Mg2+-dependent interaction of DNA with eukaryotic cells

Interaction of DNA with eukaryotic cells under conditions similar to those providing DNA adsorption onto liposomes was studied. It was revealed that mouse fibroblasts (line A9) and myeloma cells bind phage and plasmid DNA in 0.3 M sucrose solution containing Mg2+-ions. Additional pretreatment of the cells by trypsin did not affect DNA adsorption efficiency. The major part of the adsorbed DNA recovered by salt treatment of the cells, but 10-15% of DNA was found to be irreversible. Up to 50% of the irreversibly bound DNA molecules retain their linear size after treatment of cells with DNAse I. Efficiencies of DNA adsorption and irreversibly binding depend on the concentration of Mg2+ in the medium. The process of DNA irreversible binding is not inhibited by drugs affecting cell metabolism. It is assumed that DNA adsorbs onto the phospholipid domains of the cell membrane, and part of the adsorbed DNA is taken up into the interior of the cells.     

磷酸钙法转染哺乳动物细胞SP2／0及其表达产物检测

将真核细胞表达质粒以磷到钙介导法导入小鼠骨髓瘤细胞ＳＰ２／０转染细胞经超声破碎后,分别以显色法及化学发光法检测其目的蛋白（ＨＢｓＡｇ）,证实化学发光法的检测灵敏度高于目前常用的显色法,且其结果易于保存。对化学发光法的实验条件及磷酸钙转染法的有关影响因素进行了初步探讨。     

Transfer of high copy number plasmid into mammalian cells by calcium phosphate transfection

Using flow cytometry, single cell sorting, confocal microscopy and fluorescent plasmids, a thorough study of DNA uptake, DNA fate and DNA expression in mammalian cells transfected with the widely used calcium-phosphate precipitation method was executed. We show for the first time that up to 100,000 plasmid molecules can be delivered into individual cells, but also that DNA transfer into cells is a dynamic process that follows a defined kinetics of uptake and intracellular processing. Analyses by flow cytometry and confocal microscopy have also supported results suggesting endocytosis during Ca-Pi transfection. We also demonstrate that expression-enhancing treatment with glycerol during transfection did not result in increased DNA uptake. While cells with maximal DNA load appear to express the highest level of the transgene, these cells are negatively impacted in terms of growth and survival.     

磷酸钙法对哺乳动物非贴壁细胞的质粒共转染

对一种哺乳动物的非贴壁细胞Bal b/c近交系小鼠肥大细胞瘤细胞株P815进行了G418敏感浓度及甘油耐受性的测定;并通过磷酸钙法用含HBVS基因的质粒pRc/CMVS与含筛选标记基因neo的质粒pSV_2-neo进行了共转染。经G418筛选,获得了G418抗性细胞P851-S,在G418选择压力下,已连续筛选80余天,传代12次;斑点杂交和ELISA实验分别表明P815-S细胞内有HBVS基因的存在,培养上清中有HBsAg的表达。     

K+-and Mg2+-dependent hydrolysis of acetyl phosphate catalyzed by the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum

The (Ca²⁺ + Mg²⁺-ATPase of sarcoplasmic reticulum catalyzes the hydrolysis of acetyl phosphate in the presence of Mg²⁺ and EGTA and is stimulated by Ca²⁺. The Mg²⁺-dependent hydrolysis of acetyl phosphate measured in the presence of 6 mM acetyl phosphate, 5mM MgCl₂, and 2 mM EGTA is increased 2-fold by 20% dimethyl sulfoxide. This activity is further stimulated 1.6-fold by the addition of 30 mM KCl. In this condition addition of Ca²⁺ causes no further increase in the rate of hydrolysis and Ca²⁺ uptake is reduced to a low level. In leaky vesicles, hydrolysis continues to be back-inhibited by Ca²⁺ in the millimolar range. Unlike ATP, acetyl phosphate does not inhibit phosphorylation by P_i unless dimethyl sulfoxide is present. The presence of dimethyl sulfoxide also makes it possible to detect P_i inhibition of the Mg²⁺-dependent acetyl phosphate hydrolysis. These results suggest that dimethyl sulfoxide stabilizes a P_i-reactive form of the enzyme in a conformation that exhibits comparable affinities for acetyl phosphate and P_i. In this conformation the enzyme is transformed from a Ca²⁺- and Mg²⁺-dependent ATPase into a (K⁺ + Mg²⁺)-ATPase.     

Effect of Mn2+ and Mg2+ ions on DNA conformation

The DNA conformation was studied at different relation between Na+ and Me2+ (Mn2+ or Mg2+) ions in solution at the fixed total ionic strength mu. At low mu the intrinsic viscosity of DNA [eta] decreased to the limited fixed value with the increasing of Mn2+ or Mg2+ concentration (CMe2+). At higher mu greater than or equal to 0.1 M [eta] doesn't depend on CMe2+. The presence of Mn2+ in solution caused a decrease of the optical anisotropy of DNA and the value of epsilon 260 (p) independent on ionic strengths. In contrary, these parameters of DNA didn't change in solution with Mg2+-concentration. The observed differences in the effects of Mn2+ and Mg2+ on the optical properties of the macromolecule suggest that there are different modes of binding of these ions to DNA. It has been concluded, that Mn2+ interacts with bases and phosphate groups of DNA, but Mg2+--only with phosphates. The persistence length of DNA doesn't depend on Me2+ concentration under the conditions of the experiment (mu greater than or equal to 0.005 M).     

A model for the phosphorylation of the Ca2+ + Mg2+-activated ATPase by phosphate.

We have shown that changes in fluorescence intensity for the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum labelled with fluorescein isothiocyanate following the addition of Ca2+ can give the ratio of the two conformations (E1 and E2) of the ATPase. We show that the fluorescence response to Ca2+ is unaffected by Mg2+, phosphate or K+, implying that these ions bind equally well to the E1 and E2 conformations. A model is presented for phosphorylation of the ATPase by phosphate as a function of pH, Mg2+, K+ and Ca2+.     

Mg2+-, Ca2+-dependent unwinding of DNA by poly-L-glutamic acid

In order to examine a possibility that the high acidic amino acid region in the nonhistone protein HMG(1+2) is concerned with the Mg2+-, or Ca2+-dependent unwinding of DNA by the HMG(1+2) (1,2), poly-L-glutamic acid was employed as an acidic model peptide for thermal melting temperature analysis. The poly-L-glutamic acid bound to DNA either in the presence or absence of Mg2+. The poly-L-glutamic acid unwound DNA double-helix to a similar extent to HMG(1+2) in the presence of Mg2+ or Ca2+, but not in the absence of them. These results may suggest that the high acidic amino acid region in HMG(1+2) participates in Mg2+-, Ca2+-dependent unwinding of DNA double-helix.     

DNA aggregation induced by Mg2+ ions under different conditions

Cations‐induced DNA aggregation can modify the local structure of oligonucleotides and has potential applications in medicine and biotechnology. Here, we used atomic force microscopy to investigate λ‐DNA aggregation on Mg²⁺‐treated glass (Mg²⁺/glass) and in Mg²⁺ solution. Atomic force microscopy topography images showed that some DNA fragments were slightly stacked together on 10 mM Mg²⁺/glass and stacked stronger on ≥50 mM Mg²⁺/glass. They also showed that DNA aggregated stronger in Mg²⁺ solution than on Mg²⁺/glass, ie, DNAs are strongly stacked and twisted at 10 mM Mg²⁺, rolled together at 50 mM Mg²⁺, and slightly aggregated to form small particles at 100 mM Mg²⁺. At a specific condition, ie, heating λ‐DNA to 92°C, cooling down to 75°C, adding Mg²⁺, and vortexing the resulting solution, DNA strongly aggregated and formed pancake‐like shapes at 10 and 50 mM or a large aggregate at 100 mM Mg²⁺ solutions. Our results may be helpful for medical applications and gene therapy using cation‐DNA technology.     

Mg2+ restores membrane potential in rat liver mitochondria deenergized by Ca2+ and phosphate movements

Cellular ornithine biosynthesis could be expected to play a significant role in putrescine formation and hence in growth. Two enzymes are involved in ornithine biosynthesis: arginase and transamidinase. These enzyme activities were studied in two human melanoma cell lines differing in their K_m of diamine oxidase for putrescine and in their tumorigenicity in nude mice. Arginase activity accounts for the majority of ornithine formed in the highly tumorigenic cell line, while the majority of ornithine is derived from transamidinase action in the poorly tumorigenic cell line, with concomitant formation of methyl guanidine, a potent inhibitor of diamine oxidase.     

Characterization of a low-molecular-mass stimulator protein of Mg2+-independent Ca2+-ATPase: effect on phosphorylation/dephosphorylation, calcium transport and sperm-cell motility

A 14 kDa cytosolic protein purified from bovine brain homogenate has been recently reported as a stimulator of goat spermatozoa Mg2+-independent Ca2+-ATPase. In the present study, we demonstrate the formation of the [gamma-32P]ATP-labelled phosphoenzyme as the 110 kDa phosphoprotein and its rapid decomposition in presence of the stimulator protein. Together with the cross-reactivity of this 110 kDa protein with an anti-SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) 2a antibody, the ATPase can now be conclusively said to belong to the SERCA family, which is activated by the stimulator. The ability of the stimulator to enhance the Ca2+ transport has been elucidated from 45Ca2+ uptake studies and was found to be sensitive to Ca2+ channel blockers. CD revealed an alpha-helical structure of the stimulator. The amino acid analysis suggests that it is composed primarily of hydrophobic and some acidic amino acid residues. The pI of 5.1 has been re-confirmed from two-dimensional electrophoresis. Immuno-cross-reactivity studies indicate that the stimulator or similar proteins are present in cytosolic fractions of liver, kidney or testes in different species, but brain is the richest source. Proteomic analyses of its trypsinized fragments suggest its similarity with bovine THRP (thyroid hormone-responsive protein). The physiological significance of the stimulator has been suggested from its ability to activate sperm-cell motility.     

Effect of Mg2+ and Mn2+ on hydrolysis of calf thymus DNA by pancreatic deoxyribonuclease I

Divalent cations are activators for DNA hydrolysis by pancreatic deoxyribonuclease I. Apparent Vm and Km changes have been studied in presence of Mn2+ or Mg2+. The activation process modifies both Vm and Km, their relationship with Mg2+ or Mn2+ being a linear one. Deoxyribonucleotides inhibit the DNA hydrolysis, whether Mg2+ or Mn2+ is the activator; the purine deoxyribonucleotides are more effective as inhibitors than the pyrimidine ones. The effect of some derivatives of adenine has been studied: the inhibition is maximum with 5'-dAMP and minimum with adenine or adenosine. A kinetic mechanism of enzymatic activation by Mn2+ or Mg2+ is discussed.     

Restoration of active calcium transport in vesicles of an Mg2+-ATPase mutant of Escherichia coli by wild-type Mg2+-ATPase.

Strain NR70, a mutant of $\text{E. coli}$ lacking the Mg²⁺-adenosine triphosphatase (E.C. 3.6.1.3.) was previously shown to be defective in amino acid and sugar transport in whole cells and right-side-out membrane vesicles. It is shown here that the mutant is also deficient in the uptake of calcium into inverted membrane vesicles. Treatment of inverted vesicles from the wild-type strain with ethylenediamine tetraacetate removes the Mg²⁺-adenosine triphosphatase and results in an inability to transport calcium. Addition of a crude fraction containing the wild-type Mg²⁺-adenosine triphosphatase restores active uptake of calcium both to vesicles from the mutant and depleted vesicles from the wild-type.     

The effect of Mg2+ and Ca2+ on urea-catalyzed phosphorylation reactions

Summary When struvite (MgNH₄PO₄ 6H₂O) is heated with urea at 65–100°C, inorganic pyrophosphate is formed in good yield. Under similar conditions pyro-phosphate is formed much more slowly from ammonium phosphate or hydroxylapatite. The major products formed by the reaction of nucleotides with urea and either ammonium phosphate or hydroxylapatite are derivatives phosphorylated on the 2 or 3 position. With struvite, on the other hand, the main reaction is pyrophosphate bond formation. Yields of up to 25% of uridine diphosphate can be obtained at temperatures as low as 65°C.     

The effect of Mg2+ on hepatic microsomal Ca2+ and Sr2+ transport

The ATP-dependent uptake of Ca2+ by rat liver microsomal fraction is dependent upon Mg2+. Studies of the Mg2+ requirement of the underlying microsomal Ca2+-ATPase have been hampered by the presence of a large basal Mg2+-ATPase activity. We have examined the effect of various Mg2+ concentrations on Mg2+-ATPase activity, Ca2+ uptake, Ca2+-ATPase activity and microsomal phosphoprotein formation. Both Mg2+-ATPase activity and Ca2+ uptake were markedly stimulated by increasing Mg2+ concentration. However, the Ca2+-ATPase activity, measured concomitantly with Ca2+ uptake, was apparently unaffected by changes in the Mg2+ concentration. In order to examine the apparent paradox of Mg2+ stimulation of Ca2+ uptake but not of Ca2+-ATPase activity, we examined the formation of the Ca2+-ATPase phosphoenzyme intermediate and formation of a Mg2+-dependent phosphoprotein, which we have proposed to be an attribute of the Mg2+-ATPase activity. We found that Ca2+ apparently inhibited formation of the Mg2+-dependent phosphoprotein both in the absence and presence of exogenous Mg2+. This suggests that Ca2+ may inhibit (at least partially) the Mg2+-ATPase activity. However, inclusion of the Ca2+ inhibition of Mg2+-ATPase activity in the calculation of Ca2+-ATPase activity reveals that this effect is insufficient to totally account for the stimulation of Ca2+ uptake by Mg2+. This suggests that Mg2+, in addition to stimulation of Ca2+-ATPase activity, may have a direct stimulatory effect on Ca2+ uptake in an as yet undefined fashion. In an effort to further examine the effect of Mg2+ on the microsomal Ca2+ transport system of rat liver, the interaction of this system with Sr2+ was examined. Sr2+ was sequestered into an A23187-releasable space in an ATP-dependent manner by rat liver microsomal fraction. The uptake of Sr2+ was similar to that of Ca2+ in terms of both rate and extent. A Sr2+-dependent ATPase activity was associated with the Sr2+ uptake. Sr2+ promoted formation of a phosphoprotein which was hydroxylamine-labile and base-labile. This phosphoprotein was indistinguishable from the Ca2+-dependent ATPase phosphoenzyme intermediate. Sr2+ uptake was markedly stimulated by exogenous Mg2+, but the Sr2+-dependent ATPase activity was unaffected by increasing Mg2+ concentrations. Sr2+ uptake and Sr2+-dependent ATPase activity were concomitantly inhibited by sodium vanadate. In contrast to Ca2+, Sr2+ had no effect on Mg2+-dependent phosphoprotein formation. Taken together, these data indicate that Mg2+ stimulated Ca2+ and Sr2+ transport by increasing the Ca2+ (Sr2+)/ATP ratio.(ABSTRACT TRUNCATED AT 400 WORDS)