Characterization of Chromatin Subfractions Enriched in RNA from Lactating Mammary Gland of the Rat

Mammary gland chromatin from lactating rats was fractionated according to the Mg²⁺-solubility method after mild digestion with DNase II. The chromatin properties were compared between the Mg²⁺-soluble (S₂) and the Mg²⁺-insoluble (P₂) fractions. The weight ratio of RNA to DNA was much greater in the S₂ fraction than in the P₂ fraction (S₂, 0.92; P₂, 0.03). The DNA repeat length of the nucleosome was very similar between the two fractions (S₂, 193 ± 7; P₂, 1% ± 8 nucleotide pairs). A significant difference was seen in electrophoretic pattern of H1 histone between the two fractions in the acid-urea gel system. Nonhistone proteins with molecular weights higher than about 40,000 dalton were found to be enriched in the S₂ fraction.     

Effect of polyamines and cations on the in vitro methylation of histones

Na⁺ (0.05–0.15 M) increases both the rate and extent of methylation of chromosomal bound histone H4, while spermidine markedly inhibits this reaction. The effects of spermidine could be mimicked by increasing the concentration of Mg²⁺ or Ca²⁺ to 5–10 mM. At the concentrations listed above, these cations have no significant effect on the methylation of free or chromosomal bound histone H3, nor do they affect the rate or extent of methylation of soluble histone H4. Apparently, the accessibility of histone H4 to the methyltransferase is influenced by chromatin structure. Increasing concentrations of Na⁺ alter the conformation of chromatin (DNA) in such a way as to expose lysine residues in the N-terminal region of histone H4 to the methyltransferase, whereas Mg²⁺ or spermidine acts in an opposite manner.     

Characterization of a partially purified adenosine triphosphatase from a corn root plasma membrane fraction

The (K⁺,Mg²⁺)-ATPase was partially purified from a plasma membrane fraction from corn roots (WF9 × Mol7) and stored in liquid N₂ without loss of activity. Specific activity was increased 4-fold over that of the plasma membrane fraction. ATPase activity resembled that of the plasma membrane fraction with certain alterations in cation sensitivity. The enzyme required a divalent cation for activity (Co²⁺ > Mg²⁺ > Mn²⁺ > Zn²⁺ > Ca²⁺) when assayed at 3 millimolar ATP and 3 millimolar divalent cation at pH 6.3. When assayed in the presence of 3 millimolar Mg²⁺, the enzyme was further activated by monovalent cations (K⁺, NH₄⁺, Rb⁺ Na⁺, Cs⁺, Li⁺). The pH optima were 6.5 and 6.3 in the absence and presence of 50 millimolar KCl, respectively. The enzyme showed simple Michaelis-Menten kinetics for the substrate ATP-Mg, with a K_m of 1.3 millimolar in the absence and 0.7 millimolar in the presence of 50 millimolar KCl. Stimulation by K⁺ approached simple Michaelis-Menten kinetics, with a K_m of approximately 4 millimolar KCl. ATPase activity was inhibited by sodium orthovanadate. Half-maximal inhibition was at 150 and 35 micromolar in the absence and presence of 50 millimolar KCl. The enzyme required the substrate ATP. The rate of hydrolysis of other substrates, except UDP, IDP, and GDP, was less than 20% of ATP hydrolysis. Nucleoside diphosphatase activity was less than 30% of ATPase activity, was not inhibited by vanadate, was not stimulated by K⁺, and preferred Mn²⁺ to Mg²⁺. The results demonstrate that the (K⁺,Mg²⁺)-ATPase can be clearly distinguished from nonspecific phosphohydrolase and nucleoside diphosphatase activities of plasma membrane fractions prepared from corn roots.     

Characterization of Mg2+-ATPase from sheep kidney medulla: purification.

Magnesium-dependent adenosine triphosphatase has been purified from sheep kidney medulla plasma membranes. The purification, which is based on treatment of a kidney plasma membrane fraction with 0.5% digitonin in 3 mm MgCl₂, effectively separates the Mg²⁺-ATPase from (Na⁺ + K⁺)-ATPase present in the same tissue and yields the Mg²⁺-ATPase in soluble form. The purified enzyme is activated by a variety of divalent cations and trivalent cations, including Mg²⁺, Mn²⁺, Ca²⁺, Co²⁺, Fe²⁺, Zn²⁺, Eu³⁺, Gd³⁺, and VO²⁺. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme shows two bands with R_f values corresponding to molecular weights of 150,000 and 77,000. The larger peptide is phosphorylated by [γ-³²P]ATP, suggesting that this peptide may contain the active site of the Mg²⁺-ATPase. The Mg²⁺-ATPase activity is unaffected by the specific (Na⁺ + K⁺)-ATPase inhibitor ouabain.     

Isolation and properties of ion-stimulated ATPase activity associated with cauliflower plasma membranes

The association of K⁺-stimulated, Mg²⁺-dependent ATPase activity with plasma membranes from higher plants has been used as a marker for the isolation and purification of a plasma membrane-enriched fraction from cauliflower (Brassica oleraceae L.) buds. Plasma membranes were isolated by differential centrifugation followed by density gradient centrifugation of the microsomal fraction. The degree of purity of plasma membranes was determined by increased sensitivity of Mg²⁺-ATPase activity to stimulation by K⁺ and by assay of approximate marker enzymes. In the purified plasma membrane fraction, Mg²⁺-ATPase activity was stimulated up to 700% by addition of K⁺. Other monovalent cations also markedly stimulated the enzyme, but only in the presence of the divalent cation Mg²⁺. Ca²⁺ was inhibitory to enzyme activity. ATPase was the preferred substrate for hydrolysis, there being little hydrolysis in the presence of ADP, GTP, or p-nitrophenylphosphate. Monovalent cation-stimulated activity was optimum at alkaline pH. Enzyme activity was inhibited nearly 100% by AgNO₃ and about 40% by diethylstilbestrol.     

Comparative Study of the Condensation of Chicken Erythrocyte and Calf Thymus Chromatins by Di- and Multivalent Cations

Abstract

The condensation of chicken erythrocyte (CE) and calf thymus (CT) chromatins upon addition of di- and multivalent cations has been studied using turbidityJulprecipitation and electric dichroism measurements. For all the cations investigated (Mg²⁺, Tb³⁺, Co(NH₃)₆ ³⁺, spermidine Spd²⁺ and spermine Sp⁴⁺) condensation of CE chromatin occurred before the onset of aggregation, while aggregation of CT chromatin started before condensation with all cations except Mg²⁺ and Tb³⁺. Precipitation of CE chromatin required lower di- and multivalent cations concentrations than CT chromatin. The electric dichroism data for both chromatins, at low ionic strength in the absence of di- or multivalent cations, indicated that the nucleoprotein molecules were not totally decondensed but that a “precondensed” state was already present. A positive electric dichroism was observed for the most condensed chromatin fibers, in agreement with the “cross-linker” models. Tb³⁺ led to less compact condensed particles as judged from the electric dichroism observations, but electron microscopy revealed that “30 nm fibers” were formed. Very little aggregation was produced by Tb³⁺. On the contrary, spermine produced very large networks of condensed molecules, but large spheroidal particles were also observed. The condensation of CE chromatin happened without changes of solution conductivity upon cation salt addition, regardless of the condensing cation, indicating a cooperative uptake of the ions during this process.     

Coordination of magnesium with adenosine 5′-diphosphate and triphosphate

³¹P NMR chemical shifts of salts of adenosine 5′-triphosphate and diphosphate: ATPH^2?₂2(Me₄N⁺) · H₂O, ATPH^2?₂2 Na⁺ · 3.5 H₂O, ATPH^2?₂Mg²⁺ · 4 H₂O, ATPH^2?₂Ca²⁺ · 2 H₂O, ADPH^2?2(Me₄N⁺) · H₂O and ADPH^2?Mg²⁺ · 4 H₂O have been measured in 0.02 M ²H₂O solutions at 145.7 MHz (22° C) at constant p²H values (8.20 and 6.20). The results are compared with those obtained from salts of adenosine 5′-monophosphate and other simpler phosphomonoesters, e.g. AMP^2?2(Me₄N⁺), AMP^2?Mg²⁺, AMPH^?Me₄N⁺ and (AMPH^?)₂Mg²⁺. It is concluded that the effects exerted by Mg²⁺ and Ca²⁺ on the ³¹P NMR shifts of dipoly- and tripolyphosphates relative to monovalent cations are due mainly to changes in conformation of the polyphosphate chain rather than to purely electronic factors associated with the binding of divalent cations to the phospho-oxyanions. The data are consistent with the existence of the following complexes at p²H 8.20: (MgP_αP_β)ADP^? and (MgP_αP_γ)ATP^2?af (MgP_αP_β)ATP^2?af (MgP_βP_γ)ATP^2? with the latter equilibrium relatively fast in the NMR time scale. Monoprotonation of the terminal phosphate appears to weaken the Mg²⁺-polyphosphate binding, particularly at P_β of MgADPH and at P_β and P_γ of MgATPH^?. The Mg²⁺-polyphosphate binding weakens further at p²H 3.70, i.e. in MgATPH₂. Possible implications of the results in the mechanism of actomyosin Mg²⁺-ATPase in muscle contraction are discussed.     

Some characteristics of cotton pyrophosphatase activation by magnesium

The effect of Mg²⁺ ions on inducing pyrophosphatase activity of germinating cotton (Gossypium hirsutum L.) seeds was investigated. The presence of Mg²⁺ ions in the germination medium markedly shortened time for the attainment of the pyrophosphatase maximum activity (T _max). In the absence of Mg²⁺ ions in the nutrient medium, T _max comprised 6.0–6.5 days, whereas in the presence of 3–5 mM Mg²⁺, T _max was decreased to 3–4 days. An increase in the concentration of Mg²⁺ ions in the medium up to 5 mM resulted in an increase in pyrophosphatase activity. The effect of Mg²⁺ ions on the activity of a purified pyrophosphatase preparation isolated from three-day-old cotton seedlings was investigated. Mg²⁺ ions did not affect the rate of attainment of a maximum pyrophosphatase activity, but decreased the value of the Michaelis-Menten constant.     

The effect of hormones on metal and metal-ATP interactions with fat cell adenylate cyclase.

1-adrenaline, ACTH and glucagon activate the adenylate cyclase of rat adipocytes by decreasing its S_0.5(Mg²⁺) (concentration yielding 0.5 V_max) from its basal value of 11.5 to 1.2, 0.3 and 1.8 mM and by increasing its K_i(ATP^4?) from 0.03 to 0.25; 0.62 and 0.16 mM respectively. The kinetic properties of the enzyme are regulated by its state of saturation with ATP^4? or Mg²⁺; its saturation with ATP^4? and citrate^3? suppressed its basal and hormone-dependent activities. The hormone-dependent decrease in K_m and increase in V_max of the enzyme occur when shifting from suboptimal low concentrations of hormone and Mg²⁺ to optimal conditions, i.e., high concentration of hormone and low concentration of Mg²⁺. The increase in the state of saturation of the enzyme with Mg²⁺ decreases the hormone-dependent effects on V_max and results in identical values of K_m (0.14 mM) for its basal and 1-adrenaline dependent activities. CaCl₂ saturation curves at 5 mM ATP with either 5, 10 or 20 mM MgCl₂ show that the substitution of 5 mM MgCl₂ by 10 mM and 20 mM MgCl₂ increased the K_i(Ca²⁺) of the enzyme from 0.19 to 0.49 and 0.94 mM but decreased its K_i(CaATP) from 0.42 to 0.19 and 0.14 mM respectively. Only when the concentration of MgCl₂ exceeded that of ATP did 1-adrenaline and ACTH activate the enzyme by increasing its K_i(Ca²⁺), although only ACTH increased its K_i(CaATP). An increase in energy charge would decrease the intracellular concentrations of Mg²⁺ and Ca²⁺ because ATP^4? has stronger binding constants for Mg²⁺ and Ca²⁺ than ADP^3? and AMP^2?. Hence, the reported properties of the enzyme suggests that changes in energy charge may allow for metabolic feedback control of the hormonal responsiveness of the Mg²⁺, Ca²⁺, ATP^4? -sensitive adenylate cyclase.     

Arabidopsis Histone Methylase CAU1/PRMT5/SKB1 Acts as an Epigenetic Suppressor of the Calcium Signaling Gene CAS to Mediate Stomatal Closure in Response to Extracellular Calcium

Elevations in extracellular calcium ([Ca²⁺]_o) are known to stimulate cytosolic calcium ([Ca²⁺]_cyt) oscillations to close stomata. However, the underlying mechanisms regulating this process remain largely to be determined. Here, through the functional characterization of the calcium underaccumulation mutant cau1, we report that the epigenetic regulation of CAS, a putative Ca²⁺ binding protein proposed to be an external Ca²⁺ sensor, is involved in this process. cau1 mutant plants display increased drought tolerance and stomatal closure. A mutation in CAU1 significantly increased the expression level of the calcium signaling gene CAS, and functional disruption of CAS abolished the enhanced drought tolerance and stomatal [Ca²⁺]_o signaling in cau1. Map-based cloning revealed that CAU1 encodes the H4R3sme2 (for histone H4 Arg 3 with symmetric dimethylation)-type histone methylase protein arginine methytransferase5/Shk1 binding protein1. Chromatin immunoprecipitation assays showed that CAU1 binds to the CAS promoter and modulates the H4R3sme2-type histone methylation of the CAS chromatin. When exposed to elevated [Ca²⁺]_o, the protein levels of CAU1 decreased and less CAU1 bound to the CAS promoter. In addition, the methylation level of H4R3sme2 decreased in the CAS chromatin. Together, these data suggest that in response to increases in [Ca²⁺]_o, fewer CAU1 protein molecules bind to the CAS promoter, leading to decreased H4R3sme2 methylation and consequent derepression of the expression of CAS to mediate stomatal closure and drought tolerance.     

Purification and properties of tyrosinases from Vibrio tyrosinaticus

Rat liver chromatin which has been briefly sonicated is fractionated by treatment with low concentrations of magnesium ion. At 1.5 mm Mg²⁺, where approximately 20–25% of the chromatin remains soluble after low-speed centrifugation, chemical and physical analysis of the Mg-soluble and Mg-insoluble chromatin fractions show that the fractions possess markedly different properties. The Mg-soluble chromatin has more protein and RNA than the Mg-insoluble chromatin. The histone composition of the two fractions as shown by electrophoretic analysis is similar, but many of the acidic proteins are qualitatively and quantitatively different. The molecular weight of the Mg-soluble chromatin is less than that of the insoluble chromatin based on sedimentation behavior and gel filtration experiments. The soluble chromatin has nearly twice the template activity for RNA synthesis in vitro with added RNA polymerase as the Mg-insoluble chromatin and contains approximately 80% of the in vivo rapidly labeled RNA found in the total chromatin preparation. In addition the Mg-soluble chromatin has a significantly greater amount of “accessible” DNA (62%) as measured by polylysine binding than Mg-insoluble chromatin (48%). The data suggest that (a) fractionation of chromatin preparations can be achieved by titration with Mg²⁺, and (b) chromatin soluble in low concentrations of Mg²⁺ may be enriched in actively transcribed portions of the genome.     

The effect of managing nutrients in the performance of anaerobic digesters of municipal wastewater treatment plants

Is it possible to create conditions in the anaerobic digesters to control nutrients without changing the performance of a reactor? This study investigates an answer for this question. To this purpose, anaerobic reactors are operated at high concentrations of Mg²⁺ ion to harvest the nutrient ions (NH₄ ⁺ and PO₄ ^3?) in the form of struvite, that is, magnesium ammonium phosphate. The effects of this modification on the anaerobic digestion of sewage sludge were investigated in terms of chemical oxygen demand (COD) removal and cumulative CH₄ production as well as the changes in the biological diversity. The results showed that approximately 50 % of the nutrients (NH₄ ⁺ and PO₄ ^3?) were removed regardless of the method adopted for the addition of Mg²⁺ ion, slug or daily dosing. The numbers of Methanosaeta and Methanosarcina in the samples withdrawn prior to and after the addition of Mg²⁺ did not show significant difference according to the results obtained from qPCR analyses. The research results showed that the addition of Mg²⁺ into the anaerobic digesters in municipal wastewater treatment facilities may help to remove the nutrients from the effluent while recovering in their solid forms.     

Characterization of Mg2+- and (Ca2+ + Mg2+)-ATPase activity in adipocyte endoplasmic reticulum

The presence of an energy-dependent calcium uptake system in adipocyte endoplasmic reticulum (D. E. Bruns, J. M. McDonald, and L. Jarett, 1976, J. Biol. Chem.251, 7191–7197) suggested that this organelle might possess a calcium-stimulated transport ATPase. This report describes two types of ATPase activity in isolated microsomal vesicles: a nonspecific, divalent cation-stimulated ATPase (Mg²⁺-ATPase) of high specific activity, and a specific, calcium-dependent ATPase (Ca²⁺ + Mg²⁺-ATPase) of relatively low activity. Mg²⁺-ATPase activity was present in preparations of mitochondria and plasma membranes as well as microsomes, whereas the (Ca²⁺ + Mg²⁺)-ATPase activity appeared to be localized in the endoplasmic reticulum component of the microsomal fraction. Characterization of microsomal Mg²⁺-ATPase activity revealed apparent K_m values of 115 μm for ATP, 333 μm for magnesium, and 200 μm for calcium. Maximum Mg²⁺-ATPase activity was obtained with no added calcium and 1 mm magnesium. Potassium was found to inhibit Mg²⁺-ATPase activity at concentrations greater than 100 mm. The energy of activation was calculated from Arrhenius plots to be 8.6 kcal/mol. Maximum activity of microsomal (Ca²⁺ + Mg²⁺)-ATPase was 13.7 nmol ³²P/mg/min, which represented only 7% of the total ATPase activity. The enzyme was partially purified by treatment of the microsomes with 0.09% deoxycholic acid in 0.15 m KCl which increased the specific activity to 37.7 nmol ³²P/mg/min. Characterization of (Ca²⁺ + Mg²⁺)-ATPase activity in this preparation revealed a biphasic dependence on ATP with a Hill coefficient of 0.80. The apparent K_m^s for magnesium and calcium were 125 and 0.6–1.2 μm, respectively. (Ca²⁺ + Mg²⁺)-ATPase activity was stimulated by potassium with an apparent K_m of 10 mm and maximum activity reached at 100 mm potassium. The energy of activation was 21.5 kcal/mol. The kinetics and ionic requirements of (Ca²⁺ + Mg²⁺)-ATPase are similar to those of the (Ca²⁺ + Mg²⁺)-ATPase in sarcoplasmic reticulum. These results suggest that the (Ca²⁺ + Mg²⁺)-ATPase of adipocyte endoplasmic reticulum functions as a calcium transport enzyme.     

Calix[4]arene C-90 selectively inhibits Ca2+,Mg2+-ATPase of myometrium cell plasma membrane

The supramolecular compound calix[4]arene C-90 (5,11,17,23-tetra(trifluoro)methyl(phenylsulfonylimino)-methylamino-25,26,27,28-tetrapropoxycalix[4]arene) is shown to efficiently inhibit the ATP hydrolase activity of Ca²⁺,Mg²⁺-ATPase in the myometrium cell plasma membrane fraction and also in a preparation of the purified enzyme solubilized from this subcellular fraction. The inhibition coefficient I _0.5 values were 20.2 ± 0.5 and 58.5 ± 6.4 μM for the membrane fraction and the solubilized enzyme, respectively. The inhibitory effect of calix[4]arene C-90 was selective comparatively to other ATPases localized in the plasma membrane: calix[4]arene C-90 did not influence the activities of Na⁺,K⁺-ATPase and “basal” Mg²⁺-ATPase. The inhibitory effect of calix[4]arene C-90 on the Ca²⁺,Mg²⁺-ATPase activity was associated with the cooperative action of four trifluoromethylphenyl sulfonylimine (sulfonylamidine) groups oriented similarly on the upper rim of the calix[4]arene macrocycle (the calix[4]arene “bowl”). The experimental findings seem to be of importance for studies, using calix[4]arene C-90, of membrane mechanisms of regulation of calcium homeostasis in smooth muscle cells and also for investigation of the participation of the plasma membrane Ca²⁺-pump in control of electro- and pharmacomechanical coupling in myocytes.     

Effect of polyamines and divalent cations on histone H1-poly(adenosine diphosphate ribose) complex formation

When the polyamines spermine, spermidine or putrescine at 1 or 2 mM were added to HeLa cell nuclei, there was a 1- to 3-fold differential stimulation of [³H]NAD incorporation into an H1 histone-poly(ADP-ribose) complex with little effect on total poly(ADP-ribose) synthesis. At higher polyamine levels, total polymer synthesis was inhibited, but the fraction of polymer associated with H1 was still differentially increased. A differential stimulation of complex synthesis was also produced by the addition of elevated levels of Mg²⁺ or Ca²⁺. These results were not due to an effect of these agents on recovery or turnover of the H1-poly(ADP-ribose) complex but apparently are related to the chromatin condensing action of these agents since enhanced formation of the H1-poly(ADP-ribose) complex and of chromatin aggregation was closely correlated.     

Purification and properties of ATPase from an alkalophilic Bacillus

ATPase was purified from an alkalophilic Bacillus. The enzyme has a molecular weight of 410,000 and consists of five types of subunits of molecular weights of 60,000 (α), 58,000 (β), 34,000 (γ), 14,000 (δ), and 11,000 (?). The subunit structure is suggested to be α₃β₃γδ?. The enzyme is activated by Mg²⁺ and Ca²⁺. The pH optima of the enzyme with 0.1 and 2.0 mm Mg²⁺ are 9 and 6, and those with 1 and 10 mm Ca²⁺ are 8–9 and 7, respectively. Ca²⁺-ATPase hydrolyzes only ATP, whereas Mg²⁺-ATPase hydrolyzes GTP and, to a lesser extent, ATP. The values of V and K_m of the enzyme with ATP in the presence of 10 mm Ca²⁺ or 0.6 mm Mg²⁺ at pH 7.2 are 17 or 0.5 units/mg protein and 1.2 or 0.3 mm, respectively. The enzyme with Mg²⁺ is appreciably activated by HCO^?₃. Relationship of the ATPase to the active transport system in the bacterium is suggested.