Phenylalanine hydroxylase activity in foetal hepatic organ culture (original) (raw)
Related papers
Biochemical Journal, 1984
The nature of the different molecular-mass forms of phenylalanine hydroxylase in rat livers was examined by immunoprecipitation of the enzyme from crude liver extracts that had been radiolabelled by reductive methylation. The two forms of the enzyme were resolved by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and detected by fluorography. Segregation of the two forms of the enzyme was demonstrated in Sprague-Dawley rats, as would be expected if the two forms were products of allelic genes. In addition, hooded and albino Wistar rat livers contained only the slower-migrating form and Lewis rat livers contained only the faster-migrating form, and hence we suggest that the forms be referred to as W (for Wistar) and L (for Lewis). Peptide mapping showed that the W and L forms are closely related, and the difference between them appears to reside at one or other end of the polypeptide chain. The kidney contained the same forms as the liver in one-tenth the quantity, providi...
The Journal of Cell Biology, 1989
A fetal rat hepatocyte culture system has been used to study the molecular mechanisms of tyrosine aminotransferase (TAT) gene expression during development. It has previously been shown that TAT activity can be detected in 19-d, but not 15-d, gestation hepatocytes on the first day of culture (Yeoh, G. C. T., E A. Bennett, and I. T. Oliver. 1979. Biochem. J. 180:153-160). In this study enzyme activity, synthesis, and mRNA levels were determined in hepatocytes isolated from 13-, 15-, and 19-d gestation rats maintained in culture for 1, 2, or 3 d and exposed to dexamethasone. TAT expression is barely detectable in 13-d gestation hepatocytes even after 3 d in culture. Hepatocytes isolated from 15-d gestation fetuses have undetectable levels of enzyme activity and synthesis on the first day of culture; both can be assayed by days 2 1. Abbreviations used in this paper: GAPDH, glyceraldehyde-3-phosphatedehydrogenase; TAT, tyrosine aminotransferase.
Purification of rat liver phenylalanine hydroxylase by affinity chromatography
Archives of biochemistry and biophysics, 1980
Rat liver phenylalanine hydroxylase has been purified to homogeneity on a totally synthesized affinity matrix. The affinity matrix consisted of a succinylated diaminodipropylamine arm linked to Sepharose-4B, to which the cofactor, 6,'7-dimethyl-5,6,7,8tetrahydropterin, was covalently linked. The pure enzyme was eluted with buffered 50% ethylene glycol, 1 M KC1 in one step after the 50% ammonium sulfate fraction of the rat liver homogenate was applied to the affinity column. Specific activities ranging from 1.4 to 3.0 units/mg of protein were obtained. The enzyme has been shown to be homogeneous by: (i) discontinuous gel electrophoresis, and (ii) sodium dodecyl sulfate gel electrophoresis. The subunit molecular weight was determined by the same technique and was calculated to be between 51,000 and 55,000.
Phenylalanine 4-monooxygenase from bovine and rat liver: Some physical and chemical properties
Neurochemical Research, 1982
Phenylalanine 4-monooxygenase was purified from bovine liver using a modification of the procedure developed for the rat liver enzyme (Shiman, R., Gray, D. W., and Pater, A. 1979. J. Biol. Chem. 254:11300-11306). The enzyme preparation appeared essentially homogeneous on polyacrylamide gel electrophoresis under non-denaturing conditions. Electrophoresis in the presence of dodecyl sulfate revealed that about 95% of the protein had a mobility corresponding to Mr = 51,000. The remaining 5% was recovered in two minor bands corresponding to Mr of about 35,000 and 15,000 and is likely to result from limited proteolysis of the native enzyme with dissociation of the fragments on denaturation by detergent. The enzyme comigrated with the rat liver enzyme on polyacrylamide gel electrophoresis in both systems studied. No significant difference was observed between the amino acid composition of the bovine and rat liver enzyme, in the reactivity of their sulfhydryl groups or in their iron content (i.e. 1.5-3.0 iron atoms per peptide chain of Mr = 50,000). Both enzymes contained less than 0.01 copper atom per peptide chain. The enzymes were inhibited in a similar manner by the chelator bathophenanthroline disulfonate (selective for iron and copper), but not by bathocuproine disulfonate (specific for copper). The results indicate that the bovine and rat liver enzymes are closely similar and that iron, but not copper, is essential for enzyme activity. High performance size-exclusion liquid chroma-Abbreviations: Bathophenanthroline, 4,7-diphenyl-1,10-phenanthroline; bathocuproine, 2,9-
In vivo inhibition of rat liver phenylalanine hydroxylase by p-chlorophenylalanine and esculin
Biochemical Medicine, 1975
The phenylalanine hydroxylase system which is responsible for the conversion of phenylalanine (Phe) to tyrosine (Tyr), contains two enzymes, phenylalanine hydroxylase and dihydropteridine reductase (Scheme 1). The genetically linked lack of the liver phenylalanine 4hydroxylase activity (EC 1.14.16.1) is the basic biochemical defect in the human error of metabolism "phenylketonuria" (PKU) (1). The absence of this activity leads to an enhancement of the Phe concentration and consequently to the increase or appearance of some of its metabolic derivatives in the tissues and fluids of PKU patients. Koe and Weissman (2) demonstrated that the in viva administration of p-chlorophenylalanine (p-CPA) markedly inhibits the liver phenylalanine hydroxylase activity. Lipton et al. (3) proposed the use of this Escul in NADP<+)ADPH~.H+
European Journal of Biochemistry, 1997
Tyrosine aminotransferase (TyrAT) is one of several gluconeogenic enzymes which appear postnatally in humans and rodents in response to increased glucocorticoid and glucagon levels and decreased insulin. Primary cultured fetal rat hepatocytes older than day 15 of gestation (>E15) transcribe the TyrAT gene in response to the synergistic effect of dexamethasone and Nh,2'-O-dibutyryl-adenosine 3',5'-monophosphate (Bt,cAMP), whereas less mature hepatocytes (<E15) do not [Shelly, L. L. & Yeoh, G. C. T. (1991) Eur: J. Biochem. 199, 475-4811. Therefore, we consider >El5 hepatocytes, and not <El5 hepatocytes, to be determined. This study reports that 11.1 kb of sequences upstream of the TyrAT transcription start site, which include a CAMP-responsive element (CRE) and a glucocorticoid-responsive element (GRE), are required for correct developmental regulation of gene expression in determined fetal hepatocytes. In contrast, the TyrAT CRE alone does not have this capability. Dexamethasone augments basal and Bt,cAMP-stimulated activity of the TyrAT CRE alone, suggesting that synergism may be due to interaction between the glucocorticoid and CAMP-signaling pathways. However, Bt,cAMP does not further increase dexamethasone-induced activity of the 11.1 kb 5' sequences when the TyrAT CRE is removed, thus excluding interaction of Bt,cAMP with the glucocorticoid pathway. Finally, insulin inhibition of dexamethasone-induced gene transcription is shown to be conferred by TyrAT 5' sequences. This study shows that cellular components, other than those which mediate hormonal regulation of genes, are required for determination of hepatocytes with respect to TyrAT. Since this phenomenon is observed with transient transfections, it is unlikely to involve higher-order chromatin structure.
Effect of phenylalanine derivatives on the main regulatory enzymes of hepatic cholesterogenesis
Molecular and Cellular Biochemistry, 1991
Phenylalanine, phenylpyruvate and phenylacetate produced a considerable inhibition of chick liver mevalonate 5-pyrophosphate decarboxylase while mevalonate kinase and mevalonate 5-phosphate kinase were not significantly affected. Phenolic derivatives of phenylalanine produced a similar inhibition of decarboxylase activity than that found in the presence of phenyl metabolites. The degree of inhibition was progressive with increasing concentrations of inhibitors (1.25-5.00 mM). Simultaneous supplementation of different metabolites in conditions similar to those in experimental phenylketonuria (0.25 mM each) produced a clear inhibition of liver decarboxylase and 3-hydroxy-3-methylglutaryl-CoA reductase. To our knowledge, this is the first report on the in vitro inhibition of both liver regulatory enzymes of cholesterogenesis in phenylketonuria-like conditions. Our results show a lower inhibition of decarboxylase than that of reductase but suggest an important regulatory role of decarboxylase in cholesterol synthesis.
Recombinant Human Phenylalanine Hydroxylase: Novel Regulatory and Structural Properties
Archives of Biochemistry and Biophysics, 1996
Mammalian phenylalanine hydroxylase (PAH) catalyses the conversion of -phenylalanine to -tyrosine in the presence of dioxygen and tetrahydrobiopterin ; it is a highly regulated enzyme. Little is known about the rates of synthesis and degradation of PAH in i o. The enzyme has been reported to have a half-life of approx. 2 days in rat liver and 7-8 h in rat hepatoma cells, but the mechanism of its degradation is not known. In the present study it is shown that the tetrameric form of the recombinant