Studies on the effects of dietary beryllium at two different calcium concentrations in Achatina fulica (Pulmonata)

The mortality was highest in snails fed beryllium in the diet containing the sub-optimal concentration of calcium. There was no increase in weight over an 8 week period. Snails fed the diet containing the optimal concentration of calcium without added beryllium showed maximum weight increase but calcium alone may not be responsible for elevated growth rate. Dietary calcium enhancement appears to be responsible for the reduced concentration of zinc, magnesium, phosphate and beryllium in the tissues. Beryllium treatment did not affect the calcium concentration in the digestive gland tissue but increased zinc and magnesium in the shell. The results are discussed in relation to uptake studies and possible enzyme systems involved.     

Effects of inhibitors on luminal opening of Ca2+ binding sites in an E2P-like complex of sarcoplasmic reticulum Ca22+-ATPase with Be22+-fluoride

We document here the intrinsic fluorescence and 45Ca2+ binding properties of putative "E2P-related" complexes of Ca2+-free ATPase with fluoride, formed in the presence of magnesium, aluminum, or beryllium. Intrinsic fluorescence measurements suggest that in the absence of inhibitors, the ATPase complex with beryllium fluoride (but not those with magnesium or aluminum fluoride) does constitute an appropriate analog of the "ADP-insensitive" phosphorylated form of Ca2+-ATPase, the so-called "E2P" state. 45Ca2+ binding measurements, performed in the presence of 100 mm KCl, 5 mm Mg2+, and 20% Me2SO at pH 8, demonstrate that this ATPase complex with beryllium fluoride (but again not those with magnesium or aluminum fluoride) has its Ca2+ binding sites accessible for rapid, low affinity (submillimolar) binding of Ca2+ from the luminal side of SR. In addition, we specifically demonstrate that in this E2P-like form of ATPase, the presence of thapsigargin, 2,5-di-tert-butyl-1,4-dihydroxybenzene, or cyclopiazonic acid prevents 45Ca2+ binding (i.e. presumably prevents opening of the 45Ca2+ binding sites on the SR luminal side). Since crystals of E2P-related forms of ATPase have up to now been described in the presence of thapsigargin only, these results suggest that crystallizing an inhibitor-free E2P-like form of ATPase (like its complex with beryllium fluoride) would be highly desirable, to unambiguously confirm previous predictions about the exit pathway from the ATPase transmembrane Ca2+ binding sites to the SR luminal medium.     

Multidrug resistance protein 4 (ABCC4)-mediated ATP hydrolysis: effect of transport substrates and characterization of the post-hydrolysis transition state

Multidrug resistance protein 4 (MRP4/ABCC4), transports cyclic nucleoside monophosphates, nucleoside analog drugs, chemotherapeutic agents, and prostaglandins. In this study we characterize ATP hydrolysis by human MRP4 expressed in insect cells. MRP4 hydrolyzes ATP (Km, 0.62 mm), which is inhibited by orthovanadate and beryllium fluoride. However, unlike ATPase activity of P-glycoprotein, which is equally sensitive to both inhibitors, MRP4-ATPase is more sensitive to beryllium fluoride than to orthovanadate. 8-Azido[alpha-32P]ATP binds to MRP4 (concentration for half-maximal binding approximately 3 microm) and is displaced by ATP or by its non-hydrolyzable analog AMPPNP (concentrations for half-maximal inhibition of 13.3 and 308 microm). MRP4 substrates, the prostaglandins E1 and E2, stimulate ATP hydrolysis 2- to 3-fold but do not affect the Km for ATP. Several other substrates, azidothymidine, 9-(2-phosphonylmethoxyethyl)adenine, and methotrexate do not stimulate ATP hydrolysis but inhibit prostaglandin E2-stimulated ATP hydrolysis. Although both post-hydrolysis transition states MRP4.8-azido[alpha-32P]ADP.Vi and MRP4.8-azido[alpha-32P]ADP.beryllium fluoride can be generated, nucleotide trapping is approximately 4-fold higher with beryllium fluoride. The divalent cations Mg2+ and Mn2+ support comparable levels of nucleotide binding, hydrolysis, and trapping. However, Co2+ increases 8-azido[alpha-32P]ATP binding and beryllium fluoride-induced 8-azido[alpha-32P]ADP trapping but does not support steady-state ATP hydrolysis. ADP inhibits basal and prostaglandin E2-stimulated ATP hydrolysis (concentrations for half-maximal inhibition 0.19 and 0.25 mm, respectively) and beryllium fluoride-induced 8-azido[alpha-32P]ADP trapping, whereas Pi has no effect up to 20 mm. In aggregate, our results demonstrate that MRP4 exhibits substrate-stimulated ATP hydrolysis, and we propose a kinetic scheme suggesting that ADP release from the post-hydrolysis transition state may be the rate-limiting step during the catalytic cycle.     

Modulation of tubulin-nucleotide interactions by metal ions: comparison of beryllium with magnesium and initial studies with other cations.

With microtubule-associated proteins (MAPs) BeSO4 and MgSO4 stimulated tubulin polymerization as compared to a reaction mixture without exogenously added metal ion, while beryllium fluoride had no effect (E. Hamel et al., 1991, Arch. Biochem. Biophys. 286, 57-69). Effects of both cations were most dramatic at GTP concentrations in the same molar range as the tubulin concentration. We have now compared effects of beryllium and magnesium on tubulin-nucleotide interactions in both unpolymerized tubulin and in polymer. Polymer formed with magnesium had properties similar to those of polymer formed without exogenous cation, except for a 20% lower stoichiometry of exogenous GTP incorporated into the latter. In both polymers the incorporated GTP was hydrolyzed to GDP. Stoichiometry of GTP incorporation into polymers formed with beryllium or magnesium was identical, but much of the GTP in the beryllium polymer was not hydrolyzed. The beryllium polymer was more stable than the magnesium polymer. Beryllium also differed from magnesium in only weakly enhancing the binding of GTP in the exchangeable site of unpolymerized tubulin, while neither cation affected GDP exchange at the site. If both cations were present in a reaction mixture, polymer stability was little changed from that of the beryllium polymer, but most of the GTP incorporated into polymer was hydrolyzed. Six additional metal salts (AlCl3, CdCl2, CoCl2, MnCl2, SnCl2, and ZnCl2) also stimulated MAP-dependent tubulin polymerization, but enhanced polymer stability did not correlate with polymer GTP content. We postulate that enhanced polymer stability is a consequence of cation binding directly to tubulin and/or polymer while deficient GTP hydrolysis in the presence of beryllium, as well as aluminum and tin, is a consequence of tight binding of cation to GTP in the exchangeable site.     

A novel magnesium-dependent mechanism for the activation of transducin by fluoride

Activation of transducin-GDP by NaF is mainly mediated by aluminofluorde or beryllofluoride complexes acting as GTP gamma-phosphate analogs. In millimolar magnesium, NaF at concentrations above 3 mM is active even in the absence of aluminium or beryllium. This activation has a Hill coefficient of 3 with respect to F-, and its rate is linear with respect to Mg2+ concentrations above 2 mM. Upon fluoride dilution, inactivation rate is hundreds of times faster than for aluminofluoride-activated T alpha GDP. We propose that at high NaF concentrations, 3 hydrogen-bonded fluorides in the gamma-phosphate site of T alpha GDP entrap a magnesium counterion and this induces the transconformation to the T alpha GTP form.     

Regulatory Mechanisms of the Calcium Transport System of Fragmented Rabbit Sarcoplasmic Reticulum : I. The effect of accumulated calcium on transport and adenosine triphosphate hydrolysis

The rate of ATP hydrolysis decreases very rapidly during the first 2 sec of calcium uptake. It changes with time in a manner similar to that described for calcium net uptake by other workers, suggesting that the two activities are coupled. The decline in both rates may be ascribed to an inhibitory effect of accumulated calcium on calcium influx and ATPase activity for the following reasons. During the steady state, Ca-Ca and Sr-Ca exchange and the rate of ATP hydrolysis are much slower than the initial rate of net calcium uptake and the associated ATP hydrolysis. If the accumulation of free calcium is prevented by calcium-oxalate precipitation the initial rate of net calcium uptake does not decay during prolonged periods of transport. Furthermore, passive preloading of vesicles with calcium inhibits the rate of hydrolysis in proportion to the extent of preloading. The inhibition of steady-state flux is alleviated by free ATP; i.e., not chelated with magnesium, but not by free ITP.     

Effect of a beryllium complex on growth of Pseudomonas fluorescens (types R and S). II. Competition with magnesium]

A study of the inhibitory action of beryllium on the growth of Pseudomonas fluorescens reveals that the observed effect can be partly explained by competition between beryllium and magnesium in various processes which are indispensable to cellular metabolism. In addition, an "adaptation" phenomenon is observed which appears to be based on the selection of cells which are more highly resistant towards the inhibitor.     

Regular surface layer of Azotobacter vinelandii.

Washing Azotobacter vinelandii UW1 with Burk buffer or heating cells at 42 degrees C exposed a regular surface layer which was effectively visualized by freeze-etch electron microscopy. This layer was composed of tetragonally arranged subunits separated by a center-to-center spacing of approximately 10 nm. Cells washed with distilled water to remove an acidic major outer membrane protein with a molecular weight of 65,000 did not possess the regular surface layer. This protein, designated the S protein, specifically reattached to the surface of distilled-water-washed cells in the presence of the divalent calcium, magnesium, strontium, or beryllium cations. All of these cations except beryllium supported reassembly of the S protein into a regular tetragonal array. Although the surface localization of the S protein has been demonstrated, radioiodination of exposed envelope proteins in whole cells did not confirm this. The labeling behavior of the S protein could be explained on the basis of varying accessibilities of different tyrosine residues to iodination.     

Relative toxicities of particulate and soluble forms of beryllium to a rat liver parenchymal cell line in culture and possible mechanisms of uptake.

The relative toxicities of particulate beryllium phosphate, soluble beryllium sulphate and a beryllium sulphosalicylate complex to a rat liver parencymal derived cell line have been examined in culture. Due to the propensity of beryllium salts to form beryllium phosphate in solution the incubation medium used was free of inorganic phosphate. Cell death measured by the loss of cellular lactate dehydrogenase into the medium can be produced within 76 h from beryllium phosphate and beryllium sulphosalicylate or 48 h from beryllium sulphate provided the cells have, irrespective of the form of added beryllium, taken up a minimum of 2--5 nmol Be/10(6) cells. Whilst beryllium phosphate was readily taken up as a particle, beryllium complexed with excess sulphosalicylate was not so markedly accumulated by the cells except possibly by formation of small amounts of beryllium phosphate in the medium as a result of inorganic phosphate lost from the cells. The extent of beryllium uptake from beryllium sulphate quantitatively most resembled that observed for beryllium phosphate but was largely independent of beryllium phosphate formation in the medium and not accompanied by the uptake of the SO42- anion. However, the accumulation of beryllium derived from beryllium sulphate did appear to be associated with the production of a sedimentable from believed most probably to be colloidal beryllium hydroxide. The uptake of all forms of beryllium was temperature sensitive and metabolic inhibitor studies and treatment of the cells with trypsin or neuraminidase supported the view that the distinct behaviour of beryllium derived from beryllium sulphate may be related to the enhanced toxicity of this form both under the conditions used and when administered to experimental animals.     

The uptake and subsequent loss of beryllium by rat liver parenchymal and non-parenchymal cells after the intravenous administration of particulate and soluble forms

A cell isolation technique has been used to study the uptake and subsequent loss of beryllium (Be) by rat liver after intravenous administration of non-lethal doses of either particulate beryllium phosphate or the more hepatotoxic soluble BeSO₄. It has been shown that beryllium phosphate is removed from the blood predominantly by the non-parenchymal (sinusoidal) cells of the liver and to a lesser extent more slowly by the parenchymal cells. After 24 h when the parenchymal cells have reached maximal Be content there has been a 50% loss of Be from the non-parenchymal cells and a similar loss from whole liver which is reflected in an increased level of Be in the blood. The Be count of non-parenchymal cells subsequently decreases much more slowly in a manner similar to that of the parenchymal cells, both being only halved during the following week. Within 24–48 h some redistribution of Be to the spleen occurs and it is suggested that this in part may be the result of Kupffer cell death. In splenectomized animals a high proportion of this redistributed Be appears to be retaken up by the liver mainly by the parenchymal cell population. After administration of BeSO₄, which is known to form beryllium phosphate in plasma, a greater proportion of the Be is taken up slowly by the parenchymal cells and no redistribution of Be to the spleen is observed. It is suggested that this behaviour is related primarily to the smaller size and nature of the beryllium phosphate particles formed in plasma under these conditions. The rate of loss of Be from both the parenchymal and non-parenchymal cells is similar to that measured in beryllium phosphate treated animals. It has been estimated that liver cell death is produced when the cell content exceeds 2–3 nmol Be/10⁶ cells although parenchymal cells appear to be more sensitive to Be derived from BeSO₄ than preformed beryllium phosphate.     

Contrasting effect of hemodialysis and peritoneal dialysis on the inhibition of in vitro calcification by uremic serum

Our findings support the earlier observation of Yendt, Connor and Howard that uremic serum inhibits the calcification of rachitic rat cartilage in vitro. We also confirmed their studies showing that this inhibition is not the consequence of increased levels of serum magnesium or blood urea. In addition, we have shown that aqueous solutions of creatinine and uric acid in concentrations up to 20 mg./100 ml. do not cause any inhibition.Hemodialysis of uremic patients does not change the inhibitory activity of their blood. In contrast, after 24 hours of peritoneal dialysis, the blood of most patients does not inhibit calcification.The inhibitory activity of uremic serum, observed in vitro, may be important in the pathogenesis of osteomalacia in patients with renal failure. Failure of hemodialysis to alter this activity may contribute to the progression of renal osteodystrophy in patients on maintenance hemodialysis.     

Endogenous messenger ribonucleic acid-directed polypeptide chain elongation in a cell-free system from the yeast Saccharomyces cerevisiae.

An in vitro protein-synthesizing system from the yeast Saccharomyces cerevisiae has been made by a modification of the procedure for preparation of the Krebs ascites system. The protein synthetic activity is directed by endogenous messenger. Amino acid incorporation occurs over a broad range of magnesium and potassium concentration, being maximal at 6 and 85 mM, respcetively. The activity of this in vitro system is due to the elongation of polypeptides whose synthesis was initiated in vivo. The cell extract does not initiate synthesis with endogenous messenger ribonucleic acid (RNA), since 1 muM pactamycin, which blocks initiation on prokaryotic or eukaryotic ribosomes invitro, fails to decrease amino acid incorporation. Ten micromolar cycloheximide, however, inhibits incorporation by 87%. Moreover, this system is not stimulated by rabbit reticulocyte polysomal RNA, which directs the synthesis of hemoglobin in extracts of Krebs ascites cells. The translation of this messenger is not masked by high endogenous incorporation, because autoradiography of sodium dodecyl sulfate-polyacrylamide gels containing [35-S]methionine-labeled products shows that no hemoglobin is made. Preincubation of this system, which reduces the high endogenous incorporation by 80%, does not increase its capacity to be stimulated by either rabbit reticulocyte RNA or yeast polyriboadenylic acid-containing RNA. Polyuridylic acid, however, does stimulate polyphenylalanine incorporation. The failure of the yeast lysate to be stimulated by or to translate added natural messenger RNA, its insensitivity to low levels of pactamycin but inhibition by cycloheximide, and its relatively high magnesium optimum (the same as that for polyuridylic acid) suggest that it elongates but does not initiate polypeptide chains.     

Oxidation of thiols in the Ca2+-ATPase of sarcoplasmic reticulum microsomes

We recently showed that oxidative stress impairs the function of the sarcoplasmic reticulum to transport and retain calcium. Inhibition results primarily from oxidation of one or more thiol groups in the Ca2+-ATPase. We now report that thiol oxidation does not result in disulfide formation. Oxidative inhibition of Ca2+-ATPase activity was not reversed by dithiothreitol. Also, arsenite, which crosslinks dithiols, only mildly inhibited Ca2+-ATPase activity and protected against inhibition by peroxydisulfate. These data suggest the thiols susceptible to oxidation are not spatially close enough to form a disulfide. Furthermore, these thiols appear to be involved in some aspect of phosphoenzyme formation. ATP, in the presence of calcium and magnesium, protected against inhibition of Ca2+-ATPase activity by both oxidants and thiol-binding agents. Both inhibitors also decreased binding of the nucleotide analogue TNP-AMP after phosphorylation by Pi. Dithiothreitol and arsenite were protective. In conclusion, reversible redox regulation of the Ca2+-ATPase of sarcoplasmic reticulum by thiol-disulfide exchange does not occur. However, some other mechanism of redox regulation may operate because the enzyme is sensitive to oxidants, thiol-binding agents and activity can be enhanced by prolonged exposure to dithiothreitol.     

Adenine nucleotide-binding sites on mitochondrial F1-ATPase: studies of the inactive complex formed upon binding ADP at a catalytic site.

ADP-induced inhibition of mitochondrial F1-ATPase has been studied. It is shown that in the presence of magnesium and the absence of light, the photoaffinity ADP analog, 2-azido-ADP, induces a reversible inhibition of native F1 that is indistinguishable from that obtained with ADP. Photolysis of the inactive complex results in the predominant labeling of a catalytic-site peptide identified previously (Cross et al., 1987, Proc. Natl. Acad. Sci. USA 84, 5715-5719). Dissociation of the inactive complex formed between F1 and ADP is biphasic with a rapid azide-insensitive phase followed by a slow azide-sensitive phase (k approximately 3 x 10(-3) s-1). It is also shown that incubation of the ADP-inhibited enzyme with EDTA or phosphate does not result in release or migration of ADP from the catalytic site. However, it does convert the complex to a form that reactivates in the presence of 100 microM ATP at a rate too rapid to observe using manual mixing.     

Characterization of phenylmaleimide inhibition of the Ca(2+)-ATPase from skeletal-muscle sarcoplasmic reticulum

The Ca(2+)-ATPase from sarcoplasmic reticulum reacts with phenylmaleimide, producing the inhibition of the ATPase activity following a pseudo-first-order kinetic with a rate constant of 19 M(-1) s(-1). Calcium and ATP binding are not altered upon phenylmaleimide inhibition. However, the presence of millimolar calcium, and to a lesser extent magnesium, in the inhibition medium enhances the effect of phenylmaleimide, causing a higher degree of inhibition. Solubilization with C(12)E(8) does not affect the ATPase inhibition, excluding any kind of participation of the lipid bilayer. Phosphorylation with ATP in steady-state conditions as well as phosphorylation with inorganic phosphate in equilibrium conditions were strongly inhibited. Conversely, we have found that the occupancy of the phosphorylation site by ortovanadate fully protects against the inhibitory effect of phenylmaleimide, indicating a conformational transition associated with the phosphorylation reaction.     

Immunoenzymological Evidence Suggesting a Change in Conformation of Adenylic Acid Deaminase and Creatine Kinase during Substrate Combination

The kinetics of inhibition of 5′-adenylic acid deaminase and creatine-ATP transphosphorylase by their respective antibodies are studied and rate constants of combination are ascertained. It is shown that the single substrate 5′-adenylic acid (AMP) of deaminase “protects” the enzyme against antibody inhibition. However, phosphate, a competitive inhibitor of the highly specific deaminase, enhances combination with antibody. With creatine kinase, however, addition of either of the substrates, alone or in combination with the required magnesium, each of which separately bind to the enzyme, does not prevent inhibition of the enzyme by its antibody. However, the “working” enzyme combined with all substrates is “protected” against antibody inhibition.     

Paradoxical block of parathormone secretion is mediated by increased activity of G alpha subunits

The paradox of blunted parathormone (PTH) secretion in patients with severe hypomagnesemia has been known for more than 20 years, but the underlying mechanism is not deciphered. We determined the effect of low magnesium on in vitro PTH release and on the signals triggered by activation of the calcium-sensing receptor (CaSR). Analogous to the in vivo situation, PTH release from dispersed parathyroid cells was suppressed under low magnesium. In parallel, the two major signaling pathways responsible for CaSR-triggered block of PTH secretion, the generation of inositol phosphates, and the inhibition of cAMP were enhanced. Desensitization or pertussis toxin-mediated inhibition of CaSR-stimulated signaling suppressed the effect of low magnesium, further confirming that magnesium acts within the axis CaSR-G-protein. However, the magnesium binding site responsible for inhibition of PTH secretion is not identical with the extracellular ion binding site of the CaSR, because the magnesium deficiency-dependent signal enhancement was not altered on CaSR receptor mutants with increased or decreased affinity for calcium and magnesium. By contrast, when the magnesium affinity of the G alpha subunit was decreased, CaSR activation was no longer affected by magnesium. Thus, the paradoxical block of PTH release under magnesium deficiency seems to be mediated through a novel mechanism involving an increase in the activity of G alpha subunits of heterotrimeric G-proteins.     

LOX-1 mediates lysophosphatidylcholine-induced oxidized LDL uptake in smooth muscle cells

To assess the role of 14-3-3 proteins in the magnesium-dependent inhibition of nitrate reductase (NR) we tested the effect of magnesium on NR binding to 14-3-3s by coimmunoprecipitation and gel filtration. The stability of the 14-3-3 complex of NR was, unlike its activity, unaffected by magnesium. We therefore conclude that binding to 14-3-3s per se does not inhibit NR. Magnesium inhibited 14-3-3-bound NR much more strongly than 14-3-3-free NR. 14-3-3s possibly reinforce NR inhibition by magnesium.     

Specific inhibition of oxygenase activity of ribulose-1,5-diphosphate carboxylase by hydroxylamine

Ribulose-1,5-diphosphate oxygenase activity of ribulose-1,5-diphosphate carboxylase was completely inhibited by preincubation of the enzyme with 5mM hydroxylamine in presence of the substrate ribulose-1,5-diphosphate. Inhibition by hydroxylamine was uncompetitive with respect to ribulose-1,5-diphosphate and noncompetitive with respect to magnesium. Carboxylase activity was not affected by hydroxylamine. These results suggest that the two activities of the enzyme can be regulated differentially and that inhibiting the oxygenase activity does not stimulate the carboxylase activity of the enzyme. The data further suggest that the inhibition by hydroxylamine may be through its interaction with carbonyl groups of the enzyme exposed on the binding of ribulose-1,5-diphosphate to the protein.