Shirley Rogers

Liver galactose-l-phosphate uridyl transferase: activity in normal and galactosemic subjects

The kinetic characteristics of galactose-l-phosphate uridyl transferase have been determined in homogenates of human liver biopsies obtained from control subjects and in 50-fold purified enzyme preparations from liver obtained at autopsy. A standardized assay procedure employing linear kinetics was used to assess the enzyme activity in homogenates of liver biopsy specimens from five control subjects and four patients with congential galactosemia with demonstrated absence of the enzyme activity in red blood cells. Activity of control specimens ranged from 11.8 to 17.2 mmumoles of UDPgalactose formed per min mg of protein. Liver of two galactosemic patients, both Caucasian, possessed no detectable enzyme activity (less than 1-2% of normal). The tissue of two others, both Negro, who are known to be capable of metabolizing intravenously administered galactose, contained easily detectable enzyme at approximately 10% of the controls. No alternate enzymatic activity for formation of UDPgalactose was found in the liver of Negroes with galactosemia that was as active as the residual galactose-l-phosphate uridyl transferase. The data suggest that the residual liver enzyme activity accounts for the ability of Negroes with galactosemia to metabolize limited but significantly large quantities of galactose.

The concentration of red blood cell UDPglucose and UDPgalactose determined by high-performance liquid chromatography.

We have developed a sensitive method that employs high-performance liquid chromatography to separate and quantitate uridine diphosphogalactose (UDPGal) and uridine diphosphoglucose (UDPGlu) in human red blood cells. The trichloracetic acid extracts of red blood cells were chromatographed using a Dionex CarboPac anion-exchange resin and a 20-40% potassium phosphate buffer, pH 4.5, in a gradient-elution program. UDPGal and UDPGlu were detected spectrophotometrically at 254 nm. Recoveries of UDPGal and UDPGlu ranged from 96 to 106%. Under these conditions, there was exceptionally good reproducibility in stored specimens, and the method was sensitive in the low picamole range. The mean values and standard deviations in adults were 2.9 +/- 0.4 and 7.8 +/- 0.8 mumol/100 g Hgb for UDPGal and UDPGlu, respectively. The values in children were 4.5 +/- 1.2 and 10.2 +/- 1.7 mumol/100 g Hgb for UDPGal and UDPGlu, respectively. Values determined by the HPLC method are in excellent agreement with those obtained by enzyme analysis.

Characteristics of galactose-1-phosphate uridyl transferase in intestinal mucosa of normal and galactosemic humans☆

Abstract The characteristics of galactose-1-phosphate uridyl transferase have been determined in homogenates of jejunal muscle biopsies obtained from normal subjects and the enzyme activity has been assessed in tissue of five patients with congenital galactosemia plus two obligate heterozygotes for the disorder. Transferase activity in tissue from normal young adults was 12.9 ± 1.24 (mean ± SE) mμmoles UDP galactose formed/min./mg. protein. Heterozygote levels were 3.7 and 3.1 mμmoles/min./mg. protein. Of the five galactosemic children, the three Negro patients had easily detectable activity about 10 per cent of normal whereas Caucasian patients had none. The presence of some transferase in visceral tissue appears to delineate racial differences in galactosemic subjects further and may explain the ability of Negro galactosemics to adequately metabolize small amounts of galactose.

Developmental and Tissue-Specific Modulation of Rat Galactose-1-Phosphate Uridyltransferase Steady State Messenger RNA and Specific Activity Levels

ABSTRACT: To assess the role of genetic regulation as a modulating factor in the variability of rat tissue galactose-1-phosphate uridyltransferase (GALT) sp act, we have determined steady state GALT mRNA and sp act in rat liver during postnatal development. Steady state GALT mRNA levels increase from birth to d 5 and subsequently decrease toward adult levels. GALT sp act mirrors the mRNA pattern. A survey of steady state mRNA and GALT sp act of several adult rat tissues revealed marked tissue differences with a good correlation of the two parameters. Liver had the highest GALT mRNA and sp act; kidney, ovary, and heart had similar but lower mRNA and sp act; skeletal muscle and testes had the least GALT mRNA and enzyme sp act. These findings suggest that genetic regulation is important in the variable expression of GALT tissue sp act.

Nucleotide inhibition of mammalian liver galactose-1-phosphate uridylyltransferase

Abstract Mammalian liver hexose-1-phosphate uridylyltransferase (UDP-glucose:α- d -galactose-1-phosphate uridylyltransferase, EC 2.7.7.12) has been shown to be inhibited by various nucleotides. The nature of the inhibition has been examined with regard to the structural requirements of the inhibitory compounds and the possible regulatory function of intracellular nucleotides on enzyme activity. The most potent inhibitors were UTP and UDP which produced significant inhibition of the enzyme at concentrations normally found in liver tissue, UTP, UDP and UMP were linear competitive inhibitors of UDP-glucose with K i of 0.13, 0.35, and 2.3 mM, respectively, and uncompetitive inhibitors of galactose 1-phosphate UDP-glucoronic acid and UDP-mannose caused similar inhibition. The uridine moiety appeared necessary for competition with substrate UDP-glucose since substitution of cytidine, adenine or guanosine in the nucleotide as in CDP-glucose, ADP-glucose or GDP-glucose produced uncompetitive inhibition of UDP-glucose. Nucleotides containing glucose were competitive inhibitors of galactose 1-phosphate while those containing other sugars or without a sugar moiety caused uncompetitive inhibition of the sugar phosphate site.

Uridine diphosphoglucose content of human erythrocytes: assessment by conversion to uridine diphosphoglucuronate.

To settle the ongoing controversy regarding differential uridine diphosphoglucose (UDPG) and uridine diphosphogalactose (UDPGal) content of erythrocytes, which may be important in evaluating the metabolic abnormality in patients with galactosemia, we derived a combined enzymatic-high-performance liquid chromatography (HPLC) assay. Uridine diphosphoglucuronate (UDPGA), the unique product of UDPG dehydrogenase activity, was separated and quantified by HPLC in extracts of human erythrocytes. The quantity of UDPGA produced in cell filtrates incubated with the enzyme corresponds to the amount of UDPG directly determined by HPLC. The amount of UDPGA produced was independent of the enzyme purity or activity used. On the other hand, the amounts of UDPG estimated by fluorometric measurement of the production of reduced nicotinamide adenine dinucleotide varied with the enzyme purity and activity. The combined enzymatic-HPLC method confirms the direct determinations of UDPG content of normal erythrocytes. The results indicate that, under appropriate conditions, the fluorometric-based assay will give accurate estimates of UDPG, but the direct HPLC method yields consistent and correct UDPG and UDPGal determinations.

Metabolism and transport of galactose by rat intestine.

Intestinal uptake and metabolism of galactose were examined in everted jejunal rings from fasted adult rats using 0.2-28 mM sugar. After 60-min incubations, the total uptake (free tissue plus amount metabolized) of galactose ranged from 1.75 mumol/g at 0.2 mM to 21 mumol/g at 28 mM. Free tissue galactose was 17% of the former and 73% of the latter amount while that oxidized to 14CO2 represented only 6-16% of amount taken up. Compared to glucose, similar amounts of galactose are taken up at 0.2-2.0 mM, however, gllcose rtween 0.2 and 2 mM similar amounts of both sugars are metabolized, although a greater portion of the glucose is oxidized to 14CO2. Above 2.0 mM, 2-3 times more glucose is metabolized than galactose. Both uptake and metabolism showed saturability and kinetic analysis revealed two limbed Linweaver-Burk plots, suggesting operation of a high affinity low Km and a low affinity high Km system for sugar transport. In a series of in vivo studies, to assess the role of the intestine in the total body metabolism of galactose, 14C-labeled galactose injected intraperitoneally at a dose of either 50 or 300 mg into fasted normal, sham operated and enterectomized rats, no observable difference in 14CO2 production resulted in between the groups. It would thus appear that although extensive metabolism of galactose may take place in intestinal tissue in vitro, the intestine does not play a significant role in galactose disposition in vivo.

Regional Activity of Galactose-1-Phosphate Uridyltransferase in Rat Brain

ABSTRACT: The sp act of galactose-1-phosphate uridyl-transfense has been measured in individual regions of adult rat brain to see if site-specific differences in enzyme activity can aid in the understanding of brain abnormalities observed in well-treated galactosemic patients. The sp act in the cerebellum, brain stem, and midbrain were higher than in the cortex, hippocampus, and stratum. Activity in the cerebellum was 2-foW greater than that found in the cortex. Steady state levels of mRNA of the enzyme in the cerebellum were twice that of the cortex corresponding to the ratio of enzyme sp act in the two regions. Measurement of the kinetic parameters in tissue from the cerebellum and cortex revealed that the regional specificity in enzyme activity observed in the brain represents differences in the Vmax. Inhibition of the enzyme by undine and uridine triphosphate was essentially the same for all regions and was not influenced by the 2-fold differences observed in the levels of enzyme. Inhibition by uridine was significantly greater than that for uridine triphosphate.

Modulation of Rat Tissue Galactose-1-Phosphate Uridyltransferase by Uridine and Uridine Triphosphate

ABSTRACT: Uridine-containing sugar nucleotides, uridine diphosphate (UDP) -glucose and UDPgalactose are important intermediates in galactose metabolism, and tissue UDPgalactose may be a salient factor in the etiology of the long-term clinical manifestations of patients with galactose-1-phosphate uridyltransferase deficient galactosemia. Because uridine and uridine nucleotides such as uridine triphosphate (UTP) are known inhibitors of rat hepatic transferase, we have examined the effects of these compounds on the activity of the enzyme in homogenates of rat brain and ovary which are target organs of galactose toxicity in classical galactosemia. In addition, the concerted effect of uridine and UTP together on hepatic transferase has been assessed. These investigations have been prompted by considerations that uridine administration may have a therapeutic role in the long-term treatment of classical galactosemia. Both uridine and UTP have been found to be potent inhibitors of brain and ovarian transferase activity. Brain enzyme activity is more sensitive to these compounds than is that of the ovary. They are competitive inhibitors of UDPglucose in both newborn and adult brain enzyme preparations with a ki of 0.15 to 0.20 mM. Uridine and UTP at low concentrations were found to have an additive effect on rat hepatic transferase activity, which is especially significant in that uridine administration is known to increase hepatic UTP concentration. These findings suggest judicious caution should be used in giving uridine to patients with genetically limited transferase activity because the possibility exists of inhibiting small amounts of residual enzyme in the tissues of affected subjects.

Accumulation of galactonate in liver of suckling rats perfused with galactose

Livers of 14-day-old suckling rats were perfused for 30 min in the nonrecirculating (once-through) mode with media containing 0-60 mM galactose. Galactonate was detected in livers perfused with as little as 1 mM galactose and increased with higher perfusate galactose to 178 nmoles per gram of liver at 60 mM hexose. Tissue galactonate levels were as high as 60% of galactose-1-phosphate which also accumulated under the same conditions. Galactonate was also found to be present in the effluent perfusate. The perfused suckling rat liver appears to be a physiological model for assessing the role of galactose oxidation to galactonate as an alternate metabolic pathway of galactose metabolism.