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The tripolyphosphatase of comminuted muscle tissue is activated by MgC1(2) and ethylenediamine tetraacetate and inhibited by CaC1(2) and diphosphate. These results are discussed with reference to the formation of an inactive enzyme-substrate complex.
alpha-Latrotoxin, a polypeptide neurotoxin known to cause massive release of transmitter from vertebrate nerve terminals, is thought to act by forming cation-selective channels in plasma membranes. This paper describes the steady-state current carried by Ca2+, Sr2+ and Ba2+ through pores of alpha-LaTx molecules incorporated in artificial bilayer membranes made of neutral lipids. Even when the solutions separated by the membrane are identical, the I-V relations rectify strongly, the current being higher when the side to which the toxin is added is positive. The polarity of the rectification is consistent with the hypothesis that the mechanism of action of the toxin is, at least in part, that of promoting inwardly directed flow of cations, and thus, accumulation of Ca2+ and other ions in the intracellular spaces. The dependence of the I-V characteristics on voltage and Ca2+ concentration is well described by a one-site, one-ion model for a channel. Three parameters of the model are deduced: the binding constant of the site for Ca2+, K = 1.5 M-1 (or K = 7 M-1 when activities are used instead of concentrations); the "electrical" distance of the site from the toxin-containing solution, alpha = 0.3; the free energy difference between the two barrier peaks, delta F = 0.26 kT. The values of the parameters deduced by studying the channel in the presence of Ca2+ give theoretical curves that also fit the data with Sr2+ and Ba2+, indicating a low level of discrimination among these three cations.
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1. We examined the actions of mercury (Hg2+) and zinc (Zn2+) on voltage-activated calcium channel currents of cultured rat dorsal root ganglion (DRG) neurons, using the whole-cell patch clamp technique. 2. Micromolar concentrations of both cations reduced voltage-activated calcium channel currents. Calcium channel currents elicited by voltage jumps from a holding potential of -80 to 0 mV (mainly L- and N-currents) were reduced by Hg2+ and Zn2+. The threshold concentration for Hg2+ effects was 0.1 microM and that for Zn2+ was 10 microM. Voltage-activated calcium channel currents were abolished (> 80%) with 5 microM Hg2+ or 200 microM Zn2+. The peak calcium current was reduced to 50% (IC50) by 1.1 microM Hg2+ or 69 microM Zn2+. While Zn2+ was much more effective in reducing the T-type calcium channel current--activated by jumping from -80 to -35 mV--Hg2+ showed some increased effectiveness in reducing this current. 3. The effects of both cations occurred rapidly and a steady state was reached within 1-3 min. While the action of Zn2+ was not dependent on an open channel state, Hg2+ effects depended partially on channel activation. 4. While both metal cations reduced the calcium channel currents over the whole voltage range, some charge screening effects were detected with Hg2+ and with higher concentrations (> 100 microM) of Zn2+. 5. As Zn2+ in the concentration range used had no influence on resting membrane currents, Hg2+ caused a clear inward current at concentrations > or 2 microM.(ABSTRACT TRUNCATED AT 250 WORDS)
The kinetic order of addition of Mg2+ and L-malate to malic enzyme has been determined. Mg2+ is the first to bind, and probably acts as a link between the substrate and the active site.
The effect of Cd(2+) on intracellular Ca(2+) homeostasis was examined in renal epithelial A6 cells loaded with Fura-2. Cd(2+) (10 microM to 1 mM) produced a transient spike in cytosolic Ca(2+) in a dose-dependent manner. The phospholipase C inhibitor U73122 and the cation receptor agonist, neomycin, both diminish Cd(2+)-evoked increase in intracellular Ca(2+) ([deltaCa(2+)](Cd)). Further, thapsigargin, an inhibitor of intracellular Ca(2+)-ATPases, significantly reduced [deltaCa(2+)](Cd). Extending these observations, inositol-3-phosphate (IP(3)) binding studies showed that the resting level of intracellular IP(3) underwent a 1.45-fold increase when exposed to Cd(2+). Furthermore, we found that the Cd(2+)-related heavy metals, Zn(2+) and Ni(2+), were even more potent inducers of Ca(2+) mobilization and IP(3) generation than Cd(2+). It can be concluded that Cd(2+), and possibly Zn(2+) and Ni(2+), may act as agonists of a cation-sensing receptor (CSR) belonging to G-protein receptors capable of mediating IP(3) release of Ca(2+) from intracellular stores. The CSR receptor in A6 epithelia could not be stimulated with neomycin or Gd(3+), suggesting that the receptor is different from the calcium-sensing receptor.
Treatment of nucleic acid preparations from citrus exocortis viroid infected tissues with Ca2+, Co2+, Mn2+, and Zn2+ results in rapid precipitation of all large RNA molecules and selective precipitation of low-molecular-weight RNA species. Analysis of the viroid-RNA by polyacrylamide gel electrophoresis is greatly enhanced in the absence of any additional treatment with 2 M LiCl. The ratio of circular to linear molecules as well as the relative infectivity of Mn2+ -treated viroid preparations remain unaffected.
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Calcium limits the plasmic proteolysis of fibrinogen fragment D by binding to a specific site on the carboxy-terminal segment of the D gamma chain. Employing sodium dodecyl sulfate-polyacrylamide gel electrophoresis to visualize plasmic fragments, Sr2+, Ba2+, and Mn2+ were found to have an equivalent capacity to limit the degradation of fibrinogen fragment D (Mr 94,000). Mg2+, Fe2+, Co2+, and Zn2+ did not comparably limit the digestion of fragment D. Equilibrium dialysis demonstrated that Ba2+ competitively inhibited Ca2+ binding to fibrinogen, suggesting that the ions occupied the Ca2+ binding site of fibrinogen and thereby limited the plasmic digestion of fragment D. The results suggest that Ca2+, Sr2+, Ba2+, and Mn2+ limit plasmin digestion of fragment D by interacting with a Ca2+ binding site in the D domain of the fibrinogen molecule.
Phosphofructokinase (PFK) purified from rabbit skeletal muscle is fully inactivated after being frozen in liquid nitrogen for 30 s and thawed. The addition of 500 mM trehalose, sucrose, or proline to the enzyme solution prior to freezing results in a recovery of over 70% of the original activity after thawing. Slightly less stabilization is imparted by maltose and 4-hydroxyproline whereas glucose, glycine, inositol, and glycerol at concentrations up to 500 mM are relatively ineffective at protecting PFK. With 50 mM trimethylamine-N-oxide, almost 50% of the prefreeze activity is recovered, and this same level of cryoprotection is noted at concentrations up to 500 mM. The addition of ionic zinc to enzyme-organic solute mixtures prior to freezing greatly enhances the cryoprotection imparted by all of the solutes tested. This effect is not simply due to the summation of the individual cryoprotective capacities of zinc and the organic solute because in many instances a great degree of cryoprotection is noted when each component is present at a concentration at which, by itself, it is totally ineffective. In the presence of a constant 50 mM organic solute concentration, freeze-thaw stabilization of PFK is increased as the concentration of zinc is increased. When the zinc concentration is held constant (0.6 mM) and organic solute concentration varied, the maximum cryoprotection, in most cases, is noted with less than 50 mM organic solute. At higher solute concentrations the degree of enhancement decreases such that with 500 mM organic solute the addition of zinc results in only a slight increase in protection. The addition of ionic copper, cadmium, nickel, and cobalt to trehalose-PFK solutions prior to freezing also increases the percentage of activity recovered after thawing. Magnesium, manganese, and calcium are ineffective in this respect.
When Mg2+ ions were replaced by Mn2+ in the assay of Trypanosoma (Schizotrypanum) cruzi phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) the Km for D-fructose 6-phosphate (F6P) was reduced threefold while the corresponding constant for ATP was essentially unaffected. A detailed kinetic investigation showed that the apparent Km for F6P decreased monotonically with increasing free Mn2+ concentrations, from a limiting value of 5.7 mM in its absence to a limiting value of 1.1 mM in the presence of saturating concentrations of the ion; the Vmax of the enzyme was, on the other hand, not affected by the concentration of Mn2+. Conversely, it was shown that the apparent Km for Mn2+ at fixed MnATP concentrations decreased with increasing F6P concentrations, from a limiting value of 30 microM in the absence of the sugar phosphate to 9 microM at saturating concentrations of the substrate, while the apparent Vmax increased monotonically from zero to its limiting value. Both electron paramagnetic resonance and water proton longitudinal relaxation studies showed binding of one Mn2+ ion per 18,000 Da catalytic subunit of enzyme in the absence of F6P, with a dissociation constant of 57 +/- 4 microM, comparable to the apparent Km for the ion in the absence of F6P. The presence of saturating level of F6P decreases the value of the dissociation constant of Mn2+ to a limiting value of 7.9 microM in agreement with the results of the kinetic analysis. The substrate F6P decreases the enhancement of the water proton longitudinal relaxation rate in a saturable fashion, suggesting displacement of water molecules coordinated to the enzyme-bound Mn2+ ion by the sugar phosphate. Computer fitting of the several dissociation constants and relaxation enhancements for binary and ternary complexes gives a value of 7.9 mM for the dissociation constant of the enzyme-F6P complex in the absence of Mn2+ and 1.1 mM in the presence of saturating concentrations of the ion, in excellent agreement with the respective Km values of F6P extrapolated to zero and saturating Mn2+, respectively. Studies of the frequency dependence of the water proton longitudinal relaxation rate enhancements in the presence of both binary (enzyme-Mn2+) and ternary (enzyme-Mn2(+)-F6P) complexes, are most simply explained by assuming two exchangeable water molecules in the coordination sphere of the enzyme-bound Mn2+ in the binary complex, while in the ternary complex the data are consistent with the displacement of one of the water molecule from the coordination sphere with no significant alteration of the correlation time. Overall, the kinetic and binding data are consistent with the formation of an enzyme-metal-F6P bridge complex at the active site of T. cruzi phosphofructokinase, a coordination scheme which is unique among the phosphofructokinases.
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The broadening of spin-label absorption lines resulting from spin-exchange reactions that occur during collision with paramagnetic Ni2+ is diminished when Ni2+ binds to phospholipid vesicles. Subsequent addition of non-paramagnetic ions that compete for binding sites releases Ni2+ into solution and restores the line-broadening. The concentrations of various ions required to achieve this effect was used to order the ions with respect to their binding to vesicles containing phosphatidylethanolamine and phosphatidylglycerol. The relative strengths of binding for those ions studied were: Ca2+ > Mg2+ > Zn2+ > Sr2+ > Ba2+. The spin-broadening assay was also used to study the effects of two proteins on the availability of Ni2+-binding sites on the vesicles. Ribonuclease, which is thought to associate electrostatically as an extrinsic protein on the surface of vesicles, completely blocked the Ni2+-binding sites at comparatively low protein concentrations. Quantitative considerations of these data suggest the possibility that Ni2+ may bind preferenetially to phosphatidylglycerol, and that these binding sites are aggregated in the ribonuclease-containing vesicles. In contract to ribonuclease, cytochrome c does not block Ni2+-bindings sites on the phospholipid vesicles, but rather contains sites of its own that bind Ni2+, both when the protein is in solution and when it is associated with the vesicles. These results are consistent with other studies which suggest that cytochrome c becomes partially embedded in membrane bilayers and associates with phospholipid molecules through hydrophobic interactions.