Some properties of galactokinase in developing rat liver.
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ABSTRACT:
SUBMITTER: Walker DG
PROVIDER: S-EPMC1198790 | biostudies-other | 1968 Jun
REPOSITORIES: biostudies-other
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Project description:1. Galactokinase has been purified from the liver of young pigs by high-speed centrifugation, chromatography on Sephadex G-100 and DEAE-cellulose, and ammonium sulphate fractionation. 2. The enzyme preparation has a specific activity of 10-18mumoles of galactose phosphorylated/mg. of protein/min. at 37 degrees and has been purified 400-fold from the liver supernatant. 3. Purified liver galactokinase has Michaelis constants of 1x10(-4)-3x10(-4)m for galactose and 2x10(-4)m for ATP-Mg(2+), and the enzyme reaction produces equimolar amounts of galactose 1-phosphate and ADP. 4. Galactokinase phosphorylates 2-deoxygalactose and galactosamine in addition to galactose, has a pH optimum of 7.8, a Q(10) of 2, and is stimulated by cysteine and other thiols. 5. With the exception of substrate specificity, the properties of liver galactokinase are similar to galactokinase purified from yeast and Escherichia coli.
| S-EPMC1264838 | biostudies-other
Project description:1. Kinetic measurements of the forward reaction catalysed by ATP-galactose phosphotransferase were carried out with a purified preparation from pig liver. 2. The rate of reaction at pH7.8 is dependent on the concentration of MgATP(2-) rather than total ATP or magnesium chloride concentrations. 3. The effect of changes in pH on K(m) (galactose), K(m) (MgATP(2-)) and V(max.) was studied. 4. Of several possible nucleotide substrates only ATP and deoxyATP were effective. 5. The initial-velocity patterns both in the absence and presence of products were determined. 6. Galactose 1-phosphate is a non-competitive inhibitor when either galactose or MgATP(2-) was the variable substrate. 7. MgADP(-) was a non-competitive inhibitor with galactose and a competitive inhibitor with MgATP(2-) as variable substrate. 8. These results are consistent with an ordered reaction pathway in which galactose combines with an initial enzyme-MgATP(2-) complex.
| S-EPMC1270067 | biostudies-other
Project description:1. Phosphoenolpyruvate carboxykinase and pyruvate carboxylase were measured in foetal, newborn and adult rat liver extracts by a radiochemical assay involving the fixation of [(14)C]bicarbonate. 2. Pyruvate-carboxylase activity in both foetal and adult liver occurs mainly in mitochondrial and nuclear fractions, with about 10% of the activity in the cytoplasm. 3. Similar studies of the intracellular distribution of phosphoenolpyruvate carboxykinase show that more than 90% of the activity is in the cytoplasm. However, in the 17-day foetal liver about 90% of the activity is in mitochondria and nuclei. 4. Pyruvate-carboxylase activity in both particulate and soluble fractions is very low in the 17-day foetal liver and increases to near adult levels before birth. 5. Phosphoenolpyruvate-carboxykinase activity in the soluble cell fraction increases 25-fold in the first 2 days after birth. This same enzyme in the mitochondria has considerable activity in the foetal and adult liver and is lower in the newborn. 6. Kinetic and other studies on the properties of phosphoenolpyruvate carboxykinase have shown no differences between the soluble and mitochondrial enzymes. 7. It is suggested that the appearance of the soluble phosphoenolpyruvate carboxykinase at birth initiates the rapid increase in overall gluconeogenesis at this stage.
| S-EPMC1271226 | biostudies-other
Project description:1. The total activity of glycogen synthease increased 20-fold from day 17 of gestation to birth at day 22, with a further increase of 18% in the 24h after birth. Active synthase (I) rose 45-fold to a maximum at day 21, fell 40% before birth, and then increased by a similar amount 24h after birth. The fraction of synthase in the active form correlated very well with the deposition of glycogen in the liver. 2. Total phosphorylase had a similar developmental pattern of total synthease with an 18-fold increase from day 17 to day 22. The appearance of active phosphorylase showed a lag-period compared with total phosphorylase and did not increase significantly until day 20. The fraction of phosphorylase in the active form did not correlate at all with glycogen deposition or mobilization. 3. There was a close relationshp between the ratio of phosphorylase a/synthase I and the glycogen content of the liver. An increase or decrease in this ratio would result in glycogenolysis of glycogenesis respectively. 4. It is postulated that a cycle between the two enzymes under basal conditions could exist which permits a continuous turnover of glycogen. Such a system would explain why active phosphorylase is always seen, even under conditions of net glycogen synthesis. The differences in hormone sensitivity of synthase and phosphorylase would also be accounted for as only one enzyme would have to respond acutely to hormonal influences.
| S-EPMC1164232 | biostudies-other
Project description:The activities of the hepatic microsomal 2alpha-, 2beta-, 7alpha- and 18-hydroxylase systems active on 5alpha-[4-(14)C]androstane-3alpha,17beta-diol were studied in male and female rats which had been castrated at birth and at the age of 7, 13, 21, 27, 34, 43 and 55 days, treated for 5 days with 2mg of testosterone propionate/kg body weight and killed 6 days after castration. The 7alpha-hydroxylase system was affected very little by androgen treatment at all stages during development. On the other hand it was found that the rat liver passed through three phases during development with respect to androgen responsiveness as judged by changes in the activities of the 2alpha, 2beta- and 18-hydroxylase systems: a first phase (from the neonatal period up to about 19 days of age) with a relative androgen unresponsiveness in both male and female rats, a second phase (from about 27 to about 33 days of age) when male and female rats responded equally well to androgens and a final phase (from about 40 days of age) with a successively decreasing androgen responsiveness in female rats but with a retained responsiveness in male rats. The hypothesis is presented that neonatal imprinting of the liver by testicular androgen(s) determines the development and degree of androgen responsiveness of liver tissue in the rat.
| S-EPMC1168489 | biostudies-other
Project description:Inherited deficiency of galactose-1-phosphate uridyltransferase (GALT) can result in a potentially lethal disorder called classic galactosemia. Although the neonatal lethality associated with this disease can be prevented through early diagnosis and a galactose-restricted diet, the lack of effective therapy continues to have consequences: developmental delay, neurological disorders, and premature ovarian failure are common sequelae in childhood and adulthood. Several lines of evidence indicate that an elevated level of galactose-1-phosphate (gal-1-p), the product of galactokinase (GALK), is a major, if not sole, pathogenic mechanism in patients with classic galactosemia. The authors hypothesize that elimination of gal-1-p production by inhibiting GALK will relieve GALT-deficient cells from galactose toxicity. To test this hypothesis, they obtained human GALK using a bacterial expression system. They developed a robust, miniaturized, high-throughput GALK assay (Z' factor = 0.91) and used this assay to screen against libraries composed of 50,000 chemical compounds with diverse structural scaffolds. They selected 150 compounds that, at an average concentration of 33.3 microM, inhibited GALK activity in vitro more than 86.5% and with a reproducibility score of at least 0.7 for a confirmatory screen under identical experimental conditions. Of these 150 compounds, 34 were chosen for further characterization. Preliminary results indicated that these 34 compounds have potential to serve as leads to the development of more effective therapy of classic galactosemia.
| S-EPMC2705177 | biostudies-literature
Project description:PurposeGalactokinase (GALK1) deficiency is a rare hereditary galactose metabolism disorder. Beyond cataract, the phenotypic spectrum is questionable. Data from affected patients included in the Galactosemias Network registry were collected to better characterize the phenotype.MethodsObservational study collecting medical data of 53 not previously reported GALK1 deficient patients from 17 centers in 11 countries from December 2014 to April 2020.ResultsNeonatal or childhood cataract was reported in 15 and 4 patients respectively. The occurrence of neonatal hypoglycemia and infection were comparable with the general population, whereas bleeding diathesis (8.1% versus 2.17-5.9%) and encephalopathy (3.9% versus 0.3%) were reported more often. Elevated transaminases were seen in 25.5%. Cognitive delay was reported in 5 patients. Urinary galactitol was elevated in all patients at diagnosis; five showed unexpected Gal-1-P increase. Most patients showed enzyme activities ≤1%. Eleven different genotypes were described, including six unpublished variants. The majority was homozygous for NM_000154.1:c.82C>A (p.Pro28Thr). Thirty-five patients were diagnosed following newborn screening, which was clearly beneficial.ConclusionThe phenotype of GALK1 deficiency may include neonatal elevation of transaminases, bleeding diathesis, and encephalopathy in addition to cataract. Potential complications beyond the neonatal period are not systematically surveyed and a better delineation is needed.
| S-EPMC7790741 | biostudies-literature
Project description:1. The total activity of adenine phosphoribosyltransferase/liver of mice remained constant from 1 to 16 days after birth despite a fourfold increase in liver weight. The total activity of this enzyme increased fivefold from 16 to 36 days and then remained relatively constant at least until 96 days after birth. Total hypoxanthine-phosphoribosyltransferase activity/liver steadily increased between 1 and 57 days after birth. 2. The mean K(m) of 5-phosphoribosyl pyrophosphate with adenine phosphoribosyltransferase was 10.1mum between 3 and 11 days, at 64 days and at 96 days after birth. Between 17 and 51 days the mean K(m) value was 3.0mum. The K(m) of 5-phosphoribosyl pyrophosphate with hypoxanthine phosphoribosyltransferase remained constant at 28.2mum between 2 and 64 days. 3. Adenine-phosphoribosyltransferase activity was stimulated between 15 and 83% by 60mum-ATP when extracts were made between 3 and 11 days, at 64 days or at 96 days after birth. Between 17 and 51 days ATP had little stimulatory effect on the activity of this enzyme. 4. AMP competed with 5-phosphoribosyl pyrophosphate in the reaction catalysed by adenine phosphoribosyltransferase. Liver extracts containing enzyme with a low value of K(m) for 5-phosphoribosyl pyrophosphate (3mum) had a K(m)/K(i) ratio approximately half that of extracts with a high value of K(m) (10mum). 5. The results indicate that two different forms of adenine phosphoribosyltransferase can exist in mouse liver at different stages of development. The physiological significance of these findings is discussed.
| S-EPMC1270636 | biostudies-other
Project description:The Gal1p (Galactokinase) protein is known for regulation of D-galactose metabolism. It catalyzes the formation of galactose -1-phosphate from alpha - D-galactose, which is an important step in galactose catabolism. The knowledge of Gal1p protein structure, its protein interacting partners and enumeration of functional site residues will provide great insight in understanding the functional role of Gal1p. These studies are lacking in case of the Gal11p kinase enzyme. Structure of this enzyme has already been determined in S. cerevisiae, however, no structural information for this protein is available for K. lactis and E. coli. We used the homology modeling based approach to model the structures of Gal1p for K. lactis and E. coli. Furthermore, functional residues were predicted for these Gal1 proteins and the strength of interaction between Gal1p and other Gal proteins was determined by protein-protein interaction studies via patchdock software. The interaction studies revealed that the affinity for Gal1p for other Gal proteins varies in different organisms. Sequence and structural based comparison of Gal1p kinase enzyme showed that the orthologs in K.lactis and S. cervisiae are more similar to each other as compared to the ortholog in E. coli. These studies carried out by us will help in better understanding of the galactose metabolism. Our sequence and structure comparison studies revealed that Human Gal1p shows more homology for Gal1p protein of E. coli. The above studies may be applied to Human Gal1p, where it can help in gaining useful insight into Galactosemia disease.
| S-EPMC3055162 | biostudies-literature
Project description:BACKGROUND:Galactokinase catalyses the first committed step in galactose metabolism, the conversion of galactose to galactose-1-phosphate. Galactokinase deficiency is an extremely rare form of galactosaemia, and the most frequent complication reported is cataracts. Congenital hyperinsulinism (CHI) is a cause of severe hypoglycaemia in the newborn period. Galactosaemia has not previously been reported in a neonate with concomitant CHI. AIMS:To report the first case of a patient with CHI and galactokinase deficiency, and to describe the diagnostic pitfalls with bedside blood glucose testing in a neonate with combined galactokinase deficiency and CHI. PATIENTS/METHODS:A 3-day-old baby girl from consanguineous parents presented with poor feeding, irritability and seizures. Capillary blood glucose testing using bedside test strips and glucometer showed a glucose level of 18 mmol/L, but the actual laboratory blood glucose level was only 1.8 mmol/L. After discontinuation of oral feeding (stopping provision of dietary galactose), the bedside capillary blood glucose correlated with laboratory glucose concentrations. RESULTS:Biochemically the patient had CHI (blood glucose level 2.3 mmol/L with simultaneous serum insulin level of 30 mU/L) and galactokinase deficiency (elevated serum galactose level 0.62 ?mol/L). Homozygous loss of function mutations in ABCC8 and GALK1 were found, which explained the patient's CHI and galactokinase deficiency, respectively. CONCLUSION:This is the first reported case of CHI and galactokinase deficiency occurring in the same patient. Severe hypoglycaemia in neonates with CHI may go undetected with bedside blood glucose meters in patients with galactokinase deficiency.
| S-EPMC3509914 | biostudies-literature