RESUMEN

The structure-activity relationships for the inhibition of protein kinase CK2 by heparin were investigated using purified heparin fragments of different length, varying from 4 to 24 oligosaccharide sugar units. The inhibitory potency was shown to decrease concomitant with the shortening of the heparin fragment length. The fragment of 24 oligosaccharide sugar units was the most potent inhibitor with a K(i) value of 22 nM which is close to the K(i) value for the commercial heparin mixture available. Shortening of the heparin from 24 to 12 sugar units had a moderate influence on the inhibitory potency causing an increase in K(i) values up to 151 nM while fragments shorter than 12 sugar units showed a more drastic increase in K(i) values reaching up to micromolar range. The mode of inhibition was studied in respect to the protein substrate beta-casein and it was shown to be competitive for the long as well as for the short heparin fragments. In contrast, the inhibition mode in respect to a synthetic peptide substrate RRRADDSDDDDD was found to be hyperbolic partial non-competitive mixed-type. Such a kinetic model suggests that heparin binds to a site on CK2 which does not overlap with the peptide substrate binding site and that a productive enzyme complex exists where both heparin and peptide substrate are simultaneously bound. This is in contrast to the competitive inhibition model of the phosphorylation of protein substrate beta-casein where the binding of the protein substrate and inhibitor was mutually exclusive.

Asunto(s)

RESUMEN

The ER retention of lumenal proteins is achieved by a process which involves binding of escaped proteins via the C-terminal KDEL-tags to a KDEL receptor (erd2 receptor) in a post-ER compartment and return of the protein-receptor complex back to the ER. The transmembrane topology of the human KDEL receptor, which is an integral membrane protein, has been proposed. We have synthesised sets of cellulose-bound overlapping peptides covering the complete se quence of the receptor to study the interaction of the erd2 receptor with lumenal ER proteins, CaBP1 and CaBP2. At the next stage, the proposed lumenal loops of the receptor were more closely mapped. A short sequence, essential for the protein binding to the most efficient binding site of the receptor, was identified as 22KIWK25, which is in accordance with one of the proposed structural models of the receptor. The binding was of high specificity and was almost completely inhibited by KDEL-containing soluble peptides. The phosphorylation state of CaBP1/CaBP2 did not affect their binding to the KDEL receptor.

Asunto(s)

Asunto(s)

RESUMEN

The stress response to reactive oxygen species is an important defence system which can reduce their potential to induce biomolecule damage. In this investigation the effect of exposing Molt-3 lymphoblastoid cells or peripheral blood lymphocytes to a non-toxic dose of hydrogen peroxide (10 microM) was studied. Cellular response to a subsequent high dose of hydrogen peroxide (100-200 microM) was assessed by measurement of growth, viability, proliferation and DNA damage (lymphocytes only) and intracellular activities of the enzymes, superoxide dismutase, glutathione peroxidase and catalase (Molt-3 only). The results indicate that pretreatment of lymphocytes with 10 microM hydrogen peroxide can elicit a response which is protective against DNA damage normally inducible in these cells by subsequent exposure to toxic doses of hydrogen peroxide. It appears from the results with Molt-3 cells that altered activities of glutathione peroxidase may contribute to this enhanced resistance to hydrogen peroxide.

Asunto(s)

RESUMEN

There is an increased risk of developing neoplastic disease at sites of chronic inflammation. We have used a model of rat pleural cavity inflammation induced with carrageenan to obtain inflammatory cells comprising predominantly phagocytes (macrophages, monocytes and neutrophils) to examine their ability to activate chemical carcinogens. Treatment of these cells with phorbol-12-myristate-13-acetate (PMA, 15 mM) to stimulate respiratory burst, resulted in a rapid release of reactive oxygen species. Incubation of the cooked food promutagen 2-amino-3,8-dimethylimadazo-[4,5-f]quinoxaline (MeIQx) with PMA (or phorbol dibutyrate or opsonised zymosan or silica) stimulated pleural cavity phagocytes, generated highly electrophilic products which were mutagenic in an Ames Salmonella mutagenicity assay. whereas resting cells (no PMA) had negligible activity. Mutagenic activation of MeIQx by PMA stimulated cells could be reduced by inhibitors of active oxygen such as mannitol, benzoate, dimethyl sulphoxide, superoxide dismutase and catalase and by inhibition of myeloperoxidase activity. In contrast the very potent, broad spectrum cytochrome P450 inhibitor 8-methoxypsoralen had no effect, suggesting the reaction was not cytochrome P450 dependent. Activation of MeIQx by PMA stimulated cells could be abolished by the protein kinase C inhibitor staurosporin, confirming the importance of protein kinase C in the reaction. Furthermore, performing incubations in the presence of fluoride (10 mM) to directly stimulate adenylyl cyclase avoided the requirement for phorbol ester in the activation process. These data show that phagocytes stimulated to release active oxygen species can activate MeIQx to a mutagenic derivative. Mutagenic activation of MeIQx in this system was dependent upon the generation of active oxygen via signal transduction pathways involving protein kinase C and adenylyl cyclase. We suggest that the mechanism of MeIQx activation by phagocytes probably involves one electron oxidation mediated by active oxygen species.

Asunto(s)

Asunto(s)