Mechanism of interaction of myelin basic protein and S-100 protein: metal binding and fluorescence studies.

Abstract— It has been reported that myelin basic protein (MBP) forms a specific complex with S-100 protein in the presence of either Ca²⁺ or Mn²⁺, as detected by Immunoelectrophoresis. We have now studied the binding of Ca²⁺ and Mn²⁺ to these two proteins. We find that MBP binds 1 mol of Mn²⁺/mol of protein, and this binding produces an increment in its fluorescence, indicating a conformational change. Ca²⁺ does not bind to MBP nor does it affect the fluorescence of MBP. S-100 protein, as has been reported, binds about 10 mol of Ca²⁺/mol and this binding produces a conformational change. S-100 protein also has 25 binding sites for Mn²⁺, but this binding does not alter fluorescence and does not appear to affect conformation. Competitive binding experiments demonstrate that the binding sites of S-100 protein for Ca²⁺ and Mn²⁺ are independent. The alteration of electrophoretic migration in gels of S-100 protein produced by Ca²⁺ and of MBP produced by Mn²⁺ are in accord with the observations based on fluorescence. Mn²⁺ does not affect the electrophoretic mobility of S-100. These results indicate that the formation of the complex between MBP and S-100 protein in the presence of either Ca²⁺ or Mn²⁺ is due to the conformational change induced by these ions in S-100 protein, MBP, or both.     

Influence of some metal ions on the lipid content and arachidonic acid production byMortierella sp.

The influence of Ca²⁺, Mg²⁺, Mn²⁺, and Fe²⁺ ions on lipid accumulation, fatty acid composition and arachidonic acid (ARA) production byMortierella sp. S-17 was investigated. A beneficial effect of Mn²⁺ in the concentration range of 2–500 mg/L on lipogenesis was observed. The other elements at about 1 g/L repressed lipid accumulation and ARA yield. The highest yield of ARA (723 mg per liter or 148 mg per gram of dry mycelium) after incubation of the fungus in a glucose medium in the presence of 2 mg Mn²⁺ per liter was obtained. A strong inhibitory effect of Fe²⁺ (above 40 mg/L) on ARA formation was observed.     

Selective inhibition of L-glutamate and gammaaminobutyrate transport in nerve ending particles by aluminium,manganese, and cadmium chloride

AlCl₃, MnCl₂, and CdCl₂ inhibited the rates of accumulation of ¹⁴C] L-glutamate and ³H] gammaaminobutyrate (GABA) in purified rat forebrain nerve-ending particles in a dose-dependent fashion. The concentrations that would give 50% inhibition (IC₅₀) of GABA transport were 316 μM, 7.4 mM, and 1.4 mM, respectively. Ca²⁺ (1 mM) enhanced the inhibitory effect of Al³⁺ (IC₅₀ decreased to 149 μM) but antagonized that of Mn²⁺ (IC₅₀ = 10 mM) and Cd²⁺ (IC₅₀ = 2.1 mM). For glutamate transport 1 mM Ca²⁺ changed the IC₅₀ values from 299 to 224 μm for Al³⁺, 7.1 to 10 mM for Mn²⁺, and 2 to 3 mM for Cd²⁺. In contrast, the rates of accumulation of ¹⁴C] 2-deoxy-glucose and ³H] L-phenylalanine were mostly unaffected by these metal ions. The results indicate that Al³⁺, Mn²⁺, and Cd²⁺ exerted selective and differential effects on the transport systems of neurotransmitter substances in the synaptosomal membrane.     

Nutrient status of black spruce (Picea mariana [Mill.] BSP) forest soils dominated by Kalmia angustifolia L.

A study was conducted to determine soil chemistry in an uncut black spruce (Picea mariana) forest with and without the ericaceous understory shrub Kalmia angustifolia, as well as on a cut black spruce forest currently dominated by Kalmia. The organic (humus) and mineral (Ae, upper and lower B horizons) soils associated with Kalmia from uncut and cut forests, and non-Kalmia soils from uncut forest, were analyzed for selected soil properties. In general, mineral soils (B horizon) associated with Kalmia in uncut forest have lower values for organic matter (3.25%), organic nitrogen (0.66 mg·g⁻¹), Fe³⁺ (95.4 μg·g⁻¹) and Mn²⁺ (9 μg·g⁻¹), and higher values for pH (4.12) and Ca²⁺ (27 μg·g⁻¹) compared to non-Kalmia (organic matter, 3.43%; organic-N, 1.15 mg·g⁻¹; Fe³⁺, 431 μg·g⁻¹; Mn²⁺, 23.2 μg·g⁻¹; pH, 3.14; Ca²⁺, 15.6 μg·g⁻¹) and cut black spruce-Kalmia (organic matter, 3.74%; organic-N, 0.94 mg·g⁻¹; Fe³⁺, 379 μg·g⁻¹; Mn²⁺, 27 μg·g⁻¹; pH, 2.87; Ca²⁺, 25.2 μg·g⁻¹) forest. The high C:N ratio in Kalmia mineral soil from upper B (29.73) and lower B (identified as B+) (33.08) in uncut black spruce forest was recorded compared to non-Kalmia soils in B (18.17) and B+ (17.05) horizons in uncut black spruce forest. Phenolics leached out from Kalmia litter were lower in Kalmia associated soils than the non-Kalmia soils from the uncut forest, and Kalmia associated soils from the cut forest area. Results indicate that soils associated with Kalmia were nutrient poor particularly for nitrogen, phosphorus, iron and manganese, and provide some basis for the hypothesis that Kalmia has dominated microsites that were nutrient poor prior to Kalmia colonization.     

Kinetic properties of exchangeable calcium in guinea-pig heart mitochondria measured at low concentrations of free calcium and in the presence of Mg2+, ATP4− and inorganic phosphate

The inetic properties of exchangeable Ca²⁺ in isolated guinea-pig heart mitochondria were studied at 25°C in the presence of 0.9 mM free Mg²⁺, ATP, phosphate ions and 0.4 – 0.5 μM free Ca²⁺ using a ⁴⁵Ca²⁺ exchange technique. The simplest system which was found to be consistent with the data was one in which two kinetically-distinct compartments of exchangeable Ca²⁺ are present in the mitochondria. In the presence of 6 mM Na and at 0.4 μM free Ca²⁺, the fractional transfer rates for the transport of Ca²⁺ from these compartments were found to be 0.6 and 0.05 min^?1 and the quantities of exchangeable Ca²⁺ 0.04 and 0.2 μmol/g wet wt heart, respectively. The amount of ⁴⁵Ca²⁺ exchanged increased when the concentration of inorganic phosphate was increased, and decreased slightly when the concentration of free Mg²⁺ was increased from 1 mM to 3 mM. The flux of Ca²⁺ across the boundaries of both compartments was inhibited by an increase in the concentration of extramitochondrial Na⁺. The contribution of mitochondrial Ca²⁺ to compartments of kinetically-distinct exchangeable Ca²⁺ observed in intact cardiac muscle is briefly discussed.     

Effects of destruxins on free calcium and hydrogen ions in insect hemocytes

Destruxins, cyclohexadepsipeptidic mycotoxins isolated from the ento mopathogenic fungus Metarhizium anisopliae, inhibit innate insect immunity. However, their mechanism of action remains unclear. In this study, the effects ofdestruxins on changes in free calcium and hydrogen ions in the hemocytes ofExolontha serrulata, Bombyx mori and the Spodoptera litura SL1 cell line were detected using laser scanning confocal mi croscopy （LSCM）. An instant Ca2＋ influx of hemocytes induced by destruxins A and B （DA and DB） was recorded. The DA/DBdependent Ca2＋ influx was not influenced by the Ca2＋ channel inhibitors 2aminoethoxydiphenyl borane （2APB） and U73122. It also had an apparently different LSCM profile from that of the ionomycindependent Ca2＋ influx. However, the instant Ca2＋ influx was not seen in the SL1 cells; on the contrary, a slow, moderate enhancement of intracellular Ca2＋ was observed. Meanwhile, an instant intracellular free H＋ decrease aroused by DA and DB was found. DB at 20/zmol/L and DA at 690/zmol/L significantly reduced intracellular free H＋ levels. Furthermore, the vacuolar H＋ATPase （VATPase） inhibitor bafilomycin A1 had obvious effects on the decreases ofintracellular free H＋ in hemocytes. These results suggest that the mechanism of DA/DBdependent Ca2＋ influx is perhaps not related to Ca2＋ channels and ionophores; rather, the intracellular free H＋ decrease might be due to VATPase inhibition.     

Calcium transport by pigeon erythrocyte membrane vesicles

Membrane vesicles from pigeon erythrocytes show a rapid, ATP-dependent accumulation of ⁴⁵Ca²⁺. Ca²⁺ accumulation ratios greater than or approximately equal to 10⁴ are readily attained. For ATP-dependent Ca²⁺ uptake, V is 1.5 mmol · 1^?1 · min^?1 at 27°C (approx. 0.9 nmol · mg^?1 protein · min^?1), [Ca²⁺]_{$\text{1}\text{2}$} is 0.18 μM, [ATP]_{$\text{1}\text{2}$} is 30–60 μM, the Ca²⁺ uptake rate depends on [Ca²⁺]² and the dependence of uptake rate on ATP concentration implies strong ATP-ATP cooperativity. The Arrhenius activation energy is 19.1 ± 1.4 kcal/mol and the pH optimum is approx. 6.9.     

A Neuro-Comparative Study between Single/Successive Thorium Dose Intoxication and Alginate Treatment

The adult male albino rats were grouped into five groups (control group and four variably treated groups with thorium (Th) in single or successive with or without alginate treatment). The IP administration of thorium nitrate (13.6 mg/kg b.wt.) induced a regional distribution and accumulation ordered as cerebellum > cerebral cortex > brain stem > hippocampus > hypothalamus > striatum. Also, it induces a significant increase in Na⁺, Ca²⁺, and Fe³⁺ ion content and malondialdehyde (MDA) level while K⁺ ions and glutathione (GSH) level were significantly decreased. On the other hand, the daily oral administration of 5% alginate showed a significant decreasing in the accumulation of thorium in the different brain areas and mitigated its hazardous effects. By the alginate treatment, Na⁺, Ca²⁺, Fe³⁺, and level of MDA were declined while K⁺ ions and GSH level showed a significant increase. The improvement of the investigated parameters was attributed to the specific chelating, regeneration, and antioxidant properties of the alginate. So, alginate administration could ameliorate the hazardous effects of thorium nitrate.     

Release of endogenous dopamine, 3,4-dihydroxyphenylacetic acid,and amino acid transmitters from rat striatal slices

The release of endogenous dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) was measured in superfused striatal slices of the rat and the results compared with data obtained for the release of endogenous (a) DA and DOPAC in the cerebral cortex, nucleus accumbens and thalamus; (b) 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), GABA, and glutamate in the striatum; and (c) GABA, glutamate and 5-HT in the cerebral cortex. In superfused slices of all four CNS regions, there appeared to be a Ca²⁺-dependent, K⁺-stimulated release of endogenous DA. In addition, in slices of the striatum and nucleus accumbens there also appeared to be a Ca²⁺-dependent, 60 mM K⁺ stimulated release of endogenous DOPAC. In the striatum, 16 mM Mg²⁺ was as effective as 2.5 mM Ca²⁺ in promoting the 60 mM K⁺-stimulated release of DOPAC. In addition, 16 mM Mg²⁺ appeared to function as a weak Ca²⁺ agonist since it also promoted the release of DA to approximately 40% of the level attained with Ca²⁺ in the presence of 60 mM K⁺. On the other hand, in the striatum, 16 mM Mg²⁺ inhibited the Ca²⁺-dependent, 60 mM K⁺-stimulated release of GABA and glutamate. Similar Mg²⁺-inhibition was observed in the cerebral cortex not only for GABA and glutamate but also for DA and 5-HT. With the use of -methyl -tyrosine (tyrosine hydroxylase inhibitor), cocaine (uptake inhibitor) and pargyline (monoamine oxidase inhibitor), it was determined that (a) most of the released DA and DOPAC was synthesized in the slices during the superfusion; (b) DOPAC was not formed from DA which had been released and taken up; and (c) DA and DOPAC were released from DA nerve terminals. In addition, the data indicate a difference in the release process between the amino acids and the monoamines from striatal slices since Mg²⁺ inhibited the Ca²⁺-dependent, K⁺-stimulated release of GABA and glutamate and appeared to promote the release of DA and 5-HT.     

Temperature-dependent modification of divalent cation flux in the rat parotid gland basolateral membrane

Divalent cation (Mn²⁺, Ca²⁺) entry into rat parotid acinar cells is stimulated by the release of Ca²⁺ from the internal agonist-sensitive Ca²⁺ pool via a mechanism which is not yet defined. This study examines the effect of temperature on Mn²⁺ influx into internal Ca²⁺ pool-depleted acini (depl-acini, as a result of carbachol stimulation of acini in a Ca²⁺-free medium for 10 min) and passive ⁴⁵Ca²⁺ influx in basolateral membrane vesicles (BLMV). Mn²⁺ entry into deplacini was decreased when the incubation temperature was lowered from 37 to 4°C. At 4°C, Mn²⁺ entry appeared to be inactivated since it was not increased by raising extracellular [Mn²⁺] from 50 m up to 1 mm. The Arrhenius plot of depletion-activated Mn²⁺ entry between 37 and 8°C was nonlinear, with a change in the slope at about 21°C. The activation energy (Ea) increased from 10 kcal/mol (Q₁₀=1.7) at 21–37°C to 25 kcal/mol (Q₁₀=3.0) at 21-8°C. Under the same conditions, Mn²⁺ entry into basal (unstimulated) cells and ionomycin (5 m) permeabilized depl-acini exhibit a linear decrease, with E _a of 7.8 kcal/mol (Q₁₀=1.5) and 6.2 kcal/mol (Q₁₀ < 1.5), respectively. These data suggest that depletion-activated Mn²⁺ entry into parotid acini is regulated by a mechanism which is strongly temperature dependent and distinct from Mn²⁺ entry into unstimulated acini.As in intact acini, Ca²⁺ influx into BLMV was decreased (by 40%) when the temperature of the reaction medium was lowered from 37 to 4°C. Kinetic analysis of the initial rates of Ca²⁺ influx in BLMV at 37°C demonstrated the presence of two Ca²⁺ influx components: a saturable component, with K _Ca =279 ± 43 m, V_max = 3.38 ± 0.4 nmol Ca²⁺/mg protein/min, and an apparently unsaturable component. At 4°C, there was no significant change in the affinity of the saturable component, but V_max decreased by 61% to 1.3 ± 0.4 nmol Ca²⁺/mg protein/min. There was no detectable change in the unsaturable component. When BLMV were treated with DCCD (5 mm) or trypsin (1100, enzyme to membrane) for 30 min at 37°C there was a 40% decrease in Ca²⁺ influx. When BLMV were treated with DCCD or trypsin at 4°C and subsequently assayed for Ca²⁺ uptake at 37°C there was no significant loss of Ca²⁺ influx. These data suggest that the temperature sensitive high affinity Ca²⁺ flux component in BLMV is mediated by a protein which undergoes a modification at low temperatures, resulting in decreased Ca²⁺ transport.We thank Dr. Bruce Baum, Dr. Yukiharu Hiramatsu, Dr. Ofer Eidelman, and our other colleagues for their support during this work.     

The activation of bovine Factor X by bovine Factor Xa

A steady-state kinetic analysis of the activation of bovine Factor X, by bovine Factor X_a, was undertaken. The activation was found to be dependent on the presence of divalent cations; Ca²⁺ showing the greatest stimulatory effect and Mn²⁺ exhibiting a lower degree of activity for this reaction. Although Sr²⁺ and Mg²⁺ were ineffective when present alone, each contributed synergistically to the activation rate at suboptimal levels of Ca²⁺. The effect of phospholipid (phosphatidylcholine:phosphatidylserine, 4:1, w:w) on the rate of activation and on the activation pathway was investigated. Phospholipid (PL) concentrations of up to 40 μm had no effect on the activation rate; whereas, concentrations of 40–180 μm were slightly inhibitory. In the absence of PL, the major product of the activation was Factor α-X_a, while in the presence of PL, lower-molecular-weight forms of Factor X (Factor β-X) and Factor X_a (Factor β-X_a were produced. At saturating levels of Ca²⁺, the K_{m app} for the activation, at pH 7.4 and 37 °C, in the absence of PL, was found to be 0.6 ± 0.1 μm and the V was 1.7 ± 0.3 mol Factor X cleaved min^?1 mol^?1 Factor X_a. The corresponding values, in the presence of 90 μm PL, were 1.4 ± 0.2 μm and 2.2 ± 0.2 mol Factor X cleaved min^?1 mol^?1 Factor X_a.     

Voltage-operated Ca2+ channels regulate dopamine release from somata of dopamine neurons in the substantia nigra pars compacta

Dopamine (DA) neurons release DA not only from axon terminals at the striatum, but from their somata and dendrites at the substantia nigra pars compacta (SNc). Released DA may auto-regulate further DA release or modulate non-DA cells. However, the actual mechanism of somatodendritic DA release, especially the Ca²⁺ dependency of the process, remains controversial. In this study, we used amperometry to monitor DA release from somata of acutely isolated rat DA neurons. We found that DA neurons spontaneously released DA in the resting state. Removal of extracellular Ca²⁺ and application of blockers for voltage-operated Ca²⁺ channels (VOCCs) suppressed the frequency of secretion events. Activation of VOCCs by stimulation with K⁺-rich saline increased the frequency of secretion events, which were also sensitive to blockers for L- and T-type Ca²⁺ channels. These results suggest that Ca²⁺ influx through VOCCs regulates DA release from somata of DA neurons.     

Fe2+ uptake by mouse intestinal mucosa in vivo and by isolated intestinal brush-border membrane vesicles

In vivo kinetics of mucosal uptake of luminal ⁵⁹Fe²⁺ by tied segments of normal mouse duodenum are characterised by a K_m of approx. 100 μM and a V_max of approx. 9 pmol/min per mg wet weight of intestine. These values were determined at pH 7.25 in the presence of excess sodium ascorbate. Studies with luminal Fe²⁺ concentrations of 100 μM reveal: (1) uptake is relatively independent of ascorbate: Fe ratio and luminal pH and (2) uptake is potently inhibited by 1 mM Co²⁺ or Mn²⁺ and large luminal NaCl concentrations but not by Ca²⁺. 3 days of hypoxia (0.5 atmospheres) yields no significant increase in subsequent total mucosal uptake by in vivo tied segments while uptake is significantly reduced by semi-starvation. Quantitative comparison of in vivo mucosal uptake with subsequent determination of isolated brush-border membrane ⁵⁹Fe²⁺ transport in individual mice reveals a positive correlation (P < 0.01) between the two parameters. These results, in conjunction with studies of isolated mouse duodenal brush-border membrane (Simpson, R.J. and Peters, T.J. (1985) Biochim. Biophys. Acta, 814, 381–388 and (1986) Biochim. Biophys. Acta 856, 109–114) suggest that the Fe²⁺ transport properties of isolated brush-border membrane are quantitatively adequate to explain in vivo mucosal uptake in normal and hypoxic mice at Fe²⁺ concentrations up to 100 μM.     

Calcium Buffering and Free Ca2+ in Rat Brain Synaptosomes

Abstract: The effects of K⁺ depolarization and of stimulation by veratridine on apparent cytosolic free Ca²⁺ ([Ca²⁺]_cyt) and net Ca²⁺ accumulation were measured in isolated rat brain presynaptic nerve terminals (synaptosomes). [Ca²⁺]_cyt was determined with fura-2, and Ca²⁺ accumulation was measured with tracer ⁴⁵Ca. [Ca²⁺]_cyt was ～ 325 nM in synaptosomes incubated in the normal physiological salt solution under resting conditions. When [K⁺]₀, was increased from the normal 5 mM to 30 or 50 mM, ⁴⁵Ca uptake and [Ca²⁺]_cyt both increased within 1 s. Both increases were directly related to [Ca²⁺]₀ for [Ca²⁺]₀= 0.02–1.2 mM; however, the increase in ⁴⁵Ca uptake greatly exceeded the increase in [Ca²⁺]_cyt. With small Ca²⁺ loads ≤100 μmol/L of cell water, equivalent to the Ca²⁺ entry during a train of ≤60 impulses), the ⁴⁵Ca uptake exceeded the increase in [Ca²⁺]_cyt by a factor of nearly 1,000. This indicates that ～99.9% of the entering Ca²⁺ was buffered and/or sequestered within ～ 1 s. With larger Ca²⁺ loads, a larger fraction of the entering Ca²⁺ was buffered; ～99.97% of the load was buffered with loads of 250–425 μmol/L of cell water. The ratio between the total Ca²⁺ entry and the increase in [Ca²⁺]_cyt, the “calcium buffer ratio”β, was therefore ～ 3,500:1. This ratio was somewhat lower than the ratio of total intraterminal calcium: [Ca²⁺]_cyt, which ranged between ～7,300:1 and 12,800:1. When the synaptosomes were activated with 10 μM veratridine ([Ca²⁺]₀= 0.2–0.6 mM), ⁴⁵Ca influx and [Ca²⁺]_cyt increased progressively for ～10 s (β= 2,700:13,050:1) and then leveled off. Application of 10 μM tetrodotoxin before the introduction of veratridine prevented the increases in ⁴⁵Ca influx and [Ca²⁺]_cyt. Application of 10 μM tetrodotoxin after 5–10 s of exposure to veratridine caused both the [Ca²⁺]_cyt and the veratridine-stimulated ⁴⁵Ca within the terminals to decline, thereby demonstrating that the Ca²⁺ loading is reversible in the presence of extracellular Ca²⁺. These data show that synaptosomes are capable of buffering and metabolizing Ca²⁺ in a manner expected for intact neurons.     

Influence of some trace metals and stimulants on citric acid production from brewery wastes by Aspergillus niger

The addition of increasing levels of Mn²⁺, Fe³⁺, Zn²⁺, Co²⁺, Cu²⁺, Ca²⁺, sodium monofluoracetate and methanol during citric acid surface fermentation of spent grain liquor by Aspergillus niger (ATCC 9142) was investigated. For spent grain liquor the addition of 51 ppb Mn²⁺, 5 ppb Fe³⁺, 75 ppb Zn²⁺ and 4% (v/va) methanol caused a 4.9, 1.9, 10.9 and 16.8% increase in citric acid yield respectively. In all other fermentations the yield of citric acid was decreased whereas the biomass production in some cases was increased.     

Requirement of divalent cations for photoactivation of the laten water-oxidation system in intact chloroplasts from flashed leaves

The effects of divalent cations on photoactivation of the latent water-oxidation system in intact chloroplasts isolated from wheat (Triticum aestivum L.) leaves grown under intermittent flash illumination were investigated by using A23187, an ionophore for divalent cations, and the following results were obtained. (a) Photoactivation in the intact chloroplasts was inhibited by A23187, but was restored on addition of a low concentration of Mn²⁺ (10 μM). (b) A high concentration of Mn²⁺ (70 μM) was inhibitory, in contrast, for photoactivation, but the inhibition was restored by the coexistence of a suitable concentration of Ca²⁺ (5 mM). (c) The Ca²⁺-dependent restoration was inhibited by a high concentration of Mg²⁺ or Sr²⁺, but the inhibition was restored by the coexistence of Ca²⁺. (d) Kinetic analyses of these competitive effects between divalent cations revealed that: (i) High concentration of Ca²⁺ inhibits photoactivation in competition with Mn²⁺. (ii) High concentration of Mn²⁺ inhibits photoactivation in competition with Ca²⁺. (iii) High concentration of Mg²⁺ affects photoactivation by inhibiting Ca²⁺-dependent restoration in competition with Ca²⁺. Based on these results, we propose that the latent water-oxidation center has two binding sites, each specific for Mn²⁺ and Ca²⁺, and that photoactivation takes place in the center having both Mn²⁺ and Ca²⁺ on their respective binding sites.     

Calcium Uptake and Catecholamine Secretion by Cultured Bovine Adrenal Medulla Cells

Abstract: The uptake of ⁴⁵Ca²⁺ and secretion of catecholamines by primary cultures of adrenal medulla cells were studied. Nicotine, veratridine, potassium, and Ionomycin stimulate both the accumulation of ⁴⁵Ca²⁺ and the secretion of catecholamines. Nicotinic antagonists block ⁴⁵Ca²⁺ uptake induced by nicotine, tetrodotoxin blocks ⁴⁵Ca²⁺ uptake induced by veratridine, and D600 blocks uptake induced by K⁺, nicotine, and veratridine, but not ⁴⁵Ca²⁺ uptake or secretion induced by Ionomycin. The EC₅₀ for nicotine is 3 μm for catecholamine secretion and 10 μm for ⁴⁵Ca²⁺ uptake, while the EC₅₀S for veratridinestimulated uptake and secretion are approximately the same (75 μm ). Kinetic studies show that the uptake of Ca²⁺ is rapid and appears to precede the secretion of catecholamines, and that the rate of uptake declines rapidly. The uptake of ⁴⁵Ca²⁺ and secretion of catecholamines stimulated by veratridine and 50 mm -K⁺ show saturation kinetics with respect to external calcium concentrations at about 2 mm . On the other hand, the uptake of ⁴⁵ Ca²⁺ stimulated by nicotine does not become saturated at external calcium concentrations of 10 mm although the secretion of catecholamines reaches a maximum at external calcium concentrations of 2 mm . The data suggest that depolarizing agents such as veratridine and 50 mm -K⁺ stimulate ⁴⁵Ca²⁺ entry through voltage-sensitive calcium channels, while nicotinic agonists stimulate calcium entry through the acetylcholine receptor ion channels as well as through voltage-sensitive calcium channels.     

Purification and Characterization of Microbial Protease Produced Extracellularly from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> FBL-1

An ammonium sulfate precipitation of fermentation broth produced by Bacillus subtilis FBL-1 resulted in 2.9-fold increase of specific protease activity. An eluted protein fraction from the column chromatographies using DEAE-Cellulose and Sephadex G-75 had 94.2- and 94.9-fold higher specific protease activity, respectively. An SDS-PAGE revealed a band of purified protease at approximately 37.6 kDa. Although purified protease showed the highest activity at 45°C and pH 9.0, the activity remained stable in temperature range from 30 to 50°C and pH range from 7.0 to 9.0. Protease activity was activated by metal ions such as Ca²⁺, Mg²⁺, Mn²⁺, Fe²⁺, Ca²⁺ and K⁺, but 10 mM Fe³⁺ significantly inhibited enzyme activity (53%). Protease activity was inhibited by 2 mM EDTA as a metalloprotease inhibitor, but it showed good stability against surfactants and organic solvents. The preferred substrates for protease activity were found to be casein (100%) and soybean flour (71.6%).     

Characteristics of extracellular proteases produced by Bacillus laterosporus and Flavobacterium sp. isolated from gelatinfactory effluents

Forty bacterial isolates from the effluents of a gelatin factory (Jabalpur, India) were screened for protease activity and the two most potent producers were identified as Bacillus laterosporus and a Flavobacterium sp. The enzymes of both isolates were optimal at pH 8 and 60°C, with maximum activity after 90 min. The enzyme activity of B. laterosporus was suppressed by Fe²⁺, Mg²⁺, Mn²⁺ and Zn²⁺ ions but was enhanced by Ba²⁺ and Ca²⁺. That of Flavobacterium sp. was suppressed by Mg²⁺ and Mn²⁺ ions but enhanced by Ba²⁺, Ca²⁺ and Fe²⁺. The enzyme activity of the former was strongly inhibited by KCN, whereas that of the latter was only slightly inhibited by 8-hydroxyquinoline.     

The Divalent Metal Transporter Homologues SMF-1/2 Mediate Dopamine Neuron Sensitivity in Caenorhabditis elegans Models of Manganism and Parkinson Disease

Parkinson disease (PD) and manganism are characterized by motor deficits and a loss of dopamine (DA) neurons in the substantia nigra pars compacta. Epidemiological studies indicate significant correlations between manganese exposure and the propensity to develop PD. The vertebrate divalent metal transporter-1 (DMT-1) contributes to maintaining cellular Mn²⁺ homeostasis and has recently been implicated in Fe²⁺-mediated neurodegeneration in PD. In this study we describe a novel model for manganism that incorporates the genetically tractable nematode Caenorhabditis elegans. We show that a brief exposure to Mn²⁺ increases reactive oxygen species and glutathione production, decreases oxygen consumption and head mitochondria membrane potential, and confers DA neuronal death. DA neurodegeneration is partially dependent on a putative homologue to DMT-1, SMF-1, as genetic knockdown or deletion partially inhibits the neuronal death. Mn²⁺ also amplifies the DA neurotoxicity of the PD-associated protein α-synuclein. Furthermore, both SMF-1 and SMF-2 are expressed in DA neurons and contribute to PD-associated neurotoxicant-induced DA neuron death. These studies describe a C. elegans model for manganism and show that DMT-1 homologues contribute to Mn²⁺- and PD-associated DA neuron vulnerability.