Divalent Metal Ion Chelators Research Articles

Ethanol Extraction of Lactose from Nonfat Dry Milk: Production of Protein Raffinate

The efficiency of extracting lactose from NDM with aqueous ethanol was investigated under conditions where four factors were varied. The experiments were conducted in a complete random block design using three values for each of the variables: ratios of solvent to solids 10:1, 15:1, and 20:1 (wt/wt); solvent ethanol content of 62.4, 72.9, and 84.0% (wt/wt); temperature during extraction of 10, 25, and 50°C; and added divalent metal ion chelator (citrate) in the amount of 0, 10, and 20 mmol/100g NDM. The results were evaluated statistically using response surface methodology with a multivariate linear regression equation. Optimal extraction efficiency was obtained with high ratio of solvent to solid and low ethanol concentration of the solvent. High temperatures and high citrate concentrations tended to solvate proteins and make separation by filtration difficult. Milk protein with residual lactose of 2 to 4% was obtained using this one-step extraction process under optimal conditions.

Role of protein and calcium in stratum corneum cell cohesion.

The work reported here indicates that protein and calcium have roles in stratum corneum cell cohesion. A zwitterionic surfactant, 6-octadecyldimethyl ammoniohexanoate (C18AH), was found to completely disaggregate pig and human stratum corneum into intact, individual cells. This method of disaggregation provided a tool to determine the role of tissue components in cell cohesion. The C18AH disaggregation of pig and human stratum corneum was accelerated by proteolytic enzyme and ethylenediaminetetraacetate (EDTA). The C18AH disaggregation could be blocked by pretreatment of the stratum corneum with the serine-type proteolytic enzyme inhibitor phenylmethylsulfonyl fluoride (PMSF). The blockage could be overcome by addition of proteolytic enzyme or divalent metal ion chelator. These and other data indicate the importance of protein and calcium in stratum corneum cell cohesion.

Effects of calcium ion on ternary complexes formed between 4-(2-pyridylazo)resorcinol and the two-zinc insulin hexamer.

As a means for probing the microenvironment of zinc in the insulin hexamer and to investigate the effects of calcium ion on the assembly and the structure of the two-zinc insulin hexamer, the thermodynamics and kinetics of the reaction between the chromophoric divalent metal ion chelator 4-(2-pyridylazo)resorcinol (PAR) and zinc-insulin have been investigated over a wide range of conditions. For [PAR]0 much greater than [Zn2+]0 and [Zn2+]/[In] less than or equal to 0.33, the reaction leads to the sequestering and ultimate removal of all of the insulin-bound Zn2+; for [Zn2+]0 much greater than [PAR]0, two stable ternary complexes are formed where Zn2+ has ligands derived from PAR as well as from hexameric insulin. For [Zn2+]/[In] ratios below 0.33, the equilibrium distribution between the two ternary complexes is dependent on the [Zn2+]/[In] ratio. One of the complexes is assigned to the monoanion of PAR coordinated to Zn2+ that resides in a His-B10 site. The other complex is proposed to involve the coordination of (PAR)Zn to the site formed by the alpha-NH2 group of Phe-B1 and the gamma-carboxylate ion of Glu-A17 across the dimer-dimer interface on the surface of the hexamer. With either PAR or zinc-insulin in large excess, the kinetics of the PAR optical density changes are remarkably similar and biphasic. The faster step is first order in PAR and first order in insulin-bound Zn2+ (k congruent to 3 X 10(3) M-1 s-1) and involves the formation of an intermediate in which PAR is coordinated to insulin-bound zinc at the His-B10 site.(ABSTRACT TRUNCATED AT 250 WORDS)

Resolution of an ATP-metal chelate from metal-free ATP by reverse-phase high-performance liquid chromatography

The modulation of many enzymatic reactions involved in the metabolism of nucleotide phosphates such as ATP often require divalent metal ions. In the present study reverse-phase high-performance liquid chromatography (HPLC) was used to study the chelation of divalent metal ions, such as Mn 2+, Mg 2+, and Ca 2+, by ATP. The results of our study using radiolabeled [ 45Ca] showed that the metal-ATP chelate formed in solution was retained longer than the metal-free ATP due to the nonpolar groups on the column packing. Recovery of the two forms of ATP showed that the [ 45Ca] coeluted exclusively with the ATP-metal chelate. Other experiments showed that the retention time of the chelated form of the ATP was unaffected by eluent flow rate, but was affected by eluant pH and methanol concentration. The amount of ATP in the chelated form was found to be dependent on the amount of the metal in solution and that under appropriate conditions, i.e., with 0.1 m m CaCl 2 in the mobile phase, on the divalent cation as well. Thus, we found that in terms of effectiveness in chelate formation, the metal ions were Ca 2+ > Mg 2+ > Mn 2+. Recovery of the chelate and its reanalysis by HPLC revealed that the complex had dissociated. The chelate could be reformed by restoring the metal concentration to its original value and dissociated again by the addition of EDTA. The resolution of the ATP in a metal chelated form from the ATP in an unchelated form is discussed in terms of the stability of these chelates and the role of the hydrophobic groups of the column packing used in the reverse-phase HPLC in enhancement of this stability.

The stimulation of the brain alkaline phospholipase A1 attacking phosphatidylethanolamine by various salts and metal chelators.

Rat brain contains a soluble, high molecular weight phospholipase A1 of alkaline pH optimum which shows a preference for phosphatidylethanolamine as substrate. There is evidence that the same enzyme exists in liver and kidney. At low osmotic concentrations of buffer the enzyme is markedly stimulated by CaCl2. However, MgCl2 and MnCl2 are equally as effective although at concentrations above 2 mM the activation falls away with MnCl2. The phospholipase A1 is stimulated by divalent metal ion chelators (EDTA, EGTA, CDTA) and by sodium phosphate and sodium sulphate. The activity is inhibited by hexanol, benzyl alcohol, diethylether and detergents. Although the activity can be inhibited by saturated and unsaturated fatty acids, no evidence could be obtained that the activators function by counteracting the inhibitory action of fatty acids liberated at the interface of the substrate and incubation medium. It is suggested that to achieve good enzymic hydrolysis a certain type of organised hydrated phosphatidylethanolamine structure is required in which the negative zeta potential has been reduced by metallic cations in the incubation medium.

Processing of pre-ornithine transcarbamylase requires a zinc-dependent protease localized to the mitochondrial matrix

Proteolytic processing of the larger precursor of the rat liver mitochondrial matrix enzyme, ornithine transcarbamylase, has been studied in a cell free system using in vitro -synthesized precursor and various submitochondrial fractions. The protease responsible for cleavage to mature-sized enzyme fractionates with matrix marker enzymes. Maximal catalytic activity of this matrix protease requires approximately 0.1 mM Zn 2+ , a concentration known to be in the physiological range for this divalent cation in intact mitochondria. Certain other divalent metal ions (Co 2+ and Mn 2+ ) also stimulate this protease activity, while 1,10-phenanthroline, a divalent metal ion chelator, inhibits the protease. We conclude that proteolytic cleavage by a zinc-dependent protease in the mitochondrial matrix of rat liver is a required step in the conversion of pre-ornithine transcarbamylase to the mature subunit.

Pig Skin Epidermal Calmodulin: Effect on Calmodulin Deficient Phosphodiesterase

Calmodulin, a calcium-dependent modulator protein, is known to mediate a great number of Ca++-dependent processes in various tissues. Although it was originally described as a protein activator of cyclic nucleotide phosphodiesterase, the sensitivity of phosphodiesterases to this compound are suggested to be variable from tissue to tissue. In order to determine whether there was calmodulin-like activity in pig skin epidermis and to see its relationship to epidermal phosphodiesterase, we used an established calmodulin deficient phosphodiesterase system prepared from bovine heart. Calmodulin deficient phosphodiesterase prepared from bovine heart was markedly stimulated by the addition of pig skin (epidermal) boiled extract in the presence of calcium. Boiled skin extract alone had only little phosphodiesterase activity by itself. This effect of boiled skin extract on bovine heart phosphodiesterase was inhibited by the addition of EGTA, a divalent metal ion chelator of relative Ca++ specificity. At a fixed concentration of EGTA, increasing the Ca++ concentration counteracted the effect of EGTA. Pure pig skin epidermis (separated by trypsinization, NaBr, CaCl2-sucrose or NH4Cl treatment) was also shown to have heat-stable calmodulin activity. In contrast to the bovine heart phosphodiesterase, epidermal phosphodiesterase was only partially inhibited when Ca++ was removed by EGTA. The addition of boiled skin extract on the crude extract of epidermal phosphodiesterase had minimal effect on the enzyme activity. Overall results indicate that although pig skin epidermis contains significant amount of calmodulin, the regulation of phosphodiesterase may not be the main biological activity of epidermal calmodulin.

Role of Zn2+ and other divalent metal ions in human lymphokine production in vitro.

The effects of phenanthroline and of various metal ions on human leucocyte migration inhibitory factor (LIF) production were studied. Previously reported preliminary data showed that phenanthroline, a divalent metal ion chelator, reduced the elaboration o LIF in a dose-dependent manner by specifically but not by non-specifically stimulated lymphocytes. This paper shows that suppression of LIF production caused by phenanthroline could be entirely reversed by Zn2+, Ni2+ and, most effectively, by Co2+. When a battery of divalent cations were tested for direct inhibitory effects on LIF production, Cd2+ and, to a lesser extent, Cu2+ were found to be effective. Again, only specifically stimulated cells were susceptible. This profile of responses resembles greatly that seen in experiments with carboxypeptidases, indicating that a carboxypeptidase-like, probably Zn2+-dependent enzyme is active during antigen-induced lymphokine production. This metalloenzyme may be derived from activated monocytes/macrophages and, like the lymphocyte-activating factor, exerts its activity in the G1 phase of the cell cycle.

Chelation of divalent metal ions by brown algal polyphenols

High-molecular-weight, nondialyzable polyphloroglucinols from the marine brown algae Ascophyllum nodosum and Fucus vesiculosus (Phaeophyceae, Fucales) chelate the divalent metal ions Sr2+, Mg2+, Ca2+, Be2+, Mn2+, Cd2+, Co2+, Zn2+, Ni2+, Pb2+ and Cu2+ in weakly acidic aqueous solution. Values of the relative selectivity coefficients k have been calculated relative to Mn2+; the ionotropic series with the algal polyphenols are similar to those with catechol and pyrogallol. These studies may serve as a guide for the examination of metal chelation by brown algal polyphenols both in situ and after exudation into seawater.

On the mechanism of divalent metal ion chelator induced activation of the 7S nerve growth factor esteropeptidase. Thermodynamics and kinetics of activation.

The 7S nerve growth factor protein (7S NGF) is a multisubunit zinc metalloprotein containing a masked trypsin-like esteropeptidase activity. Reaction of the native 7S NGF oligomer with divalent metal ion chelators effects an approximately sevenfold activation of the esteropeptidase activity via the sequestering and dissociation of the 7S NGF-bound zinc ion (Pattison, S. E., and Dunn, M. F. (1975), Biochemistry 14, 2733; Pattison, S. E., and Dunn, M. F. (1976), Biochemistry, preceding paper in this issue). In this study, investigation of the relationship between chelator concentration and the extent of activation, as measured by the steady-state rate of hydrolysis of alpha-N-benzoyl-D,L-arginine-p-nitroanilide, has demonstrated that (a) the chelator-induced activation is a freely reversible process, (b) activated 7S NGF undergoes a slow loss of reversibility when incubated with chelator over long time-periods, (c) the affinity constant of 7S NGF for zinc ion is approximately 10(10.5) +/- 10(0.5) M(-1), (d) chelator activation depends only on the ability of the chelator to sequester zinc ion, and (e) the activation process does not involve dissociation of the 7S oligomer to smaller subunit aggregates under conditions of low ionic strength.

On the mechanism of divalent metal ion chelator induced activation of the 7S nerve growth factor esteropeptidase. Activation by 2,2',2''-terpyridine and by 8-hydroxyquinoline 5-sulfonic acid.

Our previous studies (Pattison, S. E., and Dunn, M. F. (1975), Biochemistry 14, 2733) have shown that the reaction of divalent metal ion chelators with the 140 000 mol wt mouse submaxillary nerve growth factor protein (7S NGF) activates the iota-subunit esteropeptidase activity ca. sevenfold. Ultraviolet-visible spectral studies with the chelator 2,2',2''-terpyridine (terpyridine) and fluorescence emission studies with 8-hydroxyquinoline-5-sulfonic acid (HQSA) in combination with both conventional and rapid-mixing stopped-flow kinetic techniques have been employed in the present study to investigate (a) the mechanism of the chelator-induced activation process, and (b) the identity of the divalent metal ion involved. The spectral studies confirm the presence of stoichiometrically significant amounts of tightly bound zinc ion in native 7S NGF (1-2 g-atoms of An2+/mol of 7S NGF). The kinetic studies show that the reaction of terpyridine with 7S NGF occurs via a two-step process involving first a rapid, apparent second-order step (k1 = 1 x 10(6) M-1 s-1) to form a 7S NGF-Zn2+-chelator monocomplex, then a slow step to form a bis(terpyridine)-Zn(II) complex and activated 7S NGF in an apparent first-order process (kobsd = 0.10 min-1). This rate is, within experimental error, identical with the apparent first-order rate constant for the chelator-induced activation process (monitored by the rate of change in the steady-state rate of hydrolysis of chromophoric substrate, alpha-N-benzoyl-D,L-arginine-p-nitroanilide). Kinetic studies of the reaction of HQSA with native 7S NGF show that, under the same conditions of concentration, the rate of formation of the tris(HQSA)-Zn(II) complex is identical with the rate of the HQSA-induced activation of the 7S NGF esteropeptidase. Thus, these studies unambiguously establish that zinc ion is the metal ion involved in the chelator-induced activation process, and that activation involves removal of zinc ion from native 7S NGF.

Inhibition of gluconeogenesis in rat renal cortex slices by metabolites of L-tryptophan in vitro.

The inhibitory effects of metabolites of L-tryptophan on gluconeo-genesis in rat renal cortex has been established. 1. Glucose production was inhibitied by quinolinate in vitro. The inhibition is due to the decreased phosphoenolpyruvate carboxykinase activity. As suggested for purified enzyme systems, quinolinate seems to exert its action in tissue slices by chelating divalent metal ions. The minimum effective extracellular concentration of the inhibitor was 5 X 10(-5) M with pyruvate as a precursor for gluconeo-genesis. 2. The effect of 3-hydroxyanthranilate is stronger (minimal effective concentration 10(-5) M) than that of quinolinate. 3-Hydroxyanthranilate may be effective in its original form and/or as a dimer degrandation product. The compound(s) exert a second effect in addition to blocking the phosphoenolpyruvate carboxykinase. This block is attained by conversion of 3-hydroxyanthranilate to quinolinate. The non-quinolinate mediated effect may be due to a reduced ATP regeneration. 3. It is suggested that kidney cortex responds sensitively to disturbances in ATP metabolism by reduction of glucose synthesis, when it is not the result of blocked formation of phosphoenolpyruvate.

On the contents of the cortical granules from Xenopus laevis eggs.

Abstract The extruded contents of the cortical granules in eggs of Xenopus laevis were solubilized by exposure to divalent metal ion chelators. Chelator extraction of cortical granule (CG) material from intact fertilized or artificially activated eggs was quantitated by fluorescence spectroscopy. The isolated fertilization envelope, formed upon interaction between CG material and the preexisting vitelline envelope, was also subject to extraction. An ultrastructural analysis revealed that chelator exposure resulted in the disruption of the structural integrity of the CG-derived F-component of the fertilization envelope. CG material was isolated from Xenopus ova by three procedures: (1) extrusion from artificially activated, dejellied eggs; (2) extraction of intact, fertilized eggs; and (3) extraction of isolated fertilization envelopes. Only 4–5% of the CG protein recovered by extrusion or by extraction of the intact fertilized egg could be associated with the isolated fertilization envelopes. One predominant polypeptide fraction with an identical relative mobility was demonstrated in all CG preparations upon polyacrylamide gel electrophoresis in SDS. Polymeric forms of CG protein were detected in chelator extracted preparations. The presence of an intact jelly coat during CG breakdown was a prerequisite to the transformation of the vitelline envelope to a fertilization envelope with altered physicochemical characteristics. Further, the CG-derived F-component of the fertilization envelope did not appear to play a critical role in determining the physicochemical properties of the fertilization envelope.