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.     

Gene frequencies of both forms of galactosaemia in the western Hungarian province of Vas (author's transl)]

The two enzymes of galactose metabolism, namely galactokinase and galactose-1-phosphate uridyltransferase (Gal-1-PUT), were measured in 3653 subjects aged 7 months to 84 years in der to obtain the incidence of the gene deficiency causing galactosaemia in the Western Hungarian province of Vas. To date nothing is known about these frequencies in this particular region. It was of special interest whether these enzyme defects are to be found more frequently in gypsies than in a comparable population of West Hungary. The frequency of homozygous Gal-1-PUT deficiency amounts to 1:23,500; the respective incidence of galactokinase deficiency is 1:64,000. Both enzyme deficiencies in this province are not higher than in other countries. Considerable differences were established in the gene frequencies in individual areas and for various groups of subjects with variations from 1:30,000 to 1: 127,000 for galactokinase deficiency and from 1:5,300 to 1:81,000 for Gal-1-PUT deficiency. Neither gene deficiency occurred more frequently in gypsies than in the general population. This study demonstrates that the results of such analyses are relevant only to the investigated region and the greatest possible number of subjects must be taken in order to draw reliable conclusions.     

A method for galactose-1-phosphate uridyltransferase assay and the separation of its isozymes by DEAE-cellulose column chromatography

A simplified radioactive assay for galactose-1-phosphate uridyltransferase (EC 2.7.7.12) using a small DEAE-cellulose column for the identification of the endproduct (uridine diphosphate galactose) is described. The enzyme activities in red blood cell hemolysates of normal subjects are in the range of 24 to 33 (average 30.3) μmol UDPgalactose produced per g hemoglobin per h and in fibroblasts 0.39 and 1.39 (average 0.71) nmol per mg protein per min.

Furthermore, different isozymes of red blood cell galactose-1-phosphate uridyltransferase were separated on DEAE-cellulose columns. In the case of a normal genotype, most of the enzyme activity is eluted at the earlier fractions with the low molar phosphate buffer, whereas the Duarte variant appeared at later fractions with higher molar phosphate buffer.     

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.     

Galactosemia: screening and diagnosis

Galactose is normally metabolized to glucose through the coordinated activities of three enzymes: galactokinase, galactose-1-phosphate uridyl transferase (GALT), and uridine diphospho-glucose 4-epimerase (epimerase). High concentrations of galactose and their metabolites are toxic to mammals. Hereditary deficiencies of galactokinase and of GALT and perhaps rarely of epimerase cause clinical disorders that can be prevented by early recognition and institution of a galactose-free diet. The genetics of disorders of galactose metabolisms and the methods used currently for their detection are reviewed. Future prospects in the diagnosis of these disorders are discussed.     

Positive correlation between galactose-1-phosphate uridyltransferase (GALT) and UDP-galactose-4′-epimerase (GALE) activities

Objectives

The aim of our study was to determine whether the activities of galactose-1-phosphate uridyltransferase and UDP-galactose-4′-epimerase are correlated, and in what way they may influence one another.

Design and methods

Enzyme activities were measured in red blood cells from 214 individuals.

Results

A statistically significant (p < 0.001) positive correlation was observed between GALT and GALE activities.

Conclusions

Our results suggest that GALT and GALE activities are correlated and that GALE activity has a greater impact on GALT activity than vice versa.     

Improved method of mass-screening for galactosemia

The “classical” screening method¹ for the identification of individuals suffering from galactosemia (galactose-1-phosphate uridyltransferase deficiency) has been improved. The new technique is considerably more sensitive than the previous method. It is the first screening test that permits the detection of individuals with reduced enzyme activity due to heterozygosity or to other genetic variants of the enzyme. The technique is less time consuming and reduces the possibility of human error, both of which are important factors in a mass screening laboratory.     

Prenatal diagnosis of galactosemia and properties of galactose-1-phosphate uridyltransferase in erythrocytes of galactosemic variants as well as in human fetal and adult organs

The kinetic characteristics and isoelectrofocusing patterns of uridyltransferase and the concentrations of galactose-1-phosphate in hemolysates were investigated in a family with compound variants of Duarte and classical galactosemia. There were no significant differences in Km values between the genotypes. However, the isoelectrofocusing study with thin-layer polyacrylamide gels (PAGIF) as well as with agarose gels (AGIF) showed a distinctive difference. The enzyme from the Duarte variant resolved into at least two more activity bands at pH between 5.2 and 5.4. The accumulation of galactose-1-phosphate was observed only in homozygotes for classical galactosemia. Compound heterozygotes (G-D) without any clinical manifestations did not show an accumulation of galactose-1-phosphate. The isoelectrofocusing study of the enzyme in human tissues revealed their activity resolving into multiple bands, 6-8 bands at pH 5.50-6.00 and 1-3 bands at pH 4.9-5.2. No significant differences were found in the patterns between fetal and adult liver except that the intensity of the anodic bands (pH 4.9-5.2) was weaker in fetal tissues. Prenatal diagnosis of classical galactosemia was performed in nine families by measuring the enzyme activity in cultivated amniotic fluid cells. Absence of the enzyme activity in amniotic fluid cells was found in two cases, and in four cases the heterozygosity was diagnosed by a relative low enzyme activity, 30-50% of the activity in control cells cultured in parallel.     

An improved quantitative assay of galactose-1-phosphate uridyltransferase activity in erythrocytes based on the determination of glucose 1-phosphate generation

An improved quantitative method for measuring galactose-1-phosphate uridyltransferase (EC 2.7.7.12, Gal-PUT) activity in erythrocytes was developed based on the detection of glucose 1-phosphate generated under the catalytic influence of the enzyme. This is achieved by incubating the enzyme with galactose 1-phosphate and uridyldiphosphoglucose during 15 min, followed by deproteinisation. The glucose 1-phosphate generated is quantitated subsequently by measuring NADPH formation from added NADP+ in a second incubation step with added phosphoglucomutase (EC 2.7.5.1) and glucose-6-phosphate dehydrogenase (EC 1.1.1.49). Gal-PUT activity is calculated from the increment in absorption at 340 nm. Because it is technically a simple assay that is sensitive, specific and not affected by UDPgalactose-4-epimerase (EC 5.1.3.2) activity in the erythrocytal lysates it is suggested to be the method of choice for measuring Gal-PUT activity. Activities in erythrocytes of controls varied from 264 to 556 U/kg hemoglobin; in obligate heterozygotes from 53 to 190 U/kg Hb and in homozygous deficient patients less than 5 U/kg Hb was measured at 37 degrees C.     

Galactose-1-phosphate uridyltransferase in cultured cells.

A method to measure the enzyme galactose-1-phosphate uridyltransferase in cultured cells is described. The optimun pH was 8.7 and no enzyme activity was found without preincubation with dithiothreitol. The KM values for Gal-1-P and UDPG for the wild type enzyme were found to be 0.2 mM and 0.08 mM, respectively. Values for the Duarte variant were found to be identical. No significant change in enzyme activity with time after subculture was found in either cultured skin fibroblasts or cultured amniotic fluid cells. Different transferase genotypes were clearly distinguished and reference range established. Transferase levels found in normal cultured amniotic fluid cells were the same as in normal cultured skin fibroblasts. The results of a prenatal diagnosis was obtained within 3 weeks of amniocentesis.     

Microassay for estimation of galactose and galactose-1-phosphate in dried blood specimens

A fluorometric assay for blood galactose and galactose-1-phosphate has been modified and improved to shorten the analysis time and to increase sensitivity above other published methods. The method may be useful as a quantitative screening or routine clinical test to detect infants suspected of having a defect of galactose metabolism. It can also be used to monitor blood galactose or galactose-1-phosphate levels in children with galactosemia who are on a lactose-free diet.     

Kinetics of ethanol inhibition of galactose elimination in perfused pig liver

The effect of ethanol (5--25 mM) on the galactose elimination kinetics in the intact liver was studied in the isolated perfused pig liver, using the steady-state infusion technique. Ethanol reduced galactose-Vmax on average to 0.07 mmol/min kg liver in six experiments from 0.43 mmol/min kg obtained in control experiments without ethanol. Also Km was significantly reduced from 0.23 mmol/l plasma water to 0.03 mmol/l. Ethanol increased UDP-galactose ten-fold simultaneous with a rise in hepatic outflow ratio of lactate to pyruvate to about 300 from 10; this indicates that ethanol inhibits epimerase. In experiments with increasing galactose elimination rates, the concentration of galactose-1-P increased much less than the concentration of galactose, and the phosphorylation of galactose therefore seems to be rate-limiting. In vitro galactokinase is inhibited by galactose-1-P. In the present study ethanol increased galactose-1-P five to ten times, and the reduction of Vmax and Km by ethanol could be explained by uncompetitive inhibition by galactose-1-P with Ki about 0.1 mmol/l. Ethanol decreased UDP-glucose to about 40% and UTP to less than 5%, probably due to trapping as UDP-galactose. This may depress the forward transferase reaction, and therefore the other co-substrate galactose-1-P rises--and inhibits galactokinase.     

Clinical significance of plasma galactose and erythrocyte galactose-1-phosphate measurements in transferase-deficient galactosemia and in individuals with below-normal transferase activity

We correlated the clinical symptoms of transferase-deficient galactosemia with the plasma galactose and erythrocyte galactose-1-phosphate concentrations in six galactosemic patients during dietary treatment, in a child before treatment, and in 12 individuals with below-normal erythrocyte hexose-1-phosphate uridylyltransferase activity. All the treated patients were asymptomatic. Normal galactose and either normal or above-normal galactose-1-phosphate concentrations were found. Three of these patients were clinically normal as newborns while ingesting galactose-containing foods and may resemble the asymptomatic Negro galactosemic. The clinical symptoms of galactosemia were observed in the untreated patient, who showed markedly above-normal concentrations of galactose and galactose-1-phosphate, protein and reducing substances in the urine, above-normal bilirubin and alkaline phosphatase in the plasma, with normal values for glucose, aspartate aminotransferase, alanine aminotransferase, and gamma-glutamyltransferase. Clinical improvement in this patient paralleled the decline in erythrocyte galactose-1-phosphate. The individuals with below-normal hexose-1-phosphate uridylyltransferase activity (range 7--17 U/g of hemoglobin) had normal galactose and galactose-1-phosphate concentrations and were asymptomatic.     

Galactose Metabolism in a Patient with Hereditary Galactokinase Deficiency

Abstract. The ability of a galactokinase deficient patient to metabolize galactose, galactitol and galactonate was quantitated. In galactokinase deficiency, conversion of galactose to CO₂ is minimal. Apparently the defect is extensive, involving all tissues. Galactitol and galactonate, injected intravenously, were not metabolized. The administration of C-1 and C-2 labelled galactose resulted in ¹⁴CO₂ excretory patterns similar to that observed in uridyltransferase deficient mutants. The different fates of C-1 and C-2 observed in this galactokinase deficient patient give support to the existence of a direct oxidative pathway for galactose. Galactonate, although present in urine during the period of observation following injection of radioactive galactose failed to become labelled.     

Galactose Metabolism in a Patient with Hereditary Galactokinase Deficiency

Abstract. The ability of a galactokinase deficient patient to metabolize galactose, galactitol and galactonate was quantitated. In galactokinase deficiency, conversion of galactose to CO₂ is minimal. Apparently the defect is extensive, involving all tissues. Galactitol and galactonate, injected intravenously, were not metabolized. The administration of C-1 and C-2 labelled galactose resulted in ¹⁴CO₂ excretory patterns similar to that observed in uridyltransferase deficient mutants. The different fates of C-1 and C-2 observed in this galactokinase deficient patient give support to the existence of a direct oxidative pathway for galactose. Galactonate, although present in urine during the period of observation following injection of radioactive galactose failed to become labelled.     

Glucose-6-Phosphate Dehydrogenase (G-6-PD) Hamburg, a New Variant with Chronic Nonspherocytic Haemolytic Anaemia

Abstract. A new variant of G-6-PD with chronic nonspherocytic haemolytic anaemia and very low activity, named G-6-PD Hamburg, was partially purified and biochemically characterized. It was found to have very high lability, an unusually high Km for G-6-P (2000 μM), increased utilization rates for 2-desoxy G-6-P (133%) and galactose-6-phosphate (87%) and an abnormal pH-activity curve. The electrophoretic mobility seemed to be normal. The leukocytes also revealed diminished G-6-PD activity. No impairment of bactericidal activity of neutrophilic granulocytes, as shown by a normal nitroblue tetrazolium reduction, could be demonstrated.     

Galactose-1-phosphate uridyltransferase activities in erythrocytes from a patient with galactosemia: Discrepancy between two methods

When measuring with the spectrophotometric UDP-Glu consumption test, the galactose-1-phosphate uridyltransferase (Gal-PUT) activity in erythrocyte lysates from a 22-month-old infant with a late onset form of galactosemia was found to be approximately 25% of normal. With a radiochemical assay only a very low residual activity could be detected (± 1% of normal). Preincubation of the patient's lysate with purified NADase caused a marked decrease of residual Gal-PUT activity as judged from the data obtained with the consumption test. The radiochemical assay was not influenced by a similar pre-treatment. The high level of residual activity found with the consumption test in this patient was attributed to the consumption of UDP-Glu by other reactions than Gal-PUT. Because it is a direct, simple and generally applicable assay, the radiochemical procedure is suggested to be the best method for the more detailed enzymological characterisation of the Gal-PUT deficient state in galactosemics.     

Glucose-6-phosphate dehydrogenase (G-6-PD) Hamburg, a new variant with chronic nonspherocytic haemolytic anaemia

Abstract. A new variant of G-6-PD with chronic non-spherocytic haemolytic anaemia and very low activity, named G-6-PD Hamburg, was partially purified and biochemically characterized. It was found to have very high lability, an unusually high Km for G-6-P (2000 μM), increased utilization rates for 2-desoxy G-6-P (133%) and galactose-6-phosphate (87%) and an abnormal pH-activity curve. The electrophoretic mobility seemed to be normal. The leukocytes also revealed diminished G-6-PD activity. No impairment of bactericidal activity of neutrophilic granulocytes, as shown by a normal nitroblue tetrazolium reduction, could be demonstrated.     

Direct non-radioactive assay of galactose-1-phosphate:uridyltransferase activity using high performance liquid chromatography

BackgroundGalactose-1-phosphate:uridyltransferase (GALT) catalyses the conversion of galactose-1-phosphate (Gal-1-P) and UDP-glucose (UDP-Glc) into glucose-6-phosphate and UDP-galactose (UDP-Gal). Complete, or near complete, deficiency of GALT causes classic galactosaemia. The diagnosis is confirmed by measuring GALT activity in erythrocytes. The most commonly used assays require radio labelled substrates or indirect coupled assays.MethodsGALT activity was measured in erythrocyte lysates using optimal concentrations of the substrates galactose-1-phosphate and UDP-Glc. UDP-Gal and UDP-Glc were separated using reversed-phase high performance liquid chromatography with UV detection. Clinical validity was assessed using blood samples from galactosaemic patients.ResultsUDP-Gal and UDP-Glc were separated with HPLC. The assay was linear with incubation times up 80 min and between 0 and 42.5 nmol haemoglobin. Within-day and between-day imprecision at 50, 75 and 100% enzyme activity was < 1.4% and < 2.4%, respectively. Mean GALT activity in 33 individuals was 601 ± 79 nmol UDP-Gal/(µmol Hb · h) (range 492–697). Patients with classical galactosaemia were easily detected by their extremely low activity.ConclusionsWe have developed a reliable and convenient direct method to measure GALT activity in human erythrocytes using HPLC with UV detection.     

1-磷酸鞘氨醇对血小板贮存损伤的影响

目的探讨1-磷酸鞘氨醇对手工浓缩血小板的贮存损伤(活化率、凋亡率和细胞内游离Ca2+)的影响。方法向手工浓缩血小板中分别加入0.1、0.5和1.0μmol/L 3个浓度的S1P,采用流式细胞术检测血小板22℃保存2、3、5、7 d的活化率、凋亡率和细胞内游离Ca2+。结果 0.5和1.0μmol/L的外源性S1P可以降低血小板内游离Ca2+的浓度,降低其活化率和凋亡率。结论 1-磷酸鞘氨醇对血小板的贮存损伤起到一定的抑制作用。