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Biomedical subjects

L L Houston

Publications and source records attributed to L L Houston.

At least 73 records · Page 4Linked to original sources
PubMed1982

Radioimmunoassay of ricin A- and B-chains applied to samples of ricin A-chain prepared by chromatofocusing and by DEAE Bio-Gel A chromatography.

A radioimmunoassay for ricin and ricin A- and B-chains was developed. Amounts as low as 100 pg of A-chain and 500 pg of B-chain could easily be quantitated. We showed, however, that the free chains were more reactive in the radioimmunoassay than the equivalent quantity of the individual chains when combined in intact ricin. The usefulness of the assay was demonstrated by determining the concentration of contaminating A- or B-chains in preparations of the separate polypeptides purified by DEAE Bio-Gel A chromatography and by chromatofocusing.

Chromatography, Gel↗
PubMed1982

Binding of two molecules of 4-methylumbelliferyl galactose or 4-methylumbelliferyl N-acetylgalactosamine to the B chains of ricin and Ricinus communis agglutinin and to purified ricin B chain.

The binding of 4-methylumbelliferyl galactose and 4-methylumbelliferyl N-acetylgalactosamine to ricin, Ricinus communis agglutinin, and ricin B chain was studied by fluorescence polarization and equilibrium dialysis. The binding of [3H]galactose to ricin was also studied by equilibrium dialysis. The results were consistent with binding of 2 mol of ligand to ricin (which contains 1 A chain and 1 B chain) and ricin B chain and 4 mol of ligand to the agglutinin (which contains 2 A chains and 2 B chains). There was no evidence for interaction between the 2 sites on any of the B chains.

Carbohydrates↗
PubMed1982

Transport of ricin A chain after prior treatment of mouse leukemia cells with ricin B chain.

Ricin A chain acts to inhibit protein synthesis only if it penetrates the plasma membrane, and this requires the participation of the B chain. The two chains are held together in ricin by a single disulfide bond. In this paper, it is shown that the addition of A chain to mouse leukemia cells (AKR SL3) after the cells were first reacted with purified ricin B chain also results in efficient inhibition of protein synthesis. The data indicated that B chain bound to the cell surface was capable of causing the transport of A chain. The quantities of ricin and of B chain (in the presence of a saturating amount of A chain) required to inhibit protein synthesis by 50% are nearly identical, indicating that there is little difference in the toxicity to cultured cells between ricin and the A chain-B chain heterodimer formed from purified subunits. However, if the addition of A chain to B chain-treated cells was delayed sufficiently long (90 to 120 min), B chain was no longer able to cause A chain transport and the consequent inhibition of protein synthesis. Direct binding studies indicated that only a fraction of the bound 125I-B chain was internalized during this time. No proteolytic degradation of 125I-B chain could be detected after 3 h incubation with the cells.

Animals↗
PubMed1981

Protection of rat liver 80 S ribosomes against ricin A chain inactivation by proteins extracted from rat liver and wheat germ ribosomal subunits with ammonium chloride/magnesium chloride.

Proteins extracted from wheat germ 60 S ribosomal subunits and rat liver 60 S and 40 S ribosomal subunits with 3 M NH4Cl/75 mM MgCl2 were able to prevent the ricin A chain-mediated inactivation of untreated 80 S rat liver ribosomes. The protection of polyphenylalanine synthetic capability of 80 S ribosomes was saturable and reached 100% protection in the presence of about 20 micrograms of extracted protein using a uniform set of assay conditions. No protection was observed using proteins extracted from wheat germ 40 S subunits or the core fraction of rat liver 60 S subunits or protein extracted from Escherichia coli ribosomes or ribosomal subunits. The conclusion that the protective effect of extracted 60 S subunit proteins was specific, was further strengthened by showing that unrelated proteins such as alpha-lactalbumin, bovine serum albumin and lysozyme, and polypeptides such as polylysine and poly(aspartic acid), also showed no protection. If 80 S ribosomes were first treated with ricin A chain and then incubated with proteins extracted from rat liver 60 S subunits, no protection was observed. Proteins extracted with NH4Cl/MgCl2 from 60 S rat liver subunits were applied to carboxymethylcellulose column equilibrated with 6 M urea. Stepwise elution with increasing concentrations of LiCl resulted in seven fractions. One fraction (D) contained most of the protective factor; one fraction (E) contained a lesser amount of the protective factor. Two-dimensional polyacrylamide gel electrophoresis of fraction D showed the presence of ten proteins. These data are consistent with the idea that the enzymatic target of ricin A chain is protein is nature and that fraction D contains one or more proteins that appear to act as a inhibitor against ricin A chain.

Ammonium Chloride↗
PubMed1980

Differential effects of nitrated ricin and nitrated and dithionite-reduced ricin on protein-synthesis inhibition and transmembrane tramsport in eukaryotic cells.

Ricin, was nitrated with tetranitromethane and reduced with sodium dithionite. Of the 8.0 nitro groups incorporated, 3.2 were on the A chain and 5.1 were on the B chain. Nitrated ricin1 was somewhat less active than nitrated and reduced ricin1 in inhibiting protein synthesis in vitro, but both were highly inhibitory. However, the modified toxins were less than 1% as active as ricin in inhibiting protein synthesis in cultured cells. Indirect immunofluorescence assays demonstrated tha both modified toxins were specifically bound to the cell surface and could be displaced by galactose.

Animals↗
PubMed1980

Purification of the bifunctional enzyme, imidazoleglycerolphosphate dehydratase-histidinol phosphatase, of Salmonella typhimurium.

Mn2+ precipitation, antibody affinity chromatography, and selective proteolysis have been used to purify a bifunctional core of the hisB enzyme from Salmonella typhimurium. The core is homogeneous in subunit molecular weight; however, it is heterogeneous in its charge properties, probably as a result of multiple cleavage points produced by limited proteolytic digestion. The resistance of the enzyme to irreversible denaturation by urea allowed the use of urea to elute the hisB enzyme from a column of anti-hisB IgG immobilized on Sepharose. Heterogeneous oligomeric forms of the enzyme were demonstrated by electrophoretic analysis and exist as multiples of the 46,000 molecular weight monomer.

Chemical Precipitation↗
PubMed1979

Effect of sulfhydryl reagents and protease inhibitors on sodium dodecyl sulfate-heat induced dissociation of Ricinus communis agglutinin.

Ricinus communis agglutinin dissociated to lower molecular weight forms when heated in sodium dodecyl sulfate in the absence of reducing agents, while ricin was little affected by such treatment. The data suggest that strong noncovalent bonds hold together two A-B heterodimers in the Ricinus communis agglutinin tetramer. Protease inhibitors such as diisopropylfluorophosphate, phenylmethansefulonyl fluoride, and EDTA, did not prevent the sodium dodecyl sulfate-heat induced dissociation; however, sulfhydryl specific reagents (N-ethylmaleimide, 5,5'-dithiobis (2-nitrobenzoic acid) and p-chloromercuribenzoate) were effective. Titration of the lectins in sodium dodecyl sulfate indicated that ricin contains one sulfhydryl and Ricinus communis agglutinin four sulfhydryl groups, none of which react in the presence of 8 M urea. The sulfhydryl groups that could be titrated in the intact proteins in sodium dodecyl sulfate were on the A chains.

Lectins↗
PubMed1978

Binding of ricin A chain to rat liver ribosomes: relationship to ribosome inactivation.

Ricin A chain was radioactively labeled using reductive alkylation, lactoperoxidase catalyzed iodination, and reaction with iodoacetamide or N-ethylmaleimide (NEM). The inhibition of cell-free rat liver protein synthesis by the modified A chains and the ribosome binding characteristics of each of the labeled derivatives was examined. [3H] NEW was found to quantitatively react with the A chain sulfhydryl group normally involved in a disulfide bond with the B chain in intact ricin. Labeling the protein with [3H] NEM had no effect on the in vitro inhibition of protein synthesis by the A chain. [3H] NEM-labeled A chain binds to rat liver ribosomes in a manner which is dependent on the concentrations of NaCl and Mg2+. At optimal Mg2+ concentration (5.5 mM), A chain binding to ribosomes is saturable and fully reversible either by dilution of the reaction mixture or by addition of unlabeled A chain. At 5.5 mM Mg2+, A chain was found to bind to a single site on rat liver ribosomes with a dissociation constant of 6.2 x 10(-8) M. [3H] NEM-labeled A chain did not bind to isolated 40S ribosomal subunits and bound to 60S ribosomal subunits with a 1 : 1 molar stoichiometry and a dissociation constant of 2.2 x 10(-7) M. The relationship between ribosome binding and A chain inhibition of eucaryotic protein synthesis is discussed.

Animals↗