Ronald D. Sekura

ADP-ribosylation of membrane components by pertussis and cholera toxin.

Publisher Summary Pertussis and cholera toxins are important tools to investigate functional and structural aspects of the stimulatory (Ns) and inhibitory (Ni) regulatory components of adenylyl cyclise. Cholera toxin acts on Ns by ADP-ribosylating its αs subunit. It uses NAD+ as a cosubstrate. ADP-ribosylation of Ns alters its properties so that its guanosine triphosphate (GTP) hydrolyzing capacity is inhibited and the action of GTP is potentiated. In intact cells, this leads to the increases in cyclic adenosine monophosphate (AMP) levels. Both pertussis and cholera toxin are hexameric multisubunit molecules. Cholera toxin is composed of one A and 5 B subunits; pertussis toxin is formed of one S1, one S2, one S3, two S4, and one S5 subunits. This chapter presents a set of protocols, as developed in the laboratory, that can be used to study simultaneously and comparatively the susceptibility of Ns and Ni to be ADP-ribosylated by cholera and pertussis toxin.

Aryl sulfotransferase IV from rat liver

Abstract A group of aryl sulfotransferases has been identified that catalyzes sulfate ester formation with simple phenols at an acidic pH and with several physiological metabolites at a more alkaline pH. One enzyme, aryl sulfotransferase IV, has been purified to homogeneity and found to be a protein of 61,000 daltons composed of two subunits of apparent equal size. Homogeneous preparations are active with simple phenols, organic hydroxylamines, and catecholamines as well as serotonin and its metabolites. The enzyme is also active with tyrosine methyl ester and with those peptide hormones e.g., cholecystokinin heptapeptide and some of the enkephalins, which have N-terminal tyrosine residues.

Assay of sulfotransferases

Abstract Two methods are described for the assay of sulfotransferases which are active with sulfate acceptors bearing the hydroxyl functional group. Assays were developed for enzymes which transfer sulfate from 3′-phosphoadenosine–5′-phosphosulfate (PAPS) to sterols, phenols, and simple alcohols thereby forming the corresponding sulfate esters. With a filter binding assay, useful with crude and purified enzyme preparations, a radioactive sterol substrate is used and subsequently separated from labeled product, allowing the determination of between 50 and 400 pmol of product. In a second method, [ 35 S]PAPS is used and the labeled product is separated from PAPS and inorganic sulfate by a thin-layer technique in which product migrates close to the solvent front; the assay is useful with a broad array of substrates and is more sensitive than the filter binding assay.

A hydroxysteroid sulfotransferase from rat liver

Abstract The major hydroxysteroid sulfotransferase found in the livers of female rats has been purified to homogeneity and found to catalyze the transfer of sulfate to any of several hydroxysteroids including dehydroepiandrosterone, estradiol, testosterone, and androstenediol thus forming the corresponding sulfate esters. 3′-Phosphoadenosine 5′-phosphosulfate is the sulfate donor. The reaction is competitively inhibited by both reaction products, i.e., dehydroepiandrosterone sulfate or adenosine 3′,5′-bisphosphate, and also by high concentrations of inorganic phosphate. The enzyme has a molecular weight of approximately 290,000; sodium dodecyl sulfate-gel electrophoresis reveals an apparently single species of 32,000. With standard assay conditions, no activity was observed with the purified enzyme when a phenol, a bile acid, or a hydroxamic acid was used as acceptor for sulfate.

Dual effects of pertussis toxin on lymphoid cells in culture

Pertussis toxin (Ptx), a component of Bordetella pertussis, is responsible for many of the biological activities of this bacterium, including its potent adjuvant capacity. In attempt to better understand the Ptx activity on the immune response in vivo, we have examined the effect of Ptx on certain lymphoid cell responses in vitro which could be targets for the adjuvant activity of this molecule. Ptx was found to stimulate a variety of cell responses which include (a) increased production and release of interleukin-1 (IL-1) by human monocytes and murine macrophages; (b) co-mitogenesis, in combination with IL-1, in cultures of murine thymocytes; (c) mitogenesis in cultures of various peripheral lymphocytes; (d) increased production of IL-2 in cultures of human blood lymphocytes and rodent splenocytes; and (e) elevated release of IL-3 in cultures of murine spleen cells. In addition to its stimulatory effects, however, Ptx was found to inhibit responses of both mononuclear phagocytes and lymphocytes to other stimuli. Most activities of Ptx in vitro were achieved at the optimal concentration range of 1-10 micrograms/ml, which is 100-1000 times higher than that showing adjuvant effects in vivo. Possible explanations for the dual effect of Ptx and for the discrepancy in doses optimal for the effects in vivo and in vitro are discussed.

The effects of pertussis toxin on the induction and transfer of experimental autoimmune uveoretinitis

Experimental autoimmune uveoretinitis (EAU), an intraocular inflammatory disease, is induced in experimental animals by immunization with a retinal specific antigen, S-antigen (S-Ag), emulsified in complete Freunds adjuvant (CFA). The induction of EAU is enhanced by treating S-Ag-immunized animals with Bordetella pertussis. This study examined the effects of a purified component of B. pertussis, pertussis toxin (Ptx), on EAU induction as well as the mode of action of this toxin. Treatment of Lewis rats with Ptx concurrent with S-Ag and CFA enhanced EAU induction as shown by an earlier onset of disease, increased severity of ocular changes, and the reduction of the threshold amount of S-Ag needed for EAU induction. Treatment with Ptx selectively enhanced delayed-type hypersensitivity responses to S-Ag but did not affect specific antibody production. The mode of action of Ptx was analyzed by using the adoptive transfer of EAU by sensitized lymphocytes. Ptx treatment of donor rats enhanced the capacity of lymphocytes to transfer EAU. However, Ptx treatment of recipient rats on the day of cell transfer resulted in a delay in the onset of disease. These results indicate that Ptx enhances the immunopathogenic processes of EAU by enhancing lymphocyte activation and/or increasing their pathogenic activities.

[38] Purification of Ns and Ni, the coupling proteins of hormone-sensitive adenylyl cyclases without intervention of activating regulatory ligands

Publisher Summary This chapter presents a detailed description of a procedure for the purification of N s and N i from human erythrocytes and eliminates any possible alteration of the subunit composition of these proteins as might result from the effect of these ligands to induce their subunit dissociation. The chapter explains that it is now recognized that a large number of hormones and neurotransmitters affect their target cells by modulating cyclic adenosine monophosphate (cAMP) formation in either stimulatory or inhibitory fashion. There are two of these couplings or N proteins—an N s (or G s ), mediating the effects of stimulatory hormone–receptor complexes and a N i (or G i ), mediating the effects of inhibitory or attenuating hormone–receptor complexes. The interaction of a hormone–receptor complex with an N protein then results in an increase in the proportion of the N protein in an active vs. inactive conformation or state, and activated N interacts with the catalyst C of adenylyl cyclase eliciting either an increase in catalytic activity (N s ) or an inhibition of activity (N i ). The chapter also explains that structurally, both N s and N i are αβγ heterotrimers. The activation process of both, N s and N i , is dependent on a guanine nucleotide and Mg and seems to involve not only a conformational change but also a subunit dissociation reaction whereby the αβγ heterotrimers dissociate into an activated α* subunit with guanine nucleotide bound to it (α *G ) and αβγ complex.

Suppression of the cytotoxic T-lymphocyte response in mice by pertussis toxin

Pertussis toxin (PT), the major toxin produced by Bordetella pertussis, has been reported both to enhance and to suppress immune responsiveness. These findings suggested that PT contributes to the virulence of B. pertussis through mechanisms involving immune regulation. We report that PT suppressed both the primary and the secondary cytotoxic T-lymphocyte (CTL) responses of mouse spleen cells cultured against two different allogeneic stimulator spleen cells in vitro. This suppression was dependent on the dose of PT used. PT must be present during the initial stages (within the first 24 hr) of CTL generation. Soluble factor(s) obtained from spleen cells preexposed to PT did not suppress the CTL response. Suppression of the CTL response observed was not due to depletion of the antigen by PT. The cytotoxic activity of CTL clones could not be suppressed by PT. The analysis of responder spleen cells, fractionated by anti-immunoglobulin panning techniques, provided evidence that L3T4-, Lyt 2+ cells mediate the PT-induced immunosuppression. We propose that suppression of the CTL response by PT is generated through the activation of L3T4-, Lyt 2+ suppressor T lymphocytes.

[44] Pertussis toxin: A tool for studying the regulation of adenylate cyclase

Publisher Summary This chapter discusses that regulation of adenylate cyclase is mediated, in part, through the action of two specific guanosine triphosphate (GTP) binding proteins that couple the interaction of hormones at specific receptors to modulation of catalytic activity. These proteins, termed “N s ” and “N i ” have been purified and function in expression of stimulatory and inhibitory ligands. The susceptibility of N i and N s to specific bacterial toxins, which block normal function of the coupling proteins by covalent modification, has led to a clearer understanding of the role played by these proteins in regulation of adenylate cyclase. With both proteins, toxin catalyzed modification is achieved by transfer of the ADP-ribosyl moiety of NAD to the 39 K subunit of N i or the 42 K subunit of N s . Covalent modification of N s is mediated by the action of cholera toxin. Specific modification of N i is achieved by the treatment of purified preparations of protein or cell membrane with pertussis toxin. The chapter also discusses the techniques for the preparation of pertussis toxin and its use in probing the function of N i .