Subject(s)
Immunoglobulin G/isolation & purification , Immunoglobulin Idiotypes/isolation & purification , Animals , Antigen-Antibody Complex , B-Lymphocytes/immunology , Cell Line , Chromatography, High Pressure Liquid/methods , Durapatite , Electrophoresis, Polyacrylamide Gel/methods , Hemoglobins/immunology , Humans , Hybridomas/immunology , Hydroxyapatites , Indicators and Reagents , Mice , Plasmacytoma/immunologyABSTRACT
A single-step method for purification of mouse monoclonal antibodies directly from ascitic fluids using hydroxylapatite column chromatography is described. The procedure yields highly purified IgG or IgM antibodies. The purified immunoglobulin is essentially free of contaminating mouse albumin, transferrin, and other ascites proteins, as determined by SDS-polyacrylamide gel electrophoresis. Hydroxylapatite chromatography can also separate monoclonal IgG antibodies from contaminating IgG antibodies found in ascites fluid of animals that have been immunosuppressed prior to ascites induction. Furthermore, the evidence presented here suggests that some hybridomas of SP2/0 origin synthesize an extraneous light chain resulting in the secretion of hybrid antibody molecules.
Subject(s)
Antibodies, Monoclonal/isolation & purification , Ascitic Fluid , Animals , Chromatography, Gel , Electrophoresis, Polyacrylamide Gel , Immunoglobulin G/isolation & purification , Immunoglobulin M/isolation & purification , Mice , Mice, Inbred BALB CABSTRACT
The intracellular levels of poly(ADP-ribose) in cultured mouse cells were increased in response to hyperthermic treatment (43 degrees C). When hyperthermia was combined with other stressful treatments such as with ethanol and/or an alkylating agent, a dramatic synergistic increase in polymer levels was observed. The effect of hyperthermia did not appear to be related to the presence of DNA strand breaks. A possible involvement of poly(ADP-ribose) metabolism in the general cellular response to environmental stress is suggested.
Subject(s)
Ethanol/pharmacology , Hot Temperature , Methylnitronitrosoguanidine/pharmacology , Nucleoside Diphosphate Sugars/metabolism , Poly Adenosine Diphosphate Ribose/metabolism , Animals , Cell Transformation, Neoplastic , Cells, Cultured , DNA/metabolism , Fibroblasts/drug effects , Fibroblasts/metabolism , Kinetics , Mice , Mice, Inbred BALB C , Simian virus 40/geneticsSubject(s)
Adenosine Diphosphate Ribose/analysis , Nucleoside Diphosphate Sugars/analysis , Poly Adenosine Diphosphate Ribose/analysis , Animals , Carbon Radioisotopes , Cells, Cultured , Chromatography, High Pressure Liquid/methods , Chromatography, Ion Exchange/methods , Indicators and Reagents , Radioisotope Dilution Technique , Spectrometry, Fluorescence/methods , TritiumABSTRACT
Three different immobilized boronates were compared with respect to their possible utility in studies of adenine and pyridine nucleotide metabolism. These included boronate derivatives of polyacrylamide, Sepharose, and the cation-exchange resin, Bio-Rex 70. The relative binding affinities, binding capacities, and elution properties were compared. Under the conditions utilized, the Sepharose and Bio-Rex 70 derivatives selectively retained nucleotides containing two or more sets of 1,2 cis-diol groups. Since the bulk of cellular nucleic acids contain but a single set of 1,2 cis-diol groups or less, these boronate derivatives are very useful for the isolation of the acid-soluble nucleotides, NAD and diadenosine 5',5"'-tetraphosphate, and for the acid-insoluble polymer, poly(ADP-ribose). The Bio-Rex 70 derivative had a particularly useful combination of binding selectivity, capacity, and elution characteristics. Specific applications of this resin for studies of NAD and poly(ADP-ribose) metabolism are presented.
Subject(s)
Adenine Nucleotides/isolation & purification , Boronic Acids , NAD/isolation & purification , Adenine Nucleotides/metabolism , Animals , Binding Sites , Cation Exchange Resins , Chemical Phenomena , Chemistry , Chromatography , Fibroblasts , Mice , Mice, Inbred BALB C , NAD/metabolism , Poly Adenosine Diphosphate Ribose/isolation & purification , Poly Adenosine Diphosphate Ribose/metabolism , Resins, Synthetic , Simian virus 40 , SolubilityABSTRACT
Methodology for the routine and simultaneous determination of the linear and branched residues of poly(ADP-ribose) is described. The main features of the procedure consist of the isolation of poly(ADP-ribose) by affinity chromatography; enzymatic digestion of the polymer to the unique nucleosides ribosyladenosine and diribosyladenosine which are derived from linear and branched residues, respectively; formation of fluorescent derivatives of ribosyladenosine and diribosyladenosine; and identification and quantification of these compounds by high-pressure liquid chromatography coupled with fluorescence detection. A variation on the methodology which allows the detection and quantification of ribosyladenosine and diribosyladenosine without formation of their fluorescent derivatives is also presented. Analyses of several cell lines for their capacity to synthesize poly(ADP-ribose) with a branched structure showed that the proportion of branched sites was constant (0.7-0.8%) in each of the cell lines.
Subject(s)
Nucleoside Diphosphate Sugars/isolation & purification , Poly Adenosine Diphosphate Ribose/isolation & purification , Animals , Cell Line , Chromatography, Affinity , Chromatography, High Pressure Liquid , Fluorescence , Humans , Mice , Phosphoric Diester Hydrolases , Phosphoric Monoester Hydrolases , Poly Adenosine Diphosphate Ribose/biosynthesis , Poly Adenosine Diphosphate Ribose/metabolismABSTRACT
Exposure of human fibroblasts to 5 J/m2 of UV light resulted in a rapid increase of up to 1500% in the intracellular content of poly(ADP-ribose) and a rapid depletion of its metabolic precursor, NAD. When added just prior to UV treatment, the poly(ADP-ribose) polymerase inhibitor, 3-aminobenzamide, totally blocked both the increase of poly(ADP-ribose) and decrease in NAD for up to 2.5 h. Addition of 3-aminobenzamide at the time of maximal accumulation of poly(ADP-ribose) resulted in a decrease to basal levels with a half-life of approximately 6 min. The rates of accumulation of poly(ADP-ribose) and depletion of NAD were increased in the presence of either 1-beta-arabinofuranosylcytosine or hydroxyurea. Since these agents are known to cause an additional accumulation of DNA strand breaks following UV irradiation, these data provide evidence for a mechanism in which the rate of poly(ADP-ribose) synthesis following DNA damage is regulated in intact cells by the number of DNA strand breaks. Under conditions in which the synthesis of poly(ADP-ribose) was blocked, DNA repair replication induced by UV light was neither stimulated nor inhibited.
Subject(s)
Nucleoside Diphosphate Sugars/radiation effects , Poly Adenosine Diphosphate Ribose/radiation effects , Skin/radiation effects , Ultraviolet Rays , Cells, Cultured , Cytarabine/pharmacology , DNA Repair , DNA Replication/drug effects , DNA Replication/radiation effects , Fibroblasts/metabolism , Fibroblasts/radiation effects , Humans , Hydroxyurea/pharmacology , Infant, Newborn , Male , NAD/metabolism , NAD/radiation effects , Poly Adenosine Diphosphate Ribose/metabolism , Skin/drug effects , Skin/metabolismABSTRACT
We have searched for the presence of branching in the chromosomal polymer poly(ADP-ribose) as it occurs in vivo. Treatment of the polymer with phosphodiesterase asnd phosphomonoesterase results in the conversion of internal residues to the nucleoside ribosyladenosine and the conversion of points of branching to diribosyladenosine. We have detected diribosyladenosine in digests of the polymer derived from carcinogen-treated SV40 virus-formed 3T3 cells and in normal rat liver, kidney, and spleen. The frequency of residues involved in branching varied from 0.8 to 1.6 mole % over a 50-fold range of total levels of poly(ADP-ribose). Thus, branching seems to be a general feature of poly(ADP-ribose) as it occurs in vivo.
