Acetylcholinesterase biosensor for carbaryl detection based on interdigitated array microelectrodes

In this study, an acetylcholinesterase (AChE) biosensor with superior accuracy and sensitivity was successfully developed based on interdigitated array microelectrodes (IAMs). IAMs have a series of parallel microband electrodes with alternating microbands connected together. Chitosan was used as the enzyme immobilization material, and AChE was used as the model enzyme for carbaryl detection to fabricate AChE biosensor. Electrochemical impedance spectroscopy was used in conjunction with the fabricated biosensor to detect pesticide residues. Based on the inhibition of pesticides on the AChE activity, using carbaryl as model compounds, the biosensor exhibited a wide range, low detection limit, and high stability. Moreover, the biosensor can also be used as a new promising tool for pesticide residue analysis.     

Mediator-free electrochemical biosensor based on buckypaper with enhanced stability and sensitivity for glucose detection

Here we report on a novel platform based on buckypaper for the design of high-performance electrochemical biosensors. Using glucose oxidase as a model enzyme, we constructed a biocompatible mediator-free biosensor and studied the potential effect of the buckypaper on the stability of the biosensor with both amperometry and FTIR spectroscopy. The results showed that the biosensor responses sensitively and selectively to glucose with a considerable functional lifetime of over 80 days. The fabricated enzymatic sensor detects glucose with a dynamic linear range of over 9 mM and a detection limit of 0.01 mM. To examine the efficiency of enzyme immobilization, the Michaelis–Menten constant was calculated to be 4.67 mM. In addition, the fabricated electrochemical biosensor shows high selectivity; no amperometric response to the common interference species such as ascorbic acid, uric acid and acetamidophenol was observed. The facile and robust buckypaper-based platform proposed in this study opens the door for the design of high-performance electrochemical biosensors for medical diagnostics and environmental monitoring.     

Coexistence of acid phosphatase and acid mucosubstance in the nucleoid of human blood platelet granules

Summary Fine structural localization of acid phosphatase and acid mucosubstance in the human blood platelet has shown that these two components coexist in the platelet granules. Within the granules acid phosphatase activity and acid mucosubstance both appear confined to the nucleoid region. Additionally, heavy acid phosphatase activity filled occasional small cytoplasmic bodies as well as elongated irregular cell organelles which latter appeared to correspond with atypical granules in morphologic preparations. A complex of mucosubstance with nonenzymatic protein, sequestering the lysosomal enzyme, or a complex of mucosubstance with the enzyme reversibly inactivating it, might explain the cytochemical latency of acid phosphatase in certain lysosomes and the masked dialyzed iron affinity in these sites after glutaraldehyde fixation.     

Heterogeneity of lysosomes in human fibroblasts

Summary Lysosomes are defined traditionally with the marker enzyme acid phosphatase. We showed recently that lysosomes from human fibroblasts can be separated into a light and dense fraction as well as prelysosomal population. We now provide evidence that although acid phosphatase is enriched in all three fractions, the marker enzyme in the prelysosomal compartment is qualitatively distinct from that of the lysosomes. Ultrastructural analysis showed that the acid phosphatase in the prelysosomal vesicles deposited an extremely electron-dense reaction product, entirely obliterating the lumen of the vesicle, in contrast to that of the light and dense lysosomes which deposited a fine and diffuse product scattered throughout the luminal space. Biochemical analysis showed that only 51% of the acid phosphatase in the prelysosomes was inhibited by tartrate, while 80% of that in the lysosomes was tartrate-inhibitable. Immunoprecipitation with antibodies specific for various isozymes of acid phosphatase showed that 39% of the acid phosphatase in the prelysosomes was of the lysosomal type whereas over 5007o of the acid phosphatase in the lysosomes was of this type. These results showed that acid phosphatase in the prelysosomes of human cultured fibroblasts can be distinguished from that of the lysosomes cytochemically, biochemically, and immunologically and that lysosomes, as marked by acid phosphatase, are a heterogeneous organelle.     

Isolation and properties of lysosomes from dark-grown potato shoots

Summary A method is described for the isolation of lysosomal fractions from dark-grown potato shoots using a single stage separation on a Ficoll gradient. Peaks of acid hydrolase activity consisting of acid phosphatase, phosphodiesterase, ribonuclease, carboxylic esterase and -glycerophosphatase were well separated from peaks of mitochondrial and glyoxysomal enzymes. A heavy lysosomal fraction with particle diameters from 0.1 to 1.6 and density of 1.10 g cm^-3 containing relatively low hydrolase activity was distinguishable from a light fraction with diameters 0.025 to 0.6 and density of 1.07 g cm^-3 with a higher level of hydrolase activity. Both fractions appeared heterogeneous by electron microscopy, but the fine structure of the membranes of both heavy and light lysosomes was similar. The heavy lysosomal fraction was rich in autophagic vacuoles (secondary lysosomes) containing organelles and amorphous cytoplasmic material. Both fractions were rich in ribonucleic acid.Freezing and thawing, high speed blending and ultrasonication either singly or in combination solubilised a maximum of ca. 30% of the acid phosphatase from crude lysosomal fractions derived from dark-grown potato shoots. Treatment with Triton X-100 and deoxycholate released appreciably more enzyme activity but acetone and carbon tetrachloride failed to solubilise any acid phosphatase. Only detergent treatments gave marked overrecovery of enzyme and indicated structure-linked latency. Liberation of enzyme from lysosomes varied with pH and was almost complete at both extremes of pH. Crude snake venom was rapid and effective in solubilising acid phosphatase from lysosomal preparations, purified phospholipase A was less effective and phospholipases C and D had negligible effects. Phospholipase and venom mediated release of acid phosphatase was accompanied by the coincident release of an acid end-product. Gel filtration of acid phosphatase liberated from heavy and light lysosomal fractions by snake venom digestion revealed that each of these fractions was characterised by the presence of distinct molecular forms of the enzyme. The nature of the association of acid phosphatase with potato shoot lysosomes is discussed.     

Ascorbate oxidase based amperometric biosensor for organophosphorous pesticide monitoring.

An amperometric principle based biosensor containing tissues of cucumber, rich in ascorbic acid oxidase, was used for the detection of organophosphorous (OP) pesticide ethyl paraoxon, which inhibits the activity of ascorbic acid oxidase. The optimal concentration of ascorbic acid used as substrate was found to be 5.67 mM. The biosensor response was found to reach steady state within 2 min. A measurable inhibition (> 10%) was obtained with 10 min incubation of the enzyme electrode with different concentrations of the pesticide. There was a linear relationship between the percentage of inhibition of the enzyme substrate reaction and the pesticide (ethyl paraoxon) concentration in the range 1-10 ppm with a regression value 0.9942.     

Further studies on a unique T-tubular acid phosphatase in avian skeletal muscle

Summary Whith the unique observation, using conventional cytochemistry, of acid phosphatase reaction production in the T-tubules of the posterior latissimus dorsi muscle of the chicken, the possibility of andocytosis of lysosomal enzymes by muscle cells came into question. After testing the substrate specificity of this T-tubular phosphatase, it was clear that the enzyme was not 5-nucleotidase for a typical lysosomal acid phosphatase. The T-tubular enzyme hydrolysed glucose 6-phosphate and -glycerophosphate at pH 5.0 but not cytidine-5-monophosphate which was hydrolysed by dense bodies and autophagic vacuoles. The cytochemical evidence points to a mique phosphatase present on mucle cell membranes which apparently does not belong to the vacuolar apparatus of skeletal muscle and is not 5-nucleotidase.     

A quantitative immunoelectronmicroscopic study on soluble, membrane-associated and membrane-bound lysosomal enzymes in human intestinal epithelial cells

Summary We have used quantitative immunoelectronmicroscopy to compare thein situ localization of acid -glucosidase, lysosomal acid phosphatase, -hexosaminidase and glucocerebrosidase in intestinal epithelial cells of the human duodenum. Differences between these four lysosomal enzymes were observed with respect to their presence at the apical cell surface. Transport to the apical membrane seems to be a more important intracellular route for lysosomal acid phosphatase and acid -glucosidase than it is for -hexosaminidase. The membrane associated lysosomal enzyme glucocerebrosidase is not transported to the microvilli. The studies emphasize that lysosomal enzyme transport pathways are enzyme and cell type specific.     

Evolution of acid phosphatase-1 in the genus drosophila as estimated by subunit hybridization. Interspecific tests

Summary The dimeric enzyme, acid phosphatase-1, was partially purified from eleven species of the genus Drosophila. Dissociated subunits were mixed and allowed to reassociate in forty-one interspecific combinations. In each so-called quantitative subunit hybridization test, the relative activities of the heterospecific and the two homospecific enzymes were determined by densitometry. In 34 of the 41 tests significant differences between observed and expected homospecific: heterospecific enzyme activity ratios were detected. The differences ranged from a four-fold excess of the heterospecific enzyme to over a six-fold excess of the homospecific enzymes. In order to measure the enzyme activities on a protein basis, fifteen heterospecific enzymes were purified and used as antigens in CRM tests. The antisera were diluted such that only the homologous subunit in the heterospecific enzyme complexed the acid phosphatase antibodies. The results from each CRM test show that the heterospecific enzymes is only one-half as antigenic as the homologous homospecific enzyme, when the two are adjusted to equal catalytic activities. Thus, the differences between observed and expected levels of acid phosphatase activity measured by the quantative subunit hybridization technique apparently reflect differences in the relative amounts of protein which form during subunit reassociation. The technique, then, appears to detect differences in acid phosphatase subunit affinities.The data either taken directly from the 41 interspecific tests or in terms of the average difference between each two species in third species tests were used to construct phenograms. The species relationships depicted in both phenograms were very different from their actual phylogenetic relationships. This method, then, is not useful as an evolutionary metric. The differences between observed and expected heterospecific:homospecific enzyme ratios may be due to a relatively large number of amino acid substitutions if acid phosphatase subunits pair isologously.     

Localization of tartrate-resistant acid phosphatase in human placenta

Summary Tartrate-resistant acid phosphatase is an inducible marker of cell differentiation and activation expressed by specialized cells of macrophage lineage and some activated lymphocytes. Clinically, this phosphatase is a diagnostic marker for hairy cell leukaemia and osteoclast activity. The cDNA for this enzyme has been cloned from a placental expression library, yet the cell(s) expressing the enzyme protein has not been determined with certainty. Our laboratories have developed a monoclonal antibody, 9C5, suitable for immunohistochemical localization of tartrate-resistant acid phosphatase in paraffin sections. The purpose of this study was to use antibody 9C5 to identify cells expressing tartrate-resistant acid phosphatase in sections of paraffin-embedded, normal, full-term placenta and to determine if those cells expressed other macrophage markers including CD68(PG-M1 antibody), LN5, lysozyme ₁-antitrypsin and ₁-antichymotrypsin. Histochemical localization of activity in frozen sections was compared with immunohistochemical localization in paraffin sections of the same tissue specimens. The activity and antigenicity of this enzyme were detected in decidual cells, syncytiotrophoblast, and some macrophages distributed throughout maternal and embryonic tissues, but not in neutrophils. Unlike other tissues previously examined, placenta contains significant numbers of the phosphate-positive cells that are not of macrophage origin.     

Redox modulation of reductase and phosphatase activities in human erythrocytes

Summary We have previously reported that ferricyanide reductase activity in human erythrocytes depended on glycolysis and could be modulated by several compounds including oxidants and hormones like insulin. Insulin could activate glycolysis, probably as a consequence of tyrosine phosphorylation of protein band 3, implicating phosphorylation reactions as an important signal for activation of the reductase by insulin. Reversible phosphorylation of cellular proteins is also believed to play a key role in the action of insulin. Cytosolic acid phosphatase activity has been found in human erythrocytes. To further extend initial reports, we studied the effect of modulators on the cytosolic erythrocyte acid phosphatase. Mild oxidants like ferricyanide (1 mM), vanadate (1 mM), Mn²⁺ (0.5 and 1 mM), and phenylarsine oxide (10 and 100 M) inhibited the phosphatase activity. Similarly, insulin at concentrations that stimulate ferricyanide reduction (500, 1000 IU/ml) inhibited the activity of the phosphatase enzyme. The overall results indicated that oxidants are able to inhibit the acid phosphatase and stimulate the redox enzyme. In addition, a significant negative correlation (r = –0.400; P = 0.006) was observed between phosphatase and reductase activities. The observations discussed here, together with previous ones, emphasize that a close association between reductase and phosphatase enzymes may exist and also suggest a role for redox reactions in tyrosine phosphorylation/dephosphorylation-mediated signal transduction pathways.     

Fine structural localization of acid and alkaline phosphatase activities in the absorbing cells of the duodenum of rodents

Summary The morphology of the absorbing cells of the duodenal villi in the mouse, the rat, the hamster and the guinea-pig is described. The polymorphism of the dense bodies is pointed out. The fine localization of acid and alkaline phosphatase is investigated and compared. In all the species, acid phosphatase activity is observed in the dense bodies, Golgi vesicles and rare smooth endoplasmic profiles. Alkaline phosphatase is localized on the microvilli, Golgi apparatus, some smooth endoplasmic cisternae and numerous dense bodies. The presence of an alkaline phosphatase reaction in the dense bodies, probably lysosomes, of the absorbing cells is discussed. It is assumed that this enzyme follows a catabolic pathway and is finally degraded in the lysosomes.Abbreviations used AlPase alkaline phosphatase - AcPase acid phosphatase This work was done thanks to the contract C.E.N./A.I.E.A. N 347/RB and thanks to grants from the Fonds de la Recherche scientifique fondamentale collective.     

Effect of hypoxia on phosphoesterases,oxidative and glycolytic enzymes in the rabbit common carotid artery

Summary An enzyme-histochemical study was performed on the rabbit common carotid artery at periods ranging from one to seventeen days following double-ligation and injections of human , human pre- serum lipoproteins and a physiologic saline into the lumen. The alterations in enzyme activities compared to the contralateral carotid (control) were studied for DPN diaphorase, succinic acid dehydrogenase (SDH), lactic acid dehydrogenase (LDH), glucose-6-phosphate dehydrogenase (G-6-pH DH), ATPase, AMPase, acid and alkaline phosphatase. At earlier time intervals there was a general reduction in oxidative enzyme and ATPase activities concomitant with a general increase in LDH and acid phosphatase activities. At later times oxidative enzyme and ATPase activities returned to their control levels and actually increased in the thickened intima. LDH and acid phosphatase activities remained above the control levels, especially in the thickened intima. Focal areas, presumably necrotic, demonstrated complete loss of activity for all enzymes studied. AMPase activity did not differ from the controls throughout this study, while G-6-pH DH and alkaline phosphatase activities were found only sparsely in the adventitia. The same general pattern of alteration in enzyme activity was found regardless of the substance injected into the ligated artery. Arguments for the use of this experimental model for studies on the pathogenesis of atherosclerosis are given.     

Distribution of acid phosphatase and β-glucuronidase in the hypoplastic cerebellum of jaundiced Gunn rats

Summary Activities of acid phosphatase and -glucuronidase in the cerebella of young jaundiced (j/j) and non-jaundiced (j/+; control) Gunn rats were studied with the enzyme histochemical method. The cerebellum of j/+ rats showed high acid phosphatase activities in Purkinje cells and neurons in the cerebellar nuclei. In j/j rats, a number of neurons were lost and numerous microglialike cells with a high acid phosphatase activity appeared in the hypoplastic cerebellum. Although -glucuronidase activity was rarely detected in the control cerebellum, a high enzyme activity was observed associated with microglialike cells in j/j rats. The present results provide a cytological basis for the reported differential increase in the activities of these lysosomal enzymes in the j/j rat cerebellum.     

Identification of the gene appA for the acid phosphatase (pH optimum 2.5) of Escherichia coli

Summary A strain of Escherichia coli exhibiting reduced activity of the periplasmic enzyme acid phosphoanhydride phosphohydrolase (pH 2.5 acid phosphatase) was isolated. The mutation designated appA1 was located at 22.5 min on the E. coli genetic map. Acid phosphatase purified from an appA ^–transductant showed less than ten percent of the specific activity of an isogenic appA ⁺strain. The mutant enzyme was highly thermolabile and its K_m for paranitrophenyl phosphate was increased about 20-fold. The mutant protein cross-reacted with antibody to the wild-type enzyme and had the same molecular weight and concentration in extracts as the wild-type enzyme. These findings strongly suggest that appA is the structural gene of the acid phosphatase.Abbreviations PNPP paranitrophenyl phosphate - cAMP 3-5-cyclic adenosine monophosphate - Nitrosoguanidine N-methyl-N'-nitro-N-nitrosoguanidine - TCY tetracycline - KAN kanamycin - STR streptomycin     

A study of acid phosphatase in mouse kidney using naphthol AS/BI phosphate as a substrate

Summary A kinetic study of mouse kidney acid phosphatase has been performed using an application of the histochemical method ofBurstone (1958a, b). The suitability of the use of naphthol AS/BI phosphate as a substrate for biochemical assays of acid phosphatase has been ascertained. However, the rate of inhibition of the enzyme by sodium molybdate and sodium fluoride suggests that naphthol AS/BI phosphate may represent a substrate for an acid phosphatase different from-glycerophosphatase.     

A high-molecular weight complex with acid phosphatase activity in human breast cancer

Summary The presence of a high-molecular weight complex with acid phosphatase activity in the cytosol of human mammary tumors is reported. This complex appeared in the cytosol after tissue homogenization in the presence of dithiotreitol, with or without Triton X-100 and at acidic or neutral pH. Upon gel electrophoresis, this fraction showed only one band of enzyme activity which did not enter the fine pore gel. Lubrol or n-butanol had no apparent effect on this complex, and 8 M urea or 2% sodium dodecyl sulfate did not disaggregate this large molecule. After purification by gel filtration, ammonium sulfate precipitation and ion-exchange chromatography an apparent molecular weight or 10⁶ was measured. It hydrolyzed typical acid phosphatase substrates such as p-NPP and -NP, but also ATP and PPi. Only 44% inhibition was observed with L-(+)tartrate and it was still 40% active after 1 hr incubation at 60 °C. Reduction in the presence of SDS yielded several polypeptide bands. It was also detected in some samples of normal mammary tissues, but not in normal human placenta or liver.Abbreviations AP acid phosphatase - C-AP high-molecular weight complex with acid phosphatase activity - SDS sodium dodecyl sulfate - p-NPP p-nitrophenyl phosphate - -NP -naphthyl phosphate - enzyme code number acid phosphatase or ortophosphoric monoester phosphohydrolase (EC 3.1.3.2)     

Association of Matrix Acid and Alkaline Phosphatases with Mineralization of Cartilage and Endochondral Bone

The activities of acid and alkaline phosphatases were localized by enzyme histochemistry in the chondroepiphyses of 5 week old rabbits. Using paraformaldehyde-lysine-periodate as fixative, the activity of acid phosphatase was particularly well preserved and could be demonstrated not only in osteoclasts, but also in chondrocytes as well as in the cartilage and early endochondral matrices. The acid phosphatase in the chondrocytes and the matrix was tartrate-resistant, but inhibited by 2mM sodium fluoride, whereas for osteoclasts 50–100mM sodium fluoride were required for inhibition. Simultaneous localisation of both acid and alkaline phosphatase activities was possible in tissue that had been fixed in 85% ethanol and processed immediately. In the growth plates of the secondary ossification centre and the physis, there was a sequential localisation of the two phosphatases associated with chondrocyte maturation. The matrix surrounding immature epiphyseal chondrocytes or resting/proliferating growth plate chondrocytes contained weak acid phosphatase activity. Maturing chondrocytes were positive for alkaline phosphatase which spread to the matrix in the pre-mineralising zone, in a pattern that was consistent with the known location of matrix vesicles. The region of strong alkaline phosphatase activity was the precise region where acid phosphatase activity was reduced. With the onset of cartilage calcification, alkaline phosphatase activity disappeared, but strong acid phosphatase activity was found in close association with the early mineral deposition. Acid phosphatase activity was also present in the matrix of the endochondral bone, but was only found in early spicules which had recently mineralised. The results suggest that alkaline phosphatase activity is required in preparation of mineralization, whereas acid phosphatase activity might have a contributory role during the early progression of mineral formation.     

Different tartrate sensitivity and pH optimum for two isoenzymes of acid phosphatase in osteoclasts. An electron-microscopic enzyme-cytochemical study

Summary By differentiation of substrate specificity, pH optimum range, and sensitivity to various inhibitors, 2 isoenzymes of acid phosphatase in bone cells have been studied at the electron-microscopic level. When p-nitrophenyl phosphate was used for the substrate, the demonstrable enzyme activity was affected by neither tartrate nor sodium fluoride. The reaction product, when incubated at pH 5–6, was detected in all sites along the pathway for the biosynthesis of acid phosphatase in the osteoclast, including the perinuclear space, cisternae of the endoplasmic reticulum, Golgi complex, various vesicles, and vacuoles. In the osteoclasts attached to bone, the enzymatic activity was demonstrated at the extracellular ruffled border and on the eroded bone surface. Reaction products became confined to lysosomes and extracellular ruffled border when incubated at pH 6–7. Unattached osteoclasts showed a similar intracytoplasmic localization of enzyme as the attached ones, except for the absence of the extracellular enzyme activity. The mononuclear, immature type of osteoclast also resembled the mature osteoclast in terms of enzymatic localization. Except for the osteoclasts, the acid p-nitrophenyl phosphatase activity was restricted to lysosomal vesicles in various bone cells, monocytes, and macrophages. Such activity was inhibited by adding 50 mM tartrate to the p-nitrophenyl phosphate medium. When -glycerophosphate or p-nitrocatechol sulfate was the substrate, most of the reaction product was localized intracellularly. Unlike the acid p-nitrophenyl phosphatase, the acid -glycerophosphatase or arylsulfatase activity in osteoclasts and other bone cells was inhibited completely by 10 mM tartrate or 10 mM sodium fluoride. Even preincubation of 100 mM tartrate in the buffer inhibited -glycerophosphatase activity completely, but p-nitrophenyl phosphatase activity was inhibited incompletely. Consequently, our results suggest that acid p-nitrophenyl phosphatase is a useful cytochemical marker for identification of the osteoclast family at electron-microscopic levels of resolution.     

Cytochemical evidence for the leakage of acid phosphatase through ultrastructurally intact lysosomal membranes

Synopsis Fixation under improper conditions ofin vitro cultivated cells results in an extensive diffusion of the lysosomal enzyme acid phosphatase because of the influence of a low effective osmotic pressure. In the present investigation, advantage was taken of this predictable diffusion in order to establish whether or not leakage of acid phosphatase could take place through ultrastructurally intact lysosomal membranes.In order to reveal small holes in the lysosomal membranes, secondary lysosomes were labelled with thorium dioxide particles, which were presumed to appear free in the cell sap if ruptures in the membranes larger than about Ioo Å were created.The experiments revealed that following the fixation ofin vitro cultivated human glia cells under improper conditions, mitochondria and ground cytoplasm show considerable swelling artifacts, while secondary lysosomes appear to be essentially unaffected. The lysosomes, nevertheless, apparently lost most of their content of acid phosphatase, as judged from enzyme cytochemical studies. These findings indicate that leakage of acid phosphatase from ultrastructurally intact lysosomes is possible.