1. Liver and bone alkaline phosphatase isoenzymes were solubilized with the zwitterionic detergent sulphobetaine 14, and purified to homogeneity by using a monoclonal antibody previously raised against a partially-purified preparation of the liver isoenzyme. Both purified isoenzymes had a specific activity in the range 1100-1400 mumol/min per mg of protein with a subunit Mr of 80,000 determined by SDS/polyacrylamide gel electrophoresis. Butanol extraction instead of detergent solubilization, before immunoaffinity purification of the liver enzyme, resulted in the same specific activity and subunit Mr. The native Mr of the sulphobetaine 14-solubilized enzyme was consistent with the enzyme being a dimer of two identical subunits and was higher than that of the butanol-extracted enzyme, presumably due to the binding of the detergent micelle. 2. Pure bone and liver alkaline phosphatase were used to raise further antibodies to the two isoenzymes. Altogether, 27 antibody-producing cell lines were cloned from 12 mice. Several of these antibodies showed a greater than 2-fold preference for bone alkaline phosphatase in the binding assay used for screening. No antibodies showing a preference for liver alkaline phosphatase were successfully cloned. None of the antibodies showed significant cross-reaction with placental or intestinal alkaline phosphatase. Epitope analysis of the 27 antibodies using liver alkaline phosphatase as antigen gave rise to six groupings, with four antibodies unclassified. The six major epitope groups were also observed using bone alkaline phosphatase as antigen. 3. Serum from patients with cholestasis contains soluble and particulate forms of alkaline phosphatase. The soluble serum enzyme had the same size and charge as butanol-extracted liver enzyme on native polyacrylamide-gel electrophoresis. Cellulose acetate electrophoresis separated the soluble and particulate serum alkaline phosphatases as slow- and fast-moving forms respectively. In the presence of sulphobetaine 14 all the serum enzyme migrated as the slow-moving form on cellulose acetate electrophoresis. Monoclonal anti-(alkaline phosphatase) immunoadsorbents did not bind the particulate form of alkaline phosphatase in cholestatic serum but bound the soluble form. In the presence of sulphobetaine 14 all the cholestatic serum alkaline phosphatase bound to the immunoadsorbents. 4. The electrophoretic and immunological data are consistent with both particulate and soluble forms of alkaline phosphatase in cholestatic serum being derived from the hepatocyte membrane.
Denaturation of Bordetella pertussis fimbrial preparations by guanidinium hydrochloride (GdnHCl) has been characterized using static light scattering, c.d., fluorescence and antibody recognition. The susceptibility of Fim2 + 3 (a mixed preparation of two fimbrial types) to GdnHCl was found to be highly dependent on pH; as the pH was increased from pH 7.2 to 10.5, the concentration of GdnHCl required to induce 50% denaturation was decreased. At pH 10.5, Fim2 + 3 was denatured by GdnHCl in a three-step pathway comprising: (1) formation of a pre-denaturational intermediate at less than 1.0 M-GdnHCl; (2) dissociation of the fimbrial polymer into subunits between 2 M- and 3.2 M-GdnHCl; and (3) subunit unfolding between 2.8 M- and 3.6 M-GdnHCl. A similar pathway was also found for the denaturation of the individual fimbrial types, Fim2 and Fim3, except that unfolding of either subunit commenced at a lower GdnHCl concentration (2.2 M) than that found for the mixture of fimbriae, Fim2 + 3. The second step in the denaturation pathway, dissociation into subunits, was partially reversible, but the renaturation and reassociation of fully unfolded subunits to form fimbriae-like structures was not achieved. These findings demonstrate that the GdnHCl denaturation of complex polymeric proteins is unlikely to follow a reversible two-state denaturation pathway, and support the involvement of a chaperone-like protein in the folding and assembly of the fimbriae in vivo. Measurement of the ability of anti-fimbrial monoclonal antibodies to recognize intermediates in the denaturation pathway enabled the identification of two types of epitope which were dependent on different aspects of fimbrial tertiary/quaternary structure.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.