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PubMed1989

Expression and nucleotide sequence of a plasmid-determined divalent cation efflux system from Alcaligenes eutrophus.

Resistance to cobalt, zinc, and cadmium specified by the czc determinant on plasmid pMOL30 in Alcaligenes eutrophus results from a cation efflux system. Five membrane-bound polypeptides that were expressed in Escherichia coli from this determinant under the control of a phage T7 promoter were assigned to four open reading frames identified in the nucleotide sequence of the 6881-base-pair fragment containing the czc putative operon. The contributions of the polypeptides to the cation efflux system were analyzed with deletion derivatives of the 6.9-kilobase fragment, constructed, and expressed in E. coli under the control of the phage T7 promoter and in A. eutrophus under the control of the lac promoter.

Alcaligenes↗
PubMed1997

Binding of different divalent cations to the active site of avian sarcoma virus integrase and their effects on enzymatic activity.

Retroviral integrases (INs) contain two known metal binding domains. The N-terminal domain includes a zinc finger motif and has been shown to bind Zn2+, whereas the central catalytic core domain includes a triad of acidic amino acids that bind Mn2+ or Mg2+, the metal cofactors required for enzymatic activity. The integration reaction occurs in two distinct steps; the first is a specific endonucleolytic cleavage step called "processing," and the second is a polynucleotide transfer or "joining" step. Our previous results showed that the metal preference for in vitro activity of avian sarcoma virus IN is Mn2+ > Mg2+ and that a single cation of either metal is coordinated by two of the three critical active site residues (Asp-64 and Asp-121) in crystals of the isolated catalytic domain. Here, we report that Ca2+, Zn2+, and Cd2+ can also bind in the active site of the catalytic domain. Furthermore, two zinc and cadmium cations are bound at the active site, with all three residues of the active site triad (Asp-64, Asp-121, and Glu-157) contributing to their coordination. These results are consistent with a two-metal mechanism for catalysis by retroviral integrases. We also show that Zn2+ can serve as a cofactor for the endonucleolytic reactions catalyzed by either the full-length protein, a derivative lacking the N-terminal domain, or the isolated catalytic domain of avian sarcoma virus IN. However, polynucleotidyl transferase activities are severely impaired or undetectable in the presence of Zn2+. Thus, although the processing and joining steps of integrase employ a similar mechanism and the same active site triad, they can be clearly distinguished by their metal preferences.

Amino Acid Sequence↗
PubMed1998

Divalent cations and ligands induce conformational changes that are highly divergent among beta1 integrins.

Here we show striking differences in conformational regulation among beta1 integrins. Upon manganese stimulation, a beta1 epitope defined by monoclonal antibody (mAb) 9EG7 was induced strongly (on alpha4beta1), moderately (on alpha5beta1), weakly (on alpha2beta1), or was scarcely detectable (on alpha6beta1 and alpha3beta1). Comparable results were seen for the beta1 epitope defined by mAb 15/7. Likewise, soluble ligands caused strong (alpha4beta1), moderate (alpha5beta1), weak (alpha2beta1, alpha6beta1), or minimal (alpha3beta1) induction of the 9EG7 epitope. Exchange or deletion of alpha chain cytoplasmic tails did not alter Mn2+-induced 9EG7 epitope levels. Upon removal of calcium by EGTA or EDTA, the hierarchy of 9EG7 epitope induction was similar (alpha5beta1 > alpha2beta1 > alpha6beta1 > alpha3beta1), except that EGTA reduced rather than induced 9EG7 expression on alpha4beta1. Thus in contrast to other beta1 integrins, calcium uniquely supports constitutive expression of the 9EG7 epitope on alpha4beta1. Likewise, calcium supported vascular cell adhesion molecule-stimulated 9EG7 appearance on alpha4beta1, whereas calcium inhibited ligand-induced 9EG7 epitope on other integrins. Constitutive expression of 9EG7 on alpha4beta1 was eliminated by a D698E mutation in alpha4, suggesting that Asp-698 may play a key role in maintaining atypical alpha4beta1 response to calcium. In conclusion, our results (i) demonstrate that mAb such as 9EG7 and 15/7 have limited diagnostic utility as reporters of ligand or Mn2+ occupancy for beta1 integrins, (ii) indicate pronounced differences in conformational flexibilities (alpha4beta1 > alpha5beta1 > alpha2beta1 > alpha6beta1 > alpha3beta1), (iii) allow us to hypothesize that beta1 integrins may differ markedly in conformation-dependent inside-out signaling, and (iv) have uncovered an atypical alpha4beta1 response to calcium that requires alpha4 Asp-698.

Amino Acid Sequence↗
PubMed1999

The flexibility of actin filaments as revealed by fluorescence resonance energy transfer. The influence of divalent cations.

The temperature profile of the fluorescence resonance energy transfer efficiency normalized by the fluorescence quantum yield of the donor in the presence of acceptor, f', was measured in a way allowing the independent investigation of (i) the strength of interaction between the adjacent protomers (intermonomer flexibility) and (ii) the flexibility of the protein matrix within actin protomers (intramonomer flexibility). In both cases the relative increase as a function of temperature in f' is larger in calcium-F-actin than in magnesium-F-actin in the range of 5-40 degrees C, which indicates that both the intramonomer and the intermonomer flexibility of the actin filaments are larger in calcium-F-actin than those in magnesium-F-actin. The intermonomer flexibility was proved to be larger than the intramonomer one in both the calcium-F-actin and the magnesium-F-actin. The distance between Gln41 and Cys374 residues was found to be cation-independent and did not change during polymerization at 21 degrees C. The steady-state fluorescence anisotropy data of fluorophores attached to the Gln41 or Cys374 residues suggest that the microenvironments around these regions are more rigid in the magnesium-loaded actin filament than in the calcium-loaded form.

Actins↗
PubMed1984

Effects of divalent cations and functionally related substances on the yeast to mycelium transition in Sporothrix schenckii.

In a minimal basal medium with glucose at pH 4.0 and 25 degrees C, a lowering of the magnesium and zinc concentrations or increase in the calcium concentration of the medium favoured the yeast-mycelium transition in Sporothrix schenckii. Addition of zinc (1 and 10 mM) inhibited mycelial development and induced reversion to a yeast-like morphology. EDTA and EGTA also delayed germ tube formation, possibly by their calcium-chelating effects or by altering intracellular concentrations of this or other ions. Ionophore X537A also caused a delay in germ tube formation, possibly by interfering with magnesium metabolism in these cells.

Calcium↗
PubMed1999

Effect of divalent cations on the membrane properties of the lipid A analog E5531.

To obtain information on the effects of Mg2+ on the membrane properties of the lipid A analog E5531, we determined the size, structure, zeta potential, membrane fluidity, and micropolarity of the aggregates and the permeability of the E5531 membrane after the addition of Mg2+. E5531 forms a vesicle structure and within the molar ratio of [E5531]:[Mg2+] = 1:3, Mg2+ increased the zeta potential of the E5531 membrane, but did not change the size of the aggregates (approximately 20 nm). Within that molar ratio, Mg2+ decreased the membrane fluidity and micropolarity of E5531 and increased the phase transition temperature. Above the molar ratio of [E5531]:[Mg2+] = 1:5, the size of the aggregates was increased, but at [E5531]:[Mg2+] = 1:3, the size of the aggregates was similar to that in the absence of Mg2+ (approximately 20 nm), and we could stabilize the aggregates in rat plasma.

Animals↗
PubMed2003

Structural polymorphism of telomeric DNA regulated by pH and divalent cation.

DNA oligonucleotides can form multi-stranded structures such as a duplex, triplex, and quadruplex, while the double helical structure is generally considered as the canonical structure of DNA oligonucleotides. Guanine-rich or cytosine-rich oligonucleotides, which are observed in telomere, centromere, and other biologically important sequences in vivo, can form four-stranded G-quadruplex and I-motif structures in vitro. In this study, we have investigated the effects of pH and cation on the structures and their stabilities of d(G4T4G4) and d(C4A4C4). The CD spectra and thermal melting curves of DNAs at various pHs demonstrated that acidic conditions induced a stable I-motif structure of d(C4A4C4), while the pH value did not affect the G-quadruplex structure and stability of d(G4T4G4). The CD spectra of the 1:1 mixture of d(G4T4G4) and d(C4A4C4) indicated that the acidic conditions inhibit the duplex formation between d(G4T4G4) and d(C4A4C4). Isothermal titration calorimetry measurements of the duplex formation at various pHs also quantitatively indicated that the acidic conditions inhibit the duplex formation. On the other hand, the CD spectra and thermal melting curves of DNAs in the absence and presence of Ca2+ indicated that Ca2+ induces a parallel G-quadruplex structure of d(G4T4G4) and then inhibits the duplex formation. These results lead to the conclusion that both the pH and coexisting cation can induce and regulate the structural polymorphisms the oligonucleotides in which they form the G-quadruplex, I-motif, and duplex depending on the conditions. Thus, the results reported here indicate pivotal roles of pH and coexisting cations in biological processes by regulating the conformational switching between the duplex and quadruplexes structures of the guanine-rich or cytosine-rich oligonucleotides in vivo.

Cations, Divalent↗
PubMed1972

Fractionation of the nucleus by divalent cations. Isolation of nuclear membranes.

0.3-0.5 M MgCl(2) was used to disassemble nuclei and to isolate by a single centrifugation in less than 3 hr a nuclear envelope fraction in 55-60% yield as assessed by phospholipid recovery. Its gross chemical composition was determined and its morphology was studied electron microscopically by sectioning, freeze etching, and negative staining procedures.

Animals↗
PubMed1958

Inactivation of endotoxin by a humoral component. III. Role of divalent cation and a dialyzable component.

The uniformly high potency of citrated plasma as compared with the limited capacity of serum to inactivate endotoxin in vitro was found to be a consequence of the anticoagulant employed in collecting the plasma. Addition of calcium to plasma suppressed the activity of its endotoxm detoxifying component (EDC) whereas the addition of calcium-binding anticoagulants rendered serum comparable to plasma. Dialysis of plasma resulted in a marked reduction of its EDC activity despite the concommittant elimination of calcium. EDC activity could then be fully restored upon the addition of calcium-binding anticoagulants. Resin-treated plasma, without added anticoagulant, had EDC activity equal to plasma obtained with calcium-binding anticoagulants. Following dialysis, resin-treated plasma also sustained a marked reduction in EDC activity which could be fully restored by calcium-binding anticoagulants. Restoration was also obtained with the dialysate even after ashing. These findings indicated that the suppression of EDC activity by calcium is not direct but is mediated through its effects on an anionic component of plasma which is required for inactivation of endotoxin by EDC.

Bacteria↗
PubMed1999

pH-dependent inhibition of voltage-gated H(+) currents in rat alveolar epithelial cells by Zn(2+) and other divalent cations.

Inhibition by polyvalent cations is a defining characteristic of voltage-gated proton channels. The mechanism of this inhibition was studied in rat alveolar epithelial cells using tight-seal voltage clamp techniques. Metal concentrations were corrected for measured binding to buffers. Externally applied ZnCl(2) reduced the H(+) current, shifted the voltage-activation curve toward positive potentials, and slowed the turn-on of H(+) current upon depolarization more than could be accounted for by a simple voltage shift, with minimal effects on the closing rate. The effects of Zn(2+) were inconsistent with classical voltage-dependent block in which Zn(2+) binds within the membrane voltage field. Instead, Zn(2+) binds to superficial sites on the channel and modulates gating. The effects of extracellular Zn(2+) were strongly pH(o) dependent but were insensitive to pH(i), suggesting that protons and Zn(2+) compete for external sites on H(+) channels. The apparent potency of Zn(2+) in slowing activation was approximately 10x greater at pH(o) 7 than at pH(o) 6, and approximately 100x greater at pH(o) 6 than at pH(o) 5. The pH(o) dependence suggests that Zn(2+), not ZnOH(+), is the active species. Evidently, the Zn(2+) receptor is formed by multiple groups, protonation of any of which inhibits Zn(2+) binding. The external receptor bound H(+) and Zn(2+) with pK(a) 6.2-6.6 and pK(M) 6.5, as described by several models. Zn(2+) effects on the proton chord conductance-voltage (g(H)-V) relationship indicated higher affinities, pK(a) 7 and pK(M) 8. CdCl(2) had similar effects as ZnCl(2) and competed with H(+), but had lower affinity. Zn(2+) applied internally via the pipette solution or to inside-out patches had comparatively small effects, but at high concentrations reduced H(+) currents and slowed channel closing. Thus, external and internal zinc-binding sites are different. The external Zn(2+) receptor may be the same modulatory protonation site(s) at which pH(o) regulates H(+) channel gating.

Algorithms↗
PubMed1962

Activation of deoxyribonucleases by divalent cations.

The activation of DNase I by Mg, Mn, Co, Ni, Fe, Cd, Zn, Ba, Sr, Ca, and Cu ions has been studied by several methods, at different pH and salt concentrations. Mg, Mn, and Co are the best activators for initial stages of degradation. A synergistic effect is shown only by the pair Mg-Ca. Optimal pH of action is always situated at 6.5. DNase II is activated to about the same degree by alkaline earths and Mn ions. Cd and Cu are strong inhibitors. Optimal pH is always 4.6. By titration of liberated secondary phosphate groups, two stages in the hydrolysis of DNA by DNase I are evidenced: a rapid phase activated most by Mg and a slow phase activated by Ca. Some possible mechanisms of action of both enzymes are outlined and the general influence of metal ions is discussed.

Cations, Divalent↗
PubMed1990

Divalent cation activation and inhibition of single calcium release channels from sheep cardiac sarcoplasmic reticulum.

Single Ca2+ release channels from vesicles of sheep cardiac junctional sarcoplasmic reticulum have been incorporated into uncharged planar lipid bilayers. Single-channel currents were recorded from Ca2(+)-activated channels that had a Ca2+ conductance of approximately 90 pS. Channel open probability increased sublinearly as the concentration of free Ca2+ was raised at the myoplasmic face, and without additional agonists the channels could not be fully activated even by 100 microM free Ca2+. Lifetime analysis revealed a minimum of two open and three closed states, and indicates that Ca2+ activated the channels by interacting with at least one of the closed states to increase the rate of channel opening. Correlations between adjacent lifetimes suggested there were at least two pathways between the open- and closed-state aggregates. An analysis of bursting behavior also revealed correlations between successive burst lengths and the number of openings per burst. The latter had two geometric components, providing additional evidence for at least two open states. One component appeared to comprise unit bursts, and the lifetime of most of these fell within the dominant shorter open-time distribution associated with over 90% of all openings. A cyclic gating scheme is proposed, with channel activation regulated by the binding of Ca2+ to a closed conformation of the channel protein. Mg2+ may inhibit activation by competing for this binding site, but lifetime and fluctuation analysis suggested that once activated the channels continue to gate normally.

Animals↗
PubMed1977

Salt and divalent cations affect the flexible nature of the natural beaded chromatin structure.

A natural chromatin containing simian virus 40 (SV40) DNA and histone has been used to examine changes in chromatin structure caused by various physical and chemical treatments. We find that histone H1 depleted chromatin is more compact in solutions of 0.15M NaCl or 2 mM MgCl2 than in 0.01 M NaCl or 0.6M NaCL, and is compact in 0.01 M NaCl solutions if histone H 1 is present. Even high concentrations of urea did not alter the fundamental beaded structure, consisting of 110A beads of 200 base pair content, each joined by thin DNA bridges of 50 base pairs. The physical bead observed by EM therefore contains more DNA than the 140 base pair "core particle". The natural variation in the bridge length is consistent with the broad bands observed after nuclease digestion of chromatin. Chromatin prepared for EM without fixation containing long 20A to 30A fibers possibly complexed with protein.

Cations, Divalent↗
PubMed2002

Regulation of bacterial RNase P ribozyme reaction by divalent cation and guide DNA.

The RNA subunit of bacterial ribonuclease P (RNase P) is a ribozyme which can cleave a canonical cloverleaf tRNA precursor and a hairpin RNA with a CCA-3' tag sequence as its substrate. With high concentration of Mg ion, the ribozyme as well as holo enzyme internally cleaves certain tRNAs in vitro. We denoted this unusual reaction as hyperprocessing. By controlling magnesium ion concentration for the reaction and also by forcing the RNA shape with external guide DNAs, we could regulate the hyperprocessing reaction by the bacterial RNase P enzymes. These techniques will lead the RNase P ribozyme to more designable and more applicable RNA-cleaving enzyme.

Bacteria↗
PubMed1975

Effects on ribosomes of the substitution of spermidine or divalent cations for magnesium ions.

The inactivation of rat liver ribosomes (polysomes) resulting from the replacement of bound magnesium by spermidine has been studied using a dialysis method. Inactivation of the ribosomes (polysomes) occurred when more than 40% of the Mg2+ originally bound to ribosomes in the presence of 2 mM Mg2+ was replaced by spermidine. The polypeptide-synthesizing activity of polysomes was retained partially when Mg2+ was replaced by Mn2+ by dialysis. E. coli 30S ribosomal subunits were more resistant to inactivation due to the replacement of Mg2+ by spermidine than the 50S subunits.

Animals↗
PubMed1976

Aminoacyl transfer RNA formation. VII. Lack of correlation between aminoacylation and PPi-ATP exchange catalyzed by isoleucyl-tRNA synthetase of Escherichia coli in the presence of various divalent cations.

Isoleucyl-tRNA formation and isoleucine-dependent PPi-ATP exchange catalyzed by purified isoleucyl-tRNA synthetase [EC 6.1.1.5] of Escherichia coli were studied in the presence of various amounts of either Mg2+, Ca2+, Fe2+, Ni2+, or Cu2+. In the presence of Mg2+, isoleucine-dependent PPi-ATP exchange was observed in parallel with isoleucyl-tRNA formation, while in the presence of Ca2+, isoleucyl-tRNA formation was observed without isoleucine-dependent PPi-ATP exchange. Moreover, isoleucine-dependent PPi-ATP exchange was much more in the presence of Fe2+ than in the presence of Mg2+, while little isoleucyl-tRNA was formed in the presence of Fe2+. In the presence of Ni2+ or Cu2+, neither reaction was observed. These data, indicating that formation of an isoleucyl-AMP-enzyme complex is not a necessary step in isoleucyl-tRNA formation, support the existence of a concerted mechanism of isoleucyl-tRNA formation in E. coli.

Adenosine Triphosphate↗