Glycosidases of turnip leaf tissues

Four myrosinase (β-thioglucosidase EC. 3.2.3.1) and seven disaccharase (β-fructofuranosidase, EC. 3.2.1.26) isoenzymes were isolated from turnip leaves. The most active enzymes were isolated in pure form. Myrosinase and disaccharase mol wt was 62.0 × 10³ and 69.5 × 10³ dalton, respectively, on the basis of gel filtration on Sephadex G-200. Myrosinase pH profile showed high activity between pH 5 and 7 with the optimum at pH 5.5. The purified enzyme was heat-stable for 60 min at 30°C with only loss of 24% of activity. Its activity is strongly inhibited (100%) by Pb²⁺, Ba²⁺, Cu²⁺ and Ca²⁺ ions, and activated (70%) by EDTA at 0.04M. The pure enzyme failed to hydrolyze amylose, glycogen, lactose, maltose, and sucrose. TheK _m andV _max values of myrosinase using sinigrin as specific substrate was 0.045 mM and 2.5 U, respectively. The maximal activity of disaccharase enzyme was obtained at pH 4–5 and 35–37°C. The enzyme was heat-stable at 30°C for 30 min with only 10% loss of its activity. Its activity is strongly activated (70–240%) by Ca²⁺, Ba²⁺, Cu²⁺, and EDTA at 0.01M. The enzyme activity is specific to the disaccharide sucrose and failed to hydrolyze other disaccharides (maltose and lactose). TheK _m andV _max of disaccharase were 0.123 mM and 3.33 U, respectively.     

Determination of Hepatotoxic Pyrrolizidine Alkaloids in Gynura segetum by MEKC

A novel, rapid and sensitive micellar electrokinetic capillary chromatography method was developed for the separation and determination of two hepatotoxic pyrrolizidine alkaloids in Gynura segetum (Lour.) Merr. (Jusanqi) within 8 min. The method was successfully applied to the simultaneous determination of seneciphylline and senecionine in a Jusanqi sample.     

Biochemical characteristics, metabolism, and antitumor activity of several acetylated hexosamines

We have synthesized several potential inhibitors and/or modifiers of the carbohydrate portion of plasma membrane glycoconjugates. These include fluorinated and actylated analogs of D-glucosamine, D-galactosamine, and D-mannosamine. These compounds have been tested to determine their effects on both[14C] glucosamine and [3H] leucine incorporation into glycoconjugate and on cell growth and viability using P-288 murine lymphoma cells maintained in tissue culture. The most cytotoxic agent tested was 2-acetamido-2-deoxy-1,3,4,6-tetra-O-acetyl-beta-D-glucopyranose or simply beta-pentaacetylglucosamine which prevented cell growth at 10(-4)-10(-3) M. beta-Pentaacetylglucosamine cytotoxicity was correlated with its high lipid solubility, having an octanol/water partition coefficient of 0.424 as compared with 0.278 for the alpha-anomer and 0.017 for N-acetylglucosamine. In vitro metabolism studies with [4C]- and/or [3H]-labeled pentaacetylglucosamine have indicated intracellular de-O-acetylation leading to the biosynthesis of UDP-N-acetylglucosamine, followed by the incorporation of this sugar into cellular glycoprotein. Concomitant with the formation of increased amounts of this nucleotide sugar, intracellular UTP and CTP pools fell to one third normal within 3 h after the administration of 1 mM pentaacetylglucosamine. At present it is unclear whether the cytotoxicity of beta-pentaacetylglucosamine or other similar agents is due to alterations in nucleotide and nucleotide-sugar pools causing a decrease in energy charge and polynucleotide biosynthesis or is due to a direct effect on membrane glycoconjugate biosynthesis.     

Mass-spectrometric study of some cholinesterase substrates

Conclusions The fundamental directions of mass spectrometric decomposition of molecular ions of the complex esters of N-(-hydroxyethyl) anabasine and cytisine are related to the homolytic rupture of the ethylene bridge between the amine and complex-ester portions of the molecule and the McLafferty rearrangement.Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 8, pp. 1823–1827, August, 1980.     

A potentiometric assay of cholinesterase.

The ion-selective characteristics of Orion nitrate, chloride, calcium electrode membranes and Millipore filters saturated with organic solvents (o-dichlorobenzene, 1,2-dichloroethane, 2,2-dichlorodiethyl ether and 1,1,2,2-tetrachloroethane) have been studied. The saturated membranes were not inert, but showed ion-selective qualities. Almost Nernstian behaviour was obtained for several anions. The additition of an ion exchanger scarcely affected the potentiometric behaviour for most ions, except when a selective interaction occurred.     

Characteristics of the cholinesterase of the venom of the central Asian cobra

Summary 1. By rechromatography on sulfoethyl-Sephadex C-50, an electrophoretically homogeneous preparation of cholinesterase has been obtained from the venom ofNaja oxiana Eichwald.2. The activity of the cholinesterase isolated depends on the concentration of the enzyme and the time and temperature of incubation, and also on the pH. The following must be considered the optimum conditions: time of incubation of the enzyme with the substrate 20–30 min, pH 8.0–8.5, temperature 37–38°C.3. Diisopropyl phosphorofluoridate (DIPF) in a concentration of 2 µM completely suppresses the activity of the cobra venom cholinesterase.4. The venom cholinesterase hydrolyzes acetylcholine chloride and acetylthiocholine bromide but has no effect on butyrylthiocholine bromide, in which respect it resembles the true cholinesterases.5. Preparations of cobra venom cholinesterase do not possess a lethal action and do not potentiate the activity of the neurotoxins of the same venom.Institute of Biochemistry, Academy of Sciences of the Uzbek SSR. Translated from Khimiya Prirodnykh Soedinenii, No. 6, pp. 783–789, November–December, 1972.     

Enzymatic determination of pyridostigmine in the presence of endogenous cholinesterase

An enzymatic method for the assay of pyridostigmine in body fluids is described, in which the degree of inhibition of the endogenous cholinesterase is utilized in constructing the calibration curve. Hydrogen-ion activity is measured potentiometrically (±0.1 mV). Samples are introduced directly, without any extraction, concentration or derivatization. The effect of the time of inhibition is discussed. Cholinesterase inhibition is calculated for various concentrations of pyridostigmine, and summarized graphically. The assay is applied to blood plasma from humans and animals and found to be reliable, within 20%, down to 10^?8 g mol^?1 concentrations of pyridostigmine.     

Comparative analysis of the oil and supercritical CO2 extract of Ridolfia segetum (L.) Moris

Supercritical carbon dioxide extraction allowed to obtain the volatile oil of different aerial parts of Ridolfia segetum (L.) Moris. Extraction conditions were as follows: pressure, 90 bar; temperature, 50 degrees C and carbon dioxide flow, Phi = 1.0 kg h(-1). Waxes were entrapped in the first separator set at 90 bar and -10 degrees C. The oil was recovered in the second separator working at 15 bar and 10 degrees C. The main components of the flower oil were alpha-phellandrene (19.4%), terpinolene (20.5%), piperitenone oxide (11.6%), beta-phellandrene (8.2%), (Z)-beta-ocimene (7.8%), myristicin (7.5%) and p-cymene (4.4%). The comparison with the hydrodistilled (HD) oil reveal that the significative difference was the content of sesquiterpenes which are higher in the supercritical fluid extraction (SFE) products. Collection of samples at different extraction times during supercritical extraction, allowed to monitor the change of the oil composition. Lighter compounds, as hydrocarbon monoterpenes, were extracted in shorter times than the heavier hydrocarbon and oxygenated sesquiterpenes. The oil from the steams was characterized by a high content of alpha-phellandrene (12.9%), terpinolene (11.6%), myristicin (11.0%), p-cymene (9.9%), beta-phellandrene (8.2%) and (Z)-beta-ocimene (6.0%) while the main components of the fruits were found to be myristicin (70.8%), piperitenone oxide (19.9%) and dill apiole (4.2%).     

Comparative analysis of the oil and supercritical CO2 extract of Ridolfia segetum (L.) Moris

Supercritical carbon dioxide extraction allowed to obtain the volatile oil of different aerial parts of Ridolfia segetum (L.) Moris. Extraction conditions were as follows: pressure, 90 bar; temperature, 50°C and carbon dioxide flow, Φ?=?1.0?kg?h^?1. Waxes were entrapped in the first separator set at 90?bar and ?10°C. The oil was recovered in the second separator working at 15?bar and 10°C. The main components of the flower oil were α-phellandrene (19.4%), terpinolene (20.5%), piperitenone oxide (11.6%), β-phellandrene (8.2%), (Z)-β-ocimene (7.8%), myristicin (7.5%) and p-cymene (4.4%). The comparison with the hydrodistilled (HD) oil reveal that the significative difference was the content of sesquiterpenes which are higher in the supercritical fluid extraction (SFE) products. Collection of samples at different extraction times during supercritical extraction, allowed to monitor the change of the oil composition. Lighter compounds, as hydrocarbon monoterpenes, were extracted in shorter times than the heavier hydrocarbon and oxygenated sesquiterpenes. The oil from the steams was characterized by a high content of α-phellandrene (12.9%), terpinolene (11.6%), myristicin (11.0%), p-cymene (9.9%), β-phellandrene (8.2%) and (Z)-β-ocimene (6.0%) while the main components of the fruits were found to be myristicin (70.8%), piperitenone oxide (19.9%) and dill apiole (4.2%).     

Detection of turnip crinkle virus on agarose gel electropherograms at the nanogram load level

The previous conditions for the physical characterization of turnip crinkle virus (TCV) by quantitative agarose gel electrophoresis [1, 2] were limiting the method to the microgram load level and were therefore insufficiently sensitive to satisfy the need in many areas of virology for detection of viruses containing single-stranded RNA at the nanogram level. The present report remedies that defect by presenting a technique compatible with the nanogram load level of such viruses. The technique is based on a reduction of gel thickness and on the use of silver staining.     

Surface-enhanced Raman scattering detection of cholinesterase inhibitors

A new sensitive surface-enhanced Raman scattering (SERS) assay for detection of cholinesterase inhibitors such as organophosphorous pesticides using silver colloidal nanoparticles was developed and optimized. Acetylcholinesterase (AChE) mediated the hydrolysis of acetylthiocholine to produce thiocholine, which interacted with the silver nanoparticles to give a specific SERS spectrum. Variation in enzyme activity due to inhibition was measured from changes in intensity of a characteristic peak (772 cm⁻¹) of the SERS spectrum that was directly correlated with the concentration of produced thiocholine. The method was demonstrated for the detection of paraoxon as reference AChE inhibitor. Limit of detection of paraoxon for 5 min incubation at 25 °C was 1.8 × 10⁻⁸ M. This assay can be utilized for the detection of trace amounts of any AChE inhibitor.     

Radiometric determination of nanogram quantities of cholinesterase inhibitors

Summary A radiometric method is described for the determination of cholinesterase inhibitors in nanogram quantities, which is essentially a modification of the radiometric method developed for detecting cholinesterase inhibition in small samples of human blood. The scope of the technique is demonstrated with two examples of inhibitors: 3-hydroxy-N,N-dimethyl-cis-crotonamide dimethylphosphate (Bidrin) and 2-isopropoxyphenyl-N-methylcarbamate (Arprocarb). The first compound was determined in the range 0.05–0.6 ng, the latter from 5 to 160 ng. The standard deviation was 10–14%.

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Zusammenfassung Eine radiometrische Methode zur Bestimmung von Nanogrammengen Cholinesterase-Inhibitoren wurde beschrieben, die im wesentlichen auf der radiometrischen Bestimmung der Cholinesterase im Blut beruht. Das Prinzip des Verfahrens wird an zwei Beispielen erläutert: der Bestimmung von 3-Hydroxy-N,N-dimethyl-cis-crotonamid-dimethylphosphat (Bidrin) und von 2-iso-Propoxyphenyl-N-methylcarbamat (Arprocarb). Die erstgenannte Verbindung wurde in Mengen von 0,05 bis 0,6 ng, die andere in Mengen von 5 bis 160 ng bestimmt. Die Standardabweichung betrug 10 bis 14%.

     

Enzyme electrode system for assay of serum cholinesterase

An enzyme-sensing electrode system for serum cholinesterase was prepared by coupling a pH-sensing electrode to a thin polymer membrane with a low-molecular-weightcutoff. The electrode system utilized two thin-layered solutions to form a micro electrochemical cell. One layer contained the serum; the other contained acetylcholine substrate which had been stabilized to annul the non-enzymatic decay of the substrate by using a high-molecular-weight buffer. An assay can be performed in 1.5–4.5 min. The precision and accuracy of the technique is comparable with those obtained by spectrophotometric techniques.     

Simultaneous Determination of Senecionine,Senlciphylline, and Senecionine N-Oxide in Gynura segetum by RP-HPLC

Abstract

A rapid reversed-phase high-performance liquid chromatographic (RP-HPLC) method was established for simultaneous determination of senecionine, senlciphylline, and senecionine N-oxide in a famous traditional Chinese medicine, Gynura segetum, which has been commonly used for hemostasis. The HPLC assay was performed on a Kromasil KR100-5 C₁₈ column (25 cm × 4.6 mm, 5 µm) with mobile phase composed of acetonitrile and 0.2% phosphoric acid–triethylamine within 40 min. The detection wavelength was 220 nm. All the compounds showed good linearity (r² > 0.9997). The method was reproducible with intra- and interday variation less than 2.82%. The recovery of the assay was in the range of 96.55–103.88%. The method was successfully applied to the quantification of three constituents in 15 Gynura segetum samples collected from different metropolis. The results indicated that the developed assay could be considered as a suitable quality-control method for Gynura segetum.