UDP-glucuronyltransferase activity exhibits two developmental groups in liver from foetal rhesus monkeys.

UDP-glucuronyltransferase was assayed in liver from adult rhesus monkeys and foetuses during late gestation. Activities toward 2-aminophenol, 5-hydroxytryptamine, 1-naphthol and 4-nitrophenol in the foetal liver ranged from 46 to 114% of adult values, whereas activities toward bilirubin, oestradiol and testosterone were less than 5% of adult values. This suggests that in primates UDP-glucuronyltransferase develops differentially in two clusters analogous to that in the rat.     

Development of uridine diphosphate-glucuronyltransferase activity in cultures of chick-embryo liver

1. The liver of the domestric fowl (Gallus gallus) remains capable of conjugating o-aminophenol with glucuronic acid after 8 days' culture. The pathway of o-aminophenyl glucuronide formation in cultured liver, as in fresh tissue, includes the enzyme UDP-glucuronyltransferase. 2. UDP-glucuronyltransferase activity in chick-embryo liver increases on culture from very low to adult values within 6-8 days. 3. The development of UDP-glucuronyltransferase activity in cultured chick-embryo liver requires certain serum factors in the medium. The requirements change with embryo age. Liver from embryos younger than 15 days develops enzyme activity equally well in media containing either foetal or adult serum; liver from embryos older than 16 days develops activity only with adult serum. The development of enzyme activity in liver from the older embryos appears to be stimulated by diffusible factors in adult serum and inhibited by diffusible factors in foetal serum. It is suggested that the stimulation and inhibition of enzyme formation by small, diffusible molecules may be part of the mechanism regulating UDP-glucuronyltransferase activity in vivo. 4. Liver from 19-day-old chick embryos cultured with foetal serum begins to develop UDP-glucuronyltransferase activity if transferred to an adult-serum medium. Its capacity to develop UDP-glucuronyltransferase activity in adult serum survives in a foetal-serum medium for at least 5 days, the longest period tested. 5. The activity of UDP-glucuronyltransferase reached in 19-day chick-embryo liver after 1 or 2 days with adult serum is maintained without further increase after transfer to a foetal-serum medium. After 3 days with adult serum UDP-glucuronyltransferase activity continues to increase when the tissue is transferred to a foetal-serum medium. Thus liver from 19-day-old embryos requires 3 days with adult serum before development of enzyme activity becomes independent of a continuous adult-serum environment.     

The prenatal and postnatal development of UDP-glucuronyltransferase activity towards bilirubin and the effect of premature birth on this activity in the human liver.

Liver UDP-glucuronyltransferase activity towards bilirubin was studied in a total of 88 human subjects, including foetuses, premature and full-term newborn babies, infants, children, and adults. Determination of very low enzyme activity was performed by high-pressure liquid chromatography. Prenatal and postnatal changes of the activity can be divided into four developmental phases, i.e. middle foetal, late foetal, neonatal and early infantile, and mature. The activities of the first three phases corresponded to about 0.1, 0.1-1 and 1-100%, respectively, of the mature-phase values (mean +/- S.D.: 1320 +/- 514 microgram/h per g of liver, n = 27).     

Functional heterogeneity of UDP-glucuronosyltransferase as indicated by its differential development and inducibility by glucocorticoids. Demonstration of two groups within the enzyme''s activity towards twelve substrates.

1. UDP-glucuronosyltransferase activity towards 12 substrates has been assessed in rat liver during the perinatal period. 2. Between days 16 and 20 of gestation, enzyme activities towards the substrates 2-aminophenol, 2-aminobenzoate, 4-nitrophenol, 1-naphthol, 4-methylumbelliferone and 5-hydroxytryptamine (the 'late foetal' group) surge to reach adult values, while activities towards bilirubin, testosterone, beta-oestradiol, morphine, phenolphthalein, and chloramphenicol (the 'neonatal' group) remain negligible or at less than 10% of adult values. 3. By the second postnatal day, enzyme activities towards the neonatal group have attained, or approached adult values. 4. Dexamethasone precociously stimulates in 17-day foetal liver in utero transferase activities in the late foetal, but not the neonatal group. A similar inductive pattern is found for 15-day foetal liver in organ culture. 5. It is suggested that foetal glucocorticoids, whose synthesis markedly increases between days 16 and 20 of gestation, are responsibile for triggering the simultaneous surge of all the hepatic UDP-glucuronosyltransferase activities in the late foetal group. The neonatal group of activities apparently require a different or additional stimulus for their appearance. 6. The relationship of these two groups of transferase activities to other similar groups observed during induction by xenobiotics and enzyme purification is discussed.     

Reversible ATP-induced inactivation of branched-chain 2-oxo acid dehydrogenase.

1. Antiserum was raised against purified Wistar-rat liver UDP-glucuronyltransferase. 2. UDP-glucuronyltransferase activities towards 4-nitrophenol, bilirubin, 1-naphthol and morphine were co-immunoprecipitated from solubilized Wistar-rat liver preparations. 3. UDP-glucuronyltransferase activities towards 1-naphthol, 2-aminophenol and 4-nitrophenol were precipitated from solubilized Gunn-rat liver preparations by this antiserum. 4. UDP-glucuronyltransferase activities towards 1-naphthol, 4-nitrophenol and bilirubin, from Wistar-rat liver, were slightly inhibited by antiserum, whereas 1-naphthol UDP-glucuronyltransferase activity from Gunn-rat livers was greatly inhibited. 5. Measurable Wistar-rat liver glucuronyltransferase activities in washed immunoprecipitates indicate that the enzyme(s) were not merely inhibited by antiserum. 6. Immunoglobulin G purified from this antiserum immunoprecipitated transferase activities towards 4-nitrophenol, bilirubin and 1-naphthol. 7. The washed immunoprecipitates from both rat strains, containing UDP-glucuronyltransferase activity, appear to be similar when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 8. Radial-immunodiffusion studies suggest that a smaller amount of UDP-glucuronyltransferase protein is present in Gunn-rat liver than in Wistar-rat liver. 9. The significance of these results in relation to the genetic deficiency in the Gunn rat is discussed.     

Transfer ribonucleic acid methylase activity in adult and foetal rat liver.

Foetal rat liver extracts were found to have higher tRNA methylene activities than corresponding extracts of adult liver. When the specific activities were expressed per mg of liver or per mg of protein, the foetal tRNA methylating enzymes were respectively 2.5 and 6 times higher than those of adult livers. The presence of an inhibitor in adult liver can be excluded, since the same recoveries of total tRNA methylase activity were obtained after partial purification of both adult and foetal liver extracts: yields were close to 100%. The apparent Km's for the substrates in the methylating reactions were the same when tRNA methylases from either adult or foetal liver were used: values were 0.2 muM for Escherichia coli tRNA and 2.1 muM for S-adenosyl-L-methionine. After T1-T2 ribonuclease digestion of an in vitro methylated tRNA, similar methyl nucleotide patterns were observed in foetal and adult enzymatic extracts. It is concluded that the same tRNA methylase pool is present in adult and foetal liver. In addition, it is hypothesized that the different reaction rates exhibited by these enzymes might be due to the tRNA functional requirements rather than to the presence of a tRNA methylase inhibitor.     

Liver glycogen synthase in the developing foetal rat

Kinetic constants for liver glycogen synthase (UDPglucose: glycogen 4-alpha-D-glucosyltransferase, EC 2.4.1.11) with respect to UDPglucose have been measured in foetal liver homogenates from samples taken during late gestation (days 17-22) and the first hours after birth. The V of the inactive form of glycogen synthase increased markedly in this period and there was a significant increase in V of the active enzyme to a maximum at day 20 of gestation. The Km for UDPglucose measured in the presence of glucose-6-P (total activity) did not vary greatly, mean values of 0.51 +/- 0.04 mM. Values derived for the inactive enzyme were almost identical. In contrast, Km values for active glycogen synthase in foetal livers during gestation were significantly higher than those for adult liver. Highest values were seen at day 19 of gestation (1.84 +/- 0.08 mM) followed by a steady fall to 0.55 +/- 0.05 mM in the newborn compared with a mean value of 0.48 +/- 0.04 mM for adult liver. Existence of a reduced affinity of active glycogen synthase for UDPglucose must be recognized when assaying the enzyme in foetal liver, particularly when extrapolating values to rates of glycogen synthesis in vivo. Data were obtained only after removal of an amylase-like contaminant from foetal liver samples which invalidated the radioassay of glycogen synthase. This work illustrates the care needed in the analysis of foetal tissue and the interpretation of resulting data when utilizing methods developed for adult tissue.     

Mixed function oxidase activity in the harbour seal (Phoca vitulina) from Sable Is., N.S

One adult male, eight pups (including two full term foetuses) and nine adult female harbour seals (Phoca vitulina) were analysed for indices of mixed function oxidase (MFO) activity. MFO activity was present in liver samples, but was at or below detection limits in samples of kidney, lung and pancreas. Hepatic ethoxyresorufin O-de-ethylase and benzo[a]pyrene hydroxylase activities were similar to those reported in other seals and in other mammals. Cytochromes P-450 and b5 concentrations were slightly lower than those observed in other mammals. MFO activities in newborn pups and foetuses were significantly lower than those in adult females. No qualitative differences in cytochrome P-450 isozyme distribution between foetal and adult samples could be discerned by electrophoresis.     

Thymidine kinase in rat liver during development

1. The activity of thymidine kinase in rat liver supernatant decreased with development to a value in the adult that was 1% of that in the 17-day foetus. 2. The foetal enzyme was more stable than the adult to gel filtration on Sephadex G-25 at 0 degrees . 3. The greater stability of the foetal enzyme to incubation at 45 degrees was attributable to the presence of higher concentrations of nucleotides in foetal liver supernatant. 4. The K(m) values for foetal and adult enzymes were approx. 2.5mum- and 2.1mum-thymidine respectively. 5. The foetal enzyme was more sensitive to inhibition by thymidine triphosphate. 6. The decline in enzyme activity during the neonatal period was correlated with a shift in the enzyme properties from the foetal to the adult type, and may reflect the decrease in the proportion of haemopoietic tissue in the liver.     

Hepatotropin mRNA expression in human foetal liver development and in liver regeneration

A 569 bp probe against the β-chain of hepatotropin was used to examine expression of RNA for this growth factor in human adult and foetal liver, foetal kidney and pancreas, and rat liver after partial hepatectomy. Low level expression of a 6kb RNA occurred in human adult and normal rat liver. 70% hepatectomy increased expression, peaking at 10 h and returning to near normal levels 24 h after resection. The 6 kb band was strongly expressed in human foetal liver, as compared with adult, but not in foetal kidney or pancreas, suggesting a major role for hepatotropin in both foetal development and regeneration of the liver.     

Reconstitution of purified Wistar rat liver bilirubin UDP-glucuronyltransferase into Gunn-rat liver microsomes.

1. Reconstitution of purified bilirubin UDP-glucuronyltransferase from Wistar-rat liver into Gunn-rat liver microsomes provides a better environment than phosphatidylcholine liposomes, such that the final specific activity of the Wistar-rat liver enzyme was increased up to 85 units/mg of protein. 2. Gunn- and Wistar-rat liver microsomes were equally effective for reconstitution of the purified enzyme. 3. The transferase activity does not appear to be fully expressed in the more rigid environment of foetal Wistar-rat liver microsomes. 4. These reconstitution experiments reveal a final specific activity for the purified bilirubin UDP-glucuronyltransferase consistent with the capacity of the whole rat liver to glucuronidate bilirubin and indicate that the absence of this enzyme activity in Gunn-rat liver microsomes is not due to an abnormal microenvironment.     

Transfer ribonucleic acid methylase activity in adult and fœtal rat liver

Foetal rat liver extracts were found to have higher tRNA methylase activities than corresponding extracts of adult liver. When the specific activities were expressed per mg of liver or per mg of protein, the foetal tRNA methylating enzymes were respectively 2.5 and 6 times higher than those of adult livers.The presence of an inhibitor in adult liver can be excluded, since the same recoveries of total tRNA methylase activity were obtained after partial purification of both adult and foetal liver extracts: yields were close to 100 per cent.The apparent Km's for the substrates in the methylating reactions were the same when tRNA methylases from either adult or foetal liver were used: values were 0.2 μM for Escherichia coli tRNA and 2.1 μM for S-adenosyl-l-methionine.After T₁-T₂ ribonuclease digestion of an in vitro methylated tRNA, similar methyl nucleotide patterns were observed in foetal and adult enzymatic extracts.It is concluded that the same tRNA methylase pool is present in adult and foetal liver. In addition, it is hypothesized that the different reaction rates exhibited by these enzymes might be due to the tRNA functional requirements rather than to the presence of a tRNA methylase inhibitor.     

Stimulation of defective Gunn-rat liver uridine diphosphate glucuronyltransferase activity in vitro by alkyl ketones.

Addition of alkyl ketone (10mM) to Gunn-rat liver homogenates increased UDP-glucuronyltransferase activity towards 2-aminophenol by 10--20 fold, up to enhanced values of enzyme activity observed with similarly treated Wistar-rat liver homogenates. Alkyl ketones also activate the defective enzyme purified from Gunn-rat liver. This genetic deficiency of UDP-glucuronyltransferase activity is no longer apparent when assayed in the presence of alkyl ketones.     

The distribution of enzyme and isoenzyme activities between parenchymal and haematopoietic cells in the liver of the foetal guinea pig

The hepatocyte and haematopoietic cell contents of the liver of the foetal guinea pig were measured over the latter half of gestation. Hepatocytes represented about 30% of liver volume at mid-gestation and this increased to 70-80% by term; cell volume remained fairly constant until 5-7 days before term, then more than doubled. Haematopoietic cells represented about 5% of liver volume at mid-gestation and this progressively fell to <1% by term. At 75% of gestation hepatocytes and haematopoietic cells were prepared from perfused foetal livers by collagenase digestion. Enzyme activity of the hepatocyte was, without exception, similar to that of the whole liver. In general, enzyme activity in the haematopoietic cells was similar to that in erythrocytes, with relatively low values for aldolase, glycerol 3-phosphate dehydrogenase, phosphoglycerate mutase, enolase, lactate dehydrogenase, phosphoenolpyruvate carboxykinase, fructose 1,6-bisphosphatase, isocitrate dehydrogenase, ;malic' enzyme, glutamate dehydrogenase and aspartate aminotransferase. The haematopoietic cell contribution to total enzyme activity in the foetal liver was usually much less than 10% and could thus not account for the major changes in hepatic enzyme activity over the latter half of gestation. Hepatocytes contained hexokinase isoenzymes I and III, aldolase isoenzymes A and B and pyruvate kinase isoenzymes 1, 2 and 4. The haematopoietic cells contained hexokinase isoenzyme I and two additional bands of activity with slightly greater mobility, aldolase isoenzyme A and pyruvate kinase isoenzymes 2 and 4.     

Development of fatty acid oxidation in neonatal guinea-pig liver.

The capacity of foetal and neonatal liver to oxidize short-, medium- and long-chain fatty acids was studied in the guinea pig. Liver mitochondria from foetal and newborn animals were unable to synthesize ketone bodies from octanoate, but octanoylcarnitine and palmitoylcarnitine were readily ketogenic. The ketogenic capacity at 24 h after birth was as high as in adult animals. Hepatocytes isolated from term animals were unable to oxidize fatty acids, but at 6 h after birth production of 14CO2, acid-soluble products and acetoacetate from 1-14C-labelled fatty acids was 40-50% of the rates at 24 h. At 12 h of age these rates had already reached the 24 h values and did not change during suckling in the first week of life. The activities of hepatic fatty acyl-CoA synthetases, which were minimal in the foetus or at term, increased to maximal values in 12-24 h. The data show that the capacity for beta-oxidation and ketogenesis develops maximally in this species during the first 6-12 h after birth, and appears to be partly dependent on the development of fatty acid-activating enzyme.     

The isolation and properties of phenylalanine hydroxylase from human liver

Phenylalanine hydroxylase was prepared from human foetal liver and purified 800-fold; it appeared to be essentially pure. The phenylalanine hydroxylase activity of the liver was confined to a single protein of mol.wt. approx. 108000, but omission of a preliminary filtration step resulted in partial conversion into a second enzymically active protein of mol.wt. approx. 250000. Human adult and full-term infant liver also contained a single phenylalanine hydroxylase with molecular weights and kinetic parameters the same as those of the foetal enzyme; foetal, newborn and adult phenylalanine hydroxylase are probably identical. The K(m) values for phenylalanine and cofactor were respectively one-quarter and twice those found for rat liver phenylalanine hydroxylase. As with the rat enzyme, human phenylalanine hydroxylase acted also on p-fluorophenylalanine, which was inhibitory at high concentrations, and p-chlorophenylalanine acted as an inhibitor competing with phenylalanine. Iron-chelating and copper-chelating agents inhibited human phenylalanine hydroxylase. Thiol-binding reagents inhibited the enzyme but, as with the rat enzyme, phenylalanine both stabilized the human enzyme and offered some protection against these inhibitors. It is hoped that isolation of the normal enzyme will further the study of phenylketonuria.     

Two distinct forms of glutathione transferase from human foetal liver. Purification and comparison with isoenzymes isolated from adult liver and placenta.

Isoelectric focusing of a cytosol fraction from human foetal liver revealed the existence of an acidic and a basic isoenzyme of GSH transferase. The acidic and basic forms of GSH transferase were purified in good yield by use of ion-exchange chromatography on DEAE-cellulose followed by affinity chromatography on S-hexyl-GSH coupled to epoxy-activated Sepharose 6B. The content of the acidic and the basic isoenzymes of GSH transferase together was calculated to constitute 1-2% of the soluble proteins in the hepatic cytoplasm. Physical, catalytic and immunological analyses of the acidic and the basic isoenzymes from foetal liver demonstrated unambiguously that the two forms are different structures with distinct properties. On the other hand, the results show clearly extensive similarities between the foetal acidic transferase and transferase pi from human placenta as well as between the foetal basic form and the basic isoenzymes isolated from adult liver. An exception is that both foetal enzymes seem to be considerably more efficient in catalysing the conjugation of GSH with styrene 7,8-epoxide than the corresponding adult forms of GSH transferase.     

hDlk-1: a cell surface marker common to normal hepatic stem/progenitor cells and carcinomas

Advances in stem cell biology have clarified that a tumour is a collection of heterogeneous cell populations, and that only a small fraction of tumour cells possesses the potential to self-renew. Delta-like 1 protein (Dlk-1) is a surface antigen present on foetal hepatic stem/progenitor cells but absent from mature hepatocytes in neonatal and adult rodent liver. Using a monoclonal antibody (mAb) against hDlk-1, Yanai et al. (Dlk-1, a cell surface antigen on foetal hepatic stem/progenitor cells, is expressed in hepatocellular, colon, pancreas and breast carcinomas at a high frequency. J. Biochem. 2010;148:85-92) have shown that human (h) Dlk-1 is expressed in human foetal, but not adult, liver and that 20% of all hepatocellular carcinomas (HCCs) are hDlk-1(+). Importantly, an even higher percentage of HCCs in younger patients are hDLK-1(+). These authors also found that hDlk-1 is present at high frequency in colon adenocarcinomas, pancreatic islet carcinomas and small cell lung carcinomas. Here, I discuss the implications of the expression of foetal hepatic stem/progenitor cell antigens on carcinoma cells.     

Phosphoenolpyruvate carboxykinase and pyruvate carboxylase in developing rat liver

1. Phosphoenolpyruvate carboxykinase and pyruvate carboxylase were measured in foetal, newborn and adult rat liver extracts by a radiochemical assay involving the fixation of [¹⁴C]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.