Greatly extended viability of rat brain storage vesicles in an intracellular medium based upon a non-permeant polyanion

The MgATP-stimulated accumulation of (-)-³H-nor- epinephrine (NE) by rat brain neuronal storage vesicles has been characterized in a new medium based upon polyacrylic acid (avg. MW 5,000). The medium allows careful regulation of K⁺ concentration (140 mM), has a large buffer capacity, and is non-permeant to membranes. Light scattering measurements have confirmed the osmotic stability of vesicles suspended in this medium. Vesicular accumulation of (-)-³ H-NE (Km 1 × 10^?6 M) in this system (37°) was examined under saturating (10^?5 M) and non-saturating (2 × 10^?7 M) concentrations of NE. At 10^?5 M NE, uptake saturated at 5 min and remained stable for periods up to one hour, with maximal uptake levels (pmol/mg protein) of 15.7±0.30 (37°), 3.0±0.49 (0°), 4.4±0.22 (reserpine pretreated $\text{in}$$\text{vivo}$) and 6.0±0.79 (without MgATP). At 2×10^?7 M NE uptake was biphasic with maximal uptake levels (pmol/mg protein) of 4.04±0.14 (37°), 0.19±0.01 (0°), 0.95±0.01 (reserpine) and 0.83±0.08 (without MgATP). Vesicle preparations refrigerated in this medium for 24 hrs displayed properties quite similar to those measured acutely (NE = 2.2x10^?7 M).     

Inhibition of thyrotropin binding to human thyroid plasma membranes by thyroid hormones and "reverse triiodothyronine".

Triiodothyronine, reverse triiodothyronine and thyroxine were found to inhibit ¹²⁵I labelled thyrotropin binding to human thyroid plasma membranes in vitro. Both the thyrotropin binding and the effect of the above iodoamino-acids on this binding were pH, temperature and time dependent, 50% inhibition of thyrotropin binding was observed at 2×10^?7M concentration of reverse triiodothyronine or thyroxine and at 1.1 × 10^?6M concentration of triiodothyronine. The kinetic studies of thyrotropin binding revealed that the maximal capacity of receptor sites for the pituitary hormone is unaffected by the presence of thyroid hormones. On the other hand the association and dissociation constants for thyrotropin binding changed when iodoaminoacids were present in the incubation medium /Ka 8.13 × 10⁷M^?1 vs 1.6 × 10⁸M^?1 and Kd 1.14 × 10^?8M vs 4.55 × 10^?9M respectively, depending on the pH/. The double reciprocal plots showed competitive mechanism of inhibition. The present study suggest that triiodothyronine, reverse triiodothyronine and thyroxine are able to modify the thyrotropin binding to membrane receptors.     

IgE-mediated 14C-serotonin release from rat mast cells modulated by morphine and endorphins

The formaldehyde method was used to examine the interaction of PGE₁ with morphine, β-endorphin and Met-enkephalin on rat mast cells by their effects on IgE-mediated ¹⁴C-serotonin release. PGE₁ (2×10^?8?2×10^?5 M) caused a dose-related inhibition of the mediator release 1 min after an antigen challenge, and morphine (3×10^?7?3×10^?5 M) reversed this PGE₁ effect dose-dependently and stereospecifically; naloxone (2×10^?4 M) antagonized this action of morphine. β-Endorphin (3×10^?7?10^?5 M) and Met-enkephalin (3×10^?6?10^?4 M) mimicked this morphine action dose-dependently and were antagonized by naloxone (2×10^?4 M). These results suggest that morphine and endorphins modulate immunological mediator release from rat mast cells through opioid receptors.     

Differential in vitro inhibition of polyphenoloxidase from a wild edible mushroom Lactarius salmonicolor

The polyphenol oxidase (LsPPO) from a wild edible mushroom Lactarius salmonicolor was purified using a Sepharose 4B-L-tyrosine-p-amino benzoic acid affinity column. At the optimum pH and temperature, the K_M and V_Max values of LsPPO towards catechol, 4-methylcatechol and pyrogallol were determined as 0.025 M & 0.748 EU/mL, 1.809 × 10^{? 3} M & 0.723 EU/mL and 9.465 × 10^{? 3} M & 0.722 EU/mL, respectively.

Optimum pH and temperature values of LsPPO for the three substrates above ranged between the pH 4.5–11.0 and 5–50°C. Enzyme activity decreased due to heat denaturation with increasing temperature. Effects of a variety of classical PPO inhibitors were investigated opon the activity of LsPPO using catechol as the substrate. IC₅₀ values for glutathione, p-aminobenzenesulfonamide, L-cysteine, L-tyrosine, oxalic acid, β-mercaptoethanol and syringic acid were determined as 9.1 × 10^{? 4}, 2.3 × 10^{? 4} M, 1.5 × 10^{? 4} M, 3.8 × 10^{? 7} M, 1.2 × 10^{? 4} M, 4.9 × 10^{? 4} M, and 4 × 10^{? 4} M respectively. Thus L-tyrosine was by far the most effective inhibitor. Interestingly, sulfosalicylic acid behaved as an activator of LsPPO in this study.     

δ-Aminolevulinic acid synthase of Euglena gracilis: Physical and kinetic properties

Physical and kinetic properties of δ-aminolevulinic acid synthase from wild-type and aplastidic strains of Euglena gracilis have been determined. Michaelis constants for glycine, succinyl-CoA and pyridoxal phosphate are 8.5 × 10^?3m, 2.5 × 10^?5m, and 2.9 × 10^?6m, respectively. Optimum reaction pH is 7.8, and maximal product yield during a 30-min incubation occurs at 40 °C. Activity in frozen cell extracts remains constant for 5 days, then falls slowly to one-third of the initial value after 3 months. Enzyme activity rapidly declines irreversibly in the absence of pyridoxal phosphate. Agarose gel chromatography of the native enzyme yields a single band of activity at an elution volume corresponding to a molecular weight of 138,000. δ-Aminolevulinic acid synthase obtained from green wild-type strain Z cells is identical in its physical properties to that obtained from white aplastidic mutant strain W₁₄ ZNalL cells.     

Properties and inhibition of thymidylate synthetase in Drosophila melanogaster

A quantitative comparison of the incorporation of methyl-³H-thymidine and 6-³H-deoxyuridine into the DNA of Drosophila melanogaster in the presence and in the absence of 5-fluorouracil indicated that 5-fluorouracil inhibits the reaction converting dUMP to dTMP catalysed by thymidylate synthetase (methylenetetrahydrofolate:dUrd-5′-P C-methyltransferase, E.C. 2.1.1.b). The enzyme exhibits maximal activity at pH 7·5 to 8·0 and is protected from heat inactivation by deoxyuridine monophosphate. The addition of thiol compounds to the homogenization buffer results in the enhancement of synthetase activity. The K_m values for deoxyuridine monophosphate and 5,10-methylenetetrahydrofolate are 6·8 × 10^?6 M and 8·3 × 10^?5 M, respectively. Fluorodeoxyuridine monophosphate, trifluoromethyldeoxyuridine monophosphate, and methotrexate are inhibitors of the enzyme. 5-Bromodeoxyuridine and 5-iododeoxyuridine have no inhibitory effect. The results support the contention that, under conditions which induce morphological lesions in Drosophila, fluorinated pyrimidines and methotrexate inhibit the de novo synthesis of thymidylate whereas thymidine analogues function in some other manner.     

Purification and biochemical characterization of phytocystatin from Brassica alba

Phytocystatins belong to the family of cysteine proteinases inhibitors. They are ubiquitously found in plants and carry out various significant physiological functions. These plant derived inhibitors are gaining wide consideration as potential candidate in engineering transgenic crops and in drug designing. Hence it is crucial to identify these inhibitors from various plant sources. In the present study a phytocystatin has been isolated and purified by a simple two‐step procedure using ammonium sulfate saturation and gel filtration chromatography on Sephacryl S‐100HR from Brassica alba seeds (yellow mustard seeds).The protein was purified to homogeneity with 60.3% yield and 180‐fold of purification. The molecular mass of the mustard seed cystatin was estimated to be nearly 26 000 Da by sodium dodecyl sulfate polyacrylamide gel electrophoresis as well as by gel filtration chromatography. The stokes radius and diffusion coefficient of the mustard cystatin were found to be 23A° and 9.4 × 10^?7 cm²s^?1 respectively. The isolated phytocystatin was found to be stable in the pH range of 6–8 and is thermostable up to 60 °C. Kinetic analysis revealed that the phytocystatin exhibited non‐competitive type of inhibition and inhibited papain more efficiently (K_i = 3 × 10^?7 M) than ficin (K_i = 6.6 × 10^?7 M) and bromelain (Ki = 7.7 × 10^?7 M respectively). CD spectral analysis shows that it possesses 17.11% alpha helical content. Copyright © 2016 John Wiley & Sons, Ltd.     

Phosphate transport in fertilized sea urchin eggs. I. Kinetic aspects

Properties of the fully developed phosphate transport system in the fertilized egg of the sea urchin, Strongylocentrotus purpuratus, were investigated. The rates of phosphate transport at concentrations of external phosphate of 1 to 44 μM, both in the absence and in the presence of 100 μM arsenate, exhibit typical saturation kinetics. At sea water concentrations of 2 μM phosphate, the rate of uptake is about 2 × 10^?9 μm/egg/minute at 15°C. Arsenate is a competitive inhibitor of phosphate transport, fully and immediately reversible in its effects, yielding K_i values ranging from 10.5 to 14.1 × 10^?6 M in comparison to the corresponding apparent K_M (Michaelis-Menten) constants for phosphate of 5.6 to 7.5 × 10^?6 M (pH 8.0, 15°C). The rate of arsenate uptake in a phosphate deficient medium amounts to 2.8 to 2.9 × 10^?10 μm arsenate/egg/minute at an arsenate concentration of 2.9 to 10.2 μM arsenate (HAsO₄^??), which is 9.5 and 5.6% of the rate of phosphate uptake at corresponding phosphate concentrations. Arsenate has essentially the same developmental effects at initial concentrations of 5–10 μM and 100 μM arsenate, namely no observable effects for exposure periods of 7.5 hours, although longer periods result in blockage of development at the early blastula stage. Outward flux of phosphate ions cannot be demonstrated by washing prelabelled eggs with sea water containing low or high concentrations of phosphate, even when phosphorylation has been blocked by exposing the eggs to a metabolic inhibitor. Phosphate uptake rates measured in the pH range from 5.0 to 10.0 reveal a sharp optimum at pH 8.8–8.9. Reference to the apparent pK' values of the phosphoric acid system indicate that the entering species is the HPO₄^?? ion. The effects on rates of phosphate uptake of exposure to sea water at pH values between 7 and 10 for 30 minute periods are fully reversible, but at lower pH values, reversal is delayed, and is only partial. Sodium molybdate (0.01 M), sodium pyrophosphate (1.5 × 10^?4 M), and adenosine triphosphate (1–5 × 10^?4 M) for exposure periods ranging from 40 to 180 minutes did not significantly affect phosphate uptake. Omission of Ca⁺⁺ ion from artificial sea water is without effect on phosphate uptake but the absence of both Ca⁺⁺ and Mg⁺⁺ results in profound and irreversible depression of both phosphate uptake and development. The data of this and the following paper are consistent with the conclusion that the transport of phosphate involves a surface located carrier. The apparent secondary and tertiary ionization constants of phosphoric acid in sea water (ionic strength = 0.6885) were measured, resulting in a value for pK′₂ = 6.14 and for pK′₃ = 10.99, at 15°C and phosphate at infinite dilution.     

Remediation of Explosive-Contaminated Soils: Alkaline Hydrolysis and Subcritical Water Degradation

Alkaline hydrolysis and subcritical water degradation were investigated as ex-situ remediation processes to treat explosive-contaminated soils from military training sites in South Korea. The addition of NaOH solution to the contaminated soils resulted in rapid degradation of the explosives. The degradation of explosives via alkaline hydrolysis was greatly enhanced at pH ≥12. Estimated pseudo-first-order rate constants for the alkaline hydrolysis of 2,4-dinitrotoluene (DNT), 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in contaminated soil at pH 13 were (9.6?±?0.1)×10^?2, (2.2?±?0.1)×10^?1, and (1.7?±?0.2)×10^?2 min^?1, respectively. In the case of subcritical water degradation, the three explosives were completely removed at 200–300°C due to oxidation at high temperatures and pressures. The degradation rate increased as temperature increased. The pseudo-first-order rate constants for DNT, TNT, and RDX at 300°C were (9.4?±?0.8)×10^?2, (22.8?±?0.3)×10^?2, and (16.4?±?1.0)×10^?2, respectively. When the soil-to-water ratio was more than 1:5, the extent of alkaline hydrolysis and subcritical water degradation was significantly inhibited.     

Na2CO3 Influence on DNA Double Helix Stability: Strong Anion Destabilizing Effect

Abstract

Addition of Na₂CO₃ to almost salt-free DNA solution (5·10^?5M EDTA, pH=5.7, Tm=26.5 °C) elevates both pH and the DNA melting temperature (Tm) if Na₂CO₃ concentration is less than 0.004M. For 0.004M Na₂CO₃, Tm=58 °C is maximal and pH=10.56. Further increase in concentration gives rise to a monotonous decrease in Tm to 37 °C for 1M N₂CO₃ (pH=10.57). Increase in pH is also not monotonous. The highest pH=10.87 is reached at 0.04M Na₂CO₃ (Tm=48.3 °C). To reveal the cause of this DNA destabilization, which happens in a narrow pH interval (10.56÷10.87) and a wide Na₂CO₃ concentration interval (0.004÷1M), a procedure has been developed for determining the separate influences on Tm of Na⁺, pH, and anions formed by Na₂CO₃ (HCO₃ ^? and CO₃ ^2-). Comparison of influence of anions formed by Na₂CO₃ on DNA stability with Cl^? (anion inert to DNA stability), ClO₄ ^? (strong DNA destabilizing “chaotropic” anion) and OH^? has been carried out. It has been shown that only Na⁺ and pH influence Tm in Na₂CO₃ solution at concentrations lower than 0.001M. However, the Tm decrease with concentration for [Na₂CO₃]≥0.004M is only partly caused by high pH≈10.7. Na₂CO₃ anions also exert a strong destabilizing influence at these concentrations. For 0.1M Na₂CO₃ (pH=10.84, [Na⁺]=0.2M, Tm=42.7 °C), the anion destabilizing effect is higher 20 °C. For NaClO₄ (ClO₄ ^? is a strong “chaotropic” anion), an equal anion effect occurs at much higher concentrations ～3M. This means that Na₂CO₃ gives rise to a much stronger anion effect than other salts. The effect is pH dependent. It decreases fivefold at neutral pH after addition of HCl to 0.1M Na₂CO₃ as well as after addition of NaOH for pH>11.2.     

Inelastic light scattering studies of solutions of the chloroplast coupling factor (CF1) at high and low ionic strength

The translational diffusion coefficient of CF₁ at low and high protein concentration as well as at different ionic strength (0.05 – 1.65 M) wsa determined by means of quasi-elastic light scattering experiments. The diffusion coefficient changes from D_20,w^o = 3.12 × 10^?7 cm² · sec^?1 at 0.05 M, pH 7.8, 20°C, to D_20,w^o = 3.52 × 10^?7 cm² · sec^?1 at 1.6 M, pH 7.8, 20°C. At high enzyme concentration (20 mg/ml) and under crystallization conditions (Paradies, BBRC 91: 685, 1979) CF₁ behaves as a solution of “true” hard spheres, whereas at low salt concentration the ionic atmosphere has a larger spatial extent, resulting in a higher effective hydrodynamic radius (R_H = 65 Å).     

Kinetics of phosphate uptake in excised maize root

The short term uptake of phosphate involving 10 min absorption followed by 5 min desorption, both at 30 °C, in the concentration range 1.0×10^?9 to 7.5×10^?2 M KH₂PO₄ by fresh and washed maize (Zea mays L. cv. Ganga Safed-2) roots can be described by a single isotherm having five phases (0 and I–IV) with regularly spaced kinetic constants. Almost identical kinetics were observed in both fresh and washed maize roots. The kinetics of phase 0 in the concentration range 1.0×10^?9–3.0×10^?5 M. was sigmoidal in fresh maize roots, however, in washed tissue exhibited 2 phases termed here as 0a and 0b. 0a covered the concentration range 1.0×10^?9–5.0×10^?6 M and 0b 6.0×10^?6–3.0×10^?5 M. In the concentration range 1.0×10^?4–7.5×10^?2 M four distinct phases, termed as I, II, III and IV were evident in both fresh and washed maize roots. Each phase obeyed Michaelis—Menten kinetics. The values of K_m and V_max have been estimated for each phase. The uptake isotherm was accompanied by discontinuous transitions.     

Interaction of Growth Retardants and Temperature in Growth, Flowering, Regeneration, and Auxin Activity of Begonia × cheimantha Everett

Soil application of CCC reduced stem and leaf growth in Begonia plants. This effect was evident with all concentrations tested at 18°C, whereas at 21 and 24°C no growth–retarding effect was observed with 2 × 10^?2 M CCC, and with 5 × 10^?3 M growth was even stimulated. Flowering was promoted by CCC in long day and neur–critical temperature, particularly under low light intensity in the winter. The formation of adventitious buds in leaves of plants grown at 21 and 24°C was stimulated when the plants received 5 × 10^?2 and 2 × 10^?2 M CCC, while 8 7times; 10^?2 M was inhibitory. In plants grown at 18°C bud formation was inhibited by all CCC concentrations. Root formation in the the leaves was usually stimulated by high CCC concentrations, while root elongation was reduced. The level of ether–extractable. acidic auxin (presumably IAA) in the leaves was lowered by CCC treatment of the plants, hut this required higher CCC concentrations at higt than at low temperature. When applied to detached leaves CCC stimulated bud formation at concentrations ranging from 10^?4 to 10^?2 M in leaves planted at 18 and 21°C. At 24°C budding was inhibited by 10^?2 M CCC, the lower concentrations being stimulatory also at this temperature. Root formation and growth were not much affected by CCC treatment of the leaves, but increased with the temperature. Soil application of Phosfon (4 × 10^?4 M) had no effect on growth and flowering, nov did it affect the subsequent regeneration of buds and roots in the leaves. In detached leaves Phosfon stimulated bud formation with au optimum at 10^?6 M. Root formation was stimulated by Phosfon at all temperatures, the optimal concentration being 10^?5 M, whereas root length was conversely affected. Foliar application of B-995 to intact plants and treatment of detached leaves greatly inhibited the formation of buds and had little effect on root formation. B-99D reduced the growth and delayed flowering in the plants.     

NADH-dependent cinerulose reductase in rat liver microsomes.

In the course of studies on the metabolism of a new antitumor anthracycline antibiotic, aclacinomycin A, the new keto reductase which catalyzes the reduction of keto group of L-cinerulose of aclacinomycin A to L-rhodinose was found in rat liver microsomal membrane. The enzyme requires NADH for the reduction and showed optimum pH at 7.0. Km value for aclacinomycin A, 2.1 × 10^?5 M and the concentration of NADH need to half maximal activity, 6.2 × 10^?5 M were obtained. The activity was potently inhibited by detergents, such as Triton X-100, sodium deoxycholate and sodium dodecyl sulfate.     

Abrogation by prostaglandin F2alpha of LH-stimulated cyclic AMP accumulation in isolated rat corpora lutea of pregnancy.

Corpora lutea explanted from rats on the sixth day of pregnancy responded to luteinizing hormone (LH; 5 μg/ml) in vitro with a two- to five-fold increase in cellular cyclic AMP (cAMP) concentration. The maximal cAMP level was reached within 60 min and maintained to the end of the 2 hr-incubation. On incubation with prostaglandin F_2α (PGF_2α) in addition to LH, this rise in cAMP accumulation was prevented. For significant suppression, 1.4 × 10^?5M PGF_2α was required. In the absence of LH, PGF_2α (4.2 × 10^?5M) caused no change in cellular cAMP. Addition of PGF_2α (4.2 × 10^?5M) to the incubation medium after the maximal response to LH was attained, caused the cAMP concentration to return to its basal level within 15 min. This abrogation of LH-stimulated cAMP accumulation represents the earliest and hence possibly the triggering event in PGF_2α-induced luteolysis.     

A novel chemiluminescent method for determination of phloroglucinol

It was found that the inhibition and enhancement by phloroglucinol of the chemiluminescence from the luminol–K₃Fe(CN)₆ system were dependent on the pH of luminol solution and the concentration of phloroglucinol. In Na₂CO₃–NaHCO₃ buffer, phloroglucinol exhibited strong chemiluminescent enhancement at pH 9.4. On this basis, a flow injection method was developed for the determination of phloroglucinol. The method was simple, rapid, convenient and sensitive, with a detection limit of 2.0 × 10^?9 mol/L. It is effective for determining phloroglucinol in the range of 1.0 × 10^?5–5.0 × 10^?9 mol/L. The relative standard deviation is 1.3% within one day and 3.2% between days for the determination of 5.0 × 10^?7 mol/L phloroglucinol. The method has been successfully used to determine phloroglucinol in environmental water, with satisfactory results. Copyright © 2003 John Wiley & Sons, Ltd.     

Stimulation by ATP of [3H]dopamine binding to synaptic membrane fractions of canine caudate nucleus

The rate of [³H]dopamine binding to crude synaptic membranes from canine caudate nucleus was considerably increased by 2 mM ATP, 5′-adenylylimidodiphosphate and GTP or by 1 mM 5′-guanylyl-imidodiphosphate, while strongly inhibited by 2 mM ADP and GDP. Half maximal concentrations of [³H]dopamine to bind to the membranes were 1.11 × 10^?7M and 8.75 × 10^?6M in the absence of 4 mM ATP, indicating a negative cooperativity of the dopamine receptor, and 9.25 × 10^?7 M in its presence. Hill coefficient was increased from 0.70 to 1.04 by addition of 4 mM ATP. The optimal concentration of ATP for [³H]dopamine binding was in the range of 0.5 to 5 mM.     

The isolation of pig liver monoamine oxidase

Monoamine oxidase from pig liver has been isolated and purified approximately three hundred-fold. This enzyme has a molecular weight of 1,200,000, is highly polymeric, and contains subunits of molecular weight 146,000, as determined by Sephadex chromatography. The apparent K_m at 25°C is 1.28 × 10^?6 M at pH 9.0 (0.05 M glycine) and 1.74 × 10^?5 M at pH 7.2 (0.2 M phosphate) using benzylamine as a substrate. This enzyme contains approximately 8 copper(II) ions per 1,200,000 molecular weight.     

Influx of glutamic acid in peripheral nerve. Energy,ionic, and pH dependence

The energy, ionic, and pH dependence of the influx of labelled L -glutamic acid has been studied in frog sciatic nerve

• (1) The addition of 10^?2 M D -glucose to the incubation medium was found to inhibit the total influx of glutamic acid from a 10^?6 M solution by about 40%.
• (2) The rate of influx was found to be sensitive to the temperature of the bathing medium. Influx was found to increase with temperature in a linear manner from 10°C to 34°C; above 34°C, the rate of influx dropped with increased temperature.
• (3) At room temperature, the effect of metabolic inhibitors on total influx ranged from 18% inhibition with aresenate to 62% with DNP. Increasing the incubation temperature to 34°C increased the percentage inhibition, a maximum inhibition of 86% occurring with DNP.
• (4) The cardiac glycoside ouabain (10^?3 M) slightly inhibited influx (15%).
• (5) Reduction of the sodium concentration to 2.3 mM resulted in an 82% decrease in total influx. Omission of potassium or a tenfold increase in potassium concentration had no significant effect on influx. Omission of calcium resulted in a slight stimulation of uptake (16%); a tenfold increase in calcium concentration was slightly inhibitory (15%).
• (6) No relation was found between influx and the pH of the incubation medium. Lowering the pH to 5.4 or raising it to 9.4 had no significant effect on influx.
• (7) It is concluded that L -glutamic acid influx in frog sciatic nerve is probably associated with an energy-dependent process.

     

Functional changes of mouse spermatozoa stored in vitro

In suspensions of epididymal spermatozoa in vitro at +10°C and +37°C, all nuclei-containing and mitochondria-containing structures (normal spermatozoa, spermatozoa with the bent and coiled tails, complexes of head and neck) are with propidium iodide and rhodamine 123, respectively. Intracellular ATP concentration determined by a bioluminescent method in mitochondria-containing elements of suspension decreases (significantly faster at 37°C than at 10°C) up to a certain unchangeable level (2.5 × 10^?8 M/l at 37°C and to 1.6 × 10^?8 M/l at 10°C per 10⁶ of mitochondria-containing elements). Mechanisms of spermatozoa destruction are discussed.