Trypsin is shown to generate an insecticidal toxin from the 130-kDa protoxin of Bacillus thuringiensis subsp. kurstaki HD-73 by an unusual proteolytic process. Seven specific cleavages are shown to occur in an ordered sequence starting at the C-terminus of the protoxin and proceeding toward the N-terminal region. At each step, C-terminal fragments of approximately 10 kDa are produced and rapidly proteolyzed to small peptides. The sequential proteolysis ends with a 67-kDa toxin which is resistant to further proteolysis. However, the toxin could be specifically split into two fragments by proteinases as it unfolded under denaturing conditions. Papain cleaved the toxin at glycine 327 to give a 34.5-kDa N-terminal fragment and a 32.3-kDa C-terminal fragment. Similar fragments could be generated by elastase and trypsin. The N-terminal fragment corresponds to the conserved N-terminal domain predicted from the gene-deduced sequence analysis of toxins from various subspecies of B. thuringiensis, and the C-terminal fragment is the predicted hypervariable sequence domain. A double-peaked transition was observed for the toxin by differential scanning calorimetry, consistent with two or more independent folding domains. It is concluded that the N-and C-terminal regions of the protoxin are two multidomain regions which give unique structural and biological properties to the molecule.Bacillus thuringiensis is an insect pathogen with an unusual but highly specific mode of action. During the sporulation cycle it lays down a parasporal protein crystal which is rendered toxic on ingestion by susceptible insect larvae. The major component of crystals toxic to lepidoptera is a protein (protoxin) with a molecular mass of approximately 130 kDa [l -31. Treatment with thiol reagents at basic pH solubilizes the protoxin by cleaving the disulfide bonds which stabilize the crystal. Incubation of the solubilized protoxin with proteolytic enzymes or insect gut juice produces a 58 -70-kDa proteinaseresistant toxin derived from the N-terminal portion of the molecule [4,5]. The toxin then binds to receptors in the midgut epithelium, causing cell lysis and eventual larval death [6 -81. The details of the lytic mechanism are not yet established but it appears that the toxin generates small pores or localized perturbations in the plasma membrane, causing disruption of homeostatic ion regulation [9].Large proteins are generally organized into distinct structural units referred to as domains or subdomains, but the criteria used for this classification is somewhat subjective. It is clear that the protoxin is divided into at least two major domains: the carboxyl half of the molecule which is readily attacked by proteinases, and the toxin derived from the Nterminal half which is proteinase-resistant. The toxins from
8-Hydroxydeoxyguanosine (8-OHdG) is now widely used as a sensitive marker of oxidative damage to DNA. When human granulocytes are stimulated with TPA, they release a large quantity of reactive oxygen species (superoxide, hydrogen peroxide) which might be expected to generate hydroxyl radicals (OH.) which in turn could produce 8-OHdG in the DNA. There had been considerable debate as to whether OH. is detectable in stimulated granulocytes; most workers now agree that none can be detected, unless exogenous iron is added. An earlier report had described that 8-OHdG (a marker of OH.) was increased in the DNA of TPA-stimulated, compared to control, granulocytes. We have repeated this experiment and have been unable to reproduce this finding. We conclude that the amount of 8-OHdG produced in the DNA of TPA-stimulated human granulocytes is indistinguishable from that seen in control (unstimulated) cells (less than one 8-OHdG/10(5) dG).
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