Zinc is essential for cellular proliferation. Zinc deficiency of Euglena gracilis results in arrest of cell division and deranges nucleic acid and protein metabolism pointing to a decisive role of zinc in transcription and translation. We have, therefore, investigated the role of zinc in the function of the DNA-dependent RNA polymerases of this organism. Two RNA polymerases from zinc sufficient organisms were purified first by affinity chromatography on a DNA cellulose column and subsequently separated on diethylaminoethyl (DEAE)-Sephadex A-25. The two fractions were characterized as polymerase I and II by their elution pattern from DEAE-Sephadex and sensitivity to alpha-amanitin. RNA polymerase II has a provisional molecular weight of 700 000 and contains an average of 2.2 g=atoms of zinc per mol of enzyme, but not Mn, Cu, or Fe, as measured by microwave emission spectroscopy. Chelating agents, such as 1,10-phenanthroline, 8-hydroxyquinoline, 8-hydroxyquinoline-5-sulfonic acid, and lomofungin, inhibit activity. In contrast, the nonchelating analogues, 1,7-and 4,7-phenanthroline, do not affect activity. Inhibition by 1,10-phenanthroline is instantaneous and fully reversible by dilution. 1,10-Phenanthroline also inhibits RNA polymerase I, suggesting a role of zinc in its function. The demonstration that RNA polymerase II is a zinc enzyme indicates the involvement of zinc in eukaryotic RNA synthesis and serves as a further basis for the definition of the role of this element in eukaryotic cell growth, division, and differentiation.
Calmodulin transduces Ca2+ signals by binding to and activating essential regulatory enzymes. The large number of intracellular targets for calmodulin raises the possibility that mechanisms in addition to Ca2+ may modulate calmodulin activity. Phosphocalmodulin is found in cells and tissues, and calmodulin phosphorylation is enhanced by several mitogens. Phosphorylation of calmodulin on serine/threonine residues by casein kinase II decreased its ability to activate Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II). The major effect was a 2.5-fold increase in the concentration at which half-maximal velocity (K0.5) was attained, with no apparent alteration in the Vmax, or the K0.5 for Ca2+. In contrast, calmodulin phosphorylated on tyrosine residues by the insulin receptor kinase produced an increase in the Vmax, with no alteration in the affinity for CaM-kinase II or the K0.5 for Ca2+. Direct determination by surface plasmon resonance of the dissociation constants with a synthetic peptide corresponding to the calmodulin-binding domain of CaM-kinase II revealed that phosphorylation on serine/threonine residues of calmodulin significantly decreased its affinity for the peptide, while tyrosine phosphorylation had no effect on binding. In contrast to CaM-kinase II, neither serine/threonine nor tyrosine phosphorylation of calmodulin altered its ability to activate calcineurin. These data indicate that phosphorylation of calmodulin differentially modifies its interaction with individual target enzymes. Moreover, the amino acid residues phosphorylated provide an additional level of control. These results demonstrate that phosphorylation is an in vitro regulatory mechanism in the targeting of calmodulin responses and, coupled with the stoichiometric phosphorylation of calmodulin in rat hepatocytes, suggest that it may be relevant in intact cells.
Histones, and other basic proteins, have been isolated from zinc-sufficient (+Zn) Euglena gracilis by standard chromatographic methods. These cells contain 2.46 micrograms of histones and 1.96 micrograms of DNA per 10(6) organisms. Each of the histones, H1, H3, H2A, H2B, and H4, is present in both log- and stationary-phase +Zn cells and has been characterized according to its electrophoretic mobility and molecular weight. H1 has been further identified on the basis of its amino acid composition and its cross-reactivity with calf thymus histone H1 antibodies. Similarly, H3 has been recognized as well by its specific reaction with an H3 antibody. In contrast, log-phase zinc-deficient (-Zn) cells contain H1 and H3 while H2A, H2B, and H4 are absent. All of the histones vanish in stationary-phase-Zn organisms. The DNA content increases as the -Zn cells progress from log to stationary phase, reaching a value of 4.40 micrograms/10(6) cells, double that of comparable stationary-phase +Zn organisms. A 2000-3000-dalton polypeptide whose electrophoretic behavior differs from that of the known histones constitutes over 90% of the total basic proteins of -Zn cells. On addition of zinc to stationary -Zn cells, cell division resumes, and all the histones and other basic proteins reappear. Together with previous results, the data demonstrate that zinc significantly affects the metabolism of all major chromatin components, i.e., the RNA polymerases, DNA, and histones of E. gracilis [Vallee, B.L., & Falchuk, K.H. (1981) Philos. Trans. R. Soc. London, Ser. B 294, 185-197]. The implications of these effects of zinc on chromatin structure and function are discussed.
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