatic function. The F171S mutation is close to the putative active-site nucleophile. Our data further support the notion of molecular heterogeneity of galactosemia and suggest that galactosemia mutations and GALT polymorphisms may be useful tools in highlighting different functional domains in human GALT.     

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     

Variants of galactose-1-phosphate uridyl transferase in the Greek populations

Summary The frequency of variants of galactose-1-phosphate uridyl transferase was determined among the nine Greek populations by studying a sample of 1570 unselected individuals. Average frequency of normal allele GALT=0.942, galactosemia gene GALT ^G=0.0021 and the Duarte variant gene GALT ^D=0.0548 were observed. Frequency of galactosemia heterozygotes among Greeks was similar to that in other Caucasian populations, but the frequency of the Duarte variant was considerably higher. With the exception of two populations, one with low (Epirus) and one with high (Thrace) frequencies, the polymorphism of the Duarte variant displays very similar frequencies in the various Greek population groups.Supported by Grants HD 01 974 and GM 15 253 from the U.S. Public Health Service     

Characterization of two missense mutations in human galactose-1-phosphate uridyltransferase: different molecular mechanisms for galactosemia.

We report the molecular characterization of two novel galactosemia mutations that exhibit different molecular phenotypes. Both are of the missense type with low or no residual enzyme activity. The R148W mutation results in an unstable protein, although messenger RNA is still produced. In contrast, the L195P mutation produces stable but inactive immunoreactive protein. The R148W mutation alters an amino acid that is not evolutionarily conserved, while the L195P mutation affects a well-conserved residue nine amino acids down-stream from the putative active site nucleophile. These mutations provide evidence that different mechanisms can result in galactosemia: destabilizing mutations in any given area of the protein and missense mutations in conserved domains of the enzyme resulting in low or no activity. These two mutant alleles represent the fifth and sixth galactosemia mutations and confirm the hypothesis that galactosemia results from a multiplicity of mutations at the molecular level.     

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.     

Galactose-1-phosphate uridyl transferase in density-fractionated erythrocytes

Summary Galactose-1-phosphate uridyl transferase (GALT), the deficient enzyme in classical galactosemia, was studied by Percoll-gradient age-fractionation of erythrocytes. For normal GALT, a rapid and substantial decrease in GALT activity and loss of most of two isozymes was found to occur in the reticulocyte fractions. The loss of activity was then followed by relative stabilization of both GALT-specific activity and microheterogeneity in mature and aging erythrocytes. When applied to the study of mutant GALT from galactosemic patients, the Percoll-gradient fractionation method permitted detection in the reticulocyte-enriched fractions of up to 5% of normal GALT-specific activity and an isoelectric focusing pattern essentially the same as that of normal GALT. Percoll-gradient fractionation of erythrocytes offers a simple and direct method to study characteristics of GALT activity and microheterogeneity in normal and galactosemic human erythrocytes.     

CO2 production from galactose in galactose-1-phosphate uridyl transferase-deficient Escherichia coli.

Escherichia coli K-12 deficient in galactose-1-phosphate uridyl transferase is capable of converting significant amounts of d-[1-(14)C]galactose to (14)CO(2), whereas strains deficient in other enzymes of the Leloir pathway cannot do so.     

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.     

Isoelectrofocusing of erythrocyte galactose 1 phospho uridyl transferase in a family with both galactosemia and duarte variants

Summary A family with the presence of the genes for both galactosemia and the Duarte variant is described. Galactose 1 phospho uridyl transferase has been studied not only by electrophoresis on starch gel, but also by isoelectrofocusing on thin-layer acrylamide.Normal and variant transferases were resolved into three bands, the isoelectric point of which was between 5.40 and 5.10 for the normal subjects, and between 5.25 and 4.95 for subjects with the Duarte variant.     

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.