Mechanism of Inhibition of Calcium Uptake into Sarcoplasmic Reticulum by Notexin,a Neurotoxic and Myotoxic Polypeptide

Notexin belongs to a class of snake venom neurotoxins and myotoxins that have phospholipase A₂ activity. Previous studies have shown that these toxins affect target cells differently from phospholipases that are not neurotoxic or myotoxic. Notexin inhibited the Ca²⁺ uptake into fragmented sarcoplasmic reticulum from rabbit skeletal muscle, but it did not cause an efflux of previously accumulated Ca²⁺ or inhibit the Ca²⁺–ATPase activity. It is suggested that notexin specifically binds to and decreases the conductance for Ca²⁺ of the Ca²⁺ pump and/or the conductance of a channel for an ion that facilitates Ca²⁺ transport. The K⁺ ionophore valinomycin reversed the notexin-induced inhibition of Ca²⁺ uptake into sarcoplasmic reticulum, suggesting that the molecular target of notexin could be a K⁺ channel. Two types of reconstitution experiments make it unlikely that notexin acts by degrading a minor lipid that is resistant to hydrolysis by nontoxic phospholipases A₂. Notexininactivated sarcoplasmic reticulum vesicles were reactivated (with respect to Ca²⁺ uptake) by simple solubilization with detergent and subsequent reconstitution by detergent removal. Second, notexin was still active on sarcoplasmic reticulum vesicles after >94% of the lipids were replaced by soybean phosphoglycerides during the reconstitution procedure.     

亲和层析纯化肌质网Ca2+－ATP酶

建立了一种亲和层析纯化肌质网Ca²⁺-ATP酶的方法．用非离子型去污剂C₁₂E₈ 溶解肌质网,再通过反应红－120琼脂糖亲和层析柱使肌质网Ca²⁺-ATP酶纯度从粗品中的65％提高到99％,并具有较高ATP水解活性．经SDS－聚丙烯酰胺凝胶电泳检测,为电泳纯．     

Functional Reconstitution of an ATP-Driven Ca-Transport System from the Plasma Membrane of Commelina communis L

The protein(s) that constitute(s) the ATP-driven Ca²⁺-translocator of plasma membrane enriched vesicles obtained by aqueous two-phase partitioning from leaves of Commelina communis L. has/have been solubilized and reincorporated into tightly sealed liposomes. The reconstituted Ca²⁺-transport system was studied using ATP-driven ⁴⁵Ca²⁺ import into the proteoliposomes as a measure of activity. The detergent, 3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonate proved to be the most suitable and was used at 10 millimolar concentration, i.e. just above its critical micellar concentration. The presence of additional phospholipid (2 milligrams phosphatidylcholine per milliliter) and ATP (5 millimolar) improved the solubilization and/or reconstitution. The characteristics of the reconstituted system were similar to those of the plasma membrane-bound activity, including the apparent K_m for Ca²⁺ (5.2 micromolar), inhibition by relatively high levels of vanadate (IC₅₀ = 500 micromolar) and lacking response to added calmodulin. The reconstituted transport system was very strongly inhibited by erythrosine B (IC₅₀ = 0.01 micromolar) and had a low apparent K_m for ATP (11.4 micromolar). As in the plasma membrane vesicles, the protonophore carbonylcyanide m-chlorophenyl hydrazone did not affect Ca²⁺-transport detectably in the reconstituted system. However, low levels of the Ca²⁺-ionophore A 23187 instantaneously discharged 90% of the Ca²⁺ associated with the vesicles, proving that it had been accumulated in the intravesicular volume in soluble, freely exchangeable form. Ca²⁺-transport in the reconstituted system was thus primary active, through a Ca²⁺-translocating ATPase. The system reported here may serve as a valuable tool for purifying the Ca²⁺-ATPase and for studying structural and functional aspects of the purified enzyme.     

Detergent-compatible,organic solvent-tolerant alkaline protease from Bacillus circulans MTCC 7942: Purification and characterization

Proteases are now recognized as the most indispensable industrial biocatalyst owing to their diverse microbial sources and innovative applications. In the present investigation, a thermostable, organic solvent-tolerant, alkaline serine protease from Bacillus circulans MTCC 7942, was purified and characterized. The protease was purified to 37-fold by a three-step purification scheme with 39% recovery. The optimum pH and temperature for protease was 10 and 60°C, respectively. The apparent molecular mass of the purified enzyme was 43 kD as revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The K_m and V_max values using casein-substrate were 3.1 mg/mL and 1.8 µmol/min, respectively. The protease remained stable in the presence of organic solvents with higher (>3.2) log P value (cyclohexane, n-octane, n-hexadecane, n-decane, and n-dodecane), as compared to organic solvents with lower (<3.2) log P value (acetone, butanol, benzene, chloroform, toluene). Remarkably, the protease showed profound stability even in the presence of organic solvents with less log P values (glycerol, dimethyl sulfate [DMSO], p-xylene), indicating the possibility of nonaqueous enzymatic applications. Also, protease activity was improved in the presence of metal ions (Ca²⁺, Mg²⁺, Mn²⁺); enhanced by biosurfactants; hardly affected by bleaching agents, oxidizing agents, and chemical surfactants; and stable in commercial detergents. In addition, a protease–detergent formulation effectively washed out egg and blood stains as compared to detergent alone. The protease was suitable for various commercial applications like processing of gelatinous film and as a compatible additive to detergent formulation with its operative utility in hard water.     

Solubilization and reconstitution of ca pump from corn leaf plasma membrane

The Ca²⁺ transport system of corn (Zea mays) leaf plasma membrane is composed of Ca²⁺ pump and Ca²⁺/H⁺ antiporter driven by H⁺ gradient imposed by a H⁺ pump (M Kasai, S Muto [1990] J Membr Biol 114: 133-142). It is necessary for characterization of these Ca²⁺ transporters to establish the procedure for their solubilization, isolation, and reconstitution into liposomes. We attempted to solubilize and reconstitute the Ca²⁺ pump in the present study. A nonionic detergent octaethyleneglycol monododecyl ether (C₁₂E₈) was the most effective detergent for a series of extraction and functional reconstitution of the Ca²⁺ pump among seven detergents examined. This was judged from activities of ATP-dependent ⁴⁵Ca²⁺ uptake into liposomes reconstituted with the respective detergent-extract of the plasma membrane by the detergent dilution method. C₁₂E₈-extract of the plasma membrane was subjected to high performance liquid chromatography using a DEAE anion exchange column. Ca²⁺-ATPase was separated from VO₄³⁻-sensitive Mg²⁺-ATPase. These ATPases were separately reconstituted into liposomes, and their ATP-dependent Ca²⁺ uptake was measured. The liposomes reconstituted with the Ca²⁺-ATPase, but not with the VO₄³⁻-sensitive Mg²⁺-ATPase, showed ATP-dependent Ca²⁺ uptake. Nigericin-induced pH gradient (acid inside) caused only a little Ca²⁺ uptake into liposomes reconstituted with the Ca²⁺-ATPase, suggesting that the Ca²⁺/H⁺ antiporter was not present in the preparation. These results indicate that the Ca²⁺-ATPase actually functions as Ca²⁺ pump in the corn leaf plasma membrane.     

Purification and Characterization of a Co(II)-Sensitive α-Mannosidase from Ginkgo biloba Seeds

An α-mannosidase was purified from developing Ginkgo biloba seeds to apparently homogeneity. The molecular weight of the purified α-mannosidase was estimated to be 120 kDa by SDS–PAGE in the presence of 2-mercaptoethanol, and 340 kDa by gel filtration, indicating that Ginkgo α-mannosidase may function in oligomeric structures in the plant cell. The N-terminal amino acid sequence of the purified enzyme was Ala–Phe–Met–Lys–Tyr–X–Thr–Thr–Gly–Gly–Pro–Val–Ala–Gly–Lys–Ile–Asn–Val–His–Leu–. The α-mannosidase activity for Man₅GlcNAc₁ was enhanced by the addition of Co²⁺, but the addition of Zn²⁺, Ca²⁺, or EDTA did not show any significant effect. In the presence of cobalt ions, the hydrolysis rate for pyridylaminated Man₆GlcNAc₁ was significantly faster than that for pyridylaminated Man₆GlcNAc₂, suggesting the possibility that this enzyme is involved in the degradation of free N-glycans occurring in developing plant cells (Kimura, Y., and Matsuo, S., J. Biochem., 127, 1013–1019 (2000)). To our knowledge, this is the first report showing that plant cells contain an α-mannosidase, which is activated by Co²⁺ and prefers the oligomannose type free N-glycans bearing only one GlcNAc residue as substrate.     

Extracellular Calcium Sensing by Glial Cells: Low Extracellular Calcium Induces Intracellular Calcium Release and Intercellular Signaling

Abstract: Glial cells in primary mixed cultures or purified astrocyte cultures from mouse cortex respond to reduced extracellular calcium concentration ([Ca²⁺]_e) with increases in intracellular calcium concentration ([Ca²⁺]_i) that include single-cell Ca²⁺ oscillations and propagated intercellular Ca²⁺ waves. The rate and pattern of propagation of low [Ca²⁺]_e-induced intercellular Ca²⁺ waves are altered by rapid perfusion of the extracellular medium, suggesting the involvement of an extracellular messenger in Ca²⁺ wave propagation. The low [Ca²⁺]_e-induced Ca²⁺ response is abolished by thapsigargin and by the phospholipase antagonist U73122. The low [Ca²⁺]_e-induced response is also blocked by replacement of extracellular Ca²⁺ with Ba²⁺, Zn²⁺, or Ni²⁺, and by 100 µM La³⁺. Glial cells in lowered [Ca²⁺]_e(0.1–0.5 mM) show an increased [Ca²⁺]_i response to bath application of ATP, whereas glial cells in increased [Ca²⁺]_e (10–15 mM) show a decreased [Ca²⁺]_i response to ATP. These results show that glial cells possess a mechanism for coupling between [Ca²⁺]_e and the release of Ca²⁺ from intracellular stores. This mechanism may be involved in glial responses to the extracellular environment and may be important in pathological conditions associated with low extracellular Ca²⁺ such as seizures or ischemia.     

Biochemical characterization of Ca2+/calmodulin dependent protein kinase from Candida albicans

A multifunctional Ca²⁺/calmodulin dependent protein kinase was purified approximately 650 fold from cytosolic extract of Candida albicans. The purified preparation gave a single band of 69 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis with its native molecular mass of 71 kDa suggesting that the enzyme is monomeric. Its activity was dependent on calcium, calmodulin and ATP when measured at saturating histone IIs concentration. The purified Ca²⁺/CaMPK was found to be autophosphorylated at serine residue(s) in the presence of Ca²⁺/calmodulin and enzyme stimulation was strongly inhibited by W-7 (CaM antagonist) and KN-62 (Ca²⁺/CaM dependent PK inhibitor). These results confirm that the purified enzyme is Ca²⁺/CaM dependent protein kinase of Candida albicans. The enzyme phosphorylated a number of exogenous and endogenous substrates in a Ca²⁺/calmodulin dependent manner suggesting that the enzyme is a multifunctional Ca²⁺/calmodulin-dependent protein kinase of Candida albicans.     

Regulation of the gating of the sheep cardiac sarcoplasmic reticulum ca2+-release channel by luminal Ca2+

We investigated the effects of changes in luminal [Ca²⁺] on the gating of native andpurified sheep cardiac sarcoplasmic reticulum (SR) Ca²⁺-release channels reconstituted intoplanar phospholipid bilayers. The open probability (P _o )of channels activated solely by cytosolic Ca²⁺ was greater at positive than negative holding potentials. Channels activatedsolely by 10 m cytosolic Ca²⁺ exhibited no change in steady-stateP _o or in the relationship betweenP _o and voltage when the luminal[Ca²⁺] was increased from nanomolar to millimolar concentrations. In the absence of activating concentrationsof cytosolic Ca²⁺, the channel can be activated by the phosphodiesterase inhibitor sulmazole (AR-L 115BS). However, cytosolicCa²⁺-independent activation of the channel by sulmazole requires luminal Ca²⁺. In the presence ofsulmazole, at picomolar luminal [Ca²⁺] the channel remains completely closed. Increasing the luminal [Ca²⁺]to millimolar levels markedly increases the P _o via an increase in theduration of open events. The P _o and duration of the sulmazole-activated, luminalCa²⁺-dependent channel openings are voltage dependent. In the presence of micromolar luminal Ca²⁺, theP _o and duration of sulmazole-activated openings are greater atnegative voltages. However, at millimolar luminal [Ca²⁺], long openings are also observed at positive voltages and theP _o appears to be similar at positive and negative voltages. Our findings indicate thatthe regulation of channel gating by luminal Ca²⁺ depends on the mechanism of channel activation.We would like to thank Dr Allan Lindsay for the preparation of the purified SR Ca²⁺-release channels. This work was supported by the British Heart Foundation.     

GUANYLATE CYCLASES IN CNS: ENZYMATIC CHARACTERISTICS OF SOLUBLE AND PARTICULATE ENZYMES FROM MOUSE CEREBELLUM AND RETINA

Guanylate cyclase activity is present in both soluble and particulate fractions of homogenates of mouse cerebellum and retina. Soluble guanylate cyclases in cerebellum and retina have an apparent K_m for GTP of approx 40 and 70 μM, respectively; are stimulated by Ca²⁺ and Mg²⁺ in the presence of low Mn²⁺; and do not respond to NaN₃, NH₂OH or detergent. The particulate guanylate cyclase found in brain has an apparent K_m GTP of 237 7mu;M, is not stimulated by Ca²⁺ or Mg²⁺ in the presence of low Mn²⁺, but is stimulated by NaN₃, NH₂OH, and detergent. In particulate fractions of normal retina, guanylate cyclase has two apparent K_m GTP values (42 and 225 μM); has higher activity at low concentrations of Mn²⁺ (0.5 mM) than at high concentrations (5.0 mM); is inhibited by Ca²⁺; and does not respond to NaN₃, NH₂OH, or detergent. Retinas essentially devoid of photoreceptor cells (from mice with photoreceptor dystrophy) have soluble guanylate cyclase activity which is similar to that in normal retina, but have only 4% as much particulate guanylate cyclase activity. This residual particulate guanylate cyclase has an apparent K_m GTP value of 392 μM and other properties similar to particulate guanylate cyclase from brain. These data indicate the presence of three distinguishable guanylate cyclases in CNS: (1) a soluble enzyme present in both brain and retina: (2) a particulate enzyme which is also present in brain and in the inner or neural retina: and (3) another particulate enzyme which is apparently unique and confined to retinal photoreceptor cells.     

Membrane ATPase of Bacillus subtilis. I. Purification and properties

The membrane ATPase (EC 3.6.1.3) of Bacillus subtilis can be solubilized by a shock-wash process. Two procedures for purifying the solubilized enzyme are reported. A protease inhibitor, phenylmethane sulfonylfluoride, was introduced in the solubilization and purification step.The resultant ATPase purified by density gradient centrifugation has a molecular weight of 315 000, an s_20,w of 13,4 and an ámino acid composition very similar to bacterial ATPases already studied.After exposure to polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate (SDS), or 8 M urea or SDS-urea, the purified ATPase can be dissociated in two non-identical subunits of molecular weights 59 000 (α) and 57 000 (β) with different charges.Kinetic studies showed that Ca²⁺ or Zn²⁺ are required for ATPase activity, although Mg²⁺ was uneffective. At optimal Ca²⁺ concentration, the Mg²⁺ has an inhibitory effect. The K_m for ATP is 1.3 mM. Inhibitors of the oxydative phosphorylation, of the mitochondrial ATPase and of the (Na⁺ + K⁺)-ATPase are studied.     

Solubilization,purification and reconstitution of Ca-ATPase from bovine pulmonary artery smooth muscle microsomes by different detergents: Preservation of native structure and function of the enzyme by DHPC

The properties of Ca²⁺-ATPase purified and reconstituted from bovine pulmonary artery smooth muscle microsomes {enriched with endoplasmic reticulum (ER)} were studied using the detergents 1,2-diheptanoyl-sn-phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl ether (C₁₂E₈) and Triton X-100 as the solubilizing agents. Solubilization with DHPC consistently gave higher yields of purified Ca²⁺-ATPase with a greater specific activity than solubilization with C₁₂E₈ or Triton X-100. DHPC was determined to be superior to C₁₂E₈; while that the C₁₂E₈ was determined to be better than Triton X-100 in active enzyme yields and specific activity. DHPC solubilized and purified Ca²⁺-ATPase retained the E1Ca−E1*Ca conformational transition as that observed for native microsomes; whereas the C₁₂E₈ and Triton X-100 solubilized preparations did not fully retain this transition. The coupling of Ca²⁺ transported to ATP hydrolyzed in the DHPC purified enzyme reconstituted in liposomes was similar to that of the native micosomes, whereas that the coupling was much lower for the C₁₂E₈ and Triton X-100 purified enzyme reconstituted in liposomes. The specific activity of Ca²⁺-ATPase reconstituted into dioleoyl-phosphatidylcholine (DOPC) vesicles with DHPC was 2.5-fold and 3-fold greater than that achieved with C₁₂E₈ and Triton X-100, respectively. Addition of the protonophore, FCCP caused a marked increase in Ca²⁺ uptake in the reconstituted proteoliposomes compared with the untreated liposomes. Circular dichroism analysis of the three detergents solubilized and purified enzyme preparations showed that the increased negative ellipticity at 223 nm is well correlated with decreased specific activity. It, therefore, appears that the DHPC purified Ca²⁺-ATPase retained more organized and native secondary conformation compared to C₁₂E₈ and Triton X-100 solubilized and purified preparations. The size distribution of the reconstituted liposomes measured by quasi-elastic light scattering indicated that DHPC preparation has nearly similar size to that of the native microsomal vesicles whereas C₁₂E₈ and Triton X-100 preparations have to some extent smaller size. These studies suggest that the Ca²⁺-ATPase solubilized, purified and reconstituted with DHPC is superior to that obtained with C₁₂E₈ and Triton X-100 in many ways, which is suitable for detailed studies on the mechanism of ion transport and the role of protein–lipid interactions in the function of the membrane-bound enzyme.     

Equilibrium Dialysis Measurements of the Ca2+-Binding Properties of Recombinant Radish Vacuolar Ca2+-Binding Protein Expressed in Escherichia coli

Vacuoles of radish (Raphanus sativus) contained a Ca²⁺-binding protein (RVCaB) of 43 kDa. We investigated the Ca²⁺-binding properties of the protein. RVCaB was expressed in Escherichia coli and was purified from an extract by ion-exchange chromatography, nitrocellulose membrane filtration, and gel-filtration column chromatography. Ca²⁺-binding properties of the recombinant protein were examined by equilibrium dialysis with ⁴⁵Ca²⁺ and small dialysis buttons. The protein was estimated to bind 19Ca²⁺ ions per molecule with a K _d for Ca²⁺ of 3.4 mM. Ca²⁺ was bound to the protein even in the presence of high concentrations of Mg²⁺ or K⁺. The results suggested that the protein bound Ca²⁺ with high ion selectivity, high capacity, and low affinity.     

Characteristics of magnesium-dependent, Ca2+ -stimulated endogenous protein kinase-catalyzed phosphorylation of basic proteins in myelin isolated from rat brain stem white matter

Purified myelin fraction isolated from rat brain white matter contained Mg²⁺-dependent protein kinase capable of phosphorylation of myelin basic proteins. The Mg²⁺-supported kinase was markedly stimulated (two- to fivefold) by micromolar concentrations of free Ca²⁺ with and without Triton X-100 in the assay, the degree of stimulation being greater with the detergent present. Cyclic AMP, on the other hand, failed to show any effect on phosphorylation of myelin in the absence of Triton X-100 and in the presence of Triton caused only 25–30% stimulation. The phosphorylation reaction was temperature dependent and exhibited a pH optimum at pH 6.5. Apparent affinity toward MgATP^2? was found to be about 70 μm and Ca²⁺ had no effect on this parameter. Dependence on MgCl₂ of myelin phosphorylation indicated the presence of high- and low-affinity sites toward Mg²⁺; Ca²⁺ appeared to influence the low-affinity site. Maximal level of phosphorylation was attained by 10–15 min at 30 °C and it declined at longer incubation times due to phosphatase activity present in the preparation. Stimulatory effect of Ca²⁺ on phosphorylation was not due to inhibition of phosphatase activity. Dephosphorylation experiments showed that neither cyclic AMP nor Ca²⁺ influenced the myelin phosphatase activity. Autoradiographic analysis revealed that phosphorylation of myelin basic proteins accounted for nearly 90% of total myelin phosphorylation. This was supported by the observation that the HCl extract of myelin contained 85% of total activity and comigrated with purified myelin basic proteins. Basal and Ca²⁺-stimulated phosphorylation of basic proteins were due to phosphorylation of serines mainly, although threonine was phosphorylated to a minor extent. Within myelin, Ca²⁺ and cyclic AMP kinases are differentially bound. It appears that the myelin kinase (studied in vitro) is primarily influenced by Ca²⁺ rather than cyclic AMP. Inhibitors (Type I and Type II) of cyclic nucleotide-stimulated protein kinases had no effect on the Ca²⁺-stimulated phosphorylation although basal and cyclic AMP-stimulated phosphorylation was inhibited, indicating that the Ca²⁺ kinase is a separate and distinct enzyme from the cyclic AMP-stimulated and basal kinase(s). Also, leupeptin, a protease inhibitor, did not influence basal, cyclic AMP-stimulated, or Ca²⁺-stimulated myelin phosphorylation, indicating that under the conditions used protease(s) did not alter the myelin kinase activity. The potential significance of phosphorylation of myelin basic proteins and the stimulatory action of Ca²⁺ on this reaction are discussed.     

Purification and Characterization of Phosphatidylinositol-specific Phospholipase C in Suspension-cultured Cells of Rice (Oryza sativa L.)

Phosphatidylinositol-specific phospholipase C was purified from the soluble fraction of suspension-cultured rice cells. The apparent molecular weight of rice enzyme was estimated to be 50,000 by both Sephadex G-100 gel filtration and SDS–polyacrylamide gel electrophoresis, indicating that the enzyme is composed of a single polypeptide. The enzyme had an isoelectric point of 6.3. The soluble phospholipase C had a high degree of specificity toward phosphatidylinositol and a weak activity toward phosphatidyl-inositol monophosphate, while the enzyme did not hydrolyze the other phospholipids or p-nitrophenylphosphorylcholine. V_max and K_m values were 5.0, μmol/min/mg protein and 0.3 mM, respectively. The pH dependency of the enzyme activity was sharp with an optimum of 5.2. In addition, the phospholipase C was a Ca²⁺ -dependent enzyme. The marked activation of enzyme was observed in the presence of 10 to 250, μM Ca²⁺ and higher Ca ²⁺ concentrations than 1 mM had a strong inhibitory effect. A possible regulation of the phospholipase C activity by pH and Ca²⁺ concentrations in the rice cells is discussed.     

Tyrosinase Isoenzymes: Two Melanosomal Tyrosinases With Different Kinetic Properties and Susceptibility to Inhibition by Calcium

Two forms of tyrosinase from B16 mouse melanoma were identified by nonreducing SDS-PAGE after solubilization of crude melanosomal preparations with the nonionic detergent Brij 35. These forms, named LEMT and HEMT (low and high electrophoretic mobility tyrosinase, respectively), were purified by a combination of differential detergent extraction and chromatographic techniques. They displayed tyrosine hydroxylase and dopa oxidase activity and were stereospecific and sensitive to phenylthiourea, proving that they are true tyrosinases. However, based on its kinetic parameters, HEMT is a much more efficient enzyme, Immunoprecipitation and Western blots performed with the specific antibody αPEP1, directed against the b protein carboxyl terminus, suggested that LEMT is identical to the b protein. Both forms of tyrosinase were noncompetitively inhibited by Ca²⁺ at physiologically relevant concentrations. However, the b protein was apparently more susceptible, since maximal inhibition was reached at lower Ca²⁺ concentrations for LEMT. Moreover, binding of Ca²⁺ to the tyrosinases resulted in a noticeable thermal destabilization of the enzymes, which was also more pronounced for LEMT.     

Purification and partial characterization of serine protease from thermostable alkalophilic <Emphasis Type="Italic">Bacillus laterosporus</Emphasis>-AK1

An extracellular thermostable alkaline protease isolated from Bacillus laterosporus-AK1 was purified by sephadex G-200 gel filtration and DEAE cellulose ion-exchange chromatography techniques. The purified protease showed a maximum relative activity of 100% on casein substrate and appeared as a single band on SDS-PAGE with the molecular mass of 86.29 kDa. The protease was purified to 11.1-folds with a yield of 34.3%. Gelatin zymogram also revealed a clear hydrolytic zone due to proteolytic activity, which corresponded to the band obtained with SDS-PAGE. The protease enzyme had on optimum pH of 9.0 and exhibited highest activity at 75°C. The enzyme activity was highly susceptible to the specific serine protease inhibitor PMSF, suggesting the presence of serine residues at the active sites. Enzyme activity strongly enhanced by the metal ions Ca²⁺ and Mg²⁺ and this enzyme compatible with aril detergent stability retained 75% even 1-h incubation. The purified protease remove bloodstain completely when used with Wheel detergent.     

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.     

Isolation and characterization of a cold-active,alkaline, detergent stable α-amylase from a novel bacterium Bacillus subtilis N8

A cold-active alkaline amylase producer Bacillus subtilis N8 was isolated from soil samples. Amylase synthesis optimally occurred at 15°C and pH 10.0 on agar plates containing starch. The molecular weight of the enzyme was found to be 205?kDa by performing SDS-PAGE. While the enzyme exhibited the highest activity at 25°C and pH 8.0, it was highly stable in alkaline media (pH 8.0–12.0) and retained 96% of its original activity at low temperatures (10–40°C) for 24?hr. While the amylase activity increased in the presence of β-mercaptoethanol (103%); Ba²⁺, Ca²⁺, Na⁺, Zn²⁺, Mn²⁺, H₂O₂, and Triton X-100 slightly inhibited the activity. The enzyme showed resistance to some denaturants: such as SDS, EDTA, and urea (52, 65, and 42%, respectively). N8 α-amylase displayed the maximum remaining activity of 56% with 3% NaCl. The major final products of starch were glucose, maltose, and maltose-derived oligosaccharides. This novel cold-active α-amylase has the potential to be used in the industries of detergent and food, bioremediation process and production of prebiotics.     

Cooperative interactions between calcium-binding sites on glycerinated muscle fibers the influence of cross-bridge attachment

A double isotope technique and EGTA buffers were used to measure the binding of Ca²⁺ to rabbit psoas muscle fibers extracted with detergent and glycerol. These experiments were designed to test the effect of rigor complex formation, determined by the degree of filament overlap, on the properties of the Ca²⁺-binding sites in the intact filament lattice. In the presence of 5 mM MgCl₂ (no ATP), reduction of filament overlap was associated with a reduced binding of Ca²⁺ over the entire range of free Ca²⁺ concentrations (5 · 10^?8 – 2 · 10^?5 M). With maximum filament overlap (sarcomere length 2.1–2.2 μm) the maximum bound Ca²⁺ was equivalent to 4 mol Ca²⁺/mol troponin and there was significant positive interaction between binding sites, as shown by Scatchard and Hill plots. With no filament overlap (sarcomere length 3.8–4.4 μm) the maximum bound Ca²⁺ was equivalent to 3 μmol Ca²⁺/mol troponin and graphical analysis indicated a single class of non-interacting sites. The data provide evidence that when cross-bridge attachments between actin and myosin filaments are formed not only does an additional Ca²⁺ binding site appear, but cooperative properties are imposed upon the binding sites.