ABSTRACT
The high potential, ascorbate-reducible b-type cytochrome of plant plasma membranes, named cytochrome b-561, has been purified to homogeneity from etiolated bean hypocotyls. The pure protein migrated in denaturing electrophoresis as a broad band of approximately 55 kDa, and was found to be glycosylated. Optical redox titrations of partially purified cytochrome b-561 indicated that it contains two hemes with similar spectral features, but distinct midpoint redox potentials (E(m7)+135 mV and +206 mV, respectively). The presence of two heme centers in cytochrome b-561 is consistent with its role in electron transfer across plant plasma membranes.
Subject(s)
Cytochrome b Group/isolation & purification , Fabaceae/chemistry , Plants, Medicinal , Cell Membrane/chemistry , Chromatography, Ion Exchange , Cytochrome b Group/chemistry , Electrophoresis, Polyacrylamide Gel , Heme/chemistry , Hypocotyl/chemistry , Potentiometry , SpectrophotometryABSTRACT
Seven different test systems were utilized to investigate the genetic activity of chromium compounds: infidelity of DNA replication in vitro by DNA pol alpha from calf thymus, damage of DNA detected by alkaline elution in treated mammalian cells or in DNA purified and treated in vitro, DNA repair synthesis in mammalian cells in vitro detected by autoradiography or scintillation counting after labelling with [3H]dThd, gene mutations in the Salmonella typhimurium Ames test, gene mutations (6TG resistance) in cultured hamster cells, sister-chromatid exchanges in different rodent cell cultures, and transformation to anchorage-independent growth of hamster cells in vitro (soft-agar assay). Potassium dichromate and chromium chloride were used as water-soluble Cr(VI) and Cr(III) salts. Several reference mutagens (EMS, MMS, MMC, 4NQO) were included in the single tests as positive controls. Cr(VI) was active in all the tested systems, except in the induction of DNA damage and DNA repair synthesis in cultured cells. Cr(III), on the other hand, was absolutely inactive unless a direct interaction with purified DNA was permitted by the test conditions. The relevance of data from the various tests to the understanding of the mechanisms of the genotoxic activity of chromium is discussed. Effects other than the direct interaction of Cr(III) with DNA are inferred, which can cause infidelity of the DNA polymerase functions.
Subject(s)
Chromium/toxicity , Mutagens , Mutation , Animals , Cell Adhesion/drug effects , Cell Line , Cricetinae , Cricetulus , DNA Repair/drug effects , DNA Replication/drug effects , DNA-Directed DNA Polymerase/metabolism , Drug Resistance , Lung , Mutagenicity Tests , Salmonella typhimurium/drug effects , Sister Chromatid Exchange/drug effects , Structure-Activity RelationshipABSTRACT
Treatments for 1 to 4 hr with 10-4 m potassium dichromate, a soluble hexavalent chromium salt with a strong oxidizing power, markedly reduce DNA and RNA accumulation rates in hamster fibroblasts grown in vitro (BHK line), as shown by quantitative spectrophotometric determinations. Such inhibitory action is not immediately evident on the basis of the incorporation rates of labeled nucleosides into DNA and RNA, as dichromate affects also the relative concentrations of labeled precursors in the intracellular pool. Dichromate first stimulates and then inhibits nucleoside (mostly thymidine) uptake, whereas amino acid uptake is immediately inhibited. Actual rates of macromolecular syntheses have been calculated by taking into account the induced changes of soluble precursor concentrations; sucn normalized rates point out that dichromate induces a sudden blockage of DNA replication, whereas RNA and protein syntheses are secondarily inhibited. The observed cytotoxic effects of dichromate are tentatively referred to the oxidation of cell components by hexavalent chromium and thereby to the interaction of reduced trivalent chromium with specific biological ligands on cell membrane and on DNA.
