Kinetic properties of butyrylcholinesterases immobilised on pH-sensitive field-effect transistor surface and inhibitory action of steroidal glycoalkaloids on these enzymes

The inhibitory action of steroid glycoalkaloids alpha-solanine, alpha-chaconine and tomatine on horse and human serum butyryl cholinesterases immobilized on the pH-sensitive field-effect transistors has been studied. Using acetyl- and butyryl choline as substrates, the optimal pH and the apparent kinetic parameters (< K(m) >, < V(max) >) of immobilized butyryl cholinesterases have been calculated in the absence of inhibitors. The affinity of each enzyme to glycoalkaloids has been estimated from calculation of apparent inhibition constants < K(i) > and inhibition coefficients i(0.5). Application of the studied cholinesterases for biosensoric determination of glycoalkaloids in the wide range of concentrations (10(-7)-10(-4) M) in different media has been discussed.     

Development and optimisation of biosensors based on pH-sensitive field effect transistors and cholinesterases for sensitive detection of solanaceous glycoalkaloids

Highly sensitive biosensors based on pH-sensitive field effect transistors and cholinesterases for detection of solanaceous glycoalkaloids have been developed, characterised and optimised. The main analytical characteristics of the biosensors developed have been studied under different conditions and an optimal experimental protocol for glycoalkaloids determination in model solution has been proposed. Using such a biosensor and an enzyme reversible inhibition effect, the total potato glycoalkaloids content can be determined within the range of 0.2-100 microM depending on the type of alkaloid, with lowest detection limits of 0.2 microM for alpha-chaconine, 0.5 microM for alpha-solanine and 1 microM for solanidine. The dynamic ranges for the compounds examined show that such biosensors are suitable for a quantitative detection of glycoalkaloids in real potato samples. High reproducibility, operational and storage stability of the biosensor developed have been shown.     

Determination of potato glycoalkaloids using high-pressure liquid chromatography-electrospray ionisation/mass spectrometry

A method for quantifying two toxic glycoalkaloids, alpha-solanine and alpha-chaconine, in potato (Solanum tuberosum) tuber tissue was developed using HPLC-electrospray ionisation (ESI)/MS. Potato samples were extracted with 5% aqueous acetic acid, and the extracts were subjected directly to HPLC-ESI/MS after filtration. By determining the intensities of the protonated molecules of alpha-solanine (m/z 868) and alpha-chaconine (m/z 852) using selected ion monitoring (positive ion mode), a sensitive assay was attained with detection limits of 38 and 14 ppb for the two glycoalkaloids, respectively. The high sensitivity and selectivity of MS detection effectively reduced the time of analysis thus enabling a high throughput assay of glycoalkaloids in potato tubers.     

Photo-induced synthesis of tomatidenol-based glycoalkaloids in Solanum phureja tubers

The effect of light exposure on the steroidal glycoalkaloid content of Solanum phureja tubers has been investigated and compared with that in domesticated potato (Solanum tuberosum) tubers. The results indicated that the increase in the concentration of solanidine-based glycoalkaloids, alpha-solanine and alpha-chaconine was broadly similar in both species. However, in the S. phureja tubers, light exposure also induced the synthesis of tomatidenol-based glycoalkaloids. These have been identified as alpha- and beta-solamarine. These glycoalkaloids were not detected in tubers continually stored in darkness.     

Dual specificity of sterol-mediated glycoalkaloid induced membrane disruption.

In this study the effects of the glycoalkaloids alpha-solanine, alpha-chaconine, alpha-tomatine and the aglycone solanidine on model membranes composed of PC in the absence and presence of sterols have been analysed via permeability measurements and different biophysical methods. The main result is that glycoalkaloids are able to interact strongly with sterol containing membranes thereby causing membrane disruption in a way which is specific for the type of glycoalkaloid and sterol. For this dual specificity both the sugar moiety of the glycoalkaloid and the side-chain of the sterol on position 24 turned out to be of major importance for the membrane disrupting activity. The order of potency of the glycoalkaloids was alpha-tomatine > alpha-chaconine > alpha-solanine. The plant sterols beta-sitosterol and fucosterol showed higher affinity for glycoalkaloids as compared to cholesterol and ergosterol. The mode of action of the glycoalkaloids is proposed to consist of three main steps: (1) Insertion of the aglycone part in the bilayer. (2) Complex formation of the glycoalkaloid with the sterols present. (3) Rearrangement of the membrane caused by the formation of a network of sterol-glycoalkaloid complexes resulting in a transient disruption of the bilayer during which leakage occurs.     

A major QTL and an SSR marker associated with glycoalkaloid content in potato tubers from <Emphasis Type="Italic">Solanum tuberosum </Emphasis>× <Emphasis Type="Italic">S. sparsipilum</Emphasis> located on chromosome I

New potato (Solanum tuberosum) varieties are required to contain low levels of the toxic glycoalkaloids and a potential approach to obtain this is through marker-assisted selection (MAS). Before applying MAS it is necessary to map quantitative trait loci (QTLs) for glycoalkaloid content in potato tubers and identify markers that link tightly to this trait. In this study, tubers of a dihaploid BC(1) population, originating from a cross between 90-HAF-01 (S. tuberosum(1)) and 90-HAG-15 (S. tuberosum(2) x S. sparsipilum), were evaluated for content of alpha-solanine and alpha-chaconine (total glycoalkaloid, TGA) after field trials. In addition, tubers were assayed for TGA content after exposure to light. A detailed analysis of segregation patterns indicated that a major QTL is responsible for the TGA content in tubers of this potato population. One highly significant QTL was mapped to chromosome I of the HAG and the HAF parent. Quantitative trait loci for glycoalkaloid production in foliage of different Solanum species have previously been mapped to this chromosome. In the present research, QTLs for alpha-solanine and alpha-chaconine content were mapped to the same location as for TGA content. Similar results were observed for tubers exposed to light. The simple sequence repeat marker STM5136 was closely linked to the identified QTL.     

Effects of steroidal glycoalkaloids from potatoes (Solanum tuberosum) on in vitro bovine embryo development

alpha-Solanine and alpha-chaconine are two naturally occurring steroidal glycoalkaloids in potatoes (Solanum tuberosum), and solanidine-N-oxide is a corresponding steroidal aglycone. The objective of this research was to screen potential cyto-toxicity of these potato glycoalkaloids using bovine oocyte maturation, in vitro fertilization techniques and subsequent embryonic development as the in vitro model. A randomized complete block design with four in vitro oocyte maturation (IVM) treatments (Experiment 1) and four in vitro embryo culture (IVC) treatments (Experiment 2) was used. In Experiment 1, bovine oocytes (n=2506) were matured in vitro in medium supplemented with 6 microM of alpha-solanine, alpha-chaconine, solanidine-N-oxide or IVM medium only. The in vitro matured oocytes were then subject to routine IVF and IVC procedures. Results indicated that exposure of bovine oocytes to the steroidal glycoalkaloids during in vitro maturation inhibited subsequent pre-implantation embryo development. Potency of the embryo-toxicity varied between these steroidal glycoalkaloids. In Experiment 2, IVM/IVF derived bovine embryos (n=2370) were cultured in vitro in medium supplemented with 6 microM of alpha-solanine, alpha-chaconine, solanidine-N-oxide or IVC medium only. The results showed that the pre-implantation embryo development is inhibited by exposure to these glycoalkaloids. This effect is significant during the later pre-implantation embryo development period as indicated by fewer numbers of expanded and hatched blastocysts produced in the media containing these alkaloids. Therefore, we conclude that in vitro exposure of oocytes and fertilized ova to the steroidal glycoalkaloids from potatoes inhibits pre-implantation embryo development. Furthermore, we suggest that ingestion of Solanum species containing toxic amounts of glycoalkaloids may have negative effects on pre-implantation embryonic survival.     

Inhibition of insect acetylcholinesterase by the potato glycoalkaloid alpha-chaconine.

Homogenates from several insect species were assayed for inhibition of acetylcholinesterase by the potato glycoalkaloid alpha-chaconine. Colorado potato beetle acetylcholinesterase was up to 150-fold less sensitive than other species tested. Acetylcholinesterase from an insecticide-resistant strain of Colorado potato beetles was more sensitive to inhibition than the susceptible strain. Most insect species tested had inhibitory concentrations causing a 50% reduction in activity in the 5 to 40 microM range. Sensitive insect acetylcholinesterases were similar to mammalian cholinesterases in their response to alpha-chaconine. The results indicate that pesticides and host plant resistance factors may interact at the same target. Changes in the target due to selection pressure from either pesticides or host plant resistance factors could affect the efficacy of both control strategies.     

Potato glycosterol rhamnosyltransferase, the terminal step in triose side-chain biosynthesis

Steroidal glycoalkaloids (SGAs) are potentially harmful specialty metabolites found in Solanaceous plants. Two tri-glycosylated alkaloids, alpha-chaconine and alpha-solanine accumulate in potato tubers. Expressed sequence tags (ESTs) were identified in the available database by searching for protein homology to the Sgt1 (SOLtu:Sgt1) steriodalalkaloid galactosyltransferase. The EST sequence data was used to isolate Sgt3 cDNA sequences by polymerase chain reaction (PCR) from a wounded potato tuber cDNA library. The resulting 1515bp open reading frame of Sgt3, encodes a predicted SGT3 amino acid sequence that is 18 residues longer than, 45% identical to, and 58% homologous to the SGT1 protein. The amino-terminal region of the Sgt3 cDNA was used to create an antisense transgene under control of the granule bound starch synthase, GBSS6, promoter and the ubiquitin, Ubi3, polyadenylation signal. Analysis of SGA metabolites in selected transgenic tubers revealed a dramatic decrease in the accumulation of alpha-chaconine and alpha-solanine. This decrease was compensated by an increase in beta-solanine and beta-chaconine with minor accumulation of alpha-SGAs. These results allowed the identification of the function for SGT3 as the beta-solanine/beta-chaconine rhamnosyl transferase, the terminal step in formation of the potato glycoalkaloid triose side chains.     

Enzyme Catalyzed Degradation and Formation of Peroxycarboxylic Acids

The ability of hydrolases to catalyze perhydrolysis, i.e. lysis of acyl substrates with hydrogen peroxide to form peroxycarboxylic acids, has been investigated. Lipases, esterases and cholinesterases were found to catalyze perhydrolysis but the preference of the enzymes for hydrogen peroxide relative to water as nucleophile was only 10-100 fold, even in the best cases. Hence, perhydrolysis proceeds with a very low efficiency in aqueous systems. Furthermore, all lipases, esterases and cholinesterases tested degrade peroxycarboxylic acids to the corresponding carboxylic acid and hydrogen peroxide. This reaction is most pronounced in the case of lipases while less so for cholinesterases. Consequently, cholinesterases are superior to the other hydrolases studied in catalyzing net formation of peracids in aqueous systems. In organic solvents, immobilized lipases efficiently catalyze formation of peracids from either triglycerides or the parent carboxylic acid. Proteases and phospholipase A-2 were found to neither degrade peracids nor catalyze perhydrolysis of carboxylic esters or phospholipids, respectively.     

Synergism between the potato glycoalkaloids alpha-chaconine and alpha-solanine in inhibition of snail feeding

Snails (Helix aspersa L.) were fed filter paper treated with the potato glycoalkaloids, alpha-solanine and alpha-chaconine, singly or together. In pure form, both glycoalkaloids deterred feeding, with chaconine being the more active compound. In combination, authentic solanine and chaconine interacted synergistically in their inhibition of feeding. The antifeedant activities of methanolic extracts of tuber peel of the potato varieties Majestic and Sharpe's Express presented via filter paper discs did not differ significantly from those of authentic glycoalkaloid solutions of comparable concentration and ratio. In contrast, feeding inhibition by diluted tuber peel extracts of the variety Homeguard was greater than that elicited by comparable authentic glycoalkaloid solutions suggesting additional inhibitory compound(s) in the peel of this variety. Comparison of data from peel extracts of all three potato varieties and authentic glycoalkaloids indicated that the level of feeding inhibition by the extracts was, at least in part, a consequence of a synergism between solanine and chaconine.     

Reactivation of phosphorylated cholinesterase immobilized in a gelatin membrane

The soluble and immobilized cholinesterases (acetyl cholinesterase of human blood erythrocytes (EC 3.1.1.7) and butyryl cholinesterase of equine blood serum, (EC 3.1.1.8] were inactivated by such irreversible inhibitors as diisopropyl fluorophosphate (DFP), O,O-dimethyl-O-(2,2)-dichlorovinyl) phosphate (DDVP), paraoxone, armine. The inactivated enzymes were reactivated under the effect of TMB-4 (1,1'-trimethylene-bis)-4-formyl-pyridine bromide (dioxime). The values of the reactivation rate constants proved to be equal both for the soluble and immobilized cholinesterases inactivated by the same irreversible inhibitor. The immobilized enzyme is simpler and more correct to study the reactivating action than the soluble one.     

Analysis of cholinesterase binding to a carnitine-modified EQCM sensor

A sensor for cholinesterases (ChEs) has been developed by binding carnitine via 1,6-diaminohexane to a mixed monolayer of 11-mercaptoundecanoic acid/11-mercapto-1-undecanol on the surface of a gold-coated quartz crystal. Catalytically active and organophosphate-inhibited acetyl- and butyryl-cholinesterases of different origins were tested for their binding ability to D- and L-carnitine, respectively. The binding constants were calculated by using a one-to-one binding model. Additionally, the activity of the immobilized ChE was monitored and the operational stability was investigated amperometrically.     

Monitoring changes in anthocyanin and steroid alkaloid glycoside content in lines of transgenic potato plants using liquid chromatography/mass spectrometry

Transgenic potato plants overexpressing and repressing enzymes of flavonoids biosynthesis were created and analyzed. The selected plants clearly showed the expected changes in anthocyanins synthesis level. Overexpression of a DNA encoding dihydroflavonol 4-reductase (DFR) in sense orientation resulted in an increase in tuber anthocyanins, a 4-fold increase in petunidin and pelargonidin derivatives. A significant decrease in anthocyanin level was observed when the plant was transformed with a corresponding antisense construct. The transformation of potato plants was also accompanied by significant changes in steroid alkaloid glycosides (SAG) level in transgenic potato tuber. The changes in SAGs content was not dependent on flavonoid composition in transgenic potato. However, in an extreme situation where the highest (DFR11) or the lowest (DFRa3) anthocyanin level was detected the positive correlation with steroid alkaloid content was clearly visible. It is suggested that the changes in SAGs content resulted from chromatin stressed upon transformation. A liquid chromatography/mass spectrometry (LC/MS) system with electrospray ionization was applied for profiling qualitative and quantitative changes of steroid alkaloid glycosides in tubers of twelve lines of transgenic potato plants. Except alpha-chaconine and alpha-solanine, in the extracts from dried tuber skin alpha-solamargine and alpha-solasonine, triglycosides of solasonine, were identified in minor amounts, triglycosides of solanidine dehydrodimers were also recognized.     

Comparison of rapid liquid chromatography-electrospray ionization-tandem mass spectrometry methods for determination of glycoalkaloids in transgenic field-grown potatoes

Two rapid methods for highly selective detection and quantification of the two major glycoalkaloids in potatoes, alpha-chaconine and alpha-solanine, were compared for robustness in high-throughput operations for over 1000 analytical runs using potato tuber samples from field trials. Glycoalkaloids were analyzed using liquid chromatography coupled to tandem mass spectrometry in multiple reaction monitoring mode. An electrospray interface was used in the detection of glycoalkaloids in positive ion mode. Classical reversed phase (RP) and hydrophilic interaction (HILIC) columns were investigated for chromatographic separation, ruggedness, recovery, precision, and accuracy. During the validation procedure both methods proved to be precise and accurate enough in relation to the high degree of endogenous biological variability found for field-grown potato tubers. However, the RP method was found to be more precise, more accurate, and, more importantly, more rugged than the HILIC method for maintaining the analytes' peak shape symmetry in high-throughput operation. When applied to the comparison of six classically bred potato cultivars to six genetically modified (GM) lines engineered to synthesize health beneficial inulins, the glycoalkaloid content in potato peels of all GM lines was found within the range of the six cultivars. We suggest complementing current unbiased metabolomic strategies by validating quantitative analytical methods for important target analytes such as the toxic glycoalkaloids in potato plants.     

Non-classical actions of cholinesterases: Role in cellular differentiation, tumorigenesis and Alzheimer''s disease

The cholinesterases are members of the serine hydrolase family, which utilizes a serine residue at the active site. Acetylcholinesterase (AChE) is distinguished from butyrylcholinesterase (BChE) by its greater specificity for hydrolysing acetylcholine. The function of AChE at cholinergic synapses is to terminate cholinergic neurotransmission. However, AChE is expressed in tissues that are not directly innervated by cholinergic nerves. AChE and BChE are found in several types of haematopoietic cells. Transient expression of AChE in the brain during embryogenesis suggests that AChE may function in the regulation of neurite outgrowth. Overexpression of cholinesterases has also been correlated with tumorigenesis and abnormal megakaryocytopoiesis. Acetylcholine has been shown to influence cell proliferation and neurite outgrowth through nicotinic and muscarinic receptor-mediated mechanisms and thus, that the expression of AChE and BChE at non-synaptic sites may be associated with a cholinergic function. However, structural homologies between cholinesterases and adhesion proteins indicate that cholinesterases could also function as cell-cell or cell-substrate adhesion molecules. Abnormal expression of AChE and BChE has been detected around the amyloid plaques and neurofibrillary tangles in the brains of patients with Alzheimer's disease. The function of the cholinesterases in these regions of the Alzheimer brain is unknown, but this function is probably unrelated to cholinergic neurotransmission. The presence of abnormal cholinesterase expression in the Alzheimer brain has implications for the pathogenesis of Alzheimer's disease and for therapeutic strategies using cholinesterase inhibitors.     

Effect of LSD-25 on cholinesterases of the rat brain]

The indications for investigation on the LSD-25 "in vivo" activity on brain cholinesterases of the rat, have been considered. Indications of materials and method used have been supplied too. From the results obtained it emerged no clear evidence of a statistically significant inhibition of cholinesterases due to LSD-25. The results have been discussed and it has been evidenced that the lack of action of the psychotomimetic substance on cholinesterases could be only apparent or that the discrepancy between the LSD-25 anticholinesterase "in vivo" and "in vitro" action should be ascribed to the too scarce tissue levels which can be reached with the does utilized. Apart from the hypothesis, tending to explain the results obtained, it has been considered that the LSD-25 behavioural action, is not probably carried out thanks to a cholinesterase activity.     

Anticholinesterase activity of alkyl esters of perfluoroalkyl phosphonic and perfluoroalkyl phosphinic acids

It has been demonstrated that esters (RO)2P(O)X and RO(R1)P(O)X where R and R1-alkyls, X-CF3 or C2F5, irreversibly inhibited cholinesterases. Their inhibitory effect increased with the elongation of alkyl radicals from CH3- to C4H9-, being more evident with respect to butyrylcholinesterase from horse serum than to acetylcholinesterase from human erythrocytes. It is shown that the concept on inability of esters of thiophosphoric acids to inhibit cholinesterases due to the fact that thionic sulphur (P-S) does not form a strong hydrogen bonds, cannot be applied to esters of perfluorothiophosphonic acids: (C2H5O)2P(S)CF3 inhibits cholinesterases more efficiently than (C2H5O)2P(O)CF3. One of the fluoric atoms probably forms hydrogen bond with the corresponding site of the active centre in cholinesterases, similar to phosphorylic oxygen (P-O) in case of the enzyme inhibition by esters of phosphoric acids.     

Mechanisms of cholinesterase inhibition by inorganic mercury

The poorly known mechanism of inhibition of cholinesterases by inorganic mercury (HgCl2) has been studied with a view to using these enzymes as biomarkers or as biological components of biosensors to survey polluted areas. The inhibition of a variety of cholinesterases by HgCl2 was investigated by kinetic studies, X-ray crystallography, and dynamic light scattering. Our results show that when a free sensitive sulfhydryl group is present in the enzyme, as in Torpedo californica acetylcholinesterase, inhibition is irreversible and follows pseudo-first-order kinetics that are completed within 1 h in the micromolar range. When the free sulfhydryl group is not sensitive to mercury (Drosophila melanogaster acetylcholinesterase and human butyrylcholinesterase) or is otherwise absent (Electrophorus electricus acetylcholinesterase), then inhibition occurs in the millimolar range. Inhibition follows a slow binding model, with successive binding of two mercury ions to the enzyme surface. Binding of mercury ions has several consequences: reversible inhibition, enzyme denaturation, and protein aggregation, protecting the enzyme from denaturation. Mercury-induced inactivation of cholinesterases is thus a rather complex process. Our results indicate that among the various cholinesterases that we have studied, only Torpedo californica acetylcholinesterase is suitable for mercury detection using biosensors, and that a careful study of cholinesterase inhibition in a species is a prerequisite before using it as a biomarker to survey mercury in the environment.