The Missense p.S231F Phenylalanine Hydroxylase Gene Mutation Causes Complete Loss of Enzymatic Activity In Vitro

Phenylketonuria (PKU), the most frequent disorder of amino acid metabolism, is caused by mutations in human phenylalanine hydroxylase gene (PAH), leading to deficient enzyme activity. Previously reported but uncharacterized PAH gene mutation, p.S231F (c.692C > T), was detected in Serbian patients with classical PKU. We analyzed p.S231F PAH protein in prokaryotic (Escherichia coli) and eukaryotic expression system (hepatoma cells). In both systems the mutant enzyme was unstable. Residual enzyme activity in vitro was ~1%. Mutation p.S231F PAH was not activated by pre-incubation with phenylalanine substrate. We found no GroEL/GroES chaperone effect and slightly positive effect of the (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin (BH₄) on the stabilization of the protein structure. Our findings were in accordance with severe patients’ phenotypes. In conclusion, p.S231F should be classified as a functionally null PAH gene mutation as it drastically reduces stability and activity of the PAH enzyme in vitro.     

Carcinogenic Effects in a Phenylketonuria Mouse Model

Phenylketonuria (PKU) is a metabolic disorder caused by impaired phenylalanine hydroxylase (PAH). This condition results in hyperphenylalaninemia and elevated levels of abnormal phenylalanine metabolites, among which is phenylacetic acid/phenylacetate (PA). In recent years, PA and its analogs were found to have anticancer activity against a variety of malignancies suggesting the possibility that PKU may offer protection against cancer through chronically elevated levels of PA. We tested this hypothesis in a genetic mouse model of PKU (PAH^enu2) which has a biochemical profile that closely resembles that of human PKU. Plasma levels of phenylalanine in homozygous (HMZ) PAH^enu2 mice were >12-fold those of heterozygous (HTZ) littermates while tyrosine levels were reduced. Phenylketones, including PA, were also markedly elevated to the range seen in the human disease. Mice were subjected to 7,12 dimethylbenz[a]anthracene (DMBA) carcinogenesis, a model which is sensitive to the anticancer effects of the PA derivative 4-chlorophenylacetate (4-CPA). Tumor induction by DMBA was not significantly different between the HTZ and HMZ mice, either in total tumor development or in the type of cancers that arose. HMZ mice were then treated with 4-CPA as positive controls for the anticancer effects of PA and to evaluate its possible effects on phenylalanine metabolism in PKU mice. 4-CPA had no effect on the plasma concentrations of phenylalanine, phenylketones, or tyrosine. Surprisingly, the HMZ mice treated with 4-CPA developed an unexplained neuromuscular syndrome which precluded its use in these animals as an anticancer agent. Together, these studies support the use of PAH^enu2 mice as a model for studying human PKU. Chronically elevated levels of PA in the PAH^enu2 mice were not protective against cancer.     

Preservation of high phenylalanine ammonia lyase activities in roots of Japanese Striped corn: A potential oral therapeutic to treat phenylketonuria

Phenylketonuria (PKU) is an inherited metabolic disorder caused by deficient phenylalanine hydroxylase (PAH) activity, the enzyme responsible for the disposal of excess amounts of the essential amino acid phenylalanine (Phe). Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) has potential to serve as an enzyme substitution therapy for this human genetic disease. Using 7-day-old Japanese Striped corn seedlings (Japonica Striped maize, Zea mays L. cv. japonica) that contain high activities of PAL, we investigated a number of methods to preserve the roots as an intact food and for long-term storage. The cryoprotectant effects of maple syrup and other edible sugars (mono- and oligosaccharides) were evaluated. Following thawing, the preserved roots were then examined to determine whether the rigid plant cell walls could protect the PAL enzyme from proteolysis during simulated (in vitro) digestion comprised of gastric and intestinal phases. While several treatments led to retention of PAL activity during freezing, upon thawing and in vitro digestion, root tissues that had been previously frozen in the presence of maple syrup exhibited the highest residual PAL activities (∼50% of the initial enzyme activity), in marked contrast to all of the treatments using other edible sugars. The structural integrity of the root cells, and the stability of the functional PAL tetramer were also preserved with the maple syrup protocol. These results have significance for the formulation of oral enzyme/protein therapeutics. When plant tissues are adequately preserved, the rigid cell walls constitute a protective barrier even under harsh (e.g. gastrointestinal-like) conditions.     

Predicting a clinical/biochemical phenotype for PKU/MHP patients with PAH gene mutations

Phenylketonuria (PKU) and mild hyperphenylalaninemia (MHP) are allelic disorders caused by mutations in the gene encoding phenylalanine hydroxylase (PAH). In this study, a total of 218 independent PAH chromosomes (109 unrelated patients with PKU residing in Lithuania) were investigated. All 13 exons of the PAH gene of all PKU probands were scanned for DNA alterations by denaturing gradient gel electrophoresis (DGGE). In the cases of a specific DGGE pattern recognized, mutations were identified by direct fluorescent automated sequencing or by restriction enzyme digestion analysis of relevant exons. Twenty-five different PAH gene mutations were identified in Lithuania. We estimated a connection between individual PAH locus mutations and biochemical and metabolic phenotypes in patients in whom the mutant allele acts on its own, i.e., in functionally hemizygous patients and using the assigned value (AV) method to determine the severity of both common and rare mutant alleles, as well as to check a model to predict the combined phenotypic effect of two mutant PAH alleles. The text was submitted authors English.     

Synthesis of phenylalanine ammonia-lyase in anthocyanin-containing and anthocyanin-free callus cells of Daucus carota L.

When callus cells of Daucus carota are grown on a medium containing gibberellic acid (GA₃) in a physiological concentration of 3x10^-6 M the cells cease to accumulate anthocyanins. This anthocyanin-free cell line has a very low activity of phenylalanine ammonia-lyase. After density labelling with D₂O an intensive de novo synthesis of the phenylalanine ammonia-lyase (E.C. 4.3.1.5; PAL) in the anthocyanin-containing cells does occur. 58% of the C-bound H-atoms are replaced by deuterium. The anthocyanin-free cells show only a very low enzyme synthesis which is difficult to detect with density labelling experiments. To ascertain that de novo synthesis occurs in the anthocyanin-free cells, the incorporation of ¹⁴C-labelled amino acids into the partially purified enzyme protein was measured after separation of the protein a) in CsCl gradients and b) on polyacrylamide gels. In both cases the enzyme bears ¹⁴C-label. These results suggest that in the anthocyanin-free cells de novo synthesis of PAL is still occuring but the synthesis is reduced in comparison to the anthocyanin-containing cells.Abbreviations GA₃ gibberellic acid - PAL phenylalanine ammonia-lyase (E.C.4.3.1.5) - DCb anthocyanin-containing cells - DCw anthocyanin-free cells     

Simultaneous assessment of the effects of exonic mutations on RNA splicing and protein functions

To simultaneously assess the effects of exonic mutations on RNA splicing and protein functions, we report here an intron-inclusive cDNA (Intinc) expression system. As a test model, twenty-four mutations in exon 9 of the phenylalanine hydroxylase (PAH) gene were examined in an Intinc expression plasmid composed of the PAH cDNA with the exon 9 flanked by its authentic introns. When the PAH enzyme activities from the Intinc plasmid-transfected cells were compared to those of a standard cDNA expression system, five mutations resulted in significant relative differences in PAH activities attributed to altered exon 9-inclusive mRNA levels. Two of the mutations affected exon recognition probably through splice site modifications and the remaining three affected experimentally verified exon splicing enhancer (ESE) motifs. The Intinc expression system allows not only a better link between mutation genotype to disease phenotype but also contributes to further understanding of molecular mechanisms of deleterious effects of mutations.     

Role of the second coordination sphere residue tyrosine 179 in substrate affinity and catalytic activity of phenylalanine hydroxylase

Phenylalanine hydroxylase converts phenylalanine to tyrosine utilizing molecular oxygen and tetrahydropterin as a cofactor, and belongs to the aromatic amino acid hydroxylases family. The catalytic domains of these enzymes are structurally similar. According to recent crystallographic studies, residue Tyr179 in Chromobacterium violaceum phenylalanine hydroxylase is located in the active site and its hydroxyl oxygen is 5.1 Å from the iron, where it has been suggested to play a role in positioning the pterin cofactor. To determine the catalytic role of this residue, the point mutants Y179F and Y179A of phenylalanine hydroxylase were prepared and characterized. Both mutants displayed comparable stability and metal binding to the native enzyme, as determined by their melting temperatures in the presence and absence of iron. The catalytic activity (k_cat) of the Y179F and Y179A proteins was lower than wild-type phenylalanine hydroxylase by an order of magnitude, suggesting that the hydroxyl group of Tyr179 plays a role in the rate-determining step in catalysis. The K_M values for different tetrahydropterin cofactors and phenylalanine were decreased by a factor of 3–4 in the Y179F mutant. However, the K_M values for different pterin cofactors were slightly higher in the Y179A mutant than those measured for the wild-type enzyme, and, more significantly, the K_M value for phenylalanine was increased by 10-fold in the Y179A mutant. By the criterion of k_cat/K_Phe, the Y179F and Y179A mutants display 10% and 1%, respectively, of the activity of wild-type phenylalanine hydroxylase. These results are consistent with Tyr179 having a pronounced role in binding phenylalanine but a secondary effect in the formation of the hydroxylating species. In conjunction with recent crystallographic analyses of a ternary complex of phenylalanine hydroxylase, the reported findings establish that Tyr179 is essential in maintaining the catalytic integrity and phenylalanine binding of the enzyme via indirect interactions with the substrate, phenylalanine. A model that accounts for the role of Tyr179 in binding phenylalanine is proposed.Electronic Supplementary Material Supplementary material is available in the online version of this article at Abbreviations AAAHs aromatic amino acid hydroxylases - BH₂ 7,8-dihydro-l-biopterin - BH₄ (6R)-5,6,7,8-tetrahydro-l-biopterin - CD circular dichroism - cPAH Chromobacterium violaceum phenylalanine hydroxylase - DMPH₄ 6,7-dimethyl-5,6,7,8-tetrahydropterin - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - ES-MS electrospray ionization mass spectrometry - hPAH human phenylalanine hydroxylase - ICP-AE inductively coupled plasma atomic emission - 6-MPH₄ 6-methyl-5,6,7,8-tetrahydropterin - PAH phenylalanine hydroxylase - PH₄ tetrahydropterin - PKU phenylketonuria - RDS rate-determining step - TH tyrosine hydroxylase - THA 3-(2-thienyl)-l-alanine - TPH tryptophan hydroxylase - wt wild-type     

A hypothesis on the biochemical mechanism of BH<Subscript>4</Subscript>-responsiveness in phenylalanine hydroxylase deficiency

Summary.  We describe six children with tetrahydrobiopterin (BH₄) responsive phenylalanine hydroxylase (PAH) deficiency. All patients carry two mutant alleles in the PAH gene. Cofactor deficiency was excluded. The effect of BH₄ administration was studied by correlating different oral BH₄ doses with plasma phenylalanine levels under defined protein intake. Our results indicate that oral BH₄ supplementation may be used as long-term treatment for individuals with BH₄-responsive PAH deficiency, either without or in combination with a less restrictive diet. Previous in vitro studies have demonstrated that BH₄ inhibits PAH tetramers but activates PAH dimers. This may indicate, that BH₄-responsiveness results from BH₄ induced stabilization of mutant PAH dimers. In addition, interindividual differences in the cellular folding apparatus may determine the tertiary structure and the amount of mutant PAH dimers and hence may account for divergent BH₄-responsiveness reported for the same PAH genotype. Received September 7, 2002 Accepted December 16, 2002 Published online March 17, 2003 Acknowledgements We express our gratitude to Dr. J. Zschocke for the genetic analysis of the PAH mutants, to Dr. N. Blau (Switzerland) for pterin analysis and to Dr. V. Wagner (Univ. Lübeck, Germany) for additional data on patient EM. Furthermore, we thank the technical staff of our laboratories for their accurate work throughout the project. Authors' address: Dr. Zoltan Lukacs, Metabolic Laboratory, Department of Pediatrics, University of Hamburg, Martinistr. 52, D-20246 Hamburg, Germany, Fax: +49-40-42803 6717, E-mail: Lukacs@uke.uni-hamburg.de List of abbreviations: BH₄, Tetrahydrobiopterin; DGGE, Denaturing gradient gel electrophoresis; DHPR, Dihydropteridine reductase; HPA, Hyperphenylalaninemia; MHP, Mild hyperphenylalaninemia; PAH, Phenylalanine hydroxylase; Phe, Phenylalanine; PKU, Phenylketonuria; Tyr, Tyrosine     

Demonstration and characterization of (E)-nerolidol synthase from maize: a herbivore-inducible terpene synthase participating in (3E)-4,8-dimethyl-1,3,7-nonatriene biosynthesis

Upon herbivore attack, maize (Zea mays L.) emits a mixture of volatile compounds that attracts herbivore enemies to the plant. One of the major components of this mixture is an unusual acyclic C₁₁ homoterpene, (3E )-4,8-dimethyl-1,3,7-nonatriene (DMNT), which is also emitted by many other species following herbivore damage. Biosynthesis of DMNT has been previously shown to proceed via the sesquiterpene alcohol, (E )-nerolidol. Here we demonstrate an enzyme activity that converts farnesyl diphosphate, the universal precursor of sesquiterpenes, to (3S)-(E )-nerolidol in cell-free extracts of maize leaves that had been fed upon by Spodoptera littoralis. The properties of this (E )-nerolidol synthase resemble those of other terpene synthases. Evidence for its participation in DMNT biosynthesis includes the direct incorporation of deuterium-labeled (E )-nerolidol into DMNT and the close correlation between increases in (E )-nerolidol synthase activity and DMNT emission after herbivore damage. Since farnesyl diphosphate has many other metabolic fates, (E )-nerolidol synthase may represent the first committed step of DMNT biosynthesis in maize. However, the formation of this unusual acyclic terpenoid appears to be regulated at both the level of (E )-nerolidol synthase and at later steps in the pathway. Received: 20 August 1999 / Accepted: 27 October 1999     

Rapid induction of ethylene biosynthesis in cultured parsley cells by fungal elicitor and its relationship to the induction of phenylalanine ammonia-lyase

The biosynthesis of ethylene was examined in suspension-cultured cells of parsley (Petroselinum hortense) treated with an elicitor from cell walls of Phytophthora megasperma. Untreated cells contained 50 nmol g^-1 of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), and produced ethylene at a rate of about 0.5 nmol g^-1 h^-1. Within 2 h after addition of elicitor to the culture medium, the cells started to produce more ethylene and accumulated more ACC. Exogenously added ACC did not increase the rate of ethylene production in control or elicitor-treated cells, indicating that the enzyme converting ACC to ethylene was limiting in both cases. The first enzyme in ethylene biosynthesis, ACC synthase, was very rapidly and transiently induced by the elicitor treatment. Its activity increased more than tenfold within 60 min. Density labelling with ²H₂O showed that this increase was caused by the denovo synthesis of the enzyme protein. Cordycepin and actinomycin D did not affect the induction of ACC synthase, indicating that the synthesis of new mRNA was not required. The peak of ACC-synthase activity preceded the maximal phenylalanine ammonia-lyase (PAL) activity by several hours. Exogenously supplied ethylene or ACC did not induce PAL. However, aminoethoxyvinylglycine, an inhibitor of ACC synthase, suppressed the rise in ethylene production in elicitor-treated cells and partially inhibited the induction of PAL. Exogenously supplied ACC reversed this inhibition. It is concluded that induction of the ethylene biosynthetic pathway is a very early symptom of elicitor action. Although ethylene alone is not a sufficient signal for PAL induction, the enhanced activity of ACC synthase and the ethylene biosynthetic pathway may be important for the subsequent induction of PAL.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - PAL phenylalanine ammonia-lyase     

Modification of l-phenylalanine ammonia-lyase in soybean cell suspension cultures by 2-aminooxyacetate and l-2-aminooxy-3-phenylpropionate

Suspension-cultured cells of soybean (Glycine max (L.) Merr. cv. Kanrich) produce large amounts of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), the first enzyme of phenylpropanoid metabolism, during growth. 2-Aminooxyacetic acid (AOA) and l-2-aminooxy-3-phenylpropionic acid (l-AOPP) inhibit the enzyme competitively in vitro and have been used for in vivo studies. The amount of extractable enzyme in the cells and their utilization of NO ₃ ^– and NH ₃ ⁺ are reduced upon the addition of AOA. When AOA was added at various times during growth, the appearance of additional enzyme activity was prevented but enzyme already formed was not inhibited. No evidence was obtained for the presence of an inhibitor in the extracts and AOA inhibition in vitro was readily reversible. It is conculded that AOA acts to inhibit the formation of PAL in suspension-cultured soy bean cells. In vitro inhibition of soybean PAL by l-AOPP could not be reversed; in contrast, the inhibition of maize (Zea mays L.) PAL was readily reversible. Added l-AOPP, which was rapidly taken up by the soybean cells, prevented the large increase in enzyme activity. Although PAL activity was blocked in the cultures, no appreciable increase in phenylalanine content could be detected in cell extracts. The response of soybean cell suspensions to l-AOPP addition thus differs from that of other tissues which in presence of l-AOPP show an increase in PAL activity and an accumulation of phenylalanine.Abbreviations AOA 2-aminooxyacetic acid - l-AOPP l-2-aminoxy-3-phenylpropionic acid - PAL l-phenylalanine ammonialyase (EC4.3.1.5)     

Effect of feeding precursors on phenylalanine ammonia-lyase activity and coumarin accumulation in leaves of <Emphasis Type="Italic">Matricaria chamomilla</Emphasis> L.

Four-week-old chamomile (Matricaria chamomilla) plants were exposed for 72 h to 0.01, 0.1 and 1 mM phenylalanine (Phe) or tyrosine (Tyr). Phe at all concentrations significantly increased phenylalanine ammonia-lyase (PAL) activity (by 30, 76 and 90%, respectively) as well as accumulation of coumarin-related compounds (herniarin and its precursors (Z)- and (E)-2-β-D-glucopyranosyloxy-4-methoxycinnamic acids). Free Phe content increased significantly at the highest dose tested. Lower Tyr concentrations (0.01 and 0.1 mM) significantly increased PAL activity and increased free Tyr content, however free Phe content decreased. This indicated that Tyr-mediated stimulation of PAL is coupled to Phe consumption. Notwithstanding, Tyr had no effect on coumarin accumulation. Therefore we speculate that in chamomile a regulation/signalling mechanism could be operating in the pathway leading to coumarin synthesis. The malondialdehyde accumulation, an usual marker of stress in plants, was not significantly changed by amino acid supplements, suggesting that membrane damage is not the signal causing coumarin accumulation. In parallel experiment we observed that neither lower (0.25 × full strength), nor higher (3 × full strength) nitrogen concentration of nutrient solution compared to normal (1 × full strength, 205 mg N l^-1) solution used for Phe/Tyr supply affected herniarin and GMCAs accumulation. This indicates that Phe had stimulatory effect on PAL activity and coumarin metabolism.     

Effects of testosterone and 17, β– estradiol on the polyamine metabolism in cultivated normal rat kidney epithelial cells

Summary. Ornithine decarboxylase (ODC) and diamine oxidase (DAO) are important enzymes involved in the metabolism of polyamines (putrescine, spermidine and spermine). The influence of testosterone (T) and 17, β– estradiol (E₂) on the activity of ODC and DAO was examined in cultivated normal rat kidney (NRK) epithelial cells. The results showed an increase in enzyme activities 4 hours or 12 hours after hormonal treatment. Both T and E₂ led to a significant increase (1.6-fold) in ODC protein level as compared to the controls. Cellular concentration of spermidine and spermine increased (2.2- and 2.6-fold respectively) 4 hours after T addition. A higher levels in concentrations of putrescine (1.4-fold) and spermine (1.5-fold) 12 hours after E₂ treatment were observed. These results suggest that the biosynthesis and terminal oxidation of the polyamines in NRK epithelial cells are androgen- and estrogen-mediated and depend on the hormonal sensitivity of the cells. Received April 5, 1999, Accepted December 20, 1999     

Differential activation of H+-ATPase genes by an arbuscular mycorrhizal fungus in root cells of transgenic tobacco

In arbuscular mycorrhizas, H⁺-ATPase is active in the plant membrane around arbuscules but absent from plant mutants defective in arbuscule development (Gianinazzi-Pearson et al. 1995, Can J Bot 73: S526–S532). The proton-pumping H⁺-ATPase is encoded by a family of genes in plants. Immunocytochemical studies and promoter-gusA fusion assays were performed in transgenic tobacco (Nicotiana tabacum L.) to determine whether the periarbuscular enzyme activity results from de-novo activation of plant genes by an arbuscular mycorrhizal fungus. The H⁺-ATPase protein was localized in the plant membrane around arbuscule hyphae. The enzyme was absent from non-colonized cortical cells. Regulation of seven H⁺-ATPase genes (pma) was compared in non-mycorrhizal and mycorrhizal roots by histochemical detection of β-glucuronidase (GUS) activity. Two genes (pma2, pma4) were induced in arbuscule-containing cells of mycorrhizal roots but not in non-mycorrhizal cortical tissues or senescent mycorrhiza. It is concluded that de-novo H⁺-ATPase activity in the periarbuscular membrane results from selective induction of two H⁺-ATPase genes, which can have diverse roles in plant-fungal interactions at the symbiotic interface. Received: 23 October 1999 / Accepted: 7 February 2000     

Evidence that DNA damage is associated to phenylalanine blood levels in leukocytes from phenylketonuric patients

Phenylketonuria (PKU) is an inborn error of phenylalanine (Phe) metabolism, biochemically characterized by the accumulation of Phe and its metabolites in blood and tissues of affected patients. Treatment for PKU consists of a protein restricted diet supplemented with a mixture containing essential amino acids (other than Phe) and micronutrients. In recent years several authors have studied the pathomechanisms of the disease and demonstrated the existence of lipid and protein oxidative damage in PKU patients. In this work we investigated the in vivo and in vitro effects of Phe on DNA damage determined by the alkaline comet assay using silver staining and visual scoring. We found a dose-dependent effect of Phe on DNA damage in leukocytes from normal individuals incubated with different concentrations of Phe. Additionally, by analyzing blood leukocytes from two groups of treated PKU patients based on their blood Phe levels, we verified that the DNA damage index was significantly higher in PKU patients with high Phe blood levels (DI = 68.2 ± 12.3), compared to well-treated patients and the control group (healthy individuals). Furthermore, well-treated PKU patients had greater DNA damage (DI = 44.9 ± 7.6) relatively to controls (DI = 12.7 ± 4.1). Our present in vitro and in vivo findings indicate that DNA damage occurs in peripheral blood from PKU patients and is associated to Phe blood levels.     

Functional Characterization of Novel Phenylalanine Hydroxylase p.Gln226Lys Mutation Revealed Its Non-responsiveness to Tetrahydrobiopterin Treatment in Hepatoma Cellular Model

Treatment with tetrahydrobiopterin (BH4) is the latest therapeutic option approved for patients with phenylketonuria (PKU)—one of the most frequent inborn metabolic diseases. PKU or phenylalanine hydroxylase (PAH) deficiency is caused by mutations in the PAH gene. Given that some PAH mutations are responsive to BH4 treatment while others are non-responsive, for every novel mutation that is discovered it is essential to confirm its pathogenic effect and to assess its responsiveness to a BH4 treatment in vitro, before the drug is administered to patients. We found a c.676C>A (p.Gln226Lys) mutation in the PAH gene in two unrelated patients with PKU. The corresponding aberrant protein has never been functionally characterized in vitro and its response to BH4 treatment is unknown. Computational analyses proposed that glutamine at position 226 is an important, evolutionary conserved amino acid while the substitution with lysine probably disturbs tertiary protein structure and impacts posttranslational PAH modifications. Using hepatoma cellular model, we demonstrated that the amount of mutant p.Gln226Lys PAH detected by Western blot was only 1.2% in comparison to wild-type PAH. The addition of sepiapterin, intracellular precursor of BH4, did not increase PAH protein yield thus marking p.Gln226Lys as BH4-non-responsive mutation. Therefore, computational, experimental, and clinical data were all in accordance showing that p.Gln226Lys is a severe pathogenic PAH mutation. Its non-responsiveness to BH4 treatment in hepatoma cellular model should be considered when deciding treatment options for PKU patients carrying this mutation. Consequently, our study will facilitate clinical genetic practice, particularly genotype-based stratification of PKU treatment.     

Time courses for phytochrome-induced enzyme levels in phenylpropanoid metabolism (phenylalanine ammonia-lyase,naringenin-chalcone synthase) compared with time courses for phytochrome-mediated end-product accumulation (anthocyanin,quercetin)

Time course for changes in the levels of enzymes characteristic of general phenylpropanoid metabolism (phenylalanine ammonia-lyase, PAL; EC 4.3.1.5) and of the flavonoid-glycoside branch pathway (naringenin-chalcone synthase, CHS; EC 2.3.1.74) were measured in the cotyledons of mustard (Sinapis alba L.) seedlings and compared with the rates of accumulation of related end products (anthocyanin and quercetin). Induction of enzyme levels and of end-product accumulation was carried out with red and far-red (FR) light, operating via phytochrome. The data are compatible with the concept that the phytochrome-mediated appearance of enzymes such as PAL and CHS is indeed a prerequisite for the appearance of anthocyanins and flavonols. However, there is no close correlation between enzyme levels and the rates of synthesis of end products which could justify the identification of specific rate-limiting enzymes. Rather, the data indicate that there is a second phytochrome-dependent step, beyond enzyme induction, where the actual rate of flavonoid accumulation is determined. Anthocyanin and quercetin accumulation respond differently to light. However, the relative action of continuous FR, red light pulses and stored phytochrome signal is the same in both cases. This indicates that the mode of operation of phytochrome is the same in both cases. The two syntheses differ only in the degree of responsiveness towards phytochrome. The time course for changes in CHS levels in continuous FR, i.e. under conditions of phytochrome photosteady state, is similar to the time course for PAL levels whereas the time courses in darkness, following transfer from FR to darkness, are totally different. In the case of CHS, a transient rise is observed whereas, with PAL, an instantaneous drop in enzyme level occurs after transfer from FR to darkness. It is concluded that the stored phytochrome signal operates in darkness in the case of CHS but not in the case of PAL.Abbreviations c continuous - CHS naringenin-chalcone synthase (EC 2.3.1.74) - FR far-red light (3.5 W·m^-2) - PAL phenylalanine ammonia-lyase (EC 4.3.1.5) - Pfr phytochrome (far-red absorbing) - Pr phytochrome (red absorbing) - R red light (6.8 W·m^-2) - RG9-light long-wavelength far-red light obtained with RG9 glass filter - [Pfr]/[Ptot], whereby - Ptot total phytochrome (Pr+Pfr)     

Anomalous effects of cycloheximide on phenylalanine ammonia-lyase: role of synthesis and inactivation in leaf disks of helianthus annuus*

The activity of phenylalanine ammonia-lyase (PAL) increases dramatically in leaf disks of sunflower (Helianthus annuus) cultured on 0.1 M sucrose in the dark. If disks are subsequently transferred to water, PAL activity decays rapidly. After inactivation the level of PAL can be increased again by transferring the tissue back to sucrose. The initial increase in PAL activity appears to involve an increase in the rate of PAL formation and the appearance is inhibited by cycloheximide. Inactivation of the enzyme is also inhibited by cycloheximide. A comparison of cycloheximide inhibition at different concentrations showed that inactivation was much more sensitive to the inhibitor than PAL formation. The rate of PAL inactivation was very low in fresh disks placed directly on water (t 1/2 = > 1 day) but increased greatly after culture on sucrose (t_1/2 = 2 to 4 hr). Therefore, culture appears to increase PAL inactivation as well as PAL formation. Reappearance of PAL activity after inactivation is stimulated rather than inhibited by cycloheximide. The change in effect of cycloheximide from inhibition to apparent stimulation can best be explained by the observation that (1) the turnover of PAL, both formation and inactivation, increases greatly as a result of culture on sucrose and (2) inactivation is more sensitive to cycloheximide than formation. Thus, even where an anomalous cycloheximide insensitive appearance of PAL activity occurs, a mechanism other than reactivation of the enzyme may be involved.