Cobalt hexammine inhibition of the hammerhead ribozyme

The effects of Co(NH(3))(6)(3+) on the hammerhead ribozyme are analyzed using several techniques, including activity measurements, electron paramagnetic resonance (EPR), and circular dichroism (CD) spectroscopies and thermal denaturation studies. Co(NH(3))(6)(3+) efficiently displaces Mn(2+) bound to the ribozyme with an apparent dissociation constant of K(d app) = 22 +/- 4.2 microM in 500 microM Mn(2+) (0.1 M NaCl). Displacement of Mn(2+) coincides with Co(NH(3))(6)(3+) inhibition of hammerhead activity in 500 microM Mn(2+), reducing the activity of the WT hammerhead by approximately 15-fold with an inhibition constant of K(i) = 30.9 +/- 2.3 microM. A residual 'slow' activity is observed in the presence of Co(NH(3))(6)(3+) and low concentrations of Mn(2+). Under these conditions, a single Mn(2+) ion remains bound and has a low-temperature EPR spectrum identical to that observed previously for the highest affinity Mn(2+) site in the hammerhead ribozyme in 1 M NaCl, tentatively attributed to the A9/G10.1 site [Morrissey, S. R. , Horton, T. E., and DeRose, V. J. (2000) J. Am. Chem. Soc. 122, 3473-3481]. Circular dichroism and thermal denaturation experiments also reveal structural effects that accompany the observed inhibition of cleavage and Mn(2+) displacement induced by addition of Co(NH(3))(6)(3+). Taken together, the data indicate that a high-affinity Co(NH(3))(6)(3+) site is responsible for significant inhibition accompanied by structural changes in the hammerhead ribozyme. In addition, the results support a model in which at least two types of metal sites, one of which requires inner-sphere coordination, support hammerhead activity.     

Determination of metal ion binding sites within the hairpin ribozyme domains by NMR

Cations play an important role in RNA folding and stabilization. The hairpin ribozyme is a small catalytic RNA consisting of two domains, A and B, which interact in the transition state in an ion-dependent fashion. Here we describe the interaction of mono-, di-, and trivalent cations with the domains of the ribozyme, as studied by homo- and heteronuclear NMR spectroscopy. Paramagnetic line broadening, chemical shift mapping, and intermolecular NOEs indicate that the B domain contains four to five metal binding sites, which bind Mn(2+), Mg(2+), and Co(NH(3))(6)(3+). There is no significant structural change in the B domain upon the addition of Co(NH(3))(6)(3+) or Mg(2+). No specific monovalent ion binding sites exist on the B domain, as determined by (15)NH(4)(+) binding studies. In contrast to the B domain, there are no observable metal ion interactions within the internal loop of the A domain. Model structure calculations of Mn(2+) interactions at two sites within the B domain indicate that the binding sites comprise major groove pockets lined with functional groups oriented so that multiple hydrogen bonds can be formed between the RNA and Mn(H(2)O)(6)(2+) or Co(NH(3))(6)(3+). Site 1 is very similar in geometry to a site within the P4-P6 domain of the Tetrahymena group I intron, while site 2 is unique among known ion binding sites. The site 2 ion interacts with a catalytically essential nucleotide and bridges two phosphates. Due to its location and geometry, this ion may play an important role in the docking of the A and B domains.     

Divalent metal ions tune the self-splicing reaction of the yeast mitochondrial group II intron <Emphasis Type="Italic">Sc</Emphasis>.ai5γ

Group II introns are large ribozymes, consisting of six functionally distinct domains that assemble in the presence of Mg(2+) to the active structure catalyzing a variety of reactions. The first step of intron splicing is well characterized by a Michaelis-Menten-type cleavage reaction using a two-piece group II intron: the substrate RNA, the 5'-exon covalently linked to domains 1, 2, and 3, is cleaved upon addition of domain 5 acting as a catalyst. Here we investigate the effect of Ca(2+), Mn(2+), Ni(2+), Zn(2+), Cd(2+), Pb(2+), and [Co(NH(3))(6)](3+) on the first step of splicing of the Saccharomyces cerevisiae mitochondrial group II intron Sc.ai5gamma. We find that this group II intron is very sensitive to the presence of divalent metal ions other than Mg(2+). For example, the presence of only 5% Ca(2+) relative to Mg(2+) results in a decrease in the maximal turnover rate k (cat) by 50%. Ca(2+) thereby has a twofold effect: this metal ion interferes initially with folding, but then also competes directly with Mg(2+) in the folded state, the latter being indicative of at least one specific Ca(2+) binding pocket interfering directly with catalysis. Similar results are obtained with Mn(2+), Cd(2+), and [Co(NH(3))(6)](3+). Ni(2+) is a much more powerful inhibitor and the presence of either Zn(2+) or Pb(2+) leads to rapid degradation of the RNA. These results show a surprising sensitivity of such a large multidomain RNA on trace amounts of cations other than Mg(2+) and raises the question of biological relevance at least in the case of Ca(2+).     

Metal ion stabilization of the U-turn of the A37 N6-dimethylallyl-modified anticodon stem-loop of Escherichia coli tRNAPhe

Nucleoside base modifications can alter the structures, dynamics, and metal ion binding properties of transfer RNA molecules and are important for accurate aminoacylation and for maintaining translational fidelity and efficiency. The unmodified anticodon stem-loop from Escherichia coli tRNA(Phe) forms a trinucleotide loop in solution, but Mg(2+) and dimethylallyl modification of A(37) N6 disrupt the loop conformation and increase the mobility of the loop and loop-proximal nucleotides. We have used NMR spectroscopy to investigate the binding and structural effects of multivalent cations on the unmodified and dimethylallyl-modified anticodon stem-loops from E. coli tRNA(Phe). The divalent cation binding sites were probed using Mn(2+) and Co(NH(3))(6)(3+). These ions bind along the major groove of the stem and associate with the anticodon loop on the major groove side in a nonspecific manner. Co(NH(3))(6)(3+) stabilizes the U-turn conformation of the loop in the dimethylallyl-modified molecule, and the chemical shift changes that accompany Co(NH(3))(6)(3+) binding are similar to those observed with the addition of Mg(2+). The base-phosphate and base-2'-OH hydrogen bonds that characterize the UNR U-turn motif lead to spectral signatures in the form of unusual (15)N and (1)H chemical shifts and reduced solvent exchange of the U(33) 2'-OH and N3H protons. The unmodified molecule also displays spectral features of the U-turn fold in the presence of Co(NH(3))(6)(3+), but the loop has additional conformations and is dynamic. The results indicate that charge neutralization by a polyvalent cation is sufficient to promote formation of the U-turn fold. However, base modification is necessary to destabilize competing alternative conformers even for a purine-rich loop sequence that is predicted to have strongly favorable base stacking energy.     

TRPM7 provides an ion channel mechanism for cellular entry of trace metal ions

Trace metal ions such as Zn(2+), Fe(2+), Cu(2+), Mn(2+), and Co(2+) are required cofactors for many essential cellular enzymes, yet little is known about the mechanisms through which they enter into cells. We have shown previously that the widely expressed ion channel TRPM7 (LTRPC7, ChaK1, TRP-PLIK) functions as a Ca(2+)- and Mg(2+)-permeable cation channel, whose activity is regulated by intracellular Mg(2+) and Mg(2+).ATP and have designated native TRPM7-mediated currents as magnesium-nucleotide-regulated metal ion currents (MagNuM). Here we report that heterologously overexpressed TRPM7 in HEK-293 cells conducts a range of essential and toxic divalent metal ions with strong preference for Zn(2+) and Ni(2+), which both permeate TRPM7 up to four times better than Ca(2+). Similarly, native MagNuM currents are also able to support Zn(2+) entry. Furthermore, TRPM7 allows other essential metals such as Mn(2+) and Co(2+) to permeate, and permits significant entry of nonphysiologic or toxic metals such as Cd(2+), Ba(2+), and Sr(2+). Equimolar replacement studies substituting 10 mM Ca(2+) with the respective divalent ions reveal a unique permeation profile for TRPM7 with a permeability sequence of Zn(2+) approximately Ni(2+) > Ba(2+) > Co(2+) > Mg(2+) >/= Mn(2+) >/= Sr(2+) >/= Cd(2+) >/= Ca(2+), while trivalent ions such as La(3+) and Gd(3+) are not measurably permeable. With the exception of Mg(2+), which exerts strong negative feedback from the intracellular side of the pore, this sequence is faithfully maintained when isotonic solutions of these divalent cations are used. Fura-2 quenching experiments with Mn(2+), Co(2+), or Ni(2+) suggest that these can be transported by TRPM7 in the presence of physiological levels of Ca(2+) and Mg(2+), suggesting that TRPM7 represents a novel ion-channel mechanism for cellular metal ion entry into vertebrate cells.     

Solution structure of Cobalt(III)hexammine complexed to the GAAA tetraloop, and metal-ion binding to G.A mismatches

The solution structure of a 22 nt RNA hairpin and its complex with Co(NH(3))(6)(3+) bound to the GAAA tetraloop has been determined by NMR spectroscopy. Co(NH(3))(6)(3+) has a similar geometry to Mg(H(2)O)(6)(2+) and can be used as a probe for binding sites of completely solvated magnesium ions. The hairpin contains tandem G.A mismatches, similar to the P5abc region of a group I intron, and is closed by a GAAA tetraloop. The tandem G.A mismatches are imino hydrogen bonded in contrast with the sheared G.A mismatches found in a different context in the crystal structure of the P4-P6 domain. Chemical shift changes of the imino protons upon titration of the RNA hairpin with Mg(2+) and with Co(NH(3))(6)(3+) were used to identify ion-binding sites. Paramagnetic resonance broadening upon titration with Mn(2+) was also used. The titration curves gave dissociation binding constants for the magnesium ions in the millimolar range, similar to the binding in the major groove of RNA at tandem G.U base-pairs. Although the largest chemical shift change occurred at an imino proton of one of the G.A base-pairs, no nuclear Overhauser enhancement cross-peaks between the cobalt ligand and neighboring RNA protons were seen, presumably due to the high mobility of the Co(NH(3))(6)(3+) at this site. Nuclear Overhauser enhancement cross-peaks between Co(NH(3))(6)(3+) and the GAAA tetraloop were observed, which allowed the determination of the structure of the tetraloop binding site. The Co(NH(3))(6)(3+) is bound in the major groove of the GAAA tetraloop with hydrogen bonds to guanine base N7 and to phosphate oxygen atoms of the tetraloop.     

The Role of Counterion Valence and Size in GAAA Tetraloop-Receptor Docking/Undocking Kinetics

For RNA to fold into compact, ordered structures, it must overcome electrostatic repulsion between negatively charged phosphate groups by counterion recruitment. A physical understanding of the counterion-assisted folding process requires addressing how cations kinetically and thermodynamically control the folding equilibrium for each tertiary interaction in a full-length RNA. In this work, single-molecule FRET (fluorescence resonance energy transfer) techniques are exploited to isolate and explore the cation-concentration-dependent kinetics for formation of a ubiquitous RNA tertiary interaction, that is, the docking/undocking of a GAAA tetraloop with its 11-nt receptor. Rate constants for docking (k(dock)) and undocking (k(undock)) are obtained as a function of cation concentration, size, and valence, specifically for the series Na(+), K(+), Mg(2+), Ca(2+), Co(NH(3))(6)(3+), and spermidine(3+). Increasing cation concentration acceleratesk(dock)dramatically but achieves only a slight decrease in k(undock). These results can be kinetically modeled using parallel cation-dependent and cation-independent docking pathways, which allows for isolation of the folding kinetics from the interaction energetics of the cations with the undocked and docked states, respectively. This analysis reveals a preferential interaction of the cations with the transition state and docked state as compared to the undocked RNA, with the ion-RNA interaction strength growing with cation valence. However, the corresponding number of cations that are taken up by the RNA upon folding decreases with charge density of the cation. The only exception to these behaviors is spermidine(3+), whose weaker influence on the docking equilibria with respect to Co(NH(3))(6)(3+) can be ascribed to steric effects preventing complete neutralization of the RNA phosphate groups.     

NMR structure of varkud satellite ribozyme stem-loop V in the presence of magnesium ions and localization of metal-binding sites

In the Neurospora VS ribozyme, magnesium ions facilitate formation of a loop-loop interaction between stem-loops I and V, which is important for recognition and activation of the stem-loop I substrate. Here, we present the high-resolution NMR structure of stem-loop V (SL5) in the presence of Mg(2+) (SL5(Mg)) and demonstrate that Mg(2+) induces a conformational change in which the SL5 loop adopts a compact structure with most characteristics of canonical U-turn structures. Divalent cation-binding sites were probed with Mn(2+)-induced paramagnetic line broadening and intermolecular NOEs to Co(NH(3))(6)(3+). Structural modeling of Mn(H(2)O)(6)(2+) in SL5(Mg) revealed four divalent cation-binding sites in the loop. Sites 1, 3, and 4 are located in the major groove near multiple phosphate groups, whereas site 2 is adjacent to N7 of G697 and N7 of A698 in the minor groove. Cation-binding sites equivalent to sites 1-3 in SL5 are present in other U-turn motifs, and these metal-binding sites may represent a common feature of the U-turn fold. Although magnesium ions affect the loop conformation, they do not significantly change the conformation of residues 697-699 involved in the proposed Watson-Crick base pairs with stem-loop I. In both the presence and the absence of Mg(2+), G697, A698, and C699 adopt an A-form structure that exposes their Watson-Crick faces, and this is compatible with their proposed interaction with stem-loop I. In SL5(Mg), however, U700 becomes exposed on the minor groove face of the loop in the proximity of the bases of G697, A698, and C699, suggesting that the Mg(2+)-bound conformation of stem-loop V allows additional contacts with stem-loop I. These studies improve our understanding of the role of Mg(2+) in U-turn structures and in substrate recognition by the VS ribozyme.     

Effects of cobalt hexammine on folding and self-cleavage of the Neurospora VS ribozyme

We have investigated the effects of Co(NH3)6(3+), an analog of hexahydrated Mg2+, on folding and catalysis of the Neurospora VS ribozyme. Most of the metal ion-induced changes detected by chemical modification structure probing in either metal ion are similar, but occur at approximately 33-fold lower concentrations of Co(NH3)6(3+) than Mg2+. However, Co(NH3)6(3+) is not as effective at inducing two functionally important structural changes: stabilizing the pseudoknot interaction between loops I and V, and rearranging the secondary structure of helix Ib. Comparison of the folding of the precursor and the downstream cleavage product, which lacks helix Ia, shows that helix Ia inhibits stable pseudoknot formation and rearrangement of helix Ib. The VS ribozyme does not self-cleave with Co(NH3)6(3+) as the sole polyvalent cation; however, mixed-metal kinetic experiments show that Co(NH3)6(3+) does not inhibit Mg2+-induced self-cleavage. In contrast, at sub-saturating concentrations of Mg2+, Co(NH3)6(3+) increases the rate of Mg2+-induced self-cleavage, indicating that Co(NH3)6(3+) contributes to the functionally relevant folding of the VS ribozyme.     

Effect of divalent cations on antiparallel G-quartet structure of d(G4T4G4)

The thermodynamic parameters of an antiparallel G-quartet formation of d(G4T4G4) with 1 mM divalent cation (Mg(2+), Ca(2+), Mn(2+), Co(2+), and Zn(2+)) were obtained. The thermodynamic parameters showed that the divalent cation destabilizes the antiparallel G-quartet of d(G4T4G4) in the following order: Zn(2+)>Co(2+)>Mn(2+)>Mg(2+)>Ca(2+). In addition, a higher concentration of a divalent cation induced a transition from an antiparallel to a parallel G-quartet structure. These results indicate that these divalent cations are a good tool for regulating the G-quartet structures.     

Properties of the sarcolemmal calcium ion-stimulated adenosine triphosphatase of hamster skeletal muscle

1. A sarcolemmal fraction was isolated from hamster hind-leg skeletal muscles by successive treatment with lithium bromide and potassium chloride. The membranous fraction was observed to contain a highly active Ca(2+)-stimulated ATPase (adenosine triphosphatase), a Mg(2+)-stimulated ATPase, and an Na(+)+K(+)-stimulated Mg(2+)-dependent ouabain-sensitive ATPase. 2. The Ca(2+)-stimulated ATPase activity was pH-dependent, the optimum being pH7.6. 3. Optimum activation of this enzyme was obtained with 3-4mm-Ca(2+) when 4mm-ATP was present as a substrate, and was not influenced by Na(+), K(+) or ouabain, whereas 2,4-dinitrophenol, sodium azide, oligomycin, sodium fluoride and ethanedioxybis(ethylamine)tetra-acetate were inhibitory. 4. The Ca(2+)-stimulated ATPase was markedly inhibited by thiol-blocking reagents, and cysteine was able to reverse this inhibition. 5. Various bivalent cations stimulated ATP hydrolysis by the sarcolemmal fraction in the following decreasing order of potency: Mg(2+), Ca(2+), Mn(2+), Co(2+), Sr(2+), Ba(2+), Zn(2+), Cu(2+).     

Characterization of structure and metal ions specificity of Co2+-binding RNA aptamers

Studies on RNA motifs capable of binding metal ions have largely focused on Mg(2+)-specific motifs, therefore information concerning interactions of other metal ions with RNA is still very limited. Application of the in vitro selection approach allowed us to isolate two RNA aptamers that bind Co(2+) ions. Structural analysis of their secondary structures revealed the presence of two motifs, loop E and "kissing" loop complex, commonly occurring in RNA molecules. The Co(2+)-induced cleavage method was used for identification of Co(2+)-binding sites after the determination of the optimal cleavage conditions. In the aptamers, Co(2+) ions seem to bind to N7 atoms of purines, inducing cleavage of the adjacent phosphodiester bonds, similarly as is the case with yeast tRNA(Phe). Although the in vitro selection experiment was carried out in the presence of Co(2+) ions only, the aptamers displayed broader metal ions specificity. This was shown by inhibition of Co(2+)-induced cleavages in the presence of the following transition metal ions: Zn(2+), Cd(2+), Ni(2+), and Co(NH(3))(6)(3+) complex. On the other hand, alkaline metal ions such as Mg(2+), Ca(2+), Sr(2+), and Ba(2+) affected Co(2+)-induced cleavages only slightly. Multiple metal ions specificity of Co(2+)-binding sites has also been reported for other in vitro selected or natural RNAs. Among many factors that influence metal specificity of the Co(2+)-binding pocket, chemical properties of metal ions, such as their hardness as well as the structure of the coordination site, seem to be particularly important.     

Biochemical characterization of an ATP-dependent DNA ligase from the hyperthermophilic crenarchaeon <Emphasis Type="Italic">Sulfolobus shibatae</Emphasis>

A gene encoding a putative ATP-dependent DNA ligase was identified in the genome of the hyperthermophilic archaeon Sulfolobus shibatae and expressed in Escherichia coli. The 601 amino acid recombinant polypeptide was a monomeric protein capable of strand joining on a singly nicked DNA substrate in the presence of ATP ( K(m)=34 micro mu) and a divalent cation (Mn(2+), Mg(2+), or Ca(2+)). dATP was partially active in supporting ligation catalyzed by the protein, but GTP, CTP, UTP, dGTP, dCTP, dTTP, and NAD(+) were inactive. The cloned Ssh ligase showed an unusual metal cofactor requirement; it was significantly more active in the presence of Mn(2+) than in the presence of Mg(2+) or Ca(2+). Unexpectedly, the native Ssh ligase preferred Mg(2+) and Ca(2+) rather than Mn(2+). Both native and recombinant enzymes displayed optimal nick-joining activity at 60-80 degrees C. Ssh ligase discriminated against substrates containing mismatches on the 3'-side of nick junction and was more tolerant of mismatches at the 5'-end than of those at the penultimate 5'-end. The enzyme showed little activity on a 1-nucleotide gapped substrate. This is the first biochemical study of a DNA ligase from the crenarchaeotal branch of the archaea domain.     

The requirement for bivalent cations in formation of nicotinamide–adenine dinucleotide by nicotinamide mononucleotide adenylyltransferase of pig-liver nuclei

1. The requirement for bivalent cations in catalysis of NAD formation from ATP and NMN in the presence of NMN adenylyltransferase of pig-liver nuclei was studied. Rates of NAD formation in the presence of the activating cations Cd(2+), Mn(2+), Mg(2+), Zn(2+), Co(2+) and Ni(2+) were approximately a linear function of heats of hydration of the corresponding ions. Ba(2+), Sr(2+), Ca(2+), Cu(2+) and Be(2+) did not activate the enzyme; Be(2+) inhibited the reaction in the presence of Mg(2+) and, to a greater extent, in the presence of Ni(2+). 2. Michaelis constants for NAD formation, measured in a coupled assay with NMN adenylyltransferase and alcohol dehydrogenase at pH8.0 and 25 degrees , in the presence of 3mm concentrations of the unvaried reactants, were 88+/-7mum-ATP, 42+/-4mum-NMN and 85+/-4mum-Mg(2+). The results at this pH and at pH7.5 were consistent with mechanisms in which Mg(2+)-ATP complex is a reactant and free ATP a competitive inhibitor. 3. Formation of nicotinamide-hypoxanthine dinucleotide from NMN and ITP in the presence of the transferase was also more rapid with Ni(2+) and Co(2+) than with Mg(2+).     

Cobalt- and nickel-binding property of cullin-2.

Treatment with divalent metal ions such as cobalt (Co(2+)) or nickel (Ni(2+)) result in the stabilization of hypoxia-inducible factor-1alpha (HIF1alpha). Recently, HIF1alpha was shown to be ubiquitinated by an E3-ligase complex and be subsequently targeted for proteasomal degradation. In this study, we demonstrated that Co(2+) and Ni(2+) specifically bind to cullin-2. Mutant analysis revealed that cullin-2 possesses at least three sites for the binding. Furthermore, fluorescence spectroscopy revealed that only Co(2+) and Ni(2+) have the binding activity to cullin-2, but other metal ions, including Cu(2+), Ca(2+), Mg(2+), Mn(2+), and Zn(2+), did not. Finally, we found that Co(2+) and Ni(2+) do not bind to any components of the E3-ligase other than cullin-2, suggesting that cullin-2 is a key target of Co(2+) and Ni(2+). Interestingly, Co(2+) did not affect the complex formation of the ligase, suggesting that the metal binding to cullin-2 affects the function, but not the assembly of the E3-ligase.     

Activation of DNA-hydrolyzing antibodies from the sera of autoimmune-prone MRL-lpr/lpr mice by different metal ions

We have shown previously that electrophoretically and immunologically homogeneous polyclonal IgGs from the sera of autoimmune-prone MRL mice possess DNase activity. Here we have analyzed for the first time activation of DNase antibodies (Abs) by different metal ions. Polyclonal DNase IgGs were not active in the presence of EDTA or after Abs dialysis against EDTA, but could be activated by several externally added metal (Me(2+)) ions, with the level of activity decreasing in the order Mn(2+)> or =Mg(2+)>Ca(2+)> or =Cu(2+)>Co(2+)> or =Ni(2+)> or =Zn(2+), whereas Fe(2+) did not stimulate hydrolysis of supercoiled plasmid DNA (scDNA) by the Abs. The dependencies of the initial rate on the concentration of different Me(2+) ions were generally bell-shaped, demonstrating one to four maxima at different concentrations of Me(2+) ions in the 0.1-12 mM range, depending on the particular metal ion. In the presence of all Me(2+) ions, IgGs pre-dialyzed against EDTA produced only the relaxed form of scDNA and then sequence-independent hydrolysis of relaxed DNA followed. Addition of Cu(2+), Zn(2+), or Ca(2+) inhibited the Mg(2+)-dependent hydrolysis of scDNA, while Ni(2+), Co(2+), and Mn(2+) activated this reaction. The Mn(2+)-dependent hydrolysis of scDNA was activated by Ca(2+), Ni(2+), Co(2+), and Mg(2+) ions but was inhibited by Cu(2+) and Zn(2+). After addition of the second metal ion, only in the case of Mg(2+) and Ca(2+) or Mn(2+) ions an accumulation of linear DNA (single strand breaks closely spaced in the opposite strands of DNA) was observed. Affinity chromatography on DNA-cellulose separated DNase IgGs into many subfractions with various affinities to DNA and very different levels of the relative activity (0-100%) in the presence of Mn(2+), Ca(2+), and Mg(2+) ions. In contrast to all human DNases having a single pH optimum, mouse DNase IgGs demonstrated several pronounced pH optima between 4.5 and 9.5 and these dependencies were different in the presence of Mn(2+), Ca(2+), and Mg(2+) ions. These findings demonstrate a diversity of the ability of IgG to function at different pH and to be activated by different optimal metal cofactors. Possible reasons for the diversity of polyclonal mouse abzymes are discussed.     

A study of stabilization of gluconeogenic activity in rat liver slices by calcium and manganese ions

1. The effect of some bivalent cations on gluconeogenesis by the rat liver-slice preparation has been investigated. 2. Ca(2+) and Mn(2+) stimulated glucose production from a range of substrates but not from glycerol. Mg(2+) had no effect on the rate of glucose production. 3. Ca(2+) were required to maintain phosphoenolpyruvate carboxylase activity in the slice preparation. 4. Ca(2+) and Mn(2+), but not Mg(2+), retarded the release of lysosomal enzymes from the slice into the incubation medium. 5. It is proposed that Ca(2+) and Mn(2+) stimulate glucose production by stabilizing the lysosome system in the liver-slice preparation. 6. The value of the liver-slice preparation as a means of measuring hepatic gluconeogenesis is discussed.     

Marked activation of the N-acylphosphatidylethanolamine-hydrolyzing phosphodiesterase by divalent cations.

N-Acylethanolamines including anandamide (an endogenous ligand for cannabinoid receptors) are released from N-acylphosphatidylethanolamine (N-acyl-PE) by the catalysis of a phosphodiesterase of the phospholipase D type. The enzyme was solubilized from the particulate fractions of rat heart with the aid of octyl glucoside, and partially purified by anion-exchange chromatography. The enzyme hydrolyzed N-palmitoyl-PE with a specific activity of 17 nmol/min/mg protein at 37 degrees C. The enzyme activity increased dramatically up to 30-fold by millimolar order of Ca(2+). Ca(2+) could be replaced with other divalent cations such as Co(2+), Mg(2+), Mn(2+), Ba(2+), Sr(2+) and Ni(2+). The hydrolysis of N-arachidonoyl-PE (a precursor of anandamide) was also markedly stimulated by Ca(2+).