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.     

On the nature of l-xylulose reductase deficiency in essential pentosuria

Essential pentosuria is the result of a partial deficiency of l-xylulose reductase. Red blood cells of normal individuals have been found to contain two l-xylulose reductases: a major and a minor isozyme. Red cells from pentosurics contain only one isozyme. The residual enzyme of pentosurics and the normal minor isozyme have similar Michaelis constants for l-xylulose and xylitol, similar activity responses to pH, and similar rates of migration when electrophoresed or subjected to ion-exchange chromatography. It is suggested that homozygosity for the pentosuria allele results in the absence of the major isozyme and that the residual isozyme of pentosurics is identical to the minor isozyme of normal individuals.     

Characteristics of galactokinase and galactose-1-phosphate uridyltransferase in cultivated fibroblasts and amniotic fluid cells

Summary The kinetic characteristics of galactose-1-phosphate uridyltransferase and galactokinase in cultivated fibroblasts and amniotic fluid cells were investigated. The K _m values of galactokinase for galactose at 2.0 mM ATP are 0.34 mM in amniotic fluid cells and 0.48 mM in fibroblasts. The K _m values for ATP at 0.5 mM galactose are 1.25 mM and 2.10 mM.Transferase and galactokinase activities and protein content increase logarithmically during the growth of cultivated cells. The specific activity of both enzymes also increases and reaches a maximum level 10–15 days after subculture. The specific activity of transferase increases faster than that of galactokinase in the case of amniotic fluid cells. In the case of fibroblasts the specific activity of galactokinase increases faster than that of transferase.     

Proteinase Enzyme System of Lactic Streptococci II. Role of Membrane Proteinase in Cellular Function

The effect of several environmental conditions on the structure and activity of a membrane-associated proteinase from Streptococcus lactis was investigated. The activity of the enzyme varied with pH. Before storage at 3 C, maximal activity occurred at pH 6.0, but was minimal at this pH after storage. At all pH values tested, the enzyme was inactivated after storage. After storage at 3 C, the enzyme showed gross structural alterations with a concomitant loss of activity. Gel filtration and sedimentation velocity data indicated that inactivation of the enzyme was the result of aggregation to higher molecular weight forms. p-Hydroxymercuribenzoate prevented inactivation of the enzyme during storage by preventing aggregation. Activity was correlated with disaggregation of polymer forms of the enzyme to an active monomer. The storage-inactivated enzyme could be reactivated by treatment of the enzyme with cysteine, glutathione, or ferrous ion. Glutathione enabled stored cells to produce acid at their original rate when subcultured in milk. This was attributed to the effect of glutathione on the membrane proteinase. The data suggested that the biological activity of stored cells may be dependent upon the activity of the membrane proteinase.     

Glucose regulation of specific gene expression is altered in a glucokinase-deficient mutant of Tetrahymena

Summary Expression of the galactokinase gene in Tetrahymena thermophila can be repressed by glucose, glucose analogs, and epinephrine, each apparently acting through increased intracellular levels of adenosine 3′:5′-cyc lic monophosphate (cAMP) (1). To characterize further the initial steps in the control of galactokinase gene-expression by glucose, we have analyzed mutants which are defective in the metabolism of this sugar; these mutants were selected for their resistance to the glucose analog, 2-deoxyglucose (2). In one such mutant that is deficient in glucokinase, the synthesis of galactokinase is totally resistant to repression by glucose or its analogs, while repression by exogenous catecholamines or dibutyryl cAMP is unaffected. Radiochromatographic analyses of extracts of wild-type cells incubated with [¹⁴C]-deoxyglucose reveal intracellular conversio to several deoxyglucose metabolites, principally deoxyglucose-6-P and smaller amounts of deoxyglunose 1-P and 2-deoxygluconate; extracts of glucokinase-deficient cells prepared in a similar manner contain only trace amounts of deoxyglucose-6-P. The glucose analog 3-O-methylglucose, which is transported but not phosphorylated in wild-type cells, also cannot maintain repression of galactokinase. These results establish that the transport and subsequent phosphorylation of glucose are required for glucose-initiated repression of galactokinase gene expression, possibly acting by modulation of catecholamine or cyclic AMP levels. Additionally, we show unequivocally that: (a) cells containing derepressed levels of galactokinase are repressed upon the addition of glucose by inhibition of the synthesis of new enzyme and dilution of preformed enzyme concomitant with cell division, rather than through selective inactivation or degradation of galactokinase; and (b) glycerol kinase, glucokinase and fructokinase activities also are repressed by glucose in wild-type Tetrahymena, indicating that the glucose repression phenomenon is pleiotropic. Because the glucose repression of the synthesis of each of these enzymes is abolished in cells deficient in glucokinase, the regulatory mechanisms elucidated for repression of galactokinase synthesis are likely to be of wide significance.     

Enzymes of galactose metabolism in erythrocytes and liver of inbred strains of mice.

This study compares the activity of galactokinase, galactose-1-phosphate uridyltransferase, and UDPgalactose-4-epimerase, the important enzymes in the pathway of conversion of galactose to glucose in red cells and liver of five inbred strains of mice. In the red cells, galactokinase varied over a four-fold range of activity while the other enzymes varied about two-fold. The activity of each of the enzymes varied independently of the other so that red cells of each strain had a unique pattern for the three enzymes. The red cell activity pattern was not reflected in liver tissue which showed little interstrain variation for each of the three enzymes. The ratio of liver galactokinase to uridyltransferase and epimerase was very similar in all five strains. Oxidation in vivo of ¹⁴C galactose to ¹⁴CO₂ was examined in the two strains of mice with the widest divergence of red cell galactokinase activity and no difference was found in this parameter measuring the physiological disposition of the sugar. The wide variation of the red cell enzyme activity appears to have little metabolic consequence for the animal, the oxidation of the sugar reflecting the relative constancy of the liver enzyme activity.     

Galactokinase of Bifidobacterium bifidum

Crystalline galactokinase was obtained in good yield from Bifidobacterium bifidum grown on galactose medium. This preparation moved as a single protein band in analytical disc electrophoresis and sedimented as a single symmetrical peak under ultracentrifugation. The enzyme exhibited similar physicochemical properties to galactokinase purified from glucose-grown cells of B. bifidum. The enzyme has a molecular weight of 47,000. Only galactose and ATP were effective as substrate. Km values, optimal pH, cation requirement, inhibition by SH-reagent, heat stability and product inhibition were also investigated.     

Thymidine-kinase activity of cultured cells from individuals with inherited galactokinase deficiency

Cells of a person homozygous for galactokinase deficiency and of her heterozygous parents were found to be deficient in the enzyme thymidine kinase. The decrease in thymidine-kinase activity may be the result of a qualitative alteration in the enzyme molecule. This is reflected in the apparent alteration in the sensitivity of the enzyme to trifluorothymidine. It is suggested that this relationship between the galactokinase and thymidine kinase is not fortuitous but a reflection of their interdependence as found previously in the Chinese hamster.     

Detection and Characterization of a Protein Isoaspartyl Methyltransferase Which Becomes Trapped in the Extracellular Space During Blood Vessel Injury

Injury to rat blood vessels in vivo was found to release intracellular pools of protein D-aspartyl/L-isoaspartyl carboxyl methyltransferase (PIMT) into the extracellular milieu, where it becomes trapped. This trapped cohort of PIMT is able to utilize radiolabeled S-adenosyl-L-methionine (AdoMet) introduced into the circulation to methylate blood vessel proteins containing altered aspartyl residues. As further shown in this study, methylated substrates are detected only at the specific site of injury. In vitro studies more fully characterized this endogenous PIMT activity in thoracic aorta and inferior vena cava. Methylation kinetics, immunoblotting, and the lability of methylated substrates at mild alkaline pH were used to demonstrate that both types of blood vessel contain an endogeneous protein D-aspartyl/L-isoaspartyl carboxyl methyltransferase (PIMT). At least 50% of the PIMT activity is resistant to nonionic detergent extraction, suggesting that the enzyme activity becomes trapped within or behind the extracellular matrix (ECM). Quantities of lactate dehydrogenase (LDH), another soluble enzyme of presumed intracellular origin, were found to be similarly trapped in the extracellular space of blood vessels.     

Comparative studies of galactokinase activity on mammal's red blood cells.

1. ATP: D-galactose-1-phosphotransferase activity was measured in human, pig, cow, rabbit, mouse and rat red blood cells. Mean values of galactokinase activity was markedly lower in the human and pig erythrocyte as compared to those of the other species. 2. The permeability to galactose of the red cells studied was always higher than galactose phosphorylation. 3. The affinity constants of galactokinase for galactose ranged from 119 to 291 microM and from 178 to 406 microM for ATPMg2-. 4. The thermostability values of the galactokinase of the species studied were similar. The pH-optimum is pH 7.5 for the human, mouse and rabbit enzyme and pH 8.0 for cow, pig and rat galactokinase.     

Ascidian phenoloxidase: its release from hemocytes, isolation, characterization and physiological roles

Hemocytes of the solitary ascidian Halocynthia roretzi released phenoloxidase in response to sheep red blood cells and yeast cells but not to latex beads. Phenoloxidase was also released from the hemocytes by treatments with zymosan and lipopolysaccharides but not with β1–3 glucan. EDTA scarcely inhibited the activity of phenoloxidase but inhibited the release of the enzyme. Phenoloxidase was purified from H. roretzi hemocytes by SP-Sephadex chromatography and Sephadex G-100 gel filtration. The molecular weight of the purified enzyme was estimated to be 62 000. Phenoloxidase activity was strongly inhibited by diethyldithiocarbamate, phenylthiourea and reducing agents. H. roretzi phenoloxidase was characterized as a metalloenzyme that required copper ions for the expression of full activity. The phenoloxidase showed antibacterial activity in the presence of -(3,4-dihydroxy)-phenylalanine and H. roretzi plasma. Thus, it can be concluded that phenoloxidase released from H. roretzi hemocytes functions as a humoral factor in the defense system of H. roretzi.     

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.     

Engineering of lactose metabolism inE. coli by introducing β-galactosidase/galactokinase fusion enzymes

Summary A series of plasmids encoding -galactosidase/galactokinase fusion proteins with connecting linkers of different lengths and properties separating the enzyme moieties were made.E. coli cells harbouring the genes for these bifunctional enzymes were grown on minimal media with lactose as carbon source in order to asses possible metabolic effects. Differences in growth rates were observed when the cells contained a scavenger enzyme, galactose dehydrogenase, competing with galactokinase for the galactose formed by -galactosidase.E. coli cells coding for fusion proteins with long linkers then reflected markedly slower growth rates.     

PECTINASE IN ASCLEPIAS LATEX AND ITS POSSIBLE ROLE IN LATICIFER GROWTH AND DEVELOPMENT

A pectinase with a pH optimum of 5.2 is present in the latex of the common milkweed, Asclepias syriaca L. The enzyme was partially purified from the serum fraction of fresh latex by dialysis and ammonium sulfate fractionation. Enzyme activity was detected by a viscometry assay and by the dinitrosalicylic acid assay for reducing sugars. Pectin and polygalacturonic acid could serve as substrates for the enzyme. Pectolytic activity in latex presents a basis for describing the development of the non-articulated branched laticifer system. Enzyme activity may facilitate intrusive tip growth of the laticifer among other cells by solubilizing pectic substances of the middle lamella and also may be important for loosening wall material of the laticifer itself to facilitate extension growth.     

Red blood cell galactokinase activity and presenile cataracts

Red blood cell galactokinase activity was measured in 70 patients with cataracts to assess a possible correlation between galactokinase activity levels and risk of cataract development. Among all, 15 patients developed cataracts during the first year of life, 25 patients under the age of 50 and 30 later in life. No cases of total or partial galactokinase deficiency were found. These results, taken together with the absence of cataracts in 9 patients with partial galactokinase deficiency render less certain the cause and effect relationship between partial galactokinase deficiency and the appearance of cataracts.     

Using dried blood spots stored on filter paper to measure cholinesterase activity in wild avian species

Birds of prey that are poisoned by cholinesterase inhibitors (e.g. organophosphate and carbamate insecticides) are often cared for at animal shelters, rehabilitation centres and wildlife diagnostic facilities. Plasma cholinesterase (ChE) activity is a recognized method of assessing exposure to these insecticides, but standard blood-handling protocols are difficult to follow in non-laboratory settings. The primary objective of this study was to expand upon a method for storing human blood on filter paper without the need for complicated equipment or refrigeration, and to test its utility for measurement of ChE activity in avian blood. ChE activity from whole blood, plasma, and dried blood spots was analysed from 169 wild birds and comparisons made among sample types. ChE activity measured in whole blood haemolysates and dried blood spots were significantly correlated (r=0.74, p<0.001), as was ChE activity measured in plasma and dried blood spots (r=0.68, p<0.001). This study demonstrated that monitoring pesticide exposure in birds could be conducted using elementary blood sampling, preserving and shipping techniques.     

Differential effects of dietary selenium (Se) and folate on methyl metabolism in liver and colon of rats

A previous study compared the effects of folate on methyl metabolism in colon and liver of rats fed a selenium-deficient die (<3 μg Se/kg) to those of rats fed a diet containing supranutritional Se (2 mg selenite/kg). The purpose of this study was to investigate the effects of folate and adequate Se (0.2 mg/kg) on methyl metabolism in colon and liver. Weanling, Fischer-344 rats (n=8/diet) were fed diets containing 0 or 0.2 mg selenium (as selenite)/kg and 0 or 2 mg folic acid/kg in a 2×2 design. After 70 d, plasma homocysteine was increased (p<0.0001) by folate deficiency; this increase was markedly, attenuated (p<0.0001) in rats fed the selenium-deficient diet compared to those fed 0.2 mg Se/kg. The activity of hepatic glycine N-methyltransferase (GNMT), an enzyme involved in the regulation of tissue S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH), was increased by folate deficiency (p<0.006) and decreased by selenium deprivation, (p<0.0003). Colon and liver SAH were highest (p<0.006) in rats fed deficient folate and adequate selenium. Although folate deficiency decreased liver SAM (p<0.001), it had no effect on colon SAM. Global DNA methylation was decreased (p<0.04) by selenium deficiency in colon but not liver; folate had no effect. Selenium, deficiency did not affect DNA methyltransferase (Dnmt) activity in liver but tended to decrease (p<0.06) the activity of the enzyme in the colon. Dietary folate did not affect liver or colon Dnmt. These results in rats fed adequate selenium are similar to previous results found in rats fed supranutritional selenium. This suggests that selenium deficiency appears to be a more important modifier of methyl metabolism than either adequate or supplemental selenium. The U.S. Department of Agriculture, Agriculture Research Service, Northern Plains Area, is an equal opportunity/affirmative action employer and all agency services are available without discrimination.     

Galactokinase-deficient mutants of Tetrahymena thermophila: Selection and characterization

Summary We have isolated a series of mutants of Tetrahymena thermophila which are resistant to inhibition of growth by the galactose analog, 2-deoxygalactose. These mutants were obtained after mutagenesis with nitrosoguanidine and the induction of cytogamy to permit the recovery of recessive mutations induced in the germline micronucleus. Resistance to 2-deoxygalactose is correlated with a decreased rate of growth in galactose minimal medium and greatly reduced levels of galactokinase. The resistant phenotype of the mutants is apparently due to the galactokinase deficiency, which prevents the accumulation of toxic phosphorylated metabolites of 2-deoxygalactose. Genetic analyses reveal that the 2-deoxygalactose resistance alleles segregate as single Mendelian loci. The galactokinase-deficient strains described here represent the first mutants in this organism for which the biochemical basis of the mutant phenotype is known. These mutants, as well as others isolated similarly, should be of value in the elucidation of the mechanisms governing galactokinase gene regulation and in improving techniques of selection for other recessive mutations in Tetrahymena.