Determination, proprerties and postnatal development of galactokinase in the swine liver]

1. Starting from the spectrophotometric method of Ballard optimal reaction conditions for measurements of galactokinase in piglet liver were systematically studied. These are (final conc. in the test): 100 mM triethanolamine-HCl buffer, 33 mM KCl, 16.5 mM NaF (inhibiting ATPase), 5 mM cysteine hydrochloride, 0.33 mM NADH2, 1 U pyruvate kinase and lactic dehydrogenase, 0.5 mM phosphoenolpyruvate, 1.5 mM galactose, 0.5 mM ATP and 1 mM MgCl2, final pH 7.5. 2. An optimal substrate concentration, a Mg: ATP-ratio of 2:1, pH-stability and addition of activators are important for the determination of galactokinase activity in the supernatant fraction of pig liver. 3. Using the optimized method galactokinase activity of pig liver in dependence on age, with particular reference to the perinatal period, was determined. 4. Galactokinase activity of liver of newborn piglets is 7 times that of adult pigs. In the suckling period the activity remains relatively constant at this high level and decreases remarkably immediately after weaning. 5. Galactokinase of liver of newborn piglets differs in kinetic properties (lower Km of ATP, higher maximal reaction velocity) from the enzyme of adult pigs, which is still insufficient to make sure the existence of two different forms of the enzyme.     

Purification and mechanism of action of a plant galactokinase]

The previously described galactokinase from Fenugreek seeds, has been purified by affinity chromatography on a column of galactosamine-CH Sepharose. This material ensures a more specific fixation than does ATP-Sepharose. A 400 fold purification was achieved in a single step, with a 80 per cent yield. Km's for galactose and for Mg/ATP2- complex were respectively 0.54 x 10-3 M and 5, 10-3 M. Galactose-1-phosphate is a competitive inhibitor of galactose while the inhibition for Mg-ATP2- is not a competitive one. The Mg-ADP complex is a non-competitive inhibitor of both galactose and Mg-ATP2-. Moreover, the Km of the enzyme for M-ATP2- complex is modified when 2-deoxy- and 6-deoxy-galactose are used instead of galactose. These results are consistent with an ordered sequential mechanism for this galactokinase: galactose binds to the enzyme before Mg-ATP2-, and galactose-1-phosphate is the last reaction product liberated. The affinity of the kinase for 6-deoxygalactose is lower than for 2-deoxygalactose. This observation reveals the importance of the hydroxyl in C6 position for the binding on the enzyme.     

Purification and properties of galactokinase from Saccharomyces cerevisiae.

Galactokinase (EC 2.7.1.6; ATP:D-galactose-1-phosphotransferase) was purified to homogeneity with a 50% yield from cells of Saccharomyces cerevisiae which were fully induced for the production of the galactose metabolizing enzymes. The purification was accomplished by:(a) ammonium sulfate fractionation, (b) streptomycin sulfate precipitation. (c) DEAE-cellulose chromatography, (d) hydroxylapatite chromatography, and finally (e) Bio-Gel A-0.5 m gel filtration. The resulting preparation of galactokinase was judged to be at least 95% pure by the following criteria: (a) sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (b) ultracentrifuge analysis, (c) nondissociating polyacrylamide gel electrophoresis, and (d) Bio-Gel A-0.5 m gel filtration. The purified enzyme preparation was used to determine the Km values for the two substrates, galactose and ATP, which were found to be 0.60 and 0.15 mM, respectively. Vmax was also determined and found to be 3.35 mmol/h/mg. This corresponds to a turnover rate of 3350 molecules of galactose phosphorylated/min/enzyme molecule. The effect of pH on the galactokinase-catalyzed phosphorylation of galactose was determined; the results showed the pH optimum of the reaction to be in the range of pH 8.0 to 9.0. The enzyme is highly specific for galactose since galactokinase did not appear to phosphorylate any of the other sugars tested at a rate greater than 0.5% of the rate of galactose phosphorylation. Amino acid analysis was performed on the enzyme preparation and the results were used to calculate the partial specific volume (v) of 0.736. The NH2-terminal sequence was determined for the first 3 residues. The molecular weight and subunit composition were determined by ultracentrifugation and polyacrylamide gel electrophoresis under dissociating and nondissociating conditions. The data obtained indicated that galactokinase is a monomeric protein of molecular weight 58,000.     

The molecular architecture of human N-acetylgalactosamine kinase

Galactokinase plays a key role in normal galactose metabolism by catalyzing the conversion of alpha-d-galactose to galactose 1-phosphate. Within recent years, the three-dimensional structures of human galactokinase and two bacterial forms of the enzyme have been determined. Originally, the gene encoding galactokinase in humans was mapped to chromosome 17. An additional gene, encoding a protein with sequence similarity to galactokinase, was subsequently mapped to chromosome 15. Recent reports have shown that this second gene (GALK2) encodes an enzyme with greater activity against GalNAc than galactose. This enzyme, GalNAc kinase, has been implicated in a salvage pathway for the reutilization of free GalNAc derived from the degradation of complex carbohydrates. Here we report the first structural analysis of a GalNAc kinase. The structure of the human enzyme was solved in the presence of MnAMPPNP and GalNAc or MgATP and GalNAc (which resulted in bound products in the active site). The enzyme displays a distinctly bilobal appearance with its active site wedged between the two domains. The N-terminal region is dominated by a seven-stranded mixed beta-sheet, whereas the C-terminal motif contains two layers of anti-parallel beta-sheet. The overall topology displayed by GalNAc kinase places it into the GHMP superfamily of enzymes, which generally function as small molecule kinases. From this investigation, the geometry of the GalNAc kinase active site before and after catalysis has been revealed, and the determinants of substrate specificity have been defined on a molecular level.     

Further purification and characterization of the acid α-glucosidase

1. Centrifugation of rat liver acid glucosidase, which had been purified by adsorption on dextran gel, on a density gradient of sucrose showed the enzyme to be impure. 2. Preliminary purification of the enzyme before the gel filtration improved the final degree of purity of this preparation. Disc gel electrophoresis of this preparation showed a single band of protein. 3. The sedimentation co-efficient and the molecular weight determined on a sucrose gradient were 4.9-5.1s and 76000-83000 respectively for the rat liver enzyme, and 5.6s and 97000 for the acid alpha-glucosidase purified by means of the same procedure from the human kidney. 4. The Michaelis constants of rat liver and human kidney enzyme were 4.7x10(-3)m and 13.6x10(-3)m respectively with maltose as substrate. 5. The enzyme from both tissues was inhibited by tris and by erythritol. The inhibition of the rat liver acid glucosidase by erythritol was competitive.     

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.     

Functional analysis of disease-causing mutations in human galactokinase.

Galactokinase (EC 2.7.1.6) catalyzes the first committed step in the catabolism of galactose. The sugar is phosphorylated at position 1 at the expense of ATP. Lack of fully functional galactokinase is one cause of the inherited disease galactosemia, the main clinical manifestation of which is early onset cataracts. Human galactokinase (GALK1) was expressed in and purified from Escherichia coli. The recombinant enzyme was both soluble and active. Product inhibition studies showed that the most likely kinetic mechanism of the enzyme was an ordered ternary complex one in which ATP is the first substrate to bind. The lack of a solvent kinetic isotope effect suggests that proton transfer is unlikely to be involved in the rate determining step of catalysis. Ten mutations that are known to cause galactosemia were constructed and expressed in E. coli. Of these, five (P28T, V32M, G36R, T288M and A384P) were insoluble following induction and could not be studied further. Four of the remainder (H44Y, R68C, G346S and G349S) were all less active than the wild-type enzyme. One mutant (A198V) had kinetic properties that were essentially wild-type. These results are discussed both in terms of galactokinase structure-function relationships and how these functional changes may relate to the causes of galactosemia.     

Some properties of galactokinase in developing rat liver

1. The nature of the galactokinase present in the livers of foetal, newborn and adult rats was examined by the application of several separation procedures and by measurement of a range of kinetic parameters. 2. No evidence of enzyme heterogeneity at any stage of development was found during gel filtration on Sephadex G-100, column chromatography on DEAE-cellulose or a variety of electrophoretic procedures. 3. The K(m) values, inhibition characteristics and other kinetic parameters appear to remain constant during development. 4. Rat liver galactokinase activity does not adapt to dietary changes in either the adult or the newborn rat; hence it is unlikely that the presence of galactose in milk controls the enzymic activity profile during development. 5. On the present evidence it is concluded that only one form of galactokinase is present in rat liver and that the enzymic activity is controlled by non-dietary factors.     

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.     

A galactokinase of Mycobacterium sp. 279 galactose mutant

A galactokinase and the other enzymes of a galactose catabolic pathway were found in Mycobacterium sp. 279 galactose mutant. The galactokinase was partially purified in a procedure involving ammonium sulfate precipitation, Sephadex G-100 filtration and DEAE-cellulose chromatography. The enzyme was 170-fold purified with 25% of recovery. It was most active at pH 7.8-8.0 in the presence of Mg2+, CO2+, Mn2+ or Fe2+ ions. The molecular weight of the enzyme as determined by Sephadex G-100 filtration amounted to 41,700. The apparent Michaelis constants for galactose and ATP in spectrophotometric test were 1.0 mM and 0.29 mM, respectively. Mercuric compounds at concentration of 0.4 mM completely blocked the enzyme. The galactokinase was quite stable during storage at moderatory temperatures and neutral pH but underwent rapid inactivation on heating above 50 degrees C.     

Purification and properties of malyl-coenzyme A lyase from Pseudomonas AM1

1. Malyl-CoA lyase was purified 20-fold from extracts of methanol-grown Pseudomonas AM1. 2. Preparations of the enzyme were essentially homogeneous by electrophoretic and ultracentrifugal criteria. 3. Malyl-CoA lyase has a molecular weight of 190000 determined from sedimentation-equilibrium data. 4. Within the range of compounds tested, malyl-CoA lyase is specific for (2S)-4-malyl-CoA or glyoxylate and acetyl-CoA or propionyl-CoA. 5. A bivalent cation is essential for activity, Mg(2+) or Co(2+) being most effective. 6. Malyl-CoA lyase is inhibited by (2R)-4-malyl-CoA and by some buffers, but thiol-group inhibitors are without effect. 7. Optimal activity was recorded at pH7.8. 8. An equilibrium constant of 4.7x10(-4)m was determined for the malyl-CoA cleavage reaction. 9. The Michaelis constants for the enzyme are: 4-malyl-CoA, 6.6x10(-5)m; acetyl-CoA, 1.5x10(-5)m; glyoxylate, 1.7x10(-3)m; Mg(2+), 1.2x10(-3)m.     

Purification and general properties of δ-aminolaevulate dehydratase from cow liver

1. delta-Aminolaevulate dehydratase, the enzyme catalysing the condensation of delta-aminolaevulic acid to porphobilinogen, has been prepared from cow liver and its properties have been studied. The enzyme has been purified 310-fold. 2. The purified preparation behaves as a single protein under gel filtration on Sephadex and Bio-Gel columns; it migrates as a single band in disk and starch-gel electro-phoresis at different pH values and it sediments as a single symmetrical peak in the ultracentrifuge. 3. The pH optimum for the pure enzyme was 6.8, the K(m) at pH 6.8 and 38 degrees was 1.5x10(-4)m, the isoelectric point was about pH 4.9 and the molecular weight was 140000+/-14000 by the gel-filtration method. Maximal enzyme activity was observed at 65 degrees . 4. The presence of thiol groups in the enzyme system, essential for its activity, was indicated and the total number of thiol groups was determined. 5. After the first steps of purification the enzyme required cysteine or reduced glutathione for activity.     

Purification and characterisation of lignin peroxidase from Pycnoporus sanguineus MTCC-137

Extracellular secretion of lignin peroxidase from Pycnoporus sanguineus MTCC-137 in the liquid culture growth medium amended with lignin containing natural sources has been shown. The maximum secretion of lignin peroxidase has been found in the presence of saw dust. The enzyme has been purified to homogeneity from the culture filtrate of the fungus using ultrafiltration and anion exchange chromatography on DEAE-cellulose. The purified lignin peroxidase gave a single protein band in sodium dodecylsulphate polyacrylamide gel electrophoresis corresponding to the molecular mass 40 kDa. The K(m)(, kcat) and k(cat)/K(m) values of the enzyme using veratryl alcohol and H2O2 as the substrate were 61 microM, 2.13 s(-1), 3.5 x 10(4) M(-1) s(-1) and 71 microM, 2.13 s(-1), 3.0 x 10(4) M(-1) s(-1) respectively at the optimum pH of 2.5. The temperature optimum of the enzyme was 25 degrees C.     

Kinetic studies with cytosol and mitochondrial phosphoenolpyruvate carboxykinases

1. Measurements of Michaelis constants for oxaloacetate in the reaction catalysed by liver phosphoenolpyruvate carboxykinase give values much lower than previously reported. With Mg(2+) as bivalent cation, the Michaelis constant was approx. 2.5x10(-5)m whether the enzyme used was the mitochondrial phosphoenolpyruvate carboxykinase purified from sheep liver or chicken liver or the cytosol enzyme purified from rat liver or sheep liver. 2. When Mn(2+) replaced Mg(2+) in the reaction a lower Michaelis constant of 9x10(-6)m was found, but only with the mitochondrial enzymes. 3. With all enzymes malate at high concentration was a competitive inhibitor with respect to oxaloacetate when Mn(2+) was the added cation. With Mg(2+) the inhibition by malate was competitive with the mitochondrial enzymes and non-competitive with the cytosol enzymes.     

Purification and properties of rat liver globotriaosylceramide synthase, UDP-galactose:lactosylceramide alpha 1-4-galactosyltransferase

The enzyme which catalyzes the transfer of galactose from UDP-galactose to lactosylceramide (LacCer) was obtained in a 32,000-fold purified and apparently homogeneous form from rat liver by a procedure involving affinity chromatography on UDP-hexanolamine-Sepharose and LacCer-Sepharose. The enzyme is composed of two nonidentical subunits whose apparent molecular weights are 65,000 and 22,000. Methylation and hydrolysis of the product formed by incubation of the enzyme with UDP-galactose and [3H]LacCer yielded 2,3,6-tri-O-methyl-[3H]galactose, indicating that a galactose residue was introduced to position C-4 of the terminal galactose of the LacCer. The product also specifically reacted with monoclonal antibody directed to globotriaosylceramide (Gal alpha 1-4Gal beta 1-4Glc beta 1-1Cer). This indicates that the purified enzyme is exclusively alpha 1-4-galactosyltransferase. Studies on substrate specificity indicate that the purified enzyme is highly specific for the synthesis of GbOse3Cer and is clearly distinct from the enzymes responsible for the formation of iGbOse3Cer (Gal alpha 1-3Gal beta 1-4Glc-Cer) and blood group-B substance, which possess alpha 1-3 galactosidic linkages at the nonreducing termini. The enzyme is also distinct from the alpha 1-4-galactosyltransferase which catalyzes the formation of galabiaosylceramide (Gal alpha 1-4Gal beta 1-1Cer) and IV4Gal-nLacOse4 (P1 antigen). These studies represent the first report of the properties of a highly purified alpha-galactosyltransferase catalyzing the transfer of sugar residues to glycolipids.     

Induction and characterization of -galactosidase in an extreme thermophile

A thermostable beta-galactosidase (EC 3.2.1.23; beta-dgalactoside galactohydrolase) was found to be inducible in an extreme thermophile resembling Thermus aquaticus. Enzyme induction was achieved by the addition of lactose, galactose, or the alpha-galactoside, melibiose, to growing cultures. The addition of glucose to induced cultures had a repressive effect on further enzyme synthesis. The enzyme was purified 78-fold, and the optimum temperature and pH for activity were determined to be 80 C and pH 5.0, respectively. The enzyme was activated by both manganese and ferrous iron. Sulfhydryl activation and thermal stabilization indicate that the thermophilic beta-galactosidase is a sulfhydryl enzyme. Kinetic determinations at 80 C established a K(m) of 2.0 x 10(-3)m for the chromogenic substrate o-nitrophenyl beta-d-galactopyranoside (ONPG) and a K(1) of 7.5 x 10(-3)m for lactose. The Arrhenius energy of activation (for the hydrolysis of ONPG) was calculated to be 13.7 kcal/mole. A molecular weight of 5.7 x 10(5) daltons was estimated by elution of the enzyme from Sephadex 4B.     

The Metabolism of Raffinose and Sucrose in Germinating Broad-Bean (Vicia faba) Seeds

Enzyme preparations from germinating broad-bean seeds can produceUDPG from sucrose or raffinose when incubated with UDP; in thelatter case D-galactose is also formed. This observation suggeststhat raffinose can be metabolized in two stages: (1) the hydrolyticcleavage of the trisaccharide by a-galactosidase with the releaseof galactose and sucrose; (2) a sucrose: UDP glucosyltransferase(reversed sucrose synthetase)-catalysed transfer of D-glucosefrom sucrose to UDP. The galactose liberated in stage (I) inVicia faba seeds can probably be converted to a-D-galactose-1-phosphateby galactokinase. The levels of the glucosyltransferase at variousstages of germination have been measured, the enzyme has beenpartially purified, and an estimate has been made of its apparentmolecular weight. The glucosyltransferase is specifically inhibitedby D-glucose and D-fructose and the role of this enzyme in catabolicprocesses is discussed in general terms.     

Large scale purification of myo-inositol monophosphate phosphatase from porcine brains

myo-Inositol monophosphate phosphatase (IMPase) has been purified 888-fold to apparent homogeneity from procine brains. The purification procedure involves: homogenization, ammonium sulfate fractionation, and a number of ion-exchange and gel-filtration chromatography steps. The purified enzyme exhibited a final specific activity of 932 nmol . min(-1) . mg(-1). The molecular mass of the enzyme was estimated to be 29kDa by SDS poly-acrylamide gel electrophoresis and 58 +/- 5 kDa by HPLC gel filtration in 10mM Tris-HCI, pH 7.4. Kinetic measurements have shown that the apparent K(m) value of the phosphatase for the utilization of inositol-1-phosphate and beta-glycerol phosphate are 3.20 x 10(-4) and 8 x 10(-3) M, respectively. Similar to the same enzyme isolated from bovine brains, the porcine brain enzyme has been shown to be inhibited by lithium. The K(1) was determined to be 6.38 x 10(-4) M and the inhibition is uncompetitive. (c) 1995 John Wiley & Sons, Inc.