Active Detergent-solubilized H+,K+-ATPase Is a Monomer

The H⁺,K⁺-ATPase pumps protons or hydronium ions and is responsible for the acidification of the gastric fluid. It is made up of an α-catalytic and a β-glycosylated subunit. The relation between cation translocation and the organization of the protein in the membrane are not well understood. We describe here how pure and functionally active pig gastric H⁺,K⁺-ATPase with an apparent Stokes radius of 6.3 nm can be obtained after solubilization with the non-ionic detergent C₁₂E₈, followed by exchange of C₁₂E₈ with Tween 20 on a Superose 6 column. Mass spectroscopy indicates that the β-subunit bears an excess mass of 9 kDa attributable to glycosylation. From chemical analysis, there are 0.25 g of phospholipids and around 0.024 g of cholesterol bound per g of protein. Analytical ultracentrifugation shows one main complex, sedimenting at s_20,w = 7.2 ± 0.1 S, together with minor amounts of irreversibly aggregated material. From these data, a buoyant molecular mass is calculated, corresponding to an H⁺,K⁺-ATPase α,β-protomer of 147.3 kDa. Complementary sedimentation velocity with deuterated water gives a picture of an α,β-protomer with 0.9–1.4 g/g of bound detergent and lipids and a reasonable frictional ratio of 1.5, corresponding to a Stokes radius of 7.1 nm. An α₂,β₂ dimer is rejected by the data. Light scattering coupled to gel filtration confirms the monomeric state of solubilized H⁺,K⁺-ATPase. Thus, α,β H⁺,K⁺-ATPase is active at least in detergent and may plausibly function as a monomer, as has been established for other P-type ATPases, Ca²⁺-ATPase and Na⁺,K⁺-ATPase.     

Muscle Calcium Metabolic Effects of Hypokinesia in Physically Healthy Subjects

The incompleteness of electrolyte deposition during hypokinesia (HK; diminished movement) is the defining factor of electrolyte metabolic changes, yet the effect of prolonged HK upon electrolyte deposition is poorly understood. The objective of this investigation was to determine the effect of muscle calcium (Ca⁺⁺) changes upon Ca⁺⁺ losses during prolonged HK. Studies were conducted on 20 physically healthy male volunteers during a pre-experimental period of 30 days and an experimental period of 364 days. Subjects were equally divided in two groups: control subjects (CS) and experimental subjects (ES). The CS group ran average distances of 9.2?±?1.2 km day^?l, and the ES group walked average distances of 2.3?±?0.2 km day^?l. Muscle Ca⁺⁺ contents, plasma Ca⁺⁺ concentrations, and Ca⁺⁺ losses in urine and feces were measured in the experimental and control groups of subjects. The muscle Ca⁺⁺ contents decreased (p?<?0.05), and plasma Ca⁺⁺ levels and Ca⁺⁺ losses in the urine and feces increased (p?<?0.05) in the ES group compared with their pre-experimental levels and the values in their respective CS group. Muscle Ca⁺⁺ contents and plasma Ca⁺⁺ levels and urinary and fecal Ca⁺⁺ losses did not change in the CS group compared to their pre-experimental levels. It is concluded that prolonged HK increase plasma Ca⁺⁺ concentrations and Ca⁺⁺ losses in Ca⁺⁺ deficient muscle indicating decreased Ca⁺⁺ deposition.     

A protein activator of the plasma membrane Ca++-ATPase of heart sarcolemma

A detergent extract of dog or beef heart sarcolemmal vesicles was prepared and found to have a stimulatory effect on the Ca⁺⁺-ATPase of plasma membranes from human erythrocyte and cardiac sarcolemma. A procedure is described which enriches the activating fraction. The protein nature of the preparation is illustrated by its sensitivity to boiling and to the proteolytic enzyme(s) trypsin and chymotrypsin. SDS polyacrylamide gels indicate that the protein(s) involved have a molecular weight of 56 and 60 kDa. The sarcolemmal activator can stimulate the Ca⁺⁺-ATPase activity of the isolated enzyme more than 100% in the presence of saturating amounts of calmodulin. The activation is calcium dependent, being greatest at approximately 10µm Ca⁺⁺, free, but does not change theK _m for Ca⁺⁺. A possible physiological role for the activator is discussed.     

R 24571: a new powerful inhibitor of red blood cell Ca++-transport ATPase and of calmodulin-regulated functions

Compound R 24571 (1-[bis(p-chlorophenyl)methyl]-3-[2,4-dichloro-β-(2,4-dichlorobenzyloxy)phenethyl]imidazoliniumchloride) is found to be a powerful inhibitor of red blood cell Ca⁺⁺-ATPase as well as Ca⁺⁺ transport into inside-out red blood cell vesicles with an IC₅₀-value of 0.5 and 2 μM, respectively. The inhibitory action of R 24571 is more specific on the calmodulin-dependent fraction of Ca⁺⁺-transport ATPase as compared to the basal Ca⁺⁺-transport ATPase (determined in the absence of calmodulin) and can be antagonized by increasing concentrations of calmodulin in an apparently competitive manner. With respect to other ATPases the action of R 24571 is relatively specific for red blood cell Ca⁺⁺-transport ATPase. Mg⁺⁺-ATPase requires a 40 times higher concentration for halfmaximal inhibition (IC₅₀ = 20 μM) whereas (Na⁺ + K⁺)-transport ATPase is only slightly affected in the investigated concentration range (≤20 μM).     

Technical considerations for assessing alterations in skeletal muscle sarcoplasmic reticulum Ca++-sequestration functionin vitro

A multiple measurement system for assessing sarcoplasmic reticulum (SR) Ca⁺⁺-ATPase activity and Ca⁺⁺-uptake was used to examine the effects of SR fractionation and quick freezing on rat white (WG) and red (RG) gastrocnemius muscle.In vitro measurements were performed on whole muscle homogenates (HOM) and crude microsomal fractions (CM) enriched in SR vesicles before and after quick freezing in liquid nitrogen. Isolation of the CM fraction resulted in protein yields of 0.96±0.1 and 0.99±0.1 mg/g in WG and RG, respectively. The percent Ca⁺⁺-ATPase recovery for CM compared to HOM was 14.5% (WG) and 10.1% (RG). SR Ca⁺⁺-activated Ca⁺⁺-ATPase activity was not affected by quick freezing of HOM or CM, but basal ATPase was reduced (P<0.05) in frozen HOM (5.12±0.18–3.98±0.20 mole/g tissue/min in WG and from 5.39±0.20–4.48±0.24 mole/g tissue/min in RG). Ca⁺⁺-uptake was measured at a range of physiological free [Ca⁺⁺] using the Ca⁺⁺ fluorescent dye Indo-1. Maximum Ca⁺⁺-uptake rates when corrected for initial [Ca⁺⁺]_f were not altered in HOM or CM by quick freezing but uptake between 300 and 400nM free Ca⁺⁺ was reduced (P<0.05) in quick frozen HOM (1.30±0.1–0.66±0.1 mole/g tissue/min in WG and 1.04±0.2–0.60±0.1 mole/g tissue/min in RG). Linear correlations between Ca⁺⁺-uptake and Ca⁺⁺-ATPase activity measured in the presence of the Ca⁺⁺ ionophore A23187 were r=+0.25, (P<0.05) and r=+0.74 (P<0.05) in HOM and CM preparations, respectively, and were not altered by freezing. The linear relationships between HOM and CM maximum Ca⁺⁺-uptake (r=+0.44, P<0.05) and between HOM and CM Ca⁺⁺-ATPase activity (r=+0.34, P<0.05) were also not altered by tissue freezing. These data suggest that alterations in maximal SR Ca⁺⁺-uptake function and maximal Ca⁺⁺-ATPase activity may be measured in both HOM and CM fractions following freezing and short term storage. (Mol Cell Biochem139, 41–52, 1994)     

An Endogenous Inhibitor of Ca++-ATPase from Human Placenta

Intracellular free calcium is regulated by Ca⁺⁺-ATPase, one form present on the plasma membrane (PM Ca⁺⁺-ATPase) and the other on sarcoplasmic (endoplasmic) reticulum (SR/ER Ca⁺⁺-ATPase). An endogenous inhibitor of SR Ca⁺⁺-ATPase from human placenta was shown to be present in normal placenta and the activity was not detectable in placenta from preeclamptic patients. The inhibitor was distributed in cytosol and microsomes. The inhibition of Ca⁺⁺-ATPase by this inhibitor was concentration-and time-dependent. The inhibitor neither bound to DEAE-nor CM-sepharose resins at pH 7.5 and 8.5. Furthermore, it was heat stable for 15min up to 55°C and completely destroyed at 80°C in a few minutes. It was also observed to be stable at room temperature for at least 3 months. The purification and characterization of this inhibitor would be valuable in achieving an understanding of the normal regulation of Ca⁺⁺-ATPase in the placenta during pregnancy.     

Thermodynamic and kinetic parameters of ion condensation to polynucleotides: Outer sphere complex formed by Mg++ ions

The coupling of ion binding to the single strand helix—coil transition in poly (A) and poly(C) is used to obtain information about both processes by ion titration and field-jump relaxation methods. Characterisation of the field-jump relaxation in poly(C) at various concentrations of monovalent ions leads to the evaluation of a stability constant K = 71 M^?1 for the ion binding to the polymer. The rate constant of helix formation is found to be 1.3 × 10⁷ s^?1, whereas the dissociation rate is 1.0 × 10⁶ s^?1. Similar data are presented for poly (A) and poly (dA).The interaction of Mg⁺⁺ and Ca⁺⁺ with poly (A) and poly (C) is measured by a titration method using the polymer absorbance for the indication of binding. The data can be represented by a model with independent binding “sites”. The stability constants increase with decreasing salt concentration from 2.7 × 10⁴ M^?1 at medium ionic strengths up to 2.7 × 10⁷ M^?1 at low ionic strength. The number of ions bound per nucleotide residue is in the range 0.2 to 0.3. Relaxation time constants associated with Mg⁺⁺ binding are characterised over a broad range of Mg⁺⁺ concentrations from 5 μM to 500 μM. The observed concentration dependence supports the conclusion on the number of binding places inferred from equilibrium titrations. The rate of Mg⁺⁺ and Ca⁺⁺ association to the polymer is close to the limit of diffusion control (k_R = 1 × 10¹⁰ to 2 × 10¹⁰ M^?1 s^?1). This high rate demonstrates that Mg⁺⁺ and Ca⁺⁺ ions do not form inner-sphere complexes with the polynucleotides. Apparently the distance between two adjacent phosphates is too large for a simultaneous site binding of Mg⁺⁺ or Ca⁺⁺, and inner sphere complexation at a single phosphate seems to be too weak. The data support the view that the ions like Mg⁺⁺ and Ca⁺⁺ surround the polynucleotides in the form of a mobile ion cloud without site binding.     

Effect of lipid on the access of ATP and calcium to the delipidated Ca++-ATPase of intestinal brush border membrane.

The delipidated Ca⁺⁺-ATPase prepared from intestinal brush border membranes showed a higher activity of Ca⁺⁺-independent ATPase, a lower Km value for ATP and a higher Km value for Ca⁺⁺ than its original membrane Ca⁺⁺-ATPase. The addition of phosphatidylcholine re-activated the delipidated Ca⁺⁺-ATPase to approximately 89 % of its original membrane Ca⁺⁺-ATPase activity but did not restore the affinity for Ca⁺⁺. This phospholipid raised the Km value for ATP but had little effect on the Km value for Ca⁺⁺. Palmitic acid elevated the Km value for Ca⁺⁺ but did not change the Km value for ATP. Kinetic analyses of these data suggest that the hydrocarbon chain of phosphatidylcholine is an important rate-limiting factor for the access of Ca⁺⁺ to the enzyme and the polar head groups of phosphorylcholine and ester bond may be the factor for the access of ATP.     

Low and high temperature asymmetric forms of pig kidney and rabbit muscle phosphofructokinases and reversible, temperature-dependent transitions.

Previous viscometric studies from this laboratory (Johnson, C. S., Vogtmann, L., and Deal, W. C., Jr. (1976) Biochem. Biophys. Res. Commun.73, 391–395) have shown that at 3.5 ° C, pig kidney phosphofructokinase (PFK) is markedly asymmetric and rabbit muscle PFK is moderately asymmetric. The present viscometric and ultracentrifugal studies show that both enzymes are also asymmetric at near-physiological temperatures, that both exist in high-temperature and low-temperature forms, and that the high-temperature forms of both are less asymmetric and more dissociated than the low-temperature forms. The risults also show that the transitions from low- to high-temperature forms are reversible if the exposure to 35 °C is short enough that no irreversible chemical modification occurs. For pig kidney PFK, intrinsic viscosity values of 34.0, 25.6, and 13.8 ml/g were obtained at 3.5, 20 and 35 °C, respectively, whereas rabbit muscle PFK yielded values of 6.9, 6.2, and 5.2 ml/g at the corresponding temperatures. These data clearly show a decrease in asymmetry with increase in temperature. However, both enzymes are still asymmetric at the higher temperature, inasmuch as most globular macromolecules have intrinsic viscosity values in the range of 3 to 4 ml/g, regardless of molekular weight. Studies from 1 to 45 ° C at a fixed protein concentration (4.8 mg/ml) showed that pig kidney PFK has reduced viscosity values of 51.0 ml/g (low-temperature form) and 20.4 ml/g (high-temperature form) in plateau regions of the viscosity graph at the temperature extremes; the mid-point of the transition between the two forms is at about 22–24 °C. Rabbit muscle PFK at 4.2 mg/ml reproducibly gave corresponding reduced viscosity values of 6.9 and 4.8 ml/g for the low- and high-temperature forms, respectively; the transition mid-point between the two forms is at about 16 °C. The first reported sedimentation velocity studies of rabbit muscle PFK at near-physiological temperature (35 °C) show that with near-physiological protein concentration (1.25 mg/ml), the enzyme is in a much more dissociated form, s_20,w(weight average) = 14. 5 S; s_20,w(peak leading edge) = 17 S, than that previously reported at lower temperatures, s_20,w(fastest peak) = 23–30 S. Similarly, the first sedimentation studies on the pig kidney enzyme indicate a lower sedimentation coefficient at 35 ° C (s^0.39%_20,w = 48 S) than at 3.5 ° C(65 S).     

Cation-permeable vacuolar ion channels in the moss Physcomitrella patens: a patch-clamp study

Patch-clamp studies carried out on the tonoplast of the moss Physcomitrella patens point to existence of two types of cation-selective ion channels: slowly activated (SV channels), and fast-activated potassium-selective channels. Slowly and instantaneously saturating currents were observed in the whole-vacuole recordings made in the symmetrical KCl concentration and in the presence of Ca²⁺ on both sides of the tonoplast. The reversal potential obtained at the KCl gradient (10 mM on the cytoplasmic side and 100 mM in the vacuole lumen) was close to the reversal potential for K⁺ (E _K), indicating K⁺ selectivity. Recordings in cytoplasm-out patches revealed two distinct channel populations differing in conductance: 91.6 ± 0.9 pS (n = 14) at ?80 mV and 44.7 ± 0.7 pS (n = 14) at +80 mV. When NaCl was used instead of KCl, clear slow vacuolar SV channel activity was observed both in whole-vacuole and cytoplasm-out membrane patches. There were no instantaneously saturating currents, which points to impermeability of fast-activated potassium channels to Na⁺ and K⁺ selectivity. In the symmetrical concentration of NaCl on both sides of the tonoplast, currents have been measured exclusively at positive voltages indicating Na⁺ influx to the vacuole. Recordings with different concentrations of cytoplasmic and vacuolar Ca²⁺ revealed that SV channel activity was regulated by both cytoplasmic and vacuolar calcium. While cytoplasmic Ca²⁺ activated SV channels, vacuolar Ca²⁺ inhibited their activity. Dependence of fast-activated potassium channels on the cytoplasmic Ca²⁺ was also determined. These channels were active even without Ca²⁺ (2 mM EGTA in the cytosol and the vacuole lumen), although their open probability significantly increased at 0.1 μM Ca²⁺ on the cytoplasmic side. Apart from monovalent cations (K⁺ and Na⁺), SV channels were permeable to divalent cations (Ca²⁺ and Mg²⁺). Both monovalent and divalent cations passed through the channels in the same direction—from the cytoplasm to the vacuole. The identity of the vacuolar ion channels in Physcomitrella and ion channels already characterised in different plants is discussed.     

Biochemical characteristics of the Ca2+ pumping ATPase in the peribacteroid membrane from broad bean root nodules

Ca²⁺-ATPase in the peribacteroid membrane (PBM) of symbiosomes isolated from Vicia faba root nodules was characterized in terms of its hydrolytic and transport activities. Both activities were found to be pH-dependent and exhibit pH optimum at pH 7.0. Translocation of Ca²⁺ through the PBM by the Ca²⁺-ATPase was shown to be fueled by ATP and other nucleotide triphosphates in the following order: ATP?>?ITP???GTP???UTP???CTP, the K _m of the enzyme for MgATP being about 100 μM. Ca-dependent ITP-hydrolytic activity of symbiosomes was investigated in the presence of the Ca-EGTA buffer system and showed the affinity of PBM Ca²⁺-ATPase for Ca²⁺ of about 0.1 μM. The transport activity of Ca²⁺-ATPase was inhibited by erythrosin B as well as orthovanadate, but markedly stimulated by calmodulin from bovine brain. These results allowed us to conclude that this enzyme belongs to IIB-type Ca²⁺-ATPases which are present in other plant membranes.     

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.     

Calcium-dependent phosphatidylinositol phosphorylation in lamellibranch gill lateral cilia

Summary Pure lateral (L) cilia may be separated from the remaining (R) cilia types ofMytilus edulis gill by serotonin activation after hypertonic shock. The two classes of cilia were permeabilized with 0.012% Triton X-100 and incubated with³²P-labeled ATP at low Ca⁺⁺ (10^–7 M), where L cilia beat, or in high Ca⁺⁺ (2–20 M), where L cilia arrest but R cilia are active. The labeled cilia were separated into axoneme and membrane-matrix fractions by detergent extraction, subjected to SDS-PAGE on 5–15% gels, and autoradio-graphed. Neither cilia type undergoes Ca⁺⁺-dependent phosphorylation of specific proteins, suggesting that neither Ca⁺⁺-induced arrest in L cilia nor the Ca⁺⁺ activation of other cilia is phosphorylation-dependent. However, lipid phosphorylation in L cilia is highly Ca⁺⁺-dependent. Identified by thin-layer chromatography, the phospholipid that is phosphorylated in a Ca⁺⁺-dependent manner is phosphatidylinositol 4-phosphate (PIP), yielding the 4,5-bisphosphate (PIP₂). PIP₂ increases at least 3-fold under Ca⁺⁺-arrest conditions.Aequipecten gill lateral cilia, which require higher Ca⁺⁺ levels for arrest, show even more striking changes. In both cases, the effect is maximal at micromolar Ca⁺⁺ levels. Phosphorylation of other lipids is Ca⁺⁺-independent. In the Ca⁺⁺-insensitive or activated R cilia, PIP₂ levels are intermediate, increasing only marginally with increased [Ca⁺⁺]. The formation of PIP₂ in response to Ca⁺⁺, as opposed to its breakdown to form inositol 1,4,5-trisphosphate and diacylglycerol, may be characteristic of a Ca⁺⁺ transport system. Mechanically sensitive, the L cilia arrest as a consequence of an inward flux of Ca⁺⁺ ions, acting directly on the axoneme. After Ca⁺⁺-induced arrest, the formation of PIP₂ may be involved in sequestering Ca⁺⁺ or in augmenting Ca⁺⁺ pump activity, thus reducing Ca⁺⁺ levels so that motility may resume quickly.     

Physical studies on proteins L3 and L24 from the 50 S subunit of the Escherichia coli ribosome.

Proteins L3 and L24, purified by a nondenaturing method from the 50 S ribosomal subunit of Escherichia coli A19, have been characterized. Both proteins were studied under conditions which resemble those used for reconstitution experiments. They were soluble at approximately 2–3 mg/ml and showed little or no aggregation. These proteins have s⁰_20,w values of 2.0 and 1.5 S, and D_20,w values of 7.6 × 10^?7 and 11.0 × 10^?7 cm² s^?1. Partial specific volumes at 20 °C are 0.730 and 0.740 ml g^?1 for the two proteins. The respective molecular weights determined by sedimentation equilibrium are 24,500 and 12,000. The intrinsic viscosity values for the two proteins are 6.0 and 4.0 ml g^?1. From these hydrodynamic parameters an elongated shape for L3 and a globular shape for L24 have been inferred.     

Fed-Batch Cultivation of Arthrospira platensis Using Carbon Dioxide from Alcoholic Fermentation and Urea as Carbon and Nitrogen Sources

To reduce CO₂ emissions from alcoholic fermentation, Arthrospira platensis was cultivated in tubular photobioreactor using either urea or nitrate as nitrogen sources at different light intensities (60 μmol m^?2 s^?1?≤?I?≤?240 μmol m^?2 s^?1). The type of carbon source (pure CO₂ or CO₂ from fermentation) did not show any appreciable influence on the main cultivation parameters, whereas substitution of nitrate for urea increased the nitrogen-to-cell conversion factor (Y _X/N ), and the maximum cell concentration (X _m ) and productivity (P _X ) increased with I. As a result, the best performance using gaseous emissions from alcoholic fermentation (X _m ?=?2,960?±?35 g m^?3, P _X ?=?425?±?5.9 g m^?3 day^?1 and Y _X/N ?=?15?±?0.2 g g^?1) was obtained at I?=?120 μmol m^?2 s^?1 using urea as nitrogen source. The results obtained in this work demonstrate that the combined use of effluents rich in urea and carbon dioxide could be exploited in large-scale cyanobacteria cultivations to reduce not only the production costs of these photosynthetic microorganisms but also the environmental impact associated to the release of greenhouse emissions.     

Ultracytochemical localization of Ca++-ATPase activity in the paraphyseal epithelial cells of the frog,Rana esculenta

Summary Ca⁺⁺-ATPase activity was studied ultracytochemically (cf. Ando et al. 1981) in the paraphysis cerebri of the frog. An intense reaction was demonstrated on the plasmalemma of the microvilli at the apical pole of paraphyseal cells; in contrast, the basolateral plasmalemma showed only a slight staining. In addition, mitochondria, gap junctions, cilia, and cytoplasmic elements (e.g., microfilaments) displayed Ca⁺⁺-ATPase activity. Variation of the Ca⁺⁺-concentration in the incubation medium from 0.1 mM to 100 mM altered the Ca⁺⁺-ATPase activity of the cell organelles. The substitution of Ca-by Mg-ions resulted in a conspicuous decrease in the enzyme activity, especially on the apical plasmalemma. Ca⁺⁺-ATPase activity is claimed to be involved in a number of extra-and intracellular functions. In comparison to the epithelium of the adjacent choroid plexus the paraphyseal epithelial cell is thought to be a principal Ca-ion regulator of the cerebrospinal fluid in frogs.Fellow of the Alexander von Humboldt Foundation     

Ultracytochemical localization of Ca++-ATPase activity in pituicytes of the neurohypophysis of the guinea pig

Summary Ca⁺⁺-ATPase activity (cf. Ando et al. 1981) was examined both light- and electron-microscopically in the neurohypophysis of the guinea pig. Apart from a strong activity within the walls of the blood vessels, in the parenchyma of the neurohypophysis the reaction product of the Ca⁺⁺-ATPase activity was restricted to the plasmalemma of the pituicytes. This reaction was completely dependent upon Ca⁺⁺ and the substrate, ATP; the reaction was inhibited by 0.1 mM quercetin, an inhibitor of Ca⁺⁺-ATPase. A reduction of the enzyme activity occurred by 1) adding Mg⁺⁺ to the standard incubation medium, and 2) substituting Ca⁺⁺ with Mg⁺⁺ at varing concentrations. In all experiments the neurosecretory fibers were devoid of Ca⁺⁺-ATPase activity. The function of the Ca⁺⁺-ATPase activity in the plasmalemma of the pituicytes is discussed in connection with the regulation of the extracellular Ca⁺⁺ concentration, which seems to be important with respect to the discharge of secretory material from the neurosecretory fibers.Fellow of the Alexander von Humboldt Foundation, Bonn, Federal Republic of Germany.     

Oxytocin activates calcium signaling in rat sensory neurons through a protein kinase C-dependent mechanism

In addition to its well-known effects on parturition and lactation, oxytocin (OT) plays an important role in modulation of pain and nociceptive transmission. But, the mechanism of this effect is unclear. To address the possible role of OT on pain modulation at the peripheral level, the effects of OT on intracellular calcium levels ([Ca²⁺]_i) in rat dorsal root ganglion (DRG) neurons were investigated by using an in vitro calcium imaging system. DRG neurons were grown in primary culture following enzymatic and mechanical dissociation of ganglia from 1- or 2-day-old neonatal Wistar rats. Using the fura-2-based calcium imaging technique, the effects of OT on [Ca²⁺]_i and role of the protein kinase C (PKC)-mediated pathway in OT effect were assessed. OT caused a significant increase in basal levels of [Ca²⁺]_i after application at the doses of 30 nM (n?=?34, p?<?0.01), 100 nM (n?=?41, p?<?0.001) and 300 nM (n?=?46, p?<?0.001). The stimulatory effect of OT (300 nM) on [Ca²⁺]_i was persistent in Ca²⁺-free conditions (n?=?56, p?<?0.01). Chelerythrine chloride, a PKC inhibitor, significantly reduced the OT-induced increase in [Ca²⁺]_i (n?=?28, p?<?0.001). We demonstrated that OT activates intracellular calcium signaling in cultured rat primary sensory neurons in a dose- and PKC-dependent mechanism. The finding of the role of OT in peripheral pain modification may serve as a novel target for the development of new pharmacological strategies for the management of pain.     

Process optimization of xylanase production using cheap solid substrate by Trichoderma reesei SAF3 and study on the alteration of behavioral properties of enzyme obtained from SSF and SmF

This study aimed to assess the variability in respect of titer and properties of xylanase from Trichoderma reesei SAF3 under both solid-state and submerged fermentation. SSF was initially optimized with different agro-residues and among them wheat bran was found to be the best substrate that favored maximum xylanase production of 219 U (gws)^?1 at 96 h of incubation. The mycelial stage of the fungi and intracellular accumulation of Ca⁺⁺ and Mg⁺⁺ induced maximum enzyme synthesis. Inoculum level of 10 × 10⁶ spores 5 g^?1 of dry solid substrate and water activity of 0.6 were found to be optimum for xylanase production under SSF. Further optimization was made using a Box-Behnken design under response surface methodology. The optimal cultivation conditions predicted from canonical analysis of this model were incubation time (A) = 96–99 h, inoculum concentration (B) = 10 × 10⁶ spores 5 g^?1 of dry substrate, solid substrate concentration (C) = 10–12 g flask^?1, initial moisture level (D) = 10 mL flask^?1 (equivalent to a _w  = 0.55) and the level of xylanase was 299.7 U (gws)^?1. Subsequent verification of these levels agreed (97 % similar) with model predictions. Maximum amount of xylanase was recovered with water (6:1, v/w) and under shaking condition (125 rpm). Purified xylanase from SSF showed better stability in salt and pH, was catalytically and thermodynamically more efficient over enzyme from SmF, though molecular weight of both enzymes was identical (53.8 kDa).     

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.