phosphorus-radioisotopes and sodium-arsenite

Following dosing with various levels of sodium arsenite (NaAsO2), differential [32P]-incorporation (turnover) of polypeptide fragments, generated by protease V8, from a set of four closely related stress proteins (SPs), termed 'c','b','x' and 'y', (80,000-84,000 Mr) was investigated by polyacrylamide gel (PAG) autoradiography. These SPs were physically sorted sequentially from five partitions of S-phase of a mouse lymphoma cell line (YAC-1). The fragments of each of the four SPs exhibited differential [32P]-incorporation patterns with progression in S-phase following varied dose levels of NaAsO2. The majority of V8 protease-fragments of the four SPs had identical electrophoretic mobilities. A minority of polypeptides showed varied distribution among the four SPs and these fragments revealed increased [32P]-labeling progressively in S-phase. These fragments were the most sensitive to altered dose levels of NaAsO2. It was suggested that these four SPs are discrete but structurally similar proteins. A group of other SPs (S1, S2 and S3) observed predominantly in S-phase, showed reduction in polypeptide labeling during S with varied dosing of NaAsO2. The [32P]-labeling of fragments from the SPs seems generally to follow an ordered scheme of turnover (phosphorylation-dephosphorylation) during S-phase.

Topics: Animals; Arsenic; Arsenites; Autoradiography; Biomarkers; Cell Nucleus; DNA; Electrophoresis, Polyacrylamide Gel; Heat-Shock Proteins; Hydrolysis; Lymphoma; Mice; Molecular Weight; Phosphorus Radioisotopes; Phosphorylation; S Phase; Sodium Compounds; Tumor Cells, Cultured

A portion of our research involves the investigation of biomarkers as preclinical indicators of toxic stress. Stemming from this effort, we report three unique proteins that phosphorylate and synthesize during the S-phase of lymphoma cells following chemical insult. Mouse lymphoma cell nuclei were physically sorted (using a fluorescence activated cell sorter) from partitions of the cell cycle and specific nuclear proteins in each partition were examined by gel microelectrophoresis. The changes in [32P]-incorporation by stress proteins (SPs) were examined in each of seven partitions following administration of sodium arsenite. Four SPs [80,000-84,000 relative molecular mass (Mr)], designated 'c','b','x', and 'y', underwent significant alterations in [32P]-labeling and each exhibited varying degrees of differential [32P]-incorporation in partition 3 of G1 phase, all five partitions of S-phase, and partition 1 of G2 phase of the cell cycle. Three other predominantly S-phase SPs (designated S1, S2 and S3) were phosphorylated after sodium arsenite treatment. Stress protein S1 was labeled exclusively in S-phase, while proteins S2 and S3 were labeled only in partition 3 of G1 and S-phase. Stress protein S1 possessed an identical isoelectric point, molecular mass, distribution of polypeptide fragments and immunochemical determinants as S-phase SPs found previously in mouse spleen (X') and mouse liver (LP-S). The identical biochemical characteristics of these three S-phase (SPs), found in diverse tissue types, suggest they are homologous.

Topics: Animals; Arsenic; Arsenites; Cell Nucleus; Concanavalin A; Electrophoresis, Gel, Two-Dimensional; Heat-Shock Proteins; Isoelectric Point; Isoproterenol; Liver; Lymphoma; Mice; Molecular Weight; Nuclear Proteins; Peptide Mapping; Phenobarbital; Phosphorus Radioisotopes; S Phase; Sodium Compounds; Spleen; Tumor Cells, Cultured

Exposure to a 12 h light/12 h dark (L/D) cycle for 1 month, followed by reversal to a 12 h D/12 h L (D/L) cycle stimulated within 18 h the incorporation of [3H]leucine and [32P]orthophosphoric acid into new proteins (130-25 kDa) in the G0 phase of the cell cycle of the non-regenerating and regenerating rat liver as observed in two-dimensional gel autoradiograms. Six additional proteins from the rat submaxillary gland (130-20 kDa) revealed labeling with 32P within 3 h following combined administration of isoproterenol and sodium arsenite. Labeling disappeared within 7 days for all stressed proteins.

Topics: Animals; Arsenic; Arsenites; Cell Cycle; Isoproterenol; Kinetics; Leucine; Liver; Liver Regeneration; Male; Nuclear Proteins; Phosphates; Phosphorus Radioisotopes; Rats; Sodium Compounds; Stress, Physiological; Submandibular Gland; Tritium

Cultured mouse lymphoma cells incorporated [3H]leucine and [32P]phosphate into nuclear stress proteins within 3 h after exposure to either elevated temperature (45 degrees C) or sodium arsenite. Radiolabeled proteins were detected by autoradiography after two-dimensional polyacrylamide gel electrophoresis. To determine the cell cycle stage specificity of labeling, nuclei were isolated and sorted into two cell cycle phases using a fluorescent activated cell sorter. After either heat shock or sodium arsenite treatment, the majority of [3H]leucine incorporation into stress proteins occurred during the G0 + G1 phase with minimal labeling in the G2 phase. On the other hand, 32P labeling of stress proteins occurred in both the G0 + G1 and G2 phases after exposure to sodium arsenite, while incorporation of 32P was limited after heat stress. Following sodium arsenite treatment, a distinct set of four stress proteins (80-84 kDa) was detected with [3H]leucine only in G0 + G1 phase, but with [32P]phosphate these stress proteins were labeled in both G0 + G1 and G2. There was differential [32P]phosphate labeling between proteins of the 80-84 kDa set during cell cycling. Individual proteins of this set were isolated from gel plugs after sodium arsenite or heat-shock treatment. Coelectrophoresis of proteins from the two treatment groups showed that they had similar electrophoretic mobilities. All four proteins of the 80-84 kDa set (sodium arsenite induced) possessed similar polypeptide maps after digestion with V8 protease. Cytofluorometric analysis demonstrated a reduction in the number of nuclei in both S and G2 phases of the cell cycle two h after heat shock, but not following sodium arsenite treatment. However, there was a significant depression in the number of nuclei in S and G2 4 h after exposure to sodium arsenite and very modest labeling with 32P of stress proteins was observed at this time.

Topics: Animals; Arsenic; Arsenites; Cell Line; Cell Nucleus; Heat-Shock Proteins; Hot Temperature; Leucine; Lymphoma; Mice; Molecular Weight; Phosphates; Phosphorus Radioisotopes; Sodium Compounds; Sulfhydryl Reagents; Tritium

Topics: Animals; Arsenic; Arsenites; Chick Embryo; Kinetics; Muscles; Phosphates; Phosphorus Radioisotopes; Sodium Compounds; Tropomyosin