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PubMed2004

[From gene to disease; galactosemia and galactose-1-phosphate uridyltransferase deficiency].

Classical galactosaemia (Mendelian Inheritance in Man, no 230400) is an autosomal recessive disorder of galactose metabolism caused by a deficiency of the enzyme galactose-1-phosphate uridyltransferase (GALT). The GALT enzyme is responsible for the conversion of galactose-1-phosphate with UDP glucose to glucose-1-phosphate and UDP galactose. The gene encoding for GALT is located on chromosome 9p13. Patients present with hepatomegaly, liver failure, food intolerance, hypoglycaemia, muscle hypotonia, sepsis and cataract. Treatment involving the total restriction of lactose-containing foods is life-saving but many patients develop late complications such as problems of mental development, disorders of motor function, disorders of speech and hypergonadotrophic hypogonadism.

Galactosemias↗
PubMed1972

Specific radioactive labeling of terminal n-acetylgalactosamine of glycosphingolipids by the galactose oxidase-sodium borohydride method.

The galactose oxidase-sodium borohydride method was used to specifically label the terminal N-acetylgalactosamine of three glycosphingolipids, Gm2-ganglioside, asialo-Gm2-ganglioside, and globoside. All of the compounds showed a minimum of 95% radiopurity, and generally more than 90% of the total radioactivity was located in the terminal galactosamine moiety. Globoside and asialo-Gm2-ganglioside were labeled to high specific activities comparable with those of the sphingolipids with a terminal galactose moiety, labeled with the same procedure. These labeled compounds were well suited as substrates for the study of specific sphingolipid N-acetylgalactosaminidase. Gm2-ganglioside, however, was a poor substrate for galactose oxidase, and its specific activity was only a small percentage of the others. Furthermore, because of the low specific activity of the galactosamine moiety, it was necessary to pretreat Gm2-ganglioside with unlabeled sodium borohydride to reduce the nonspecific labeling of other portions of the molecule. The use of labeled sodium borohydride of a very high specific activity may yield specifically labeled Gm2-ganglioside suitable for metabolic studies. Thus, the method is useful for labeling not only terminal galactose but also terminal N-acetylgalactosamine of glycosphingolipids.

Alcohol Oxidoreductases↗
PubMed1995

Independent modulation of galactose-specific receptor expression in rat liver cells.

The expression of galactose-specific receptors on liver cells from rats at the end of pregnancy and from estrogen-treated animals was studied. The number and distribution of binding sites were estimated on hepatocytes and Kupffer and endothelial cells in vitro as well as in situ by means of protein-gold complexes. Hepatocytes and endothelial cells from pregnant rats showed an increased binding activity of at least three times for hepatocytes and one and a half times for endothelial cells with respect to normal rat livers. The increase in the hepatocyte receptor expression was paralleled by an increase in the level of its specific messenger RNA (mRNA). On Kupffer cells, a decreased number of binding sites, at least three times less than control values, was measured. The correlation between the altered hormonal level during pregnancy and the expression of galactose binding sites was examined in hepatocytes and Kupffer cells isolated from virgin rats treated with the synthetic estrogen diethylstilbesterol. In estrogen-treated rats both the binding sites and the specific mRNA of hepatocytes increased as compared with vehicle-treated or untreated animals. In contrast, in Kupffer cells both the estrogen treatment as well as vehicle-only injection led to a significant reduction in the expression of binding sites as compared with virgin untreated animals. To establish whether the decrease of galactose binding sites in Kupffer cells was related to the activation of macrophages or to the removal of plasma membrane caused by enhanced nonspecific phagocytosis, in situ binding experiments were performed after lipopolysaccharide (LPS)-stimulation or latex-bead phagocytosis. Nonspecific phagocytosis does not affect the binding activity, which instead appears strongly reduced after LPS injection. These findings suggest an independent response of galactose-specific receptor expression systems in the different types of liver cells to modulating agents.

Animals↗
PubMed1994

Characterization and activation of naturally occurring abortive complexes of UDP-galactose 4-epimerase from Escherichia coli.

UDP-galactose 4-epimerase catalyzes the interconversion of UDP-galactose and UDP-glucose. The enzyme from Escherichia coli is a dimeric protein with an overall molecular weight of 79,000 that contains NAD+ very tightly but noncovalently bound in the enzymatic active site. NAD+ is the coenzyme for epimerization and is transiently reduced to NADH in the course of catalysis. All samples of highly purified UDP-galactose 4-epimerase contain significant amounts of NADH, and that purified after overexpression in E. coli cells contains a substantial amount of NADH. To the degree that NADH replaces enzyme bound NAD+ in the coenzyme binding site, the epimerase activity is decreased. The extinction coefficient at 345 nm for NADH in its binding site is estimated to be 3.3 mM-1 cm-1. 31P NMR spectroscopic and enzymatic analyses reveal that UDP-glucose, UDP-galactose, UDP, and UMP are gradually released from the purified enzyme upon addition of UMP or P1-5'-uridine-P2-methyl diphosphate (MeUDP). It is concluded that NADH associated with the purified enzyme is a component of inactive, abortive complexes (E-NADH-uridine nucleotide) that contain tightly bound uridine nucleotides in place of the epimerization intermediate UDP-4-keto-alpha-D-hexoglucopyranose. These complexes are produced in vivo in the course of bacterial growth. The enzymatic activity of purified epimerase is increased by reaction with 1,2-naphthoquinone-4-sulfonate, which oxidizes the NADH to NAD+. Compositionally defined abortive complexes (E-NADH-uridine nucleotide) containing UMP, UDP, or UDP-hexoses (Glc/Gal) have been prepared in vitro and subjected to activation by 1,2-naphthoquinone-4-sulfonate. All are activated at rates comparable to that for the purified enzyme, although those containing UMP and UDP-hexose are more readily activated than those containing UDP. The activity of the reactivated enzyme approaches that of the most highly active epimerase that has been reported from E. coli.

Carbohydrate Epimerases↗
PubMed1976

Pore diffusion model for a two-substrate enzymatic reaction: application to galactose oxidase immobilized on porous glass particles.

An analysis of the pore diffusion model involving a two-substrate enzymatic reaction is presented. The resulting equations have been applied to the case of galactose oxidase catalyzed oxidation of galactose when the enzyme is immobilized on porous glass particles. The physical constants of the system were obtained by theoretical predictions and the enzyme concentration in the porous medium was derived from the experimental results. The calculations were performed with the assumption that the kinetic parameters of the enzyme remain unchanged upon immobilization. The theoretically calculated effectiveness factors were compared with the experimental effectiveness factors determined from the batch kinetic experiments and were found to be in agreement. The results are presented as effectiveness factor plots graphed as functions of bulk galactose and oxygen concentrations. The model was extended in order to study the effect of external mass transfer coefficients and pore enzyme concentrations on the effectiveness factors.

Adsorption↗
PubMed1992

The synthesis of high-specific-activity UDP-[6-3H]galactose, UDP-N-[6-3H]acetylgalactosamine, and their corresponding monosaccharides.

Tritiated uridine-5'-diphosphogalactose (UDP-[3H]Gal) has been widely used to study oligosaccharide biosynthesis and structure. It can be synthesized either chemically or enzymatically using galactose oxidase to oxidize the hydroxyl moiety at C-6 to an aldehyde (6-aldo-UDP-Gal), which is then reduced back to the alcohol with tritiated sodium borohydride. Although the enzymatic approach is simple and efficient, there are several problems associated with it. First, incomplete oxidation to the aldehyde reduces the final specific activity. Second, if the galactose oxidase is not removed from the 6-aldo-UDP-Gal prior to reduction, the resulting UDP-[6-3H]Gal can be reoxidized to 6-aldo-UDP-[6-3H]Gal. We present evidence for the occurrence of this compound in one commercially obtained preparation of UDP-[6-3H]Gal. Finally, if an excess of 6-aldo-UDP-Gal is used for good yield, it is necessary to quench the reduction with nonradioactive borohydride, again reducing the final specific activity. We have devised a rapid, inexpensive, and efficient synthesis of UDP-[6-3H]Gal that circumvents all of these problems. Galactose oxidase is used to produce 6-aldo-UDP-Gal and the completeness of this reaction is confirmed on polyethyleneimine (PEI) cellulose TLC plates. The 6-aldo-UDP-Gal is purified on silica gel 60 TLC plates. This purified compound is then reduced with tritiated sodium borohydride, with the aldehyde present in excess. Unreacted 6-aldo-UDP-Gal is then purified away from the product UDP-[6-3H]Gal by chromatography on PEI cellulose. Radiochemically pure UDP-[6-3H]Gal with a specific activity of 10 Ci/mmol was obtained using the above scheme.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromatography, High Pressure Liquid↗
PubMed1985

Galactose-rich glycoproteins are on the cell surface of herpes virus-infected cells. 1. Surface labeling and serial lectin binding studies of Asn-linked oligosaccharides of glycoprotein gC.

Cell-surface glycoproteins of mock-infected and herpes simplex virus type 1 (HSV-1)-infected BHK-21 and HEp-2 cells were radiolabeled by incubation with galactose oxidase followed by reduction with NaB3H4. The incorporation of radiolabel into glycoconjugates in both BHK-21 and HEp-2 cells was increased several fold following infection with HSV, showing an increase in surface-exposed Gal residues in the infected cells. This was further confirmed by an increase in binding of cell-surface-labeled glycoproteins gC and gB from HSV-infected BHK-21 cells to Ricinus communis agglutinin I, which is specific for beta-D-Gal residues. Prior treatment of cells with Clostridium perfringens neuraminidase enhanced the surface radiolabeling by the galactose oxidase/NaB3H4 method: HEp-2 cells exhibited over sixfold enhancement in labeling, while BHK-21 cells showed only a slight increase. HSV glycoprotein gC was the predominant cell-surface glycoprotein radiolabeled by the galactose oxidase/NaB3H4 method in virus-infected BHK-21 cells. The glycoprotein gC was purified by immunoaffinity column chromatography on monoclonal anti-gC-antibody-Sepharose. The radiolabel in the glycopeptides of gC was resistant to beta elimination, showing that it was associated only with Asn-linked oligosaccharides. A serial lectin affinity chromatography of glycopeptides on columns of concanavalin A-Sepharose, lentil (Lens culinaris) lectin-Sepharose, and Ricin I-agarose allowed the assignment of minimal oligosaccharide structures bearing terminal Gal residues in gC.

Animals↗
PubMed1990

Construction and characterization of a recombinant tripartite enzyme, galactose dehydrogenase/beta-galactosidase/galactokinase.

The in-frame gene fusion between 3 enzymes, galactose dehydrogenase, beta-galactosidase and galactokinase, is described. The purified artificial tripartite enzyme displayed all three enzymic activities. Two major forms of the hybrid protein were found, consisting of 4 and 8 subunits respectively, but other forms could also be identified. Each subunit was made up of one monomer each of galactose dehydrogenase, beta-galactosidase and galactokinase. Proximity effects exhibited by the hybrid enzyme could be demonstrated using [14C]galactose as a reporter molecule.

Base Sequence↗
PubMed2002

Galactose dialdehyde as potential protein cross-linker: proof of principle.

Combined enzymatic oxidation of D-galactose by D-galactose oxidase [EC 1.1.3.9] in water, amination with butylamine, and oxalic acid catalyzed Amadori rearrangement in methanol yielded 1,6-bis(butylamino)-1,6-dideoxy-erythro-hexo-2,5-diulose, demonstrating how in situ formed galacto-hexodialdose can be used to cross-link protein residues. The various species formed during this three-step conversion are present as bicyclic structures in solution as established by 13C labeling and in situ NMR spectroscopy of the reaction mixtures. Using protein (gelatin) instead of butylamine, distinct Amadori product formation was observed using 99% enriched D-(1-(13)C)- and D-(2-(13)C)-galactose.

Aldehydes↗
PubMed2001

Kinetics of the coupled reaction catalysed by a fusion protein of beta-galactosidase and galactose dehydrogenase.

The mechanistic implications of the kinetic behaviour of a fusion protein of beta-galactosidase and galactose dehydrogenase have been analysed in view of predictions based on experimentally determined kinetic parameter values for the galactosidase and dehydrogenase activities of the protein. The results show that the time course of galactonolactone formation from lactose in the coupled reaction catalysed by the fusion protein can be most satisfactorily accounted for in terms of a free-diffusion mechanism when consideration is given to the mutarotation of the reaction intermediate galactose. It is concluded that no tenable kinetic evidence is available to support the proposal that the fusion protein catalyses galactonolactone formation from lactose by a mechanism involving channelling of galactose.

Diffusion↗
PubMed2004

Stereoselective hydrogen abstraction by galactose oxidase.

The fungal enzyme galactose oxidase is a radical copper oxidase that catalyzes the oxidation of a broad range of primary alcohols to aldehydes. Previous mechanistic studies have revealed a large substrate deuterium kinetic isotope effect on galactose oxidase turnover whose magnitude varies systematically over a series of substituted benzyl alcohols, reflecting a change in the character of the transition state for substrate oxidation. In this work, these detailed mechanistic studies have been extended using a series of stereospecifically monodeuterated substrates, including 1-O-methyl-alpha-D-galactose as well as unsubstituted benzyl alcohol and 3- and 4-methoxy and 4-nitrobenzyl derivatives. Synthesis of all of these substrates was based on oxidation of the alpha,alpha'-dideuterated alcohol to the corresponding (2)H-labeled aldehyde, followed by asymmetric hydroboration using alpha-pinene/9-BBN reagents to form the stereoisomeric alcohols. Products from enzymatic oxidation of each of these substrates were characterized by mass spectrometry to quantitatively evaluate the substrate dependence of the stereoselectivity of the catalytic reaction. For all of these substrates, the selectivity for pro-S hydrogen abstraction was at least 95%. This selectivity appears to be a direct consequence of constraints imposed by the enzyme on the orientation of substrates bearing a branched beta-carbon. Steady state analysis of kinetic isotope effects on V/K has resolved individual contributions from primary and alpha-secondary kinetic isotope effects in the reaction, providing a test for the involvement of an electron transfer redox equilibrium in the oxidation process. Multiple isotope effect measurements utilizing simultaneous labeling of the substrate and solvent have contributed to refinement of the relation between proton transfer and hydrogen atom transfer steps in substrate oxidation.

Aldehyde Oxidase↗
PubMed1976

Galactose metabolism in regenerating rat liver.

1. Rats trained on a controlled lighting and feeding schedule were subjected to partial hepatectomy or sham operation. 2. After a large period of about 6h the activity of UDP-galactose 4-epimerase increased threefold, reaching a maximum 4 days after partial hepatectomy, and returned to normal values within a fortnight. 3. The enzyme pattern of the UDP-galactose-glycoprotein galactosyltransferase was biphasic, one peak appearing at 20 h, the second at 72 h after partial hepatectomy. 4. The rise in enzyme activities could be blocked by the injection of actinomycin D, and the Km values for UDP-glucose and UDP-galactose were nearly identical in regenerating and adult liver. It is therefore concluded that the increase in enzyme activity is due to synthesis de novo of enzyme protein.

Animals↗
PubMed1978

The labelling of galactose residues in hepatitis B surface antigen glycoprotein.

The 20 to 25 nm particles of hepatitis B surface antigen were purified from the serum of a carrier chimpanzee. Five major polypeptide species were revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Treatment of the particles with neuraminidase (EC 3.2.1.18) and galactose oxidase (EC 1.1.3.9) followed by reduction with tritiated sodium borohydride labelled galactose residues in a single glycoprotein with an apparent mol. wt. of 28 000. The glycoprotein was not labelled when neuraminidase treatment was omitted, indicating that the galactose residues are in subterminal positions in the oligosaccharide chains. There was no significant incorporation of radiolabel into lipid. The serological activity of the antigen, as measured by a competitive double-antibody radioimmunoprecipitation assay, was not altered by the labelling procedure nor by exposure to neuraminidase alone.

Animals↗
PubMed1962

Galactose metabolism. I. Pathway of carbon in fermentation by Streptococcus faecalis.

Fukuyama T. T. (University of Pennsylvania, Philadelphia) and D. J. O'Kane. Galactose metabolism. I. Pathway of carbon in fermentation by Streptococcus faecalis. J. Bacteriol. 84:793-796. 1962.-The pathway by which galactose-1-C(14) is fermented in Streptococcus faecalis was investigated using dried-cell preparations. Lactic acid, acetic acid, formic acid, ethanol, and carbon dioxide were the end products formed, with lactic acid representing approximately 50% of the sugar fermented. The distribution of radio-activity indicated that the fermentation follows the Embden-Meyerhof route, suggesting that the difference in the formation of the products of glucose and galactose fermentation must be attributed to alternate routes of pyruvic acid metabolism. Differences in pH did not account for the dissimilar fermentation patterns.

Acetates↗
PubMed1978

Factor VIII/von Willebrand factor protein. Galactose a cryptic determinant of von Willebrand factor activity.

The normal Factor VIII/von Willebrand factor protein has the ability to agglutinate or aggregate normal platelets in the presence of ristocetin (von Willebrand factor activity). Removal of greater than 95% of the sialic acid from this protein by neuraminidase did not affect the von Willebrand factor or procoagulant activity. However, oxidation of the penultimate galactose of the asialo Factor VIII/von Willebrand factor protein with galactose oxidase resulted in a progressive loss of von Willebrand factor activity with no effect on procoagulant activity. Reduction of the 6-aldehydo intermediate by potassium borohydride caused full regeneration of von Willebrand factor activity. These studies confirm the identification of the intact penultimate galactose moiety as a critical determinant of von Willebrand factor activity.

Blood Coagulation Factors↗
PubMed1982

Dynamic changes in the organophosphate profile of the experimental galactose-induced cataract.

Dynamic changes in lens organophosphate metabolites during 24 hr incubation in 30 mM galactose media were measured with phosphorus-31 nuclear magnetic resonance spectroscopy. The following phosphates were quantitated from the intact crystalline lens: adenosine triphosphate (ATP), adenosine diphosphate (ADP), inorganic orthophosphate, alpha-glycerophosphate, phosphorylated hexoses and trioses, nicotinamide adenine dinucleotide, uridine diphosphoglucose and uridine diphosphogalactose, glycerol-3-phosphorylethanolamine and 3-phosphorylcholine, and an unidentified phosphorus-containing molecule. The temporal sequences of metabolic events that define the dynamic rates of accumulation or depletion of lens organophosphates reveal that the first event in the decline of the tissue upon galactose incubation is a net consumption of ATP, which occurs as a sigmoidal function with time and which is typified by a characteristic half-life of 18 hr. Alpha-glycerophosphate accumulated at an increasing rate with time, whereas ADP, inorganic orthophosphate, and the other organophosphates were essentially unchanged. Cataract formation in the subcapsular and superficial cortical regions was visible after 16 hr incubation in the experimental buffer. These findings support the hypothesis that alterations in the organophosphate levels of the lens are contributing factors to the initial formation of the experimental galactose cataract.

Adenosine Diphosphate↗
PubMed1990

Prevention of retinal vessel changes associated with diabetic retinopathy in galactose-fed dogs by aldose reductase inhibitors.

Vascular changes associated with early diabetic retinopathy that include the selective degeneration of pericytes, the formation of microaneurysms and acellular capillaries, and vessel dilation have been experimentally investigated in age- and sex-matched beagle dogs fed a 30% galactose diet and treated with or without the aldose reductase inhibitors sorbinil and/or M79175. Eyes from dogs in each group were periodically enucleated during a 36-month period and their retinal capillaries were examined as trypsin-digested flat preparations. These studies reveal that the destruction of retinal pericytes to form pericyte ghosts is the earliest observable retinal vessel change occurring after 19 to 21 months of galactose feeding. By 24 months, both an irregular distribution of endothelial cell nuclei near pericyte ghosts and the presence of acellular capillaries containing neither endothelial cells nor pericytes can be observed. This was followed by the histologic appearance of microaneurysms after 27 months and the funduscopic appearance of intraretinal hemorrhages after 33 months. Varicose enlargements of capillaries were also observed in the trypsin-digested preparations from dogs fed galactose for 33 to 36 months. All of these changes are linked to the initial aldose reductase-associated destruction of pericytes. The onset and progression of these retinal changes were retarded in a dose-dependent manner with aldose reductase inhibitors.

Aldehyde Reductase↗
PubMed1987

Galactose elimination kinetics in sepsis. Correlations of hepatic blood blow with function.

To study hepatic blood flow with clearance techniques during sepsis, it is essential to work within the limitations of the test being applied. Based on galactose elimination kinetics, this study validates galactose clearance at low concentrations as an estimate of effective hepatic blood flow in a rat peritonitis model of cecal ligation and puncture. Hepatic function as determined by galactose elimination capacity fell 25% at ten hours after induction of peritonitis, which correlated closely with the 20% reduction in effective hepatic blood flow at the same time point despite a normal cardiac output. The pattern of reduced flow and reduced function is consistent with intrahepatic flow redistribution. Inadequate flow at the microvascular level with secondary cellular injury may explain the liver dysfunction observed during sepsis.

Animals↗