Isolation and characterization of a myeloma--spleen-cell hybrid producing antibody to phenylalanine hydroxylase.
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ABSTRACT:
SUBMITTER: Cotton RG
PROVIDER: S-EPMC1162277 | biostudies-other | 1980 Dec
REPOSITORIES: biostudies-other
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Project description:A monoclonal antibody directed against monkey liver phenylalanine hydroxylase was produced by using a rat-myeloma--rat-spleen-cell-fusion system. This antibody showed the interesting property of increasing mammalian phenylalanine hydroxylase activity more than 2-fold. Perhaps monoclonal antibodies with this effect on other enzyme or proteins could be developed.
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Project description:The liver enzyme phenylalanine hydroxylase is responsible for conversion of excess phenylalanine in the diet to tyrosine. Phenylalanine hydroxylase is activated by phenylalanine; this activation is inhibited by the physiological reducing substrate tetrahydrobiopterin. Phosphorylation of Ser16 lowers the concentration of phenylalanine for activation. This review discusses the present understanding of the molecular details of the allosteric regulation of the enzyme.
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Project description:Phenylalanine hydroxylase was prepared from rat liver and purified 200-fold to about 90% purity. All the enzymic activity of the liver appeared in a single protein of mol.wt. approx. 110000, but omission of dithiothreitol and of a preliminary filtration step to remove lipids resulted in partial conversion into a second enzymically active protein of mol.wt. approx. 250000. The K(m) and V(max.) values of the enzyme for phenylalanine, p-fluorophenylalanine and dimethyltetrahydropterin were measured; p-chlorophenylalanine inhibited the enzyme by competing with phenylalanine. Disc gel electrophoresis at pH7.2 showed a single protein band containing all the enzymic activity, but at pH8.7 the enzyme dissociated into two inactive fragments of similar but not identical molecular weight. The molecule of phenylalanine hydroxylase contained two atoms of iron, one atom of copper and one molecule of FAD; molybdenum was absent. Treatment with chelating agents showed that both non-haem iron and copper were necessary for enzymic activity. The molecule contained five thiol groups, and thiol-binding reagents inhibited the enzyme. Catalase or peroxidase enhanced enzymic activity fivefold; it is postulated that catalase (or other peroxidase) plays a part in the hydroxylation reaction independent of the protection by catalase of enzyme and cofactor from inactivation by a hydroperoxide.
| S-EPMC1166337 | biostudies-other
Project description: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.
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Project description:The loss of heterologous protein expression is one of the major problems faced by industrial cell line developers and has been reported by several authors. Therefore, the understanding of the mechanisms involved in the generation of stable and high producer cell lines is a critical issue, especially for those processes based on long term continuous cultures. We characterized two recombinant NS0 myeloma cell lines expressing Nimotuzumab, a humanized anti-human epidermal growth factor receptor (EGFR) antibody. The hR3/H7 clone is a stable producer obtained from the unstable hR3/t16 clone. The unstable clone was characterized by a bimodal distribution of intracellular immunoglobulin staining using flow cytometry. Loss of antibody production was due to the emergence of a non-producer cell subpopulation that increased with cell generation number. Immunoglobulin heavy chain (HC) and light chain (LC) ratio (HC/LC) was lower for the unstable phenotype. Proteomic maps using two dimensional gel electrophoresis (2DE) were obtained for both clones, at initial cell culture time and after 40 generations. Fifteen proteins potentially associated with the phenomenon of production stability were identified. The hR3/H7 stable clone showed an up-regulated expression pattern for most of these proteins. The regulation of recombinant antibody production by the host NS0 myeloma cell line most likely involves simultaneously cellular processes such as DNA transcription, mRNA processing, protein synthesis and folding, vesicular transport, glycolysis and energy production, according to the proteins identified in the present proteomic study.
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Project description:Phenylketonuria (PKU) is a loss-of-function inborn error of metabolism. As many other inherited diseases the main pathologic mechanism in PKU is an enhanced tendency of the mutant phenylalanine hydroxylase (PAH) to misfold and undergo ubiquitin-dependent degradation. Recent alternative approaches with therapeutic potential for PKU aim at correcting the PAH misfolding, and in this respect pharmacological chaperones are the focus of increasing interest. These compounds, which often resemble the natural ligands and show mild competitive inhibition, can rescue the misfolded proteins by stimulating their renaturation in vivo. For PKU, a few studies have proven the stabilization of PKU-mutants in vitro, in cells, and in mice by pharmacological chaperones, which have been found either by using the tetrahydrobiopterin (BH(4)) cofactor as query structure for shape-focused virtual screening or by high-throughput screening of small compound libraries. Both approaches have revealed a number of compounds, most of which bind at the iron-binding site, competitively with respect to BH(4). Furthermore, PAH shares a number of ligands, such as BH(4), amino acid substrates and inhibitors, with the other aromatic amino acid hydroxylases: the neuronal/neuroendocrine enzymes tyrosine hydroxylase (TH) and the tryptophan hydroxylases (TPHs). Recent results indicate that the PAH-targeted pharmacological chaperones should also be tested on TH and the TPHs, and eventually be derivatized to avoid unwanted interactions with these other enzymes. After derivatization and validation in animal models, the PAH-chaperoning compounds represent novel possibilities in the treatment of PKU.
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Project description:Liver phenylalanine hydroxylase (PheH) is an allosteric enzyme that requires activation by phenylalanine for full activity. The location of the allosteric site for phenylalanine has not been established. NMR spectroscopy of the isolated regulatory domain (RDPheH(25-117) is the regulatory domain of PheH lacking residues 1-24) of the rat enzyme in the presence of phenylalanine is consistent with formation of a side-by-side ACT dimer. Six residues in RDPheH(25-117) were identified as being in the phenylalanine-binding site on the basis of intermolecular NOEs between unlabeled phenylalanine and isotopically labeled protein. The location of these residues is consistent with two allosteric sites per dimer, with each site containing residues from both monomers. Site-specific variants of five of the residues (E44Q, A47G, L48V, L62V, and H64N) decreased the affinity of RDPheH(25-117) for phenylalanine based on the ability to stabilize the dimer. Incorporation of the A47G, L48V, and H64N mutations into the intact protein increased the concentration of phenylalanine required for activation. The results identify the location of the allosteric site as the interface of the regulatory domain dimer formed in activated PheH.
| S-EPMC4817173 | biostudies-literature
Project description:The laboratory mouse represents an important model for the study of phenylalanine metabolism and the pathochemistry of phenylketonuria, yet mouse phenylalanine hydroxylase (PAH) has not been extensively studied. We report the cloning and sequencing of a mouse PAH cDNA, the expression of enzymic activity from the mouse PAH cDNA clone and the identification of mouse PAH and human PAH by two-dimensional PAGE of liver samples. These data confirm the expected homology of mouse PAH and human PAH and suggest differences in the primary sequence and the phosphorylation state of the two enzymes.
| S-EPMC1131302 | biostudies-other
Project description:Phenylalanine hydroxylase (PheH) is a pterin-dependent, mononuclear nonheme iron(II) oxygenase that uses the oxidative power of O2 to hydroxylate phenylalanine to form tyrosine. PheH is a member of a superfamily of O2-activating enzymes that utilizes a common metal binding motif: the 2-His-1-carboxylate facial triad. Like most members of this superfamily, binding of substrates to PheH results in a reorganization of its active site to allow O2 activation. Exploring the energetics of each step before O2 activation can provide mechanistic insight into the initial steps that support the highly specific O2 activation pathway carried out by this metalloenzyme. Here the thermal stability of PheH and its substrate complexes were investigated under an anaerobic environment by using differential scanning calorimetry. In context with known binding constants for PheH, a thermodynamic cycle associated with iron(II), tetrahydrobiopterin (BH4), and phenylalanine binding to the active site was generated, showing a distinctive cooperativity between the binding of BH4 and Phe. The addition of phenylalanine and BH4 to PheH·Fe increased the stability of this enzyme (ΔTm of 8.5 (±0.7) °C with an associated δΔH of 43.0 (±2.9) kcal/mol). The thermodynamic data presented here gives insight into the complicated interactions between metal center, cofactor, and substrate, and how this interplay sets the stage for highly specific, oxidative C-H activation in this enzyme.
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Project description:Analytical ultracentrifugation has been used to analyze the oligomeric structure of the isolated regulatory domain of phenylalanine hydroxylase. The protein exhibits a monomer-dimer equilibrium with a dissociation constant of ~46 μM; this value is unaffected by the removal of the 24 N-terminal residues or by phosphorylation of Ser16. In contrast, phenylalanine binding (Kd = 8 μM) stabilizes the dimer. These results suggest that dimerization of the regulatory domain of phenylalanine hydroxylase is linked to allosteric activation of the enzyme.
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