Protein kinase C was measured in the cytoskeletal fraction of lymphocytes, platelets and HL60 cells, by specific binding of PHjphorbol dibutyrate and by immunoblotting with antibody to a consensus sequence in the regulatory domain of a-, p-and y-isozymes of protein kinase C. Treatment of cells for 40 min with a combination of zinc (2-50 ,eM), zinc ionophore pyrithione and unlabelled phorbol dibutyrate (200 nM) caused up to a ten-fold increase in cytoskeletal protein kinase C and a corresponding decrease in other cellular compartments. Omission of any of the reagents resulted in much less or no translocation. These effects were inhibited by l,lO-phenanthroline, which chelates zinc, and were not seen with calcium. Increase in cytoskeletal protein kinase C persisted for several hours and appeared to involve attachment of the enzyme to actin microfilaments. We propose that zinc, like calcium, regulates the distribution of PKC in cells. However, unlike calcium which controls the binding of PKC to the lipid component on cell membranes, zinc controls the distribution of PKC to membrane cytoskeleton, possibly actin.Zinc ionophore; Phorbol ester; Protein kinase C; Cytoskeleton
Bacteroids of Bradyrhizobium japonicum strain CB1809, unlike CC705, do not have a high level of constitutive nitrate reductase (NR; EC 1.7.99.4) in the soybean (Glycine max. Merr.) nodule. Ex planta both strains have a high activity of NR when cultured on 5 mM nitrate at 2% O2 (v/v). Nitrite reductase (NiR) was active in cultured cells of bradyrhizobia, but activity with succinate as electron donor was not detected in freshly-isolated bacteroids. A low activity was measured with reduced methyl viologen. When bacteroids of CC705 were incubated with nitrate there was a rapid production of nitrite which resulted in repression of NR. Subsequently when NiR was induced, nitrite was utilized and NR activity recovered. Nitrate reductase was induced in bacteroids of strain CB1809 when they were incubated in-vitro with nitrate or nitrite. Increase in NR activity was prevented by rifampicin (10 μg· ml(-1)) or chloramphenicol (50 μg·ml(-1)). Nitrite-reductase activity in bacteroids of strain CB1809 was induced in parallel with NR. When nitrate was supplied to soybeans nodulated with strain CC705, nitrite was detected in nodule extracts prepared in aqueous media and it accumulated during storage (1°C) and on further incubation at 25°C. Nitrite was not detected in nodule extracts prepared in ethanol. Thus nitrite accumulation in nodule tissue appears to occur only after maceration and although bacteroids of some strains of B. japonicum have a high level of a constitutive NR, they do not appear to reduce nitrate in the nodule because this anion does not gain access to the bacteroid zone. Soybeans nodulated with strains CC705 and CB1809 were equally sensitive to nitrate inhibition of N2 fixation.
In the presence of pyrithione, which was used as a Zn *+ ionophore, Znzf (l&l00 PM) increased phorbol ester binding by intact B-CLL cells in a dose-dependent fashion. Zn pyrithione increased 2-fold the number of phorbol ester receptors in B-cells (0.74 to 1.4 pmol/lV cells), neutrophil polymorphs (0.2 to 0.51 pmol/lW cells) and platelets (91 to 209 pmol/lOiO cells). Fractionation of cells after treatment with Zn pyrithione showed that increased binding of PDBu occurred in the particulate fraction of cells and this was accompanied by loss of phorbol ester receptors from the cytosol. These data are compatible with a role for Zn in the subcellular distribution and activation of protein kinase C.
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