Evidence for the presence of chromatin-bound deoxyribonucleic acid phosphatase-exonuclease in Yoshida sarcoma ascites cells

An enzyme which cleaves the phosphoester bond of 3′-phosphoryl termini of DNA was isolated and purified from the chromatin of Yoshida sarcoma cells. The DNA phosphatase is specific for only 3′-phosphorylated DNA with a lesser activity for its single stranded form. Phosphoester bonds of various nucleotides, 3′-phosphorylated RNA and 5′-phosphorylated DNA were not hydrolysed by the enzyme. The DNA phosphatase required 10 mM MgCl₂, and was inactivated by 70 % with 1 mM ?-chloromercuribenzoate and completely by heat treatment at 70° for 5 min. Furthermore, an exonuclease activity could not be separated from the purified DNA phosphatase.     

Magnesium reduces nickel inhibition of DNA polymerization

The activities of DNA polymerization and DNA ligation in extract of Chinese hamster ovary cells were both stimulated by MgCl₂. DNA polymerization was stimulated by MgCl₂ above 0.25 mM, whereas, MgCl₂ above 2 mM was required to stimulate DNA ligation. The activity of DNA polymerization maintained a plateau at MgCl₂ 1–12 mM, whereas DNA ligation reached a maximal activity at MgCl₂ 6 mM and decreased thereafter. NiCl₂ 0.1-0.2 mM also had a stimulatory effect on DNA polymerization, but was much less potent than MgCl₂. However, nickel ion (Ni²⁺) had no detectable stimulating effect on the activity of DNA ligation. In the presence of MgCl₂, the activities of DNA polymerization and DNA ligation decreased with increasing concentration of NiCl₂. Ni₂₊ inhibition of DNA polymerization was reduced by increasing the concentration of MgCl2, but increasing the concentration of MgCl₂ did not reduce Ni²⁺ inhibition of DNA ligation. Preincubating cell extract with MgCl₂ decreased the Ni²⁺ inhibition of DNA polymerization but not DNA ligation. These results suggest that Ni²⁺ may compete with magnesium ion (Mg²⁺) to reduce DNA polymerization, but this mechanism seems not applicable to Ni²⁺ inhibition of DNA ligation.     

Incorporation of 14C-Tyrosine into Soluble Ribonucleic Acid from Baker’s Yeast

The incorporation of ¹⁴C-tyrosine into S-RNA catalyzed by a partially purified tyrosine activating enzyme from baker’s yeast was observed. The maximum incorporation was shown in the presence of 5 μmoles of ATP, 10 μmoles of MgCl₂ and 10~100 μmoles of KCl in the reaction mixture of total volume of 1ml, at pH 7.8 when 1.2 mg of S-RNA, 0.1 μmole of ¹⁴C-tyrosine and 400 μg of enzyme protein were used. Beyond the concentration of ATP, MgCl₂ and KCl described above, the tendency of inhibition was observed. The incorporation was strongly inhibited by pCMB and reactivated by cysteine. Manganese and calcium ions were effective as substitutes for magnesium. S-RNA used was prepared from whole baker’s yeast cell with phenol, but S-RNA obtained from the supernatant of the ground yeast had lost its incorporating activity.     

Spontaneous Disaggregation of Methanosarcina mazei S-6 and Its Use in the Development of Genetic Techniques for Methanosarcina spp

When monomethylamine was the growth substrate, spontaneous disaggregation of Methanosarcina mazei S-6 commenced at the mid-exponential phase and resulted in the formation of a suspension containing 10⁸ to 10⁹ free cells per ml. Free cells were osmotically fragile and amenable to extraction of DNA. Hypertonic media for the manipulation and regeneration of free cells into aggregates were developed, and plating efficiencies of 100% were achieved for M. mazei S-6 and LYC. Free cells of strain S-6 required MgCl₂ (10 mM) for growth, whereas aggregates did not. Specific growth rates of strains S-6 and LYC were increased by MgCl₂. Treatment with pronase caused sphere formation and removal of the protein wall of cells of strain S-6, but protoplasts could not be regenerated. The disaggregating enzyme produced by strain S-6 facilitated the preparation of suspensions of free cells of some strains of Methanosarcina barkeri. Although this provided a means of extracting high-molecular-weight DNA from M. barkeri, less than 0.1% of free cells were viable.     

Inhibitor of pyrimidine metabolism from tumor tissues

Inhibitors of normal rat liver 5′-nucleotidase and dUMP kinase $\text{in vitro}$ were found in rapidly proliferating tissues, such as Yoshida sarcoma. Two inhibitors were separated from Yoshida sarcoma by zone electrophoresis, gel filtration on Sephadex G-200 and DEAE-cellulose column chromatography. One inhibited both 5′-nucleotidase and dUMP kinase, while the other inhibited only dUMP kinase. These inhibitors were not detectable in normal rat liver. They were induced in regenerating rat liver and present in rapidly proliferating tissues, such as Yoshida sarcoma and Ehrlich ascites tumor and rat marrow cells. These inhibitors were heat labile. One had a large molecular weight (500,000>) and the other a small molecular weight ($\text{Ca}\text{.}$ 50,000).     

Escherichia coli ß-galactosidase is heterogeneous with respect to a requirement for magnesium

Commercially obtained E. coli ß-galactosidase was stored at 25 °C in buffer containing 1 mM MgCl₂ and in buffer containing no added MgCl₂. Samples were removed at set times and the activity of individual enzyme molecules assayed. When stored in the presence of 1 mM magnesium, the number of active molecules did not change over a 2.5-h period. When stored in the absence of added MgCl₂, over half the enzyme molecules became inactive within the first hour. However, those molecules which retained activity remained active for the duration of the experiment. This indicates that there may exist two populations of E. coli ß-galactosidase, one which requires storage in the presence of the higher concentration of Mg²⁺ in order to remain active. There was no observed correlation between this requirement for magnesium and reaction rate. Additionally, the presence of the 1 mM MgCl₂ was found to decrease the average activity of the ß-galactosidase molecules under the conditions employed.     

Lecithin Biosynthetic Enzymes of Onion Stem and the Distribution of Phosphorylcholine-Cytidyl Transferase among Cell Fractions

Enzymatic activities of the cytidine 5′-diphosphate choline pathway for lecithin biosynthesis were demonstrated in homogenates of onion stem (Allium cepa). Choline kinase activity was present in the postmicrosomal supernatant, with less than 3% sedimenting with the particulate fractions. Phosphorylcholine-cytidyl transferase was distributed among all fractions, and phosphorylcholine-glyceride transferase was predominantly found in the particulate fraction.     

The interaction of calcium and magnesium ions with deoxyribonucleic acid.

Conductance and equilibrium dialysis studies are reported for the aqueous systems (native calf thymus) DNA-CaCl₂ and DNA-MgCl₂ at various pH values and ionic strengths at 25 °C. Discontinuities occur in the conductance curves at mole ratios of Ca²⁺ and Mg²⁺ to nucleate phosphorus of 0.125, 0.30, and 0.50. The dialysis results show the formation of complexes of stoichiometry 0.50 and 1.00 mol Ca²⁺ or Mg²⁺/mol nucleate phosphorus (2:1 and 1:1 complexes), the latter only in neutral or alkaline solutions, in agreement with the conductance discontinuity at 0.50. The other discontinuities may be due to preferential binding in the formation of the 2:1 complex. Binding constants for the 2:1 complexes are evaluated. Absorption-temperature profiles have been determined for “native” and dialysed DNA in the presence of NaCl, CaCl₂, and MgCl₂. For dialysed DNA at 26 ° C and 260 nm the decrease of absorbance with increased salt concentration was halted for MgCl₂ and CaCl₂ at a concentration corresponding to the formation of the 2:1 complex. The absorbance of “native” DNA did not decrease. T_m and the reciprocal of the hypochromic rise ($\text{1}\text{h}$) increased linearly with log (salt concn). Values of T_m were the same at 230, 260, and 280 nm, but h was greater at 230 and 280 than at 260 nm, which may be due to the existence of alternating blocks of (A + T) and (G ? C) pairs. The entropy of transition was in the order Ca > Mg ? Na.     

Evidence for in vivo Light-induced Synthesis of Ribulose-1,5-diP Carboxylase and Phosphoribulokinase in Greening Barley Leaves

When actinomycin D, puromycin, streptomycin, chloramphenicol, and cycloheximide, known inhibitors of protein synthesis, were applied to leaves of intact seedlings or detached leaves of barley prior to their greening, the same general response resulted: the light-induced increase in activity of ribulose 1,5-diphosphate carboxylase was prevented while that of phosphoribulokinase was only partially suppressed; synthesis of chlorophyll was arrested. This is taken as preliminary evidence that de novo synthesis of protein may be responsible for the observed increase in ribulose-1,5-diphosphate carboxylase activity during greening. However, other factors may be involved with the light-induced stimulation of phosphoribulokinase.

Carbohydrate metabolites and substrates of the enzymes failed to induce the formation of ribulose-1,5-diphosphate carboxylase and phosphoribulokinase in the dark. No evidence was found for the presence of inhibitors in etiolated seedlings or activators in illuminated leaves of barley. Carboxylase activity almost equal to that of the illuminated water control was stimulated by MgCl₂ in the dark; MgCl₂ had no effect on the activity of the kinase.

     

Enzymatic characteristics of pp60v-src isolated from vanadium-treated transformed cells

The transforming protein of Rous sarcoma virus (RSV) typically appears as a single phosphorylated polypeptide designated pp60^v-src, pp60^v-src possesses a protein kinase activity specific for tyrosine residues on select protein substrates. Treatment of RSV-transformed cells with vanadium ions resulted in the appearance of an electrophoretic variant of pp60^v-src and was paralleled by a significant increase in the src kinase specific activity in purified enzyme preparations. Both the normal (standard) src kinase and the src kinase preparations obtained from vanadium-treated cells exhibited similar optimal activity profiles for MgCl₂, KCl, and pH. Furthermore, their site specificities of phosphorylation of the substrates casein and vinculin were the same. The reaction kinetic profile of the standard src kinase showed a nonlinear pattern, while the vanadium enzyme exhibited conventional linear Michaelis-Menten kinetics. These results are discussed with respect to the possible functional regulation of pp60^v-src activity by a vanadium-sensitive protein phosphatase activity.     

The fine structure of euchromatin and centromeric heterochromatin in Tenebrio molitor chromosomes

The fine structure of constitutive heterochromatin and euchromatin was compared in electron microscope whole-mount preparations of Tenebrio molitor (Insecta, Coleoptera) spermatocyte nuclei. Tenebrio molitor pachytene chromosomes display extended segments of centromeric heterochromatin and thus are especially suitable for this purpose. When nuclei were incubated in solutions containing different concentrations of NaCl or of MgCl₂, two levels of chromatin fine structures were observed in the euchromatic segments: nucleosome fibers (0.1 mM–20 mM NaCl) and supranucleosomal fibers with 28 nm in diameter (40 mM–100 mM NaCl, 0.2 mM–1.0 mM MgCl₂). The fine structure in the heterochromatic segments was the same as that in the euchromatic segments in all NaCl concentrations and in MgCl₂ concentrations up to 0.4 mM. In higher MgCl₂ concentrations the heterochromatin remained more compact than the euchromatin and consisted of 37-nm-thick fibers in 0.6 mM MgCl₂ and of 65-nm-thick fibers in 1.0 mM MgCl₂. After the 37-nm and the 65-nm fibers had been dispersed in Mg²⁺-free solutions they could be recondensed by incubation in 0.6 mM and 1.0 mM MgCl₂, respectively. It is concluded that a Mg²⁺-sensitive component of the heterochromatin is responsible for the folding of the nucleosome chain to heterochromatin-specific supranucleosomal structures.     

Reversal of antizyme-induced inhibition of ornithine decarboxylase by cations in barley seedlings

Ornithine decarboxylase (ODC) forms a stable complex with its antizyme (Az), a non-competitive protein inhibitor of ODC. The complex formation of ODC with Az occurs very rapidly and is dissociated by high salt concentrations e.g., 10% ammonium sulfate. When ODC and Az were mixed in the presence of increasing concentrations of Mg²⁺, a relief of ODC inhibition by Az was obtained. Complete relief of inhibition occurred at 2.0 mM of MgCl₂. Other bivalent cations Ca²⁺, Ba²⁺, Co²⁺, Mn²⁺, Zn²⁺ as well as the monocations Na⁺ and K⁺ caused similar effect. The polyamines putrescine, spermidine and spermine also caused relief of the in vitro inhibition of ODC by Az. Therefore, the in vivo inactivation of ODC by forming the ODC-Az complex is dependent on the intracellular amounts of salt and polyamines.     

Inactivation of (Na+ + K+)-stimulated ATPase by a cytotoxic protein from cobra venom in relation to its lytic effects on cells

The mechanism of action of the cytotoxic protein P₆ isolated from cobra venom (Naja naja) which shows preferential cytotoxicity particularly to Yoshida sarcoma cells has been studied by its effects on the membrane-bound enzyme (Na⁺ + K⁺)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) of a variety of cell systems. Evidence obtained with Yoshida sarcoma cells, dog and human erythrocytes and three tissue culture cell lines KB (human oral carcinoma), Hela (human cervix carcinoma) and L-132 (human lung embryonic) shows that inhibition of (Na⁺ + K⁺)-ATPase by the P₆ protein can be correlated with its lytic activity. (Na⁺ + K⁺)-ATPase of Yoshida sarcoma membrane fragments inactivated by P₆ protein could be reconstituted by the addition of phosphatidylserine and phosphatidic acid. It is conceivable that lysis of cells by the P₆ protein may be due to an imbalance of K⁺ and Na⁺ in the cell which leads to swelling and disintegration of the membrane structure. Observations indicate that the P₆ protein combines with membrane constituents of susceptible cells. The overall evidence suggests that both the specificity of its protein structure and the highly basic nature of the P₆ protein are factors which enable it to compete with the lipid moiety maintaining the (Na⁺ + K⁺)-ATPase of the susceptible cells in proper conformation for activity.     

Studies on Glyoxalase Inhibitor Isolation of a New Active Agent,MS-3, from a Mushroom Culture

Cultured broths were screened by measuring the inhibition of glyoxalase, using 105,000 g supernatant of rat liver homogenates as a crude enzyme. An active agent, MS–3 (C₂₁H₂₄O₇), was isolated from a cultured mushroom. ID₅₀ value of MS–3 against the crude glyoxalase was 12 mcg/ml. MS–3 has low toxicity and inhibited the growth of Yoshida rat sarcoma cells in cell culture. The production, isolation, and properties of MS–3 are described.     

Translation of maternal messenger ribonucleoprotein particles from sea urchin in a cell-free system from unfertilized eggs and product analysis

Maternal mRNP particles were isolated from the postribosomal supernatant fluid of unfertilized sea urchin eggs. They were translated in a cell-free system derived from unfertilized eggs. The translation of these particles required the presence of 12 mM MgCl₂, which is considered very high. The same high Mg²⁺ requirement was observed when mRNP particles were translated in a cell-free system from morula embryos. In contrast, mRNA extracted from mRNP particles is translated at 3 mM MgCl₂. This concentration of Mg²⁺ is known to be optimal for initiation of mRNA translation. Likewise, a rabbit globin mRNA is faithfully translated into α and β globin chains in a cell-free system from eggs at 3, but not at 12, mM MgCl₂. The translational products directed by mRNP or by mRNA derived from mRNP were examined in two gel systems and were found to be very similar. In both cases, histones were identified as part of the translational product. This indicated that the translation of mRNP in high Mg²⁺ is not due to nonspecific binding of these particles to ribosomes. The rates of globin synthesis in a cell-free system derived from eggs is comparable to that of morula ribosomes and to that reported for translation of globin with mouse liver and reticulocyte ribosomes, indicating that unfertilized sea urchin egg ribosomes do not possess a translational inhibitor and that no deficiency in initiation factors for mRNA translation could explain the low rate of protein synthesis in unfertilized sea urchin eggs.     

Effect of Magnesium on Chlorophyll-Protein Complexes

The effect of Mg²⁺ during the isolation of chlorophyll-protein complexes was studied in two moss species (Pleurozium schreberi and Ceratodon purpureus) and New Zealand spinach (Tetragonia expansa). When 2 mM MgCl₂ was included in all the extraction and separation phases, the proportions of chlorophyll-protein complex I. were very small in all plants studied. The withdrawal of Mg²⁺ considerably increased the proportions of CP I. The most pronounced increase in the chlorophyll present as CP I was found when Mg²⁺ was withdrawn from the gel, and this also increased the mobility of the CP II complex and free pigment zone. Exclusion of Mg²⁺ from the running buffer had very little effect. Although Mg²⁺ had little effect on the relative amount of chlorophyll in CP II, the withdrawal of Mg²⁺ from all the extraction and separation phases caused formation of polymers of CP II. In the mosses, the formation of polymers of CP II seemed to be more obvious in the species with large grana. Absence of Mg²⁺ from all the extraction and separation phases sometimes also produced a polymer of CP I.     

Subcellular localization of ribonucleotide reductase in Novikoff hepatoma and regenerating rat liver

The subcellular localization of ribonucleotide reductase was ascertained in Novikoff heptoma and normal and regenerating rat tissue. Over 90% of the cellular ribonucleotide reductase is found to be associated with a membrane fraction derived from the postmicrosomal supernatant after centrifugation at 78,000g for 18 hr which bands at 1.3 m sucrose in a discontinuous sucrose gradient. The properties of this particular ribonucleotide reductase are similar to those reported for mammalian ribonucleotide reductase. This membrane fraction, which contains ribonucleotide reductase, had been previously shown to contain a DNA polymerase whose activity is related to cell proliferation. The association of these two enzymes involved in DNA synthesis leads to the suggestion that there may exist a complex of enzymes involved in deoxynucleotide and DNA synthesis in this membrane fraction.     

Binding to the high-affinity substrate site of the (Na+ + K+)-dependent ATPase

The (Na⁺ + K⁺)-dependent ATPase exhibits substrate sites with both high affinity (K _m near 1 µM) and low affinity (K _m near 0.1 mM) for ATP. To permit the study of nucleotide binding to the high-affinity substrate sites of a canine kidney enzyme preparation in the presence as well as absence of MgCl₂, the nonhydrolyzable - imido analog of ATP, AMP-PNP, was used in experiments performed at 0–4°C by a centrifugation technique. By this method theK _D for AMP-PNP was 4.2 µM in the absence of MgCl₂. Adding 50 µM MgCl₂, however, decreased theK _D to 2.2 µM; by contrast, higher concentrations of MgCl₂ increased theK _D until, with 2 mM MgCl₂, theK _D was 6 µM. The half-maximal effect of MgCl₂ on increasing theK _D occurred at approximately 1 mM. This biphasic effect of MgCl₂ is interpreted as Mg²⁺ in low concentrations favoring AMP-PNP binding through formation at the high-affinity substrate sites of a ternary enzyme-AMP-PNP-Mg complex; inhibition of nucleotide binding at higher MgCl₂ concentrations would represent Mg²⁺ acting through the low-affinity substrate sites. NaCl in the absence of MgCl₂ increased AMP-PNP binding, with a half-maximal effect near 0.3 mM; in the presence of MgCl₂, however, NaCl increased theK _D for AMP-PNP. KCl decreased AMP-PNP binding in the presence or absence of MgCl₂, but the simultaneous presence of a molar excess of NaCl abolished (or masked) the effect of KCl. ADP and ATP acted as competitors to the binding of AMP-PNP, although a substrate for the K⁺-dependent phosphatase reaction also catalyzed by this enzyme,p-nitrophenyl phosphate, did not. This lack of competition is consistent with formulations in which the phosphatase reaction is catalyzed at the low-affinity substrate sites.     

Dielectric relaxation of DNA solutions. II

Real and imaganiry parts of complex dielectric constant of dilute solutions of DNA in 10^?3M NaCl with molecular weight ranging from 0.4 × 10⁶ to 4 × 10⁶ were measured at frequencies from 0.2 Hz to 30 kHz. Dielectric increments Δε were obtained from Cole-Cole plots and relaxation times τ_D from the loss maximum frequency. The τ_D of all samples agrees well with twice of the maximum viscoelastic relexation time in the Zimm theory, indicating that the low-frequency dielectric relaxiation should be ascribed to be the rotation of DNA. The rms dipole moment, which was obtained from Δε, agree well with that calculated from the counterion fluctuation theory. The dielectric increment was found to be greatly depressed in MgCl₂, which is resonably interpreted in terms of a strong binding of Mg⁺⁺ ions with DNA.