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At least 19 recordsLinked to original sources
PubMed1985

Regulation of galactokinase gene expression in Tetrahymena thermophila. I. Intracellular catecholamine control of galactokinase expression.

The addition of glucose to the medium of Tetrahymena thermophila results in a 7-fold repression of galactokinase (EC 2.7.1.6; ATP:D-galactose-1-phosphotransferase). The presence of millimolar amounts of the catecholamines dopa, dopamine, norepinephrine, and epinephrine or the hormone glucagon also results in the repression of galactokinase in the absence of glucose. The addition of millimolar amounts of adrenergic agonists (isoproterenol, tyramine, 2-amino-6,7-dihydroxytetrahydronaphthalene) results in significant repression of galactokinase in the absence of glucose; concentrations of 2-amino-6,7-dihydroxytetrahydronaphthalene less than or equal to 10(-4) M result in a derepression of galactokinase specific activity. Addition of adrenergic antagonists (propranolol, dichloroisoproterenol) have no effect on galactokinase activity at concentrations less than 10(-4) M but do arrest cell growth at greater concentrations. The addition of the cAMP analogs caffeine or theophylline in millimolar amounts results in repression of galactokinase activity; however, cell growth is greatly slowed or completely arrested at these concentrations. Analysis of the repression response of several mutants demonstrates that mutants deficient in catecholamine biosynthesis are altered in their regulation of galactokinase. Measurements of intracellular cAMP levels for 0-24 h following the addition of several of the above compounds to exponentially growing cells did not demonstrate any change over this period. Measurement of intracellular cAMP levels for 24 h following the addition of glucose or galactose to exponentially growing wild-type and mutant cell strains did not demonstrate any difference in cAMP concentrations over this period although a wide range of galactokinase activity was exhibited. Starvation of wild-type cells prior to the addition of glucose in minimal medium without added carbohydrate resulted in a significant increase in cAMP following the addition of glucose. This increase is demonstrated to be dependent upon the ability of the cells to resume division after the arrest of growth and is not correlated with galactokinase regulation. These results support the conclusion that cAMP is not involved in the repression of galactokinase gene expression initiated by glucose or hormone-like effectors and demonstrate the participation of an adrenergic control system in galactokinase regulation which is subordinate to the regulation by glucose. A possible model is discussed.

1-Methyl-3-isobutylxanthine↗
PubMed1995

Comparison of the enzymatic activities of human galactokinase GALK1 and a related human galactokinase protein GK2.

The GALK1 cDNA encoding human galactokinase was recently cloned and its cognate GALK1 gene shown to be involved in galactokinase deficient galactosemia. Previously, a separate human galactokinase cDNA, GK2, was cloned by complementation of a galactokinase deficient yeast mutant; however, the galactokinase activity of GK2 was not demonstrated in mammalian cells. To compare the relative galactokinase activity of GALK1 and GK2, their corresponding cDNAs were expressed in COS cells. Northern blot analysis indicated that both cDNAs were transcribed into mRNA transcripts of the expected size; however, only the GALK1 cDNA produced high levels of galactokinase activity. This result would suggest that GALK1 is the major enzyme for galactose metabolism while the role of GK2 remains uncertain.

Base Sequence↗
PubMed1985

Regulation of galactokinase gene expression in Tetrahymena thermophila. II. Identification of 3,4-dihydroxyphenylalanine as a primary effector of adrenergic control of galactokinase expression.

Intracellular concentrations of catecholamines were determined in wild-type and mutant Tetrahymena thermophila, using the highly sensitive techniques of high-performance liquid chromatography and electro-chemical detection. Catecholamines were determined in these cell strains grown under various steady-state conditions, including those which initiate and maintain repression of galactokinase gene expression. Wild-type cells grown in defined minimal medium supplemented with 1% glycerol, exhibiting derepressed galactokinase synthesis, were found to contain considerable quantities of dopa (3,4-dihydroxyphenylalanine) and dopamine, but no detectable levels of either norepinephrine or epinephrine. Analyses of wild-type cells revealed a strong positive correlation between the internal concentration of dopa and expression of the galactokinase gene, both of which are regulated by exogenous carbohydrates, catecholamine agonists, or dibutyryl-cAMP; an analogous relationship between intracellular dopamine concentrations and galactokinase activity was not found. In addition, a correlation between intracellular dopa content and the phenotypic expression of galactokinase in various mutants deficient in the catecholamine biosynthetic pathway or in glucokinase further confirms the role of dopa as a primary effector in the regulation of galactokinase gene expression.

Animals↗
PubMed1985

Cloning and expression of Clostridium pasteurianum galactokinase gene in Escherichia coli K-12 and nucleotide sequence analysis of a region affecting the amount of the enzyme.

The Clostridium pasteurianum galactokinase gene was cloned by complementation, of the galK locus, into Escherichia coli. Restriction enzyme analysis subcloning and Tn5 mutagenesis indicated that the gene was located on a 1.8 X 10(3) base-pair ClaI-Sau3A fragment that encoded a polypeptide of approximately 40 Mr. Although the C. pasteurianum and the E. coli galactokinases have similar subunit molecular weights, Southern hybridization analysis indicated no strong homology between their genes. Even though this clone showed a low level of galactokinase expression, the Gal+ phenotype, provided by the clostridial galactokinase, was unstable in E. coli, and the gene was frequently inactivated by the spontaneous acquisition of insertion sequences. A second clone containing this gene on a large restriction fragment was isolated by hybridization. This clone was unable to grow on galactose-containing media due to the overproduction of galactokinase. Comparison of the plasmids from these two clones revealed that the second contained an additional 300 base-pairs located at one end of the galactokinase gene. Appropriate operon fusions with a promoter-less E. coli galactokinase gene indicated that these additional 300 base-pairs had promoter activity in E. coli. The DNA sequence of this region which lies upstream of the C. pasteurianum galactokinase gene was determined and compared with that from several clones producing high, low or undetectable amounts of galactokinase. The reasons for the high and low level expression and for the instability of the C. pasteurianum galactokinase in E. coli are discussed. The presence of the galactokinase suggests that galactose is used in C. pasteurianum through the Leloir pathway via galactose 1-phosphate.

Base Sequence↗
PubMed1979

Cloning and expression of the yeast galactokinase gene in an Escherichia coli plasmid.

This report describes the construction and isolation of a plasmid, derived from pBR322, which carries a BglII restriction fragment of DNA containing the galactokinase gene from Saccharomyces cerevisiae. This was accomplished by the following procedure: (1) Purified galactokinase mRNA, labelled with 125I, was hybridized to BglII digests of yeast DNA employing Southern's filter transfer technique to identify a restriction fragment containing the galactokinase gene. (2) This fragment was partially purified by agarose gel electrophoresis, ligated into the BamHI site of pBR322 and transformed into Escherichia coli to generate a clone bank containing the galactokinase gene. (3) This bank was screened by in situ colony hybridization with galactokinase mRNA resulting in the identification of a plasmid carrying this gene. This plasmid DNA hybridized with the galactokinase mRNA to the same extent in the presence of absence of a large excess of unlabelled mRNA from cells that were not induced for galactokinase synthesis, while the same amount of unlabelled galactose-induced mRNA reduced the hybridization by 95%. When this plasmid was introduced into an E. coli strain deleted for the galactose operon it caused the synthesis of low levels of yeast galactokinase activity.

DNA, Recombinant↗
PubMed1991

Normal expression of thymidine kinase and O6-methylguanine-DNA methyltransferase in cultured fibroblasts from individuals with hereditary galactokinase deficiency.

Expression of the enzymes galactokinase, thymidine kinase, and O6-methylguanine-DNA methyltransferase is occasionally coordinately regulated in human cell lines. We have measured the activities of these three enzymes in extracts of fibroblasts from individuals with hereditary galactokinase deficiency. These cells do not express measurable galactokinase activity. The levels of O6-methylguanine-DNA methyltransferase were in the normal range in cells from three galactokinase-deficient individuals. The activity of thymidine kinase in the affected cells was in the normal range for two of the three individuals. The reduced thymidine kinase activity in the third individual reflected the extremely poor growth of the cells in culture. Immortalization of one galactokinase-deficient cell line resulted in loss of O6-methylguanine-DNA methyltransferase activity, but the galactokinase and thymidine kinase levels remained unchanged. The data indicate that the loss of galactokinase activity in these individuals is the consequence of an alteration of gene expression which does not involve coordinate silencing with the thymidine kinase and methyltransferase loci.

Cell Line↗
PubMed1981

Characterization of galactose-1-phosphate uridyl-transferase and galactokinase in human organs from the fetus and adult.

Some properties of galactose-1-phosphate uridyltransferase (EC 2.7.7.12) and galactokinase (EC 2.7.1.6) were investigated in human organs, i.e., in liver kidney, skeletal muscle, lung, spleen, heart and brain from fetuses as well as liver, kidney and skeletal muscle tissues from adults. (1) Galactose-1-phosphate uridyltransferase (transferase) is quite stable when stored below 4 degrees C, and can be frozen from a couple of months without noticeable loss of activity. Galactokinase is relatively stable as long as the cell structure is intact. In cell homogenates its activity decreases very fast, especially under freezing conditions. (2) The pH optimum of transferase in all human tissues is at pH values between 8.2 and 8.4 except in erythrocytes in which it is at a higher pH value. Maximal activity of galactokinase is observed at approximately 8.2 in all human tissues. (3) The Km values of transferase are similar in all human organs, and the values in fetal tissues are not significantly different from those in adult tissues. In the case of galactokinase also no distinct tissue variations are observed in Km values. However, galactokinase affinity for both substrates is considerably higher in adult organs than in fetal organs. (4) Transferase and galactokinase activity in human liver is resolved into two major components on DEAE-cellulose columns. It seems that transferase and galactokinase exist in human tissues as more than two isoenzyme constituents.

Adult↗
PubMed2002

Clinical features of galactokinase deficiency: a review of the literature.

Galactokinase deficiency (McKusick 230200) is a rare autosomal recessive inborn error of galactose metabolism. Cataract and, rarely, pseudotumor cerebri caused by galactitol accumulation seem to be the only consistently reported abnormalities in this disorder. We performed a literature search to obtain information on the clinical spectrum of galactokinase deficiency. A total of 25 publications were traced describing 55 galactokinase-deficient patients. Cataract was reported in most patients. Clinical abnormalities other than cataract were reported in 15 (35%) out of 43 cases on which information was available. However, all symptoms were reported infrequently and a causal relationship with the galactokinase deficiency is unlikely. As cataract and pseudotumor cerebri appear to be the sole complications of galactokinase deficiency, the outcome for patients with galactokinase deficiency is much better than for patients with classical galactosaemia (McKusick 230400), a more common autosomal recessive disorder of galactose metabolism caused by galactose-1-phosphate uridyltransferase (GALT; EC 2.7.7.12) deficiency. Long-term follow-up of patients with this disorder has shown that, in spite of a severely galactose-restricted diet, most patients develop abnormalities such as a disturbed mental and/or motor development, dyspraxia and hypergonadotropic hypogonadism. Endogenous production of galactose has been considered an important aetiological factor. Although damage may well occur in utero, available evidence suggests that damage will continue after birth. Inhibition of galactokinase may then be a promising approach for controlling damage in GALT-deficient patients.

Carbohydrate Metabolism, Inborn Errors↗
PubMed1992

Cloning of a human galactokinase gene (GK2) on chromosome 15 by complementation in yeast.

A human cDNA encoding a galactokinase (EC 2.7.1.6) was isolated by complementation of a galactokinase-deficient (gal1-) strain of Saccharomyces cerevisiae. This cDNA encodes a predicted protein of 458 amino acids with 29% identity to galactokinase of Saccharomyces carlsbergensis. Previous studies have mapped a human galactokinase gene (GK1) to chromosome 17q23-25, closely linked to thymidine kinase. The galactokinase gene that we have isolated (GK2) is located on chromosome 15. The relationship between the disease locus for galactokinase deficiency galactosemia, which is responsible for cataracts in newborns and possibly presenile cataracts in adults, and the two galactokinase loci is unknown.

Amino Acid Sequence↗
PubMed1986

Cataracts in patients heterozygous for galactokinase deficiency.

The role of heterozygous galactokinase deficiency in the development of presenile cataracts is presently undetermined. Erythrocyte galactokinase activity was measured from 95 normal Caucasian subjects and from 39 Caucasian patients who had developed idiopathic bilateral cataracts between ages 20 and 55. The diagnosis of heterozygous galactokinase deficiency was based on the following criteria: galactokinase activity more than 2.0 SD below the control population mean; when available, familial evidence for heterozygous galactokinase activity was used as additional evidence. Three of 39 patients (1/13) with cataracts were found to be carriers of the galactokinase deficiency allele (P less than 0.001). Two heterozygotes had high dietary galactose intake suggesting a possible relationship between a high galactose diet and cataract formation. Dietary information was unavailable for the third heterozygote. We conclude that there is a high prevalence of heterozygous galactokinase deficiency existing in patients less than 55 yr of age with cataracts, and recommend that adults at risk restrict their consumption of dairy products.

Adolescent↗
PubMed1969

Galactose-1-phosphate uridyltransferase and galactokinase activity in cultured human diploid fibroblasts and peripheral blood leukocytes. I. Analysis of transferase genotypes by the ratio of the activities of the two enzymes.

The specific activities of galactokinase and galactose-1-phosphate uridyltransferase were determined in peripheral blood leukocytes directly after separation from whole blood, and in cultured skin fibroblasts at various times during the subculture growth period. Growth curves were obtained for fibroblasts based on three different parameters: direct cell counts, total protein, and total deoxyribonucleic acid (DNA) content. At the time in culture when the specific activity of both enzymes was maximal and least variable, the ratio of transferase to galactokinase correlated well with the transferase genotypes of the original tissue donors. Leukocyte transferase: galactokinase ratios gave a similar distribution pattern. Whereas transferase activity in both fibroblasts and leukocytes was similar, galactokinase was approximately three times as active in fibroblasts as in leukocytes. All fibrobast cell strains tested had similar galactokinase activity regardless of transferase genotype.The kinetic properties of fibroblast galactokinase were examined. Galactose-1-phosphate inhibits galactokinase activity in both normal and galactosemic cell strains, whereas other glycolytic intermediates have no effect. There was no detectable transferase activity in eight galactosemic (Gt(G)/Gt(G)) cell strains when transferase activity was maximal in cell strains of other transferase genotypes. Inhibitors responsible for the absence of transferase activity could not be demonstrated. In addition, transferase activity in galactosemic cell lysates was not observed in cells during logarithmic growth; measurable uridine diphosphate galactose (UDPgal) pyrophosphorylase activity was found in human diploid fibroblast cultures, as well as significant levels of endogenous uridine triphosphate (UTP) in lysates of fibroblast cultures.

Black People↗
PubMed1979

Expression of the yeast galactokinase gene in Escherichia coli.

In Saccharomyces cerevisiae the genes for three of the enzymes involved in galactose metabolism are tightly linked near the centromere of chromosome II (Douglas and Hawthorne, 1964). However, the molecular mechanisms which control the expression of these genes are not well understood. A DNA fragment containing at least one of these yeast genes, the galactokinase gene (gal1), has been joined to the bacterial plasmid pBR322 and maintained in an Escherichia coli strain that carries a deletion in its own galactokinase gene, galK. The presence of the yeast gene was demonstrated by (i) complementation of the E. coli galactokinase deletion, (ii) by hybridization of the cloned DNA fragment to restriction enzyme digests of total yeast DNA and (iii) by assaying for yeast galactokinase activity in bacterial cell extracts. The yeast DNA fragment is 4700 base pairs long, and enables the host E. coli K-12 strain to grow in minimal medium containing galactose as the sole carbon source with a generation time of 14.3 h. The yeast galactokinase activity in the bacterial extracts is 0.7% of the bacterial galactokinase activity found in wild-type E. coli fully induced with fucose.

DNA↗
PubMed1995

Cloning of the galactokinase cDNA and identification of mutations in two families with cataracts.

Galactokinase is an essential enzyme for the metabolism of galactose and its deficiency causes congenital cataracts during infancy and presenile cataracts in the adult population. We have cloned the human galactokinase cDNA, which maps to chromosome 17q24, and show that the isolated cDNA expresses galactokinase activity in bacteria and mammalian cells. We also describe two different mutations in this gene in unrelated families with galactokinase deficiency and cataracts. The availability of the cloned galactokinase gene provides an important reference to identify mutations in patients with galactokinase deficiency and cataracts.

Adult↗
PubMed2003

Molecular structure of galactokinase.

Galactokinase plays a key role in normal galactose metabolism by catalyzing the ATP-dependent phosphorylation of alpha-D-galactose to galactose 1-phosphate. In humans, mutations in the galactokinase gene can lead to the diseased state referred to as Type II galactosemia. Here we describe the three-dimensional structure of galactokinase from Lactococcus lactis determined to 2.1-A resolution. As expected from amino acid sequence alignments, galactokinase adopts a similar topology to that observed for members of the GHMP superfamily. The N-terminal domain is characterized by a five-stranded mixed beta-sheet while the C-terminal motif is dominated by two distinct four-stranded anti-parallel beta-sheets. The structure was solved in the presence of alpha-D-galactose and inorganic phosphate. These ligands are wedged between the N- and C-terminal domains. Amino acid side chains responsible for anchoring the sugar ligand to the protein include Arg36, Glu42, Asp45, Asp183, and Tyr233. Both Arg36 and Asp183 are strictly conserved in the amino acid sequences available in the literature thus far for galactokinases. Interestingly, the carboxylate side chain of Asp183 is positioned within 3.5 A of the C-1 hydroxyl group of galactose, whereas the guanidinium group of Arg36 is situated between both the C-1 hydroxyl group and the inorganic phosphate. Most likely these residues play key roles in catalysis. The structure of galactokinase described here serves as a model for understanding the functional consequences of point mutations known to result in Type II galactosemia in humans.

Adenosine Triphosphate↗
PubMed1995

Mouse galactokinase: isolation, characterization, and location on chromosome 11.

Elevated galactose levels can be caused by several enzyme defects, one of which is galactokinase. Galactokinase deficiency cause congenital cataracts during infancy and presenile cataracts in the adult population. We have isolated the mouse cDNA for galactokinase, which shares extensive amino acid sequence homology, 88% identity, with a recently cloned human galactokinase. It is expressed in all tissues examined. In an interspecific backcross analysis galactokinase maps to the distal region of mouse chromosome 11, a region that is homologous to human chromosome 17q22-25. The availability of the mouse gene provides an opportunity to make a knockout model for galactokinase deficiency.

Alleles↗
PubMed2000

Novel mutations in 13 probands with galactokinase deficiency.

Galactokinase is an essential enzyme in the metabolism of galactose. Patients with deficiencies in galactokinase exhibit early-onset cataracts. We examined the sequence of the human galactokinase gene (GK1) from 13 patients exhibiting galactokinase deficiency and identified 12 novel mutations. One of the mutations occurred in six of the 13 probands examined, and the remaining 11 were unique mutations. Expression of each of the mutant GK1 genes in Xenopus oocytes resulted in very low galactokinase activity levels. These results provide important information regarding the types of GK1 mutations that occur in the human population.

Base Sequence↗
PubMed2000

Disruption of galactokinase signature sequence in gal3p of Saccharomyces cerevisiae does not lead to loss of signal transduction function.

Gal3p of Saccharomyces cerevisiae is a 520-amino-acid residue protein, which activates the GAL genes in the presence of galactose by relieving the repression of Gal80p. It shows significant amino acid sequence homology to galactokinases but does not possess galactokinase activity. Deletion mutants of Gal3p were generated to identify the role of N-terminal amino acid residues required for function. The mutant versions of Gal3p could be detected on a Western blot. The Gal3p mutant lacking N-terminal 50-amino-acid residues which is disrupted for galactokinase signature sequence was found to be functional. These results suggest that the evolutionarily conserved galactokinase signature sequence present in known galactokinases may not have a role in Gal3p function.

Amino Acid Sequence↗