Isolation of phenylalanine hydroxylase-stimulating monoclonal antibody by rat-myeloma--rat-spleen-cell fusion.
Ontology highlight
ABSTRACT:
SUBMITTER: Choo KH
PROVIDER: S-EPMC1162262 | biostudies-other | 1980 Nov
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Application of the technique of myeloma--spleen-cell fusion [Kohler & Milstein (1975) Nature (London) 256, 495--497] has allowed the isolation of a cell colony that produced a monoclonal antibody against monkey liver phenylalanine hydroxylase. The antibody exhibited cross-reactivity against hepatic phenylalanine hydroxylase from other mammalian species, including human, rat and mouse. Cross-reactivity was established by (a) enzyme-inhibition assay, (b) double-immunodiffusion reaction, and (c) two-dimensional polyacrylamide-gel-electrophoretic analysis of immunoprecipitate. The various properties of the monoclonal antibody and its use in the study of mammalian phenylalanine hydroxylase are presented.
| S-EPMC1162277 | biostudies-other
Project description:Phenylalanine hydroxylase purified from rat liver shows positive co-operativity in response to variations in phenylalanine concentration when assayed with the naturally occurring cofactor tetrahydrobiopterin. In addition, preincubation of phenylalanine hydroxylase with phenylalanine results in a substantial activation of the tetrahydrobiopterin-dependent activity of the enzyme. The monoclonal antibody PH-1 binds to phenylalanine hydroxylase only after the enzyme has been preincubated with phenylalanine and is therefore assumed to recognize a conformational epitope associated with substrate-level activation of the hydroxylase. Under these conditions, PH-1 inhibits the activity of phenylalanine hydroxylase; however, at maximal binding of PH-1 the enzyme is still 2-3 fold activated relative to the native enzyme. The inhibition by PH-1 is non-competitive with respect to tetrahydropterin cofactor. This suggests that PH-1 does not bind to an epitope at the active site of the hydroxylase. Upon maximal binding of PH-1, the positive co-operativity normally expressed by phenylalanine hydroxylase with respect to variations in phenylalanine concentration is abolished. The monoclonal antibody may therefore interact with phenylalanine hydroxylase at or near the regulatory or activator-binding site for phenylalanine on the enzyme molecule.
| S-EPMC1135591 | biostudies-other
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: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:Monoclonal antibody PH7 has specificity for the phosphorylated form of the human liver phenylalanine hydroxylase and negligible reactivity towards the dephosphorylated form of the native enzyme by enzyme-linked immunoassay. PH7 binds specifically to the phosphorylated form of the liver enzyme after SDS/polyacrylamide-gel electrophoresis and transfer to nitrocellulose. Competitive blocking assays have been applied in conjunction with reversed-phase h.p.l.c. of purified tryptic fragments of human liver phenylalanine hydroxylase to localize the epitope. The major immunoreactive tryptic peptide cross-reacting with PH7 had an amino acid analysis corresponding to the first 41 amino acids of the human liver phenylalanine hydroxylase sequence and included the serine residue that is thought to be the phosphorylation site. The monoclonal antibody recognized the phosphorylated form of the synthetic decapeptide corresponding to the local phosphorylation-site sequence Gly-Leu-Gly-Arg-Lys-Leu-Ser(P)-Asp-Phe-Gly, but not the dephosphodecapeptide. Thermolysin digestion of the peptide demonstrated the monoclonal antibody bound to the pentapeptide Leu-Ser(P)-Asp-Phe-Gly. Monoclonal antibody PH7 recognized the phosphodecapeptide at concentrations 10(3)-fold higher than with phenylalanine hydroxylase, compared with 10(4)-10(7)-fold higher for other phosphopeptides and phosphoproteins. The results demonstrate that monoclonal antibody PH7 has specificity for the phosphorylated form of phenylalanine hydroxylase at the phosphorylation site.
| S-EPMC1148042 | biostudies-other
Project description:Macrophages can promote tumor development. Preclinically, targeting macrophages by colony-stimulating factor 1 (CSF1)/CSF1 receptor (CSF1R) monoclonal antibodies (mAbs) enhances conventional therapeutics in combination treatments. The physiological distribution and tumor uptake of CSF1R mAbs are unknown. Therefore, we radiolabeled a murine CSF1R mAb and preclinically visualized its biodistribution by PET. CSF1R mAb was conjugated to N-succinyl-desferrioxamine (N-suc-DFO) and subsequently radiolabeled with zirconium-89 (89Zr). Optimal protein antibody dose was first determined in non-tumor-bearing mice to assess physiological distribution. Next, biodistribution of optimal protein dose and 89Zr-labeled isotype control was compared with PET and ex vivo biodistribution after 24 and 72 h in mammary tumor-bearing mice. Tissue autoradiography and immunohistochemistry determined radioactivity distribution and tissue macrophage presence, respectively. [89Zr]Zr-DFO-N-suc-CSF1R-mAb optimal protein dose was 10 mg/kg, with blood pool levels of 10 ± 2% injected dose per gram tissue (ID/g) and spleen and liver uptake of 17 ± 4 and 11 ± 4%ID/g at 72 h. In contrast, 0.4 mg/kg of [89Zr]Zr-DFO-N-suc-CSF1R mAb was eliminated from circulation within 24 h; spleen and liver uptake was 126 ± 44% and 34 ± 7%ID/g, respectively. Tumor-bearing mice showed higher uptake of [89Zr]Zr-DFO-N-suc-CSF1R-mAb in the liver, lymphoid tissues, duodenum, and ileum, but not in the tumor than did 89Zr-labeled control at 72 h. Immunohistochemistry and autoradiography showed that 89Zr was localized to macrophages within lymphoid tissues. Following [89Zr]Zr-DFO-N-suc-CSF1R-mAb administration, tumor macrophages were almost absent, whereas isotype-group tumors contained over 500 cells/mm2. We hypothesize that intratumoral macrophage depletion by [89Zr]Zr-DFO-N-suc-CSF1R-mAb precluded tumor uptake higher than 89Zr-labeled control. Translation of molecular imaging of macrophage-targeting therapeutics to humans may support macrophage-directed therapeutic development.
| S-EPMC8716378 | biostudies-literature
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.
| S-EPMC3271142 | biostudies-literature
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.
| S-EPMC1166338 | biostudies-other
Project description:Multiple myeloma is the second most common hematologic malignancy. Current treatment strategies are mainly based on immunomodulatory drugs, proteasome inhibitors or combination of both. Novel agents added to these backbone treatments represent a promising strategy in treatment of newly diagnosed as well as relapsed and refractory multiple myeloma patients. In this respect, the incorporation of monoclonal antibodies into standard-of-care regimens markedly improved prognosis of myeloma patients during the last years. More specifically, monoclonal anti-CD38 antibodies, daratumumab and isatuximab, have been implemented into treatment strategies from first-line treatment to refractory disease. In addition, the monoclonal anti-SLAM-F7 antibody elotuzumab in combination with immunomodulatory drugs has improved the clinical outcomes of patients with relapsed/refractory disease. Belantamab mafodotin is the first approved antibody drug conjugate directed against B cell maturation antigen and is currently used as a monotherapy for patients with advanced disease. This review focuses on clinical efficacy and safety of monoclonal antibodies as well as antibody drug conjugates in multiple myeloma.
| S-EPMC8037134 | biostudies-literature
Project description:2015 was a groundbreaking year for the multiple myeloma community partly due to the breakthrough approval of the first two monoclonal antibodies in the treatment for patients with relapsed and refractory disease. Despite early disappointments, monoclonal antibodies targeting CD38 (daratumumab) and signaling lymphocytic activation molecule F7 (SLAMF7) (elotuzumab) have become available for patients with multiple myeloma in the same year. Specifically, phase 3 clinical trials of combination therapies incorporating daratumumab or elotuzumab indicate both efficacy and a very favorable toxicity profile. These therapeutic monoclonal antibodies for multiple myeloma can kill target cells via antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and antibody-dependent phagocytosis, as well as by direct blockade of signaling cascades. In addition, their immunomodulatory effects may simultaneously inhibit the immunosuppressive bone marrow microenvironment and restore the key function of immune effector cells. In this review, we focus on monoclonal antibodies that have shown clinical efficacy or promising preclinical anti-multiple myeloma activities that warrant further clinical development. We summarize mechanisms that account for the in vitro and in vivo anti-myeloma effects of these monoclonal antibodies, as well as relevant preclinical and clinical results. Monoclonal antibody-based immunotherapies have already and will continue to transform the treatment landscape in multiple myeloma.
| S-EPMC6698817 | biostudies-literature