ABSTRACT
A histidine-tagged, carboxy-terminal fragment of the murine double minute 2 gene product, p90(MDM2), was purified by Ni--NTA chromatography and preparative gel electrophoresis. The purified MDM2 fragment was used to generate polyclonal antisera that recognize multiple species of MDM2 proteins, including the inhibitor of p53, p90(MDM2), as well as the activator of p53, p76(MDM2). The antibodies are useful for Western analysis, immunoprecipitation, and immunofluorescence.
Subject(s)
Histidine , Immune Sera/immunology , Nuclear Proteins , Proto-Oncogene Proteins/immunology , Proto-Oncogene Proteins/isolation & purification , Animals , Antibody Specificity , Blotting, Western , Chromatography, Affinity , Escherichia coli , Fluorescent Antibody Technique , Immune Sera/biosynthesis , Mice , Nickel/metabolism , Peptide Fragments/genetics , Peptide Fragments/immunology , Peptide Fragments/isolation & purification , Peptide Fragments/metabolism , Peptides/metabolism , Precipitin Tests , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-mdm2 , Rabbits , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolismABSTRACT
Shewanella putrefaciens MR-1 can reduce a diverse array of compounds under anaerobic conditions, including manganese and iron oxides, fumarate, nitrate, and many other compounds. These reductive processes are apparently linked to a complex electron transport system. Chromium (Cr) is a toxic and mutagenic metal and bacteria could potentially be utilized to immobilize Cr by reducing the soluble and bioavailable state, Cr(VI), to the insoluble and less bioavailable state, Cr(III). Formate-dependent Cr(VI) reductase activity was detected in anaerobically grown cells of S. putrefaciens MR-1, with highest specific activity in the cytoplasmic membrane. Both formate and NADH served as electron donors for Cr(VI) reductase, whereas L-lactate or NADPH did not support any activity. The addition of 10 micromol l(-1) FMN markedly stimulated formate-dependent Cr(VI) reductase, and the activity was almost completely inhibited by diphenyliodonium chloride, an inhibitor of flavoproteins. Cr(VI) reductase activity was also inhibited by p-chloromercuriphenylsulphonate, azide, 2-heptyl-4-hydroxyquinolone-N-oxide, and antimycin A, suggesting involvement of a multi-component electron transport chain which could include cytochromes and quinones. Cr(V) was detected by electron paramagnetic resonance (EPR) spectroscopy, suggesting a one-electron reduction as the first step.
Subject(s)
Electron Transport/physiology , Intracellular Membranes/enzymology , Oxidoreductases/metabolism , Shewanella putrefaciens/enzymology , Anaerobiosis , Electron Spin Resonance Spectroscopy , Enzyme Inhibitors/pharmacology , Flavin Mononucleotide/metabolism , Formates/metabolism , NAD/metabolism , Oxidoreductases/antagonists & inhibitors , Shewanella putrefaciens/metabolismABSTRACT
The mdm2 oncogene is expressed at elevated levels in a variety of human tumors, and its product inactivates the p53 tumor suppressor protein. MDM2 forms an autoregulatory loop with p53, because the mdm2 gene contains a promoter that is responsive to p53. Synthesis of MDM2 protein increases in a p53-dependent manner in response to DNA-damaging agents such as UV light. Although this increase likely results from enhanced transcription, the amount of MDM2 protein does not correspond to the amount of p53 protein in cells exposed to UV light. Here we show that the p53-specific internal promoter in the mdm2 gene is induced after exposure to UV light, whereas the upstream constitutive promoter is not induced. The amount of the mdm2 transcript does not parallel the ability of p53 to bind DNA, indicating that transcription is regulated at a step distinct from activation of the DNA-binding function of p53.
