The quantitative determination of phenylalanine hydroxylase in rat tissues. Its developmental formation in liver

A sensitive method was developed for determining the phenylalanine hydroxylase activity of crude tissue preparations in the presence of optimum concentrations of the 6,7-dimethyl-5,6,7,8-tetrahydropterin cofactor (with ascorbate or dithiothreitol to maintain its reduced state) and substrate. Tissue distribution studies showed that, in addition to the liver, the kidney also contains significant phenylalanine hydroxylase activity, one-sixth (in rats) or half (in mice) as much per g as does the liver. The liver and the kidney enzyme have similar kinetic properties; both were located in the soluble phase and were inhibited by the nucleo-mitochondrial fraction. Phenylalanine hydroxylase, like most rat liver enzymes concerned with amino acid catabolism, develops late. On the 20th day of gestation, the liver (and the kidney) is devoid of phenylalanine hydroxylase and at birth contains 20% of the adult activity. During the second postnatal week of development, when the phenylalanine hydroxylase activity was about 40% of the adult value, an injection of cortisol doubled this value. Cortisol had no significant effect on phenylalanine hydroxylase in adult liver or on phenylalanine hydroxylase in kidney at any age.

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Comparison of Different Methods for the Determination of Phenylalanine Hydroxylase Activity in Rat Liver and Euglena gracilis

Zeitschrift für Naturforschung C ◽

10.1515/znc-1984-7-808 ◽

1984 ◽

Vol 39 (7-8) ◽

pp. 728-733 ◽

Cited By ~ 1

Author(s):

Rita M. Fink ◽

Erich F. Elstner

Keyword(s):

Rat Liver ◽

Euglena Gracilis ◽

Phenylalanine Hydroxylase ◽

Hplc Method ◽

Enzymic Activity ◽

Hydroxylase Activity ◽

Product Separation ◽

Tyrosine Concentration ◽

Layer Chromatography

Abstract Three different methods for the determination of phenylalanine hydroxylase activity have been compared: a) Differential photometric assay of the increase in tyrosine concentration in the presence of phenylalanine; b) Product separation by thin layer chromatography and scintillation counting of the [14C]tyrosine formed;c) HPLC separation and spectrofluorometric quantification of derivatized amino acids. A comparison of the activities of phenylalanine hydroxylase in rat liver and Euglena gracilis clearly showed that only rat liver contains this enzymic activity as shown by methods b) and c) although pseudo-activity of Euglena gracilis preparations was found during the spectrophotometric test a). The HPLC method proved to be the fastest, most reliable and convenient method for direct tyrosine determination and thus for measuring phenylalanine hydroxylase activity.

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In vivo Determination of Phenylalanine Hydroxylase Activity Using Heptadeutero-Phenylalanine and Comparison to the in vitro Assay Values1

Inborn Errors of Metabolism in Man - Monographs in Human Genetics ◽

10.1159/000401619 ◽

2015 ◽

pp. 108-113 ◽

Cited By ~ 6

Author(s):

Friedrich K. Trefz ◽

Klaus Bartholom� ◽

Horst Bickel ◽

Peter Lutz ◽

Hildgund Schmidt

Keyword(s):

Phenylalanine Hydroxylase ◽

In Vitro Assay ◽

Hydroxylase Activity ◽

Vitro Assay

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Effect of glucagon on phenylalanine metabolism and phenylalanine-degrading enzymes in the rat

Biochemical Journal ◽

10.1042/bj1420231 ◽

1974 ◽

Vol 142 (2) ◽

pp. 231-245 ◽

Cited By ~ 16

Author(s):

Larry M. Brand ◽

Alfred E. Harper

Keyword(s):

Urinary Excretion ◽

Oxidation Rate ◽

Phenylalanine Hydroxylase ◽

Dihydropteridine Reductase ◽

Aminotransferase Activity ◽

Hydroxylase Activity ◽

Phenylalanine Metabolism ◽

Rate Limiting ◽

Reduced State

Glucagon administered subcutaneously to rats for 10 days had no significant effect on liver phenylalanine hydroxylase activity, but induced liver dihydropteridine reductase more than twofold. In rats administered a phenylalanine load orally, glucagon treatment stimulated oxidation and depressed urinary phenylalanine excretion. These responses could not be related to an effect of glucagon on hepatic tyrosine–α-oxoglutarate aminotransferase activity. Even in rats with phenylalanine hydroxylase activity depressed to 50% of control values by p-chlorophenylalanine administration, glucagon treatment increased the phenylalanine-oxidation rate substantially. Although hepatic phenylalanine–pyruvate aminotransferase was increased tenfold in glucagon-treated rats, glucagon treatment did not increase urinary excretion of phenylalanine transamination products by rats given a phenylalanine load. Glucagon treatment did not affect phenylalanine uptake by the gut or liver, or the liver content of phenylalanine hydroxylase cofactor. It is suggested that dihydropteridine reductase is the rate-limiting enzyme in phenylalanine degradation in the rat, and that glucagon may regulate the rate of oxidative phenylalanine metabolism in vivo by promoting indirectly the maintenance of the phenylalanine hydroxylase cofactor in its active, reduced state.

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The regulation of phenylalanine hydroxylase in rat tissues in vivo. Substrate- and cortisol-induced elevations in phenylalanine hydroxylase activity

Biochemical Journal ◽

10.1042/bj1540619 ◽

1976 ◽

Vol 154 (3) ◽

pp. 619-624 ◽

Cited By ~ 9

Author(s):

O Greengard ◽

J A. Delvalle

Keyword(s):

Phenylalanine Hydroxylase ◽

Enzyme Concentration ◽

Rat Tissues ◽

Hydroxylase Activity ◽

Adult Rats ◽

No Inhibition ◽

Hormonal Induction ◽

Positive Regulators

Injections of phenylalanine increased a 2.5-fold in 9 h the hepatic phenylalanine hydroxylase activity of 6-day-old or adult rats that had been pretreated (24h earlier) with p-chlorophenylalanine; without such pretreatment, phenylalanine did not raise the enzyme concentration. This difference is paralleled by the much greater extent to which the injected phenylalanine accumulated in livers of the pretreated compared with the normal animals. The hormonal induction of hepatic phenylalanine hydroxylase activity obeyed different rules: an injection of cortisol was without effect on adult livers but caused a threefold rise in phenylalanine hydroxylase activity of immature ones, both without and after pretreatment with p-chlorophenylalanine. In the latter instance, the effects of cortisol, and of phenylalanine were additive. Actinomycin inhibited the cortisol- but not the substrate-induced increase of phenylalanine hydroxylase, whereas puromycin inhibited both. The results indicate that substrate and hormone, two potential positive regulators of the amount of the hepatic (but not the renal) phenylalanine hydroxylase, act independently by two different mechanisms. The negative effector, p-chlorophenylalanine, also appears to interact with the synthetic (or degradative) machinery rather than with the existing phenylalanine hydroxylase molecules: 24h were required in vivo for an 85% decrease to ensue, and no inhibition occurred in vitro when incubating the enzyme with p-chlorophenylalanine or with liver extracts from p-chlorophenylalanine-treated rats.

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Simultaneous HPLC Analysis of Tryptophan Hydroxylase Activity and Serotonin Metabolism in Rat Pineal Gland: Determination of Its Kinetic Properties

Journal of Liquid Chromatography ◽

10.1080/10826079408013511 ◽

1994 ◽

Vol 17 (13) ◽

pp. 2939-2950 ◽

Cited By ~ 2

Author(s):

F. J. Hernández-Dáaz ◽

P. Abreu ◽

A. T. Dáaz-Cruz ◽

R. Alonso

Keyword(s):

Pineal Gland ◽

Hplc Analysis ◽

Tryptophan Hydroxylase ◽

Kinetic Properties ◽

Serotonin Metabolism ◽

Hydroxylase Activity ◽

Tryptophan Hydroxylase Activity ◽

Rat Pineal Gland

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Determination of Na(+)-K(+)-ATPase alpha- and beta-isoforms and kinetic properties in mammalian liver

AJP Cell Physiology ◽

10.1152/ajpcell.1992.262.6.c1491 ◽

1992 ◽

Vol 262 (6) ◽

pp. C1491-C1499 ◽

Cited By ~ 28

Author(s):

Y. Sun ◽

W. J. Ball

Keyword(s):

Western Blot ◽

Rat Liver ◽

Blot Analysis ◽

Western Blot Analysis ◽

Polyclonal Antibodies ◽

Liver Microsomes ◽

Kinetic Properties ◽

Rat Tissues ◽

Rat Liver Microsomes ◽

Kidney Enzyme

While Western blot analysis clearly revealed the presence of the alpha- and beta-subunits of Na(+)-K(+)-ATPase in a variety of rat tissues, beta was not readily detectable in liver. This observation was consistent with a previous report indicating that Na(+)-K(+)-ATPase immunoprecipitated from rat liver gives no clear evidence for the presence of a beta-subunit (Hubert et al. Biochemistry 25: 4156-4163, 1986). However, Western blot analysis of density gradient-purified lamb and rat liver microsomes showed the presence of a protein with an approximate molecular mass of 42 kDa that was immunoreactive with beta-specific polyclonal antibodies as well as beta-directed monoclonal antibodies. Deglycosylation of this protein by N-glycosidase F generated a core protein (beta c, M(r) approximately 32,000) that had the identical electrophoretic mobility as the beta c protein of the purified kidney enzyme. Isoform-specific monoclonal and synthetic peptide-directed polyclonal antibodies were used to demonstrate the presence of only the alpha 1- and beta 1-proteins in the liver and the presence of beta 2 in rat brain. Functional studies then showed that although both rat and lamb liver enzymes had sensitivities to cardiac glycoside inhibition similar to that of their corresponding kidney enzyme, the lamb liver enzyme had higher affinities for Na+, K+, and ATP than the kidney enzyme.

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