Internation of zinc ions with DNA-dependent RNA polymerases A,B and C isolated from calf thymus

Purified DNA-dependent RNA polymerases A, B and C isolated from calf thymus contain a significant amount of zinc. Atomic absorption spectroscopy revealed the presence of 6.7, 5.35 and 2.6–4.1 g-atoms of zinc per mole of polymerase A, B and C, respectively. These enzymes are inhibited by treatment with 1,10-phenanthroline at concentrations varying from 10^-5 to 10^-4 M. However, the addition of zinc ions does not restore fully the activity of 1,10-phenanthroline treated enzymes. Exogenous zinc ions reducein vitro an overall RNA synthesis catalysed by RNA polymerases from calf thymus. In addition to the sites which bind zinc in a specific and stoichiometric way these enzymes possess other classes of binding sites with high and low affinity. Occupancy by exogenous zinc of these additional binding sites inhibits polymerase activity.     

The role of Zn(II) in transcription by T7 RNA polymerase

Homogeneous T7 RNA polymerase contains from 2–4 gm atoms of zinc per mole of M.W. 107,000. Inactivated molecules which can be separated from the active molecules by repeated chromatography contain less zinc, from 0.4 to 1 gm at per mole. Instability of the enzyme makes it difficult to relate maximal activity to a specific stoichiometry of Zn. The enzyme is inhibited by 1,10-phenanthroline, EDTA, CN^?, SH^?, N₃^? and by incubation with Chelex resin. Zinc is retained on gel filtration, but can be removed by dialysis for 96 hr against 5 mM 1,10-phenanthroline which totally inactivates the enzyme. Catalytic activity requires the presence of thiol reagents. Preparations with low activity can be activated by exogenous Zn ions.     

E. gracilis RNA polymerase I: a zinc metalloenzyme.

$\text{E. gracilis}$ DNA dependent RNA polymerase I has been purified to homogeneity. α-amanitin, over the concentration range 0.05 to 200 μg/ml, does not affect its activity, consistent with its being classified as an RNA polymerase I. Based on a molecular weight of 624,000 daltons the enzyme contains 2.2 g atom of Zn but no Mn, Cu, Fe, as determined by microwave excitation emission spectrometry. Zinc is essential for activity since the chelating agent, 1,10-phenanthroline, inhibits enzymatic function but its non-chelating analogue, 4,7-phenanthroline is ineffective. Thus, like the RNA polymerase II, zinc is a catalytically essential component of $\text{E. gracilis}$ RNA polymerase I (1).     

1,10-Phenanthroline-cuprous ion complex, a potent inhibitor of DNA and RNA polymerases.

The inhibition by 1,10-phenanthroline of $\text{E. coli}$ DNA polymerase I has recently been attributed to the formation in the assay mixtures of a unique and effective inhibitor, the 2:1 1,10-phenanthroline-cuprous ion complex (1). We have now found that this coordination complex is also an effective inhibitor of $\text{E. coli}$ DNA dependent RNA polymerase, Micrococcus luteus DNA dependent DNA polymerase, and T-4 DNA dependent DNA polymerase. This conclusion is based either on the requirement of a thiol for 1,10-phenanthroline inhibition or on the reversal of 1,10-phenanthroline inhibition by the non-inhibitory cuprous ion specific chelating agent 2,9-dimethyl-1,10-phenanthroline. 2,2′,2″-Terpyridine is also very effective at relieving 1,10-phenanthroline inhibition. The reversal of 1,10-phenanthroline inhibition should be attempted before it is claimed that 1,10-phenanthroline inhibits any polymerases by coordinating a zinc ion at the active site.     

Comparative effects of 1, 10-phenanthroline and 1, 7-phenanthroline on DNA and RNA synthesis in mouse spleen

When 1, 10-phenanthroline at 10^?4 mole/kg was administered intraperitoneally to C3H mice, a significant decrease of (³²P) Na₂HPO₄ incorporation into splenic DNA and RNA was noted within 15 min. The same dose or higher was required to significantly inhibit the incorporation of (5-³H) uridine and (methyl-³H) thymidine into splenic nucleic acid. 1, 10-phenanthroline also decrease the incorporation of the ³²P into DNA and RNA in 6C3HED ascites tumor. 1, 7-phenanthroline, a non-chelating analogue at 10^?4 mole/kg, less effectively altered the rate of the ³²P incorporation into splenic nucleic acid within 15 min, but significantly inhibited the incorporation within 1 hr.     

The effect of pH on the structure and activity of yeast RNA polymerase I

The analysis of the effect of pH upon the rate of polymerization indicates that the activity of yeast RNA polymerase I is optimal between pH 7.5 and 9 and depends on the ionization state of two groups with apparent pK_a values of 6.5 and 10. Yeast RNA polymerase I is extremely labile at acid pH. Below pH 5 the enzyme is irreversibly inactivated by [H⁺], with a second-order rate constant of 1.6 × 10^?4m^?1 min^?1. Sucrose gradient sedimentation and gel electrophoresis analysis of the enzyme inactivated at acid pH indicates the sequential dissociation of several enzyme subunits. The polypeptides of 44,000 and 24,000 daltons dissociate first from the enzyme core followed by the dissociation of the polypeptides of 48,000 and 36,000 daltons.     

Solubilization and characterization of RNA polymerase from a higher plant

RNA polymerase has been solubilized from sugar beet chromatin. With calf thmus or sugar beet DNA as template enzyme activity was linear with respect to protein concentration and required the presence of all four nucleoside triphospahates, added DNA and divalent metal ions. The enzyme exhibited a sharp Mn²⁺ optimum of 1·25 mM and a Mg²⁺ optimum at 10mM. The Mn²⁺/Mg²⁺ activity ratio (activity at optimum concentrations) was 2·0 with an optimum salt concentration of 50 mM. Based on data including inhibition with α-amanitin (0·025 μg/ml), the majority of the total activity appeared to be RNA polymerase I. Subsequent fractionation by DEAE-Sephadex column chromatography resulted in one peak of activity eluted with 0·18 M (NH₄)₂SO₄.     

RNA polymerase II from wheat germ contains tightly bound zinc

Atomic absorption studies indicate that the DNA-dependent RNA polymerase II from wheat germ contains about 7 tightly bound zinc atoms per enzyme molecule. This value has been repeatedly obtained with a number of enzyme preparations subjected to varying conditions of purification and dialysis. However, prolonged dialysis of the enzyme with the metal chelator $\text{o}$-phenanthroline results in the loss of enzyme activity and extraction of the bound zinc. Other metals including copper, cobalt, manganese, magnesium, chromium, nickel and iron were not present in significant amounts.     

RNA POLYMERASE ACTIVITY IN THE SUPERIOR CERVICAL GANGLION OF THE NEONATAL RAT: THE EFFECT OF NERVE GROWTH FACTOR

The activity of RNA polymerase in the superior cervical ganglia of the neonatal rat has been studied. The characteristics of the activity with respect to enzyme concentration, temperature, time, and ion concentrations are presented. Ionic conditions of the assay favoring polymerase I and polymerase II were each studied in the presence of specific inhibitors of DNA-dependent RNA synthesis. The K_m for GTP under either polymerase I or polymerase II conditions was found to be 10^?5M, and the relative amounts of poly(A)-containing RNA synthesized under both conditions was 4-5%. A decrease in the activity of RNA polymerase with age has been observed. The time course of increased activity following treatment of the animals in vivo or treatment of the isolated ganglia in organ culture with nerve growth factor is presented. The increase in activity observed after the administration of nerve growth factor is discussed in terms of possible mechanisms of action of nerve growth factor in this tissue.     

Synthesis of RNA in isolated polytene nuclei from salivary gland cells of Chironomus thummi

The incorporation of [³H]UTP into RNA by isolated polytene salivary gland nuclei of Chironomus thummi was investigated under different incubation conditions; the labeled RNA fractions were characterized by electrophoresis. The results suggested that at two characteristic ionic conditions most of the RNA synthesized was the product of RNA polymerase I or RNA polymerase II as distinguished by their differential sensitivities to α-amanitin. Electrophoretical analysis of the RNA synthesized under conditions favouring polymerase I showed that this RNA population consisted mainly of four distinct molecular weight fractions within a range between 2.8 × 10⁴ and 2.5 × 10⁶. Under conditions favouring polymerase II two fractions were detected: one with a broad molecular weight distribution around 0.4 × 10⁶ containing considerable amounts of poly(A)-bearing RNA molecules, and a second with a peak at a molecular weight of 2.8 × 10⁴.     

Limiting rates of ligand association to alcohol dehydrogenase.

Detailed stopped-flow kinetic studies of the association of 2,2-bipyridine, 1,10-phenanthroline, and 5-chloro-1,10-phenanthroline to the zinc ion at the active site of alcohol dehydrogenase have demonstrated that a process with a limiting rate constant of about 200 s^?1 restricts the binding of the bidentate chelating agents to the free enzyme. The formation of the enzyme-ligand complexes has been followed by means of the characteristic absorption spectra of the resulting complexes or by the displacement of the fluorescent dye, auramine O. Monodentate ligands, upon binding to the free enzyme or enzyme-NAD⁺ and enzyme-NADH complexes, do not exhibit a comparable limiting rate. In analogy with simple inorganic systems, these observations have been interpreted in terms of the rate limiting dissociation of an inner sphere water molecule following the rapid formation by the bidentate ligand of an outer sphere complex. The displacement of a water molecule from the zinc ion by 1,10-phenanthroline has been observed in crystallographic studies which have also established that the zinc ion in the enzyme-1,10-phenanthroline complex is pentacoordinate. Monodentate ligands, which are substrate analogs, do not exhibit limiting rates because displacement of water is not required for their addition to a coordinate position which is apparently vacant in the free enzyme. If a water molecule remains bound to the zinc ion in the kinetically competent ternary complex, it could play an essential role in the proton transfer reaction accompanying catalysis.     

Synthesis,characterization, DNA interaction,and cytotoxicity of novel Pd(II) and Pt(II) complexes

Four complexes [Pd(L)(bipy)Cl]·4H₂O (1), [Pd(L)(phen)Cl]·4H₂O (2), [Pt(L)(bipy)Cl]·4H₂O (3), and [Pt(L)(phen)Cl]·4H₂O (4), where L = quinolinic acid, bipy = 2,2’-bipyridyl, and phen = 1,10-phenanthroline, have been synthesized and characterized using IR, ¹H NMR, elemental analysis, and single-crystal X-ray diffractometry. The binding of the complexes to FS-DNA was investigated by electronic absorption titration and fluorescence spectroscopy. The results indicate that the complexes bind to FS-DNA in an intercalative mode and the intrinsic binding constants K of the title complexes with FS-DNA are about 3.5?×?10⁴ M^?1, 3.9?×?10⁴ M^?1, 6.1?×?10⁴ M^?1, and 1.4?×?10⁵ M^?1, respectively. Also, the four complexes bind to DNA with different binding affinities, in descending order: complex 4, complex 3, complex 2, complex 1. Gel electrophoresis assay demonstrated the ability of the Pt(II) complexes to cleave pBR322 plasmid DNA.     

Neonatal exposure to bisphenol A advances pubertal development in female rats

Neonatal exposure to bisphenol A (BPA) is hypothesized to advance pubertal development. However, the effects of neonatal BPA exposure on pubertal development has not been described. In this study, female Sprague‐Dawley rats were exposed to 0.05, 0.5, 5, or 10 mg·kg^?1·day^?1 BPA, or corn oil vehicle alone from postnatal day 1 (PND1) to PND10 via subcutaneous injection. We evaluated day of vaginal opening (DVO), ovarian morphology, serum hormone concentrations, and hypothalamic expression of Gnrh1 and Kiss1 in female rats at PND35. DVO was significantly advanced in rats exposed to 5 and 10 mg·kg^?1·day^?1 BPA. Serum hormone concentrations increased as BPA dose increased. Additionally, hypothalamic Gnrh1 and Kiss1 expression were increased with BPA exposure; rats exposed to 10 mg·kg^?1·day^?1 BPA had significantly upregulated hypothalamic Gnrh1 and Kiss1 expressions in terms of both messenger RNA and protein levels. Our results suggest that exposure to a 10 mg·kg^?1·day^?1 dose of BPA might advance pubertal development significantly. In addition, within the range of 0 to 10 mg·kg^?1·day^?1, neonatal exposure to BPA may affect pubertal development in a dose‐dependent manner.     

PHYSIOLOGICAL VARIATION AND ELECTROPHORETIC BANDING PATTERNS OF GENETICALLY DIFFERENT SEASONAL POPULATIONS OF SKELETONEMA COSTATUM (BACILLARIOPHYCEAE)1

Clones of Skeletonema costatum (Grev.) Cl. isolated from Narragansett Bay, R.I., during different seasons were grouped according to their electrophoretic banding patterns. The growth rates, pg chlorophyll · cell^?1, carbon uptake · cell^?1· h^?1, and carbon uptake · pg chl^?1· h^?1 were measured at 20°C, in a 14:10 h L:D cycle at 180 μE · m^?2· s^?1. Statistically significant sources of variation were found among groups of clones in growth rate, pg chl · cell^?1, and carbon uptake · pg chl^?1· h^?1. It was concluded that there is a significant relationship between the physiological characteristics of clones isolated from populations in different seasons and patterns of genetic variation inferred from the electrophoretic studies. However, genetic diversity detected by banding patterns tends to underestimate the total genetic diversity in natural populations. The groups of clones most common in summer bloom populations had significantly higher growth rates, lower values of pg chl · cell^?1, and higher rates of carbon uptake · pg chl^?1· h^?1 at 20°C than did the group of clones most common in winter bloom populations. However, differences among groups in these parameters at 20°C alone cannot account for the seasonal cycling of genetically variable populations of Skeletonema in Narragansett Bay. The range of growth rates among clones of this species is 0.1–5.0 divisions · d^?1 under a single set of temperature and light conditions. Chlorophyll concentrations range from 0.2–1.7 pg chl · cell^?1 and carbon uptake · pg chl^?1· h^?1 varies by a factor of 7 among clones. The range of physiological variation in this species means that it is difficult to use laboratory studies of single clones to analyze the responses of natural populations of Skeletonema.     

Zinc as a co-factor for an enzyme involved in exsheathment of Haemonchus contortus

The activity of concentrated exsheathing fluid of Haemonchus contortus against isolated sheaths was not inhibited by ethylenediamine tetra-acetic acid (EDTA), 10^?2 M, even when the concentrations of Mg and Mn were < 4 × 10^?4 M and < 0·9 × 10^?6 M respectively. Purified or diluted solutions of exsheathing fluid, even in the presence of Mg²⁺, 10^?3 M, were inhibited. Leucine aminopeptidase (LAP) in exsheathing fluid was active even at concentrations of Mg < 1·3 × 10^?5M. Concentrated solutions were partially inhibited by EDTA, 10^?2 M, at low concentrations of Mg; inhibition was increased in diluted and purified preparations.1,10-phenanthroline (Ophen) strongly inhibited exsheathing activity (Zn < 1 × 10^?6 M). When Zn²⁺, 10^?3 M was added, the inhibition was abolished. The hydrolysis of l-leucinamide was greatly increased in the presence of Ophen, 10^?4 M; this effect was abolished by adding Zn²⁺, 10^?3 M.It is suggested that exsheathing fluid from at least some ‘strains’ of H. contortus contains a Zn metallo-enzyme, probably LAP, which is involved in the process of exsheathment.     

Protection of mouse spermatogonia against x-ray induced translocations

Adult male C57BL mice were exposed to 75, 150, 300 or 450 R X-rays with or without pre-treatment with Adeturon (S-2-aminoethyl-isothiuronium bromide hydrobromide (AET) adenosine triphosphate, 500 mg/kg b.w.). Twelve weeks later, primary spermatocytes were examined cytologically at diakinesis-metaphase I for persisting chromosomal translocations, namely multivalents in the form of rings or chains.For the dose range studied, regression analysis indicated that the data were best fitted to the equation Y = aD + bD² with coefficients for translocated-cell and translocations-per-cell yields, respectively, a = 1.57·10^?2 and 1.59·10^?2 and b = ?2.29·10^?5 and ?2.09·10^?5, for Adeturon protected irradiated animals vs.a = 1.80·10^?2 and 2.05·10^?2, and b = ?0.94·10^?5 and ?1.19·10^?5, in non-protected irradiated animals.Adeturon protection of heritable structures in mouse germ cells showed a dose reduction factor of about 2.     

Exopeptidase activity in eggs of Trichostrongylus colubriformis (Nematoda)

Summary

A supernatant from eggs of the ruminant nematode Trichostrongylus colubriformis contained an enzyme that was similar to leucine aminopeptidase (LAP), based on hydrolysis of the substrate L-leucine β-naphthylamide to β-naphthylamine. A Michaelis-Menten constant (K _m) of 0.155 mM was obtained. Rate of hydrolysis of 16 substrates revealed that L-phenylalanine and L-tyrosine β-naphthylamides were hydrolyzed most readily while seven additional substrates were hydrolyzed at lesser rates. The optimum pH for enzymatic activity was 6.75–7.5. Enzymatic activity was lost by heating the egg supernatant to 60°C for 5 min or freezing at 0°C for 28 days. Addition of millimolar concentrations of the chlorides of zinc, manganese and magnesium to the egg supernatant had no stimulatory effect on enzyme activity while 10 and 100 mM concentrations significantly reduced activity. Ethylenediamine tetraacetic acid at 10^?4 M had no effect on enzymatic activity. Activity was inhibited by 10^?4 M 1,10-phenanthroline, but the inhibition was reversed by zinc chloride at 10^?3 M. Di-isopropylphosphofluoridate at 10^?3 M reduced enzymatic activity moderately. Enzyme activity in egg supernatant increased 2.2-fold from 21 days to 60–90 days of a primary infection in the host while a 3.3-fold increase was found in primary versus secondary infections.     

Interaction of elongation factor EF-Tu with γ-amides of GTP and β-amides of GDP bearing the azidoaryl group or the chloroethylaminoaryl group placed at the terminal phosphate

New types of azidoaryl analogs of GTP: γ-(4-azido)anilide of GTP (I), γ-(N-(4-azidobenzyl)-N-methyl)amide of GTP (II) and of GDP: β-(4-azido)anilide of GDP (III), β-(N-(4-azidobenzyl)-N-methyl)amide of GDP (IV) have been synthesized by treatment of the nucleotide in aqueous solution with N-cyclohexyl-N′-β-(4-methylmorpholinium)- ethylcarbodiimidep-toluene sulfonate and the respective amine. The analog of GTP bearing at the γ-phosphate an alkylating 2-chloroethylamino group: γ-(4-N-(2-chloroethyl)-N-methylaminobenzyl)amide of GTP (V) was prepared by the method described previously for the preparation of the analog of ATP (Knorre, D.G., Kurbatov, V.A. and Samukov, V.V. (1976) FEBS Lett. 70, 105–108). Azidoaryl analogs of GTP and GDP as well as the chloroethylaminoaryl analog of GTP compete with GDP in the formation of the binary complex EF-Tu·GDP with the respective K_i values 3.9·10^?7 M (I), 2.9·10^?8 M (II), 6.9·10^?7 M (III), 5.0·10^?7 M (IV) and 3.8·10^?8 M (V) relative to GDP. constants of the complexes of the radioactively-labeled GTP analogs I, II and V with elongation factor Tu were calculated to be 8.5·10^?6 M, 3.4·10^?7 M and 4.6·10^?8 M, respectively, or approx. 1740-, 70- and 9-times greater than that of GDP. GTP analogs I, II and V were found to substitute GTP in the stimulation of EF-Tu-dependent binding of aminoacyl-tRNA to the ribosome-mRNA complex.