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At least 685 records · Page 38Linked to original sources
PubMed1982

Carnitine acyltransferase activities in rat liver and heart measured with palmitoyl-CoA and octanoyl-CoA. Latency, effects of K+, bivalent metal ions and malonyl-CoA.

1. Liver carnitine acyltransferase activities with palmitoyl-CoA and octanoyl-CoA as substrates and heart carnitine palmitoyltransferase were measured as overt activities in whole mitochondria or in mitochondria disrupted by sonication or detergent treatment. All measurements were made in sucrose/KCl-based media of 300 mosmol/litre. 2. In liver mitochondria, acyltransferase measured with octanoyl-CoA, like carnitine palmitoyltransferase, was found to have latent and overt activities. 3. Liver acyltransferase activities measured with octanoyl-CoA and palmitoyl-CoA differed in their response to changes in [K+], Triton X-100 treatment and, in particular, in their response to Mg2+. Mg2+ stimulated activity with octanoyl-CoA, but inhibited carnitine palmitoyltransferase. 4. The effects of K+ and Mg2+ on liver overt carnitine palmitoyltransferase activity were abolished by Triton X-100 treatment. 5. Heart overt carnitine palmitoyltransferase activity differed from the corresponding activity in liver in that it was more sensitive to changes in [K+] and was stimulated by Mg2+. Heart had less latent carnitine palmitoyltransferase activity than did liver. 6. Overt carnitine palmitoyltransferase in heart mitochondria was extremely sensitive to inhibition by malonyl-CoA. Triton X-100 abolished the effect of low concentrations of malonyl-CoA on this activity. 7. The inhibitory effect of malonyl-CoA on heart carnitine palmitoyltransferase could be overcome by increasing the concentration of palmitoyl-CoA.

Acyl Coenzyme A↗
PubMed1983

Low-molecular-weight constituents of isolated insulin-secretory granules. Bivalent cations, adenine nucleotides and inorganic phosphate.

The concentrations of Zn2+, Ca2+, Mg2+, Pi and adenine nucleotides were determined in insulin-secretory granules prepared from a transplantable rat insulinoma. Differential and density-gradient centrifugation analyses revealed that Zn2+ in this tissue was principally localized in the secretory granule, a second major fraction being found in association with cytosolic proteins. Pi was principally recovered in the latter fraction, whereas Ca2+ and Mg2+ were more widely distributed. Intragranular ion-distribution experiments suggested that Zn2+ was complexed mainly to insulin and its precursor forms and remained in the granule in an insoluble state. The Zn2+/insulin ratio (0.54) was greater than that expected for insulin molecules having two centrally co-ordinated Zn2+ atoms/hexamer, but less than the maximal Zn2+-binding capacity of the molecule. Most of the granular Ca2+, Mg2+ and Pi was released in a soluble form when granules were disrupted by sonication. Simulation in vitro of the ionic composition of the granule suggested that up to 90% of its Ca2+ was complexed to Pi and adenine nucleotides. Granular macromolecules also bound Ca2+, as shown by equilibrium-dialysis studies of granule lysates. However, such binding was displaced by Mg2+. Examination of the efflux of Ca2+ from granules incubated in iso-osmotic suspensions at 37 degrees C suggested that the passive permeability of the granule membrane to Ca2+ was very low. Nevertheless, more than 50% of the granular Ca2+ was rapidly released in an ionized form on hypo-osmotic or detergent-induced disruption of the granule membrane. This may represent a potentially mobilizable pool of Ca2+ in vivo.

Adenine Nucleotides↗
PubMed1983

Interactions between retinyl phosphate and bivalent cations.

In the presence of Mn(II) ions, the u.v. absorption spectrum of retinyl phosphate (Ret-P) solubilized in Triton X-100 micelles, phosphatidylcholine liposomes or rat liver microsomes exhibited a shift from the maximum of 330 nm to 287 nm. The effect of Mn(II) was reversed by adding EDTA or phosphate buffer. The same spectral change was found in the presence of poly-L-lysine in place of Mn(II) ions. The e.s.r. spectrum of Mn(II) in the presence or in the absence of Ret-P clearly showed that approx. 75% of the initial concentration of Mn(II) ions is bound to Ret-P when the molar ratio of Ret-P to Mn(II) ions is 4:1; no such binding occurred in the presence of retinol or retinoic acid. The appearance of two isosbestic points at 303 and 368 nm, in the presence of Mn(II) ions, suggests the existence of an equilibrium between an Mn(II)-bound monomer and an Mn(II)-bound dimer of Ret-P in Triton X-100 micelles. The same effect on the u.v.-absorption spectrum of Ret-P was also induced by Co(II), Cr(II), Zn(II) and Fe(II), but not by Mg2+ or Cu(II). The formation of the 'metachromatic complex' between Ret-P and Mn(II) or Co(II) inhibited the synthesis of retinyl phosphate mannose (Ret-P-Man) from exogenous and endogenous Ret-P and guanosine diphosphate [14C]mannose when bovine serum albumin was added after the metal ion. However, the order of addition did not influence Ret-P-Man synthesis in incubations containing MgCl2, which does not form the metachromatic complex with Ret-P. These results suggest that the bioavailability of proteins, polyamines and metal ions may control the extent to which Ret-P can be mannosylated in the intact membrane.

Animals↗
PubMed1984

Conformational changes induced by binding of bivalent cations to oncomodulin, a paravalbumin-like tumour protein.

When Mg2+ was added to rat oncomodulin, a paravalbumin-like tumour protein, changes in the c.d. spectrum and tyrosine fluorescence intensity were observed. The addition of Ca2+ resulted in even greater changes in these spectra. The fluorescence excitation spectra of apo- and Mg-oncomodulin were superimposable, whereas that of Ca-oncomodulin was markedly different. The u.v.-absorption spectrum of the Ca2+ form also showed major differences from those of the other two forms. These observations indicate that Ca2+ induced a significant and specific conformational change in the protein that was not observed on binding Mg2+. In contrast, the conformational change induced by either Mg2+ or Ca2+ was identical in the homologous rat parvalbumin. This Ca2+-specific conformational change may be the basis for oncomodulin's Ca2+-dependent protein/protein interaction.

Animals↗
PubMed1986

Studies on the bivalent-cation-activated ATPase activities of highly purified human platelet surface and intracellular membranes.

Membrane-bound Ca2+-ATPases are responsible for the energy-dependent transport of Ca2+ across membrane barriers against concentration gradients. Such enzymes have been identified in sarcoplasmic reticulum of muscle tissues and in non-muscle cells in both surface membranes and endoplasmic-reticulum-like intracellular membrane complexes. In a previous study using membrane fractionation by density-gradient and free-flow electrophoresis, we reported that the intracellular membranes of human blood platelets were a major storage site for Ca2+ and involved in maintaining low cytosol [Ca2+] in the unactivated cell. In the present report we demonstrated that the intracellular membranes also exhibit a high-affinity Ca2+-ATPase which appears to be kinetically associated with the Ca2+-sequestering process. We found that both the surface membrane and the intracellular membrane exhibited a basal Mg2+-ATPase activity, but Ca2+ activation of this enzyme was confined only to the intracellular membrane. Use of Ca2+-EGTA buffers to control the extravesicle [Ca2+] allowed a direct comparison of the Ca2+-ATPase and the Ca2+-uptake process over a Ca2+ range of 0.01 microM to 1.0 mM, and it was found that both properties were maximally expressed in the range of external [Ca2+] 1-50 microM, with concentrations greater than 100 microM showing substantial inhibition. Double-reciprocal plots for the Ca2+-ATPase activity and Ca2+ uptake gave apparent Km values for Ca2+ of 0.15 and 0.13 microM respectively. However, similar plots for ATP with the enzyme revealed a discontinuity (two affinity sites, with Km 20 and 145 microM), whereas plots for the Ca2+ uptake gave a single Km value for Ca2+, 1.1 microM. Phosphorylation studies during Ca2+ uptake using [gamma-32P]ATP revealed two components of 90 and 95 kDa phosphorylated at extravesicle [Ca2+] of 3 microM. The Ca2+-ATPase activity, Ca2+ uptake and phosphorylation were all almost completely inhibited in the presence of 500 microM-Ca2+. Similar studies using mixed membranes revealed four other phosphoproteins (50, 40, 20 and 18 kDa) formed in addition to the 90 and 95 kDa components. The findings are discussed in the context of platelet Ca2+ mobilization for function and the mechanisms whereby Ca2+ homoeostasis is controlled in the unactivated cell.

Adenosine Triphosphatases↗
PubMed1987

Guanine-nucleotide and hormone regulation of polyphosphoinositide phospholipase C activity of rat liver plasma membranes. Bivalent-cation and phospholipid requirements.

The effect of the GTP analogue guanosine 5'-[gamma-thio]triphosphate (GTP[S]) on the polyphosphoinositide phospholipase C (PLC) of rat liver was examined by using exogenous [3H]phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]. GTP[S] stimulated the membrane-bound PLC up to 20-fold, with a half-maximal effect at approx. 100 nM. Stimulation was also observed with guanosine 5'-[beta gamma-imido]triphosphate, but not with adenosine 5'-[gamma-thio]triphosphate, and was inhibited by guanosine 5'-[beta-thio]diphosphate. Membrane-bound PLC was entirely Ca2+-dependent, and GTP[S] produced both a decrease in the Ca2+ requirement and an increase in activity at saturating [Ca2+]. The stimulatory action of GTP[S] required millimolar Mg2+. [8-arginine]Vasopressin (100 nM) stimulated the PLC activity approx. 2-fold in the presence of 10 nM-GTP[S], but had no effect in the absence of GTP[S] or at 1 microM-GTP[S]. The hydrolysis of PtdIns(4,5)P2 by membrane-bound PLC was increased when the substrate was mixed with phosphatidylethanolamine, phosphatidylcholine or various combinations of these with phosphatidylserine. With PtdIns(4,5)P2, alone or mixed with phosphatidylcholine, GTP[S] evoked little or no stimulation of the PLC activity. However, maximal stimulation by GTP[S] was observed in the presence of a 2-fold molar excess of phosphatidylserine or various combinations of phosphatidylethanolamine and phosphatidylserine. Hydrolysis of [3H]phosphatidylinositol 4-phosphate by membrane-bound PLC was also increased by GTP[S]. However, [3H]phosphatidylinositol was a poor substrate, and its hydrolysis was barely affected by GTP[S]. Cytosolic PtdIns(4,5)P2-PLC exhibited a Ca2+-dependence similar to that of the membrane-bound activity, but was unaffected by GTP[S]. It is concluded that rat liver plasma membranes possess a Ca2+-dependent polyphosphoinositide PLC that is activated by hormones and GTP analogues, depending on the Mg2+ concentration and phospholipid environment. It is proposed that GTP analogues and hormones, acting through a guanine nucleotide-binding protein, activate the enzyme mainly by lowering its Ca2+ requirement.

Adenosine Triphosphate↗
PubMed1990

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.

Animals↗
PubMed1992

Disruption of prosomes by some bivalent metal ions results in the loss of their multicatalytic proteinase activity and cancels the nuclease resistance of prosomal RNA.

Prosomes are ribonucleoprotein particles constituted by a variable set of about 20 proteins found associated with untranslated mRNA. In addition, they contain a small RNA, the presence of which has been an issue of controversy for a long time. The intact particles have a multicatalytic proteinase (MCP) activity and are very stable; we have never observed autodigestion of the particle by its intrinsic proteinase activity. Surprisingly it was found that Zn2+ and Cu2+ ions at concentrations of 0.1-1 mM disrupt the prosome particles isolated from HeLa cells and duck erythroblasts and abolish instantaneously its MCP activity, without altering the two-dimensional electrophoretic pattern of the constituent proteins. Fe2+, however, seems to induce autodegradation rather than dissociation of the prosome constituents. Most interestingly, protein or oligopeptide substrates protect the particle and its proteinase activity from disruption by Zn2+ or Cu2+. Nuclease-digestion assays reveal that the prosomal RNA, which is largely resistant in the intact particle, becomes digestible after dissociation of prosomes by Zn2+. These data give, for the first time, unambiguous proof of the presence of an RNA in the particle. Furthermore, they demonstrate a structure-function relationship between the complex and its enzyme activity, which seems to be based on the particle as an entity and not on the single constituent proteins.

Animals↗
PubMed1994

The effect of calcium-store depletion and refilling with various bivalent cations on tyrosine phosphorylation and Mn2+ entry in fura-2-loaded human platelets.

To investigate the possible involvement of tyrosine phosphorylation in the process of store-regulated Ca2+ entry, ionomycin (in the presence of EGTA) was used to deplete the intracellular Ca2+ stores of fura-2-loaded human platelets, and the effect of refilling with Ca2+, Ba2+ or Sr2+ evaluated. Depletion of the intracellular Ca2+ stores resulted in an increase in protein tyrosine phosporylation. This increase is reversed when the stores were refilled in Ca2+ or Sr2+, but not Ba2+. Refilling of the stores with Ca2+ or Sr2+, but not Ba2+, suppressed Mn2+ entry. These findings support the hypothesis that tyrosine phosphorylation plays a role in mediating store-regulated Ca2+ entry in human platelets and provides evidence for tyrosine phosphatase activity regulated by the Ca2+ content of the intracellular stores.

Barium↗
PubMed1996

Differentiation of BC3H1 smooth muscle cells changes the bivalent cation selectivity of the capacitative Ca2+ entry pathway.

Differentiation of BC3H1 cells leads to expression of a variety of proteins characteristic of smooth muscle and to changes in the behaviour of intracellular Ca2+ stores. Treatment of both differentiated and undifferentiated cells with thapsigargin (2 microM) emptied their intracellular Ca2+ stores, and in the presence of extracellular Ca2+ caused an increase in cytosolic [Ca2+] that rapidly reversed after its removal. The amplitudes of these capacitative Ca2+ entry signals were 101 +/- 8 nM (n = 42) in differentiated cells and 188 +/- 16 nM (n = 35) in undifferentiated cells. Mn2+ entry in thapsigargin-treated cells, measured by recording the quenching of cytosolic fura 2 fluorescence, was 374 +/- 26% (n = 34) and 154 +/- 7% (n = 41) of control rates in differentiated and undifferentiated cells, respectively. Empty stores caused Ba2+ entry to increase to 282 +/- 20% (n = 8) of its basal rate in differentiated cells and to 187 +/- 20% (n = 8) in undifferentiated cells. Rates of Ca2+ extrusion, measured after rapid removal of extracellular Ca2+ from cells in which capacitative Ca2+ entry had been activated, were similar in differentiated (t1/2 = 23 +/- 2 s, n = 7) and undifferentiated (23 +/- 1 s, n = 6) cells. The different relationships between capacitative Ca2+ and Mn2+ signals are not, therefore, a consequence of more active Ca2+ extrusion mechanisms in differentiated cells, nor are they a consequence of different fura 2 loadings in the two cell types. We conclude that during differentiation of BC3Hl cells, the cation selectivity of the capacitative pathway changes, becoming relatively more permeable to Mn2+ and Ba2+. The change may result either from expression of a different capacitative pathway or from modification of the permeation properties of a single pathway.

Adenosine Triphosphate↗
PubMed1997

Bivalent cations stabilize yeast alcohol dehydrogenase I.

The thermostability of yeast alcohol dehydrogenase (ADH) I is strongly dependent on the presence of NaCl, a salt that is almost neutral on the Hofmeister scale, which suggests that solvent-accessible electrostatic repulsion might play a role in the inactivation of the enzyme. Moreover, CaCl2 and MgCl2 are able to stabilize the enzyme at millimolar concentrations. Ca2+ stabilizes yeast ADH I by preventing the dissociation of the reduced form of the enzyme and by preventing the unfolding of the oxidized form of the enzyme. An analysis of several chimaeric ADHs suggests that Ca2+ is fixed by the Asp-236 and Glu-101 side chains in yeast ADH I, but that Ca2+ can be displaced by replacing Met-168 by an Arg residue, as suggested by a three-dimensional model of the enzyme structure. These results indicate that electrostatic repulsion can cause protein unfolding and/or dissociation. It is proposed that yeast ADH I binds Mg2+ in vivo.

Alcohol Dehydrogenase↗
PubMed1997

Regulation by bivalent cations of phospholipid binding to the C2A domain of synaptotagmin III.

Synaptotagmins are Ca2+-and phospholipid-binding proteins of synaptic vesicles that might function as Ca2+ receptors for neurotransmitter release via their first C2 (C2A) domain. Here we describe the effect of Mg2+ on phospholipid binding to the C2A domains of multiple synaptotagmins (II-VI), and demonstrate that only synaptotagmin III can bind negatively charged phospholipids [phosphatidylserine (PS) and phosphatidylinositol] in a Mg2+-dependent manner. The Mg2+-dependent interaction with PS was found to have an EC50 of approx. 30 microM Mg2+, which is comparable to that of Sr2+ and Ba2+ (EC50 values of approx. 10 microM). This binding property of the C2A domain is specific to synaptotagmin III, because none of the C2A domains of other proteins, such as rabphilin 3A, Doc2alpha, Doc2beta or Gap1(m), showed phospholipid binding activity in the presence of 1 mM Mg2+. Our results suggest that synaptotagmin III is involved in presynaptic functions different from those of synaptotagmins I and II.

Amino Acid Sequence↗
PubMed1977

Plasma calcitonin in chronic renal failure: relation to other factors of importance in bivalent ion metabolism.

1. Plasma concentrations of human calcitonin were measured in groups of patients with chronic renal failure, treated either conservatively or by haemodialysis, and compared with a normal group of persons. 2. Plasma calcitonin was statistically significantly elevated in both groups with renal failure. 3. When the data from the three groups were pooled, plasma calcitonin was found to be inversely correlated with total calcium and directly correlated with plasma creatinine.

Calcitonin↗
PubMed2001

Bivalent cations and amino-acid composition contribute to the thermostability of Bacillus licheniformis xylose isomerase.

Comparative analysis of genome sequence data from mesophilic and hyperthermophilic micro-organisms has revealed a strong bias against specific thermolabile amino-acid residues (i.e. N and Q) in hyperthermophilic proteins. The N + Q content of class II xylose isomerases (XIs) from mesophiles, moderate thermophiles, and hyperthermophiles was examined. It was found to correlate inversely with the growth temperature of the source organism in all cases examined, except for the previously uncharacterized XI from Bacillus licheniformis DSM13 (BLXI), which had an N + Q content comparable to that of homologs from much more thermophilic sources. To determine whether BLXI behaves as a thermostable enzyme, it was expressed in Escherichia coli, and the thermostability and activity properties of the recombinant enzyme were studied. Indeed, it was optimally active at 70-72 degrees C, which is significantly higher than the optimal growth temperature (37 degrees C) of B. licheniformis. The kinetic properties of BLXI, determined at 60 degrees C with glucose and xylose as substrates, were comparable to those of other class II XIs. The stability of BLXI was dependent on the metallic cation present in its two metal-binding sites. The enzyme thermostability increased in the order apoenzyme < Mg2+-enzyme < Co2+-enzyme approximately Mn2+-enzyme, with melting temperatures of 50.3 degrees C, 53.3 degrees C, 73.4 degrees C, and 73.6 degrees C. BLXI inactivation was first-order in all conditions examined. The energy of activation for irreversible inactivation was also strongly influenced by the metal present, ranging from 342 kJ x mol(-1) (apoenzyme) to 604 kJ x mol(-1) (Mg2+-enzyme) to 1166 kJ x mol(-1) (Co2+-enzyme). These results suggest that the first irreversible event in BLXI unfolding is the release of one or both of its metals from the active site. Although N + Q content was an indicator of thermostability for class II XIs, this pattern may not hold for other sets of homologous enzymes. In fact, the extremely thermostable alpha-amylase from B. licheniformis was found to have an average N + Q content compared with homologous enzymes from a variety of mesophilic and thermophilic sources. Thus, it would appear that protein thermostability is a function of more complex molecular determinants than amino-acid content alone.

Aldose-Ketose Isomerases↗