Mg2+ -dependent inactivation / H+ -dependent activation equilibrium of chloroplast F1-ATPase

The catalytic part of chloroplast thylakoid ATPase, the chloroplast coupling factor CF1, is reversibly inactivated during incubation in the presence of Mg2+. The inactivation has two phases. Its fast phase occurs at basic pH of the incubation medium (k = 6 min-1), while the slow phase ( k = 0.1-0.2 min-1) depends on pH only slightly throughout the studied range (5.5-9.0). As followed from changes in the inactivation effect of magnesium ions, Mg2+ affinity for the enzyme decreases dramatically with decreasing medium pH. The pH-dependence of Mg2+ dissociation apparent constant suggests that the binding/dissociation equilibrium is determined by protonation/deprotonation of specific acid-base groups of the enzyme. The analysis of pH-dependence plots gives the equilibrium constant of magnesium dissociation (3-9 M) and the dissociation constant of the protonated groups pK 5.8-6.7). Sodium azide is known to stabilize the inactive CF1-MgADP complex; when added to the incubation medium it diminishes the Mg2+ dissociation constant and has no effect on the dissociation constant of the acid-base groups. At lower pH, Mg2+-inactivated CF1-ATPase reactivates. Octyl glucoside accelerates the reactivation, while Triton-100 affects it only slightly. The reactivation rate of membrane-bound CF1 (thylakoid ATPase) inactivated by preincubation with Mg2+ in the presence of gramicidin is a few times higher than that of isolated CF1. These results suggest that the reactivation of isolated and membrane-bound CF1-ATPase is determined by protonation of a limited number of acid-base groups buried in the enzyme molecule.     

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.     

Platelet adhesion to collagen. Factors affecting Mg2(+)-dependent and bivalent-cation-independent adhesion.

Platelet adhesion to collagens immobilized on plastic has been measured, with the following results. (1) Human, but not rabbit, platelets adhered readily to pepsin-extracted monomeric collagens in an Mg2(+)-dependent manner. (2) Rabbit platelets adhered to a monomeric collagen extracted without pepsin by a process that was cation-independent; human platelet adhesion to this collagen exhibited a cation-independent element. (3) Human platelet adhesion to polymeric collagens, including intact native fibres and those reconstituted from pepsin-extracted monomeric collagens, exhibited appreciable cation-independence; adhesion of rabbit platelets to these collagens occurred only by a cation-independent process; pepsin treatment of the intact fibres caused a reduction in cation-independent binding. Two mechanisms of adhesion can therefore be distinguished, one Mg2(+)-dependent, expressed by human, but not rabbit, platelets, the other cation-independent and exhibited by platelets of both species. Mg2(+)-dependent and cation-independent adhesion sites are located within the triple helix of collagen, but the latter sites are only expressed in collagen in polymeric form. In neither case is the helical conformation of the sites essential for their binding activity. Cation-independent adhesion sites are also located in the pepsin-sensitive non-helical telopeptides of collagen and can be expressed in both monomeric and polymeric collagens. Chemical modification of collagen lysine residues indicates that specific lysine residues may be involved in Mg2(+)-dependent adhesion. Adhesion using human citrated platelet-rich plasma is Mg2(+)-independent. Plasma contains factors, conceivably the adhesive proteins fibronectin and von Willebrand factor, that promote the Mg2(+)-independent mechanism.     

Proton-translocating Mg2+-dependent ATPase activity in insulin-secretory granules

Insulin-secretory granules isolated from a pancreatic islet-cell tumour by centrifugation on Percoll density gradients exhibited a membrane-associated Mg(2+)-dependent ATPase activity. In granule suspensions incubated in iso-osmotic media, activity was increased 2-3-fold by carbonyl cyanide p-trifluoromethoxyphenylhydrazone, the combination of valinomycin, nigericin and K(2)SO(4) or by the addition of a detergent. Permeant anions also increased Mg(2+)-dependent ATPase activity under iso-osmotic conditions when combined with K(+) and nigericin, or NH(4) (+). It was deduced that a major component of the activity was coupled to the translocation of protons into the granule interior. The granule membrane appeared poorly permeable to H(+), K(+), NH(4) (+) and SO(4) (2-) but permeable, in increasing order, to phosphate or acetate, Cl(-), I(-) and SCN(-). Like the proton-translocating ATPase of mammalian mitochondria the granule enzyme when membrane-bound was inhibited by up to 85% by tributyltin or NN'-dicyclohexylcarbodi-imide and was solubilized in a tributyltin-insensitive form after extraction with dichloromethane. It was clearly not a mitochondrial contaminant as evidence by the distribution of marker proteins on density gradients. Unlike mitochondrial activity it was insensitive to oligomycin, efrapeptin, atractyloside, azide and oxyanions. Its properties, however, were indistinguishable from those of the proton-translocating ATPase found in the chromaffin granules of the adrenal medulla. Moreover, insulin granules and chromaffin granules exhibited similar levels of activity. This indicated that in spite of the differences in their internal composition, granules from tissues involved in polypeptide and amine hormone secretion possess catalytic components in common. Only a minor role for the ATPase in amine transport in insulin granules was apparent. Rather, its presence here may relate to the process of secretory vesicle morphogenesis or to the exocytotic mechanism.     

Characterization of Na(+)-dependent Mg2+ efflux from Mg2(+)-loaded rat erythrocytes

Na(+)-dependent Mg2+ efflux from Mg2(+)-loaded rat erythrocytes was determined from the increase of extracellular Mg2+ concentration or decrease of intracellular Mg2+ content, as measured by means of atomic absorption spectrophotometry. Mg2+ efflux was specifically combined with the uptake of Na+ at a stoichiometric ratio of 2Na+:1Mg2+, indicating electroneutral Na+/Mg2+ antiport. Na+/Mg2+ antiport depended on intracellular ATP and was inhibited by amiloride and quinidine, but was insensitive to strophanthin. Net Mg2+ efflux was only occurring at increased concentration of intracellular Mg2+ ([Mg2+]i), and stopped when the physiological Mg2+ content was reached. Intracellular Mg2+ acted cooperatively with a Hill coefficient of 2.4, which may indicate gating of Na+/Mg2+ antiport at increased [Mg2+]i. At increased intracellular Na+ concentration, Na+ competed with intracellular Mg2+ for Mg2+ efflux and Na+ could leave the rat erythrocyte via this transport system. Na+/Mg2+ antiport was working asymmetrically with respect to extra- and intracellular Na+ and Mg2+, and did not perform net Mg2+ uptake.     

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.     

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.     

Ca2+-stimulated, Mg2+-dependent ATPase in bovine thyroid plasma membranes

An isolated plasma membrane fraction from bovine thyroid glands contained a Ca2+-stimulated, Mg2+-dependent adenosine triphosphatase ((Ca2+ + Mg2+)-ATPase) activity which was purified in parallel to (Na+ + K+)-ATPase and adenylate cyclase. The (Ca2+ + Mg2+)-ATPase activity was maximally stimulated by approx. 200 microM added calcium in the presence of approx. 200 microM EGTA (69.7 +/- 5.2 nmol/mg protein per min). In EGTA-washed membranes, the enzyme was stimulated by calmodulin and inhibited by trifluoperazine.     

Mg2+ inhibition of Na2+-stimulated Ca2+ release from brain mitochondria

Magnesium has been shown to modulate the Na+-stimulated release of Ca2+ (Na/Ca exchange) from brain mitochondria. The presence of 5 mM MgCl2 extramitochondrially inhibits the Na/Ca exchange as much as 70%. Additionally, Na+-stimulated Ca2+ release is enhanced by the presence of divalent chelators, this stimulation also being inhibited by the addition of excess Mg2+. The inhibitory effect of Mg2+ and the enhancement by chelating agents were both reversible. Heart mitochondria exhibit a similar enhancement of Na/Ca exchange by chelators and inhibition by MgCl2, though not as pronounced.     

The RNA-induced silencing complex is a Mg2+-dependent endonuclease

In the Drosophila and mammalian RNA interference (RNAi) pathways, target RNA destruction is catalyzed by the siRNA-guided, RNA-induced silencing complex (RISC). RISC has been proposed to be an siRNA-directed endonuclease, catalyzing cleavage of a single phosphodiester bond on the RNA target. Although 5' cleavage products are readily detected for RNAi in vitro, only 3' cleavage products have been observed in vivo. Proof that RISC acts as an endonuclease requires detection of both 5' and 3' cleavage products in a single experimental system. Here, we show that siRNA-programmed RISC generates both 5' and 3' cleavage products in vitro; cleavage requires Mg(2+), but not Ca(2+), and the cleavage product termini suggest a role for Mg(2+) in catalysis. Moreover, a single phosphorothioate in place of the scissile phosphate blocks cleavage; the phosphorothioate effect can be rescued by the thiophilic cation Mn(2+), but not by Ca(2+) or Mg(2+). We propose that during catalysis, a Mg(2+) ion is bound to the RNA substrate through a nonbridging oxygen of the scissile phosphate. The mechanism of endonucleolytic cleavage is not consistent with the mechanisms of the previously identified RISC nuclease, Tudor-SN. Thus, the RISC-component that mediates endonucleolytic cleavage of the target RNA remains to be identified.     

Highly conserved modified nucleosides influence Mg2+-dependent tRNA folding

Transfer RNA structure involves complex folding interactions of the TΨC domain with the D domain. However, the role of the highly conserved nucleoside modifications in the TΨC domain, rT₅₄, Ψ₅₅ and m⁵C₄₉, in tertiary folding is not understood. To determine whether these modified nucleosides have a role in tRNA folding, the association of variously modified yeast tRNA^Phe T-half molecules (nucleosides 40–72) with the corresponding unmodified D-half molecule (nucleosides 1–30) was detected and quantified using a native polyacrylamide gel mobility shift assay. Mg²⁺ was required for formation and maintenance of all complexes. The modified T-half folding interactions with the D-half resulted in K_ds (rT₅₄ = 6 ± 2, m⁵C₄₉ = 11 ± 2, Ψ₅₅ = 14 ± 5, and rT₅₄,Ψ₅₅ = 11 ± 3 µM) significantly lower than that of the unmodified T-half (40 ± 10 µM). However, the global folds of the unmodified and modified complexes were comparable to each other and to that of an unmodified yeast tRNA^Phe and native yeast tRNA^Phe, as determined by lead cleavage patterns at U₁₇ and nucleoside substitutions disrupting the Levitt base pair. Thus, conserved modifications of tRNA’s TΨC domain enhanced the affinity between the two half-molecules without altering the global conformation indicating an enhanced stability to the complex and/or an altered folding pathway.     

Mg2+-dependent/poly(ADP-ribose)-sensitive endonuclease

A novel endonuclease from adult hen liver nuclei has been purified to a homogeneous state through salt extraction, ammonium sulfate fractionation, gel filtration, acetone fractionation, and successive chromatography of 1) hydroxyapatite and DNA Sepharose and 2) hydroxyapatite and isoelectric focusing. The endonuclease has a pH optimum at 9.0 and requires Mg2+ for activity. The enzyme hydrolyzes more rapidly in the order of polynucleotide: denatured DNA = rRNA greater than poly(dA) = poly(dT) greater than poly(dC) = poly(dG) greater than native DNA. This endonuclease degrades denatured DNA about 20 times more rapidly than does the native DNA. The products contain 5'-phosphoryl and 3'-hydroxyl termini and all four deoxynucleotides are present while dGMP is predominant. The enzyme cleaves the circular duplex PM2 DNA, endonucleotically, via single strand scission. The isoelectric point is 10.2 +/- 0.2 and the molecular weight is 43,000 +/- 2,000, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. Pyridoxal 5'-phosphate and 2,3-butanedione inhibit the catalytic activity, respectively. The inhibition of DNA binding activity was also seen with former, but not with the latter. Purified Mg2+-dependent alkaline endonuclease was used to investigate the nature of poly(ADP-ribose) inhibition of the enzyme. In contrast to the Ca2+/Mg2+-dependent endonuclease (Yoshihara, K., Tanigawa, Y., Burzio, L., and Koide, S. S. (1975) Proc. Natl. Acad. Sci. U. S. A. 72, 289-293), ADP-ribosylation of the endonuclease protein was not observed. When 100 ng of the poly(ADP-ribose) having four to five ADP-ribose units per molecule were added to the nuclease assay system (total volume of 0.2 ml) 14% inhibition was observed, and increase in the chain length increased the inhibition. When 100 ng of poly(ADP-ribose) consisting of 20 or more units of the ADP-ribose per mol were added, the inhibition was over 95%. The possible role of the poly(ADP-ribose)-sensitive endonuclease is discussed.     

A monoclonal antibody to the calmodulin-binding (Ca2+ + Mg2+)-dependent ATPase from pig stomach smooth muscle inhibits plasmalemmal (Ca2+ + Mg2+)-dependent ATPase activity.

A monoclonal antibody (2B3) directed against the calmodulin-binding (Ca2+ + Mg2+)-dependent ATPase from pig stomach smooth muscle was prepared. This antibody reacts with a 130,000-Mr protein that co-migrates on SDS/polyacrylamide-gel electrophoresis with the calmodulin-binding (Ca2+ + Mg2+)-ATPase purified from smooth muscle by calmodulin affinity chromatography. The antibody causes partial inhibition of the (Ca2+ + Mg2+)-ATPase activity in plasma membranes from pig stomach smooth muscle, in pig erythrocytes and human erythrocytes. It appears to be directed against a specific functionally important site of the plasmalemmal Ca2+-transport ATPase and acts as a competitive inhibitor of ATP binding. Binding of the antibody does not change the Km of the ATPase for Ca2+ and its inhibitory effect is not altered by the presence of calmodulin. No inhibition of (Ca2+ + Mg2+)-ATPase activity or of the oxalate-stimulated Ca2+ uptake was observed in a pig smooth-muscle vesicle preparation enriched in endoplasmic reticulum. These results confirm the existence in smooth muscle of two different types of Ca2+-transport ATPase: a calmodulin-binding (Ca2+ + Mg2+)-ATPase located in the plasma membrane and a second one confined to the endoplasmic reticulum.     

Mg2+-dependent folding of a large ribozyme without kinetic traps

The folding kinetics of the catalytic domain of Bacillus subtilis ribonuclease P is analyzed here by fluorescence and catalytic activity. The folding pathway is apparently free of kinetic traps, as indicated by a decrease in folding rates upon the addition of urea. We apply Mg2+ and urea chevron analysis to fully describe the folding and unfolding kinetics of this ribozyme. A folding scheme containing two kinetic intermediates completely accounts for the free energy, the Mg2+ Hill coefficient and the surface buried in the equilibrium transition. At saturating Mg 2+concentrations, folding is limited by a barrier that is independent of Mg2+ and urea. These results describe the first trap-free folding pathway of a large ribozyme and indicate that kinetic traps are not an obligate feature of RNA folding.