Application of Radioisotopes in Biochemical Analyses: Metal Binding Proteins and Metal Transporters

Abstract

IntroducitionRadioisotopes (RI) such as 3 H, 14 C, 32 P, and 45 Ca are excellent tools in biological research. Most RI are used as tracers in studies of primary and secondary metabolism, drug metabolism, transcription, translation, post-translational modifications such as protein phosphorylation, association of proteins with metals, and transport of metals across biomembranes. Furthermore, some experiments have used neutrons for mutagenesis of microorganisms, animals, and plants. Recent progress in the biological sciences has resulted in novel probes and labeling reagents, which has decreased the need for RI. Experiments with RI require experimental space specialized for RI, careful experimental procedures, and training. Although these are disadvantages, RI are still useful and powerful tools with high resolution compared with non-RI methods. Here, we describe the advantages of RI in biochemical assays, and detailed experimental procedures of metal-binding assays and membrane transport measurements of metal cations, especially calcium and zinc. Advantages of radioisotopes as tracersMost metabolic pathways that are described in biochemistry textbooks, in various organisms including humans, plants, and microorganisms, could not have been determined without RI such as 14 C, 35 S, 32 P, and 3 H. Biochemical experiments with RI provide information on the fates of metabolites, nutrients, and inorganic ions at each periodic stage of living organisms or cells. In the early era of molecular biology, 32 P was used as an essential tool in a large number of laboratories to determine DNA sequences and to identify target DNAs or mRNAs. Phosphorylation of serine and/or tyrosine residue s i s a key covalent modification of proteins. 32 P ([-32 P]ATP) is still used to investigate this biochemical process. 35 Antibodies specific to phosphoserine, phosphotyrosine, or peptides containing phosphorylated amino acid residues are prepared and used. The accuracy and sensitivity depend on the quality and specificity of the antibodies. In most cases, researchers must pay attention to artifactual signals. In contrast, labeling proteins with RI provides clear and quantitative information on protein phosphorylation. In RI methods, proteins are phosphorylated with [-32 P]ATP in cell free systems (in vitro) or in experiments using cells, tissues, or organisms (in vivo), and then proteins are extracted and separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Proteins in the gel are transferred onto a transfer membrane such as polyvinylidine fluoride (PVDF). The membrane is dried and subjected to autoradiography by contact with an X-ray film at 80C for a few days. Imaging plates are now generally used for the detection of phosphorylated proteins. Imaging plates have several advantages compared with autoradiography: (i) high sensitivity (quick detection), (ii) good linearity between the content of 32 P and the signal, (iii) digital imaging, and (iv) no requirement of a dark room. 3. Reflection of natural con...

Electrodialytic Handling of Radioactive Metal Ions for Preparation of Tracer Reagents

“…Radioisotopes are widely used as a tracer in biochemistry and clinical diagnosis . Because of low background and highly sensitive gamma­(γ)-ray detection technology, radioisotopes can be determined even at a pico- or femtomolar level.…”