Comparison of two extraction methods for evaluation of volatile constituents patterns in commercial whiskeys: Elucidation of the main odour-active compounds

An analytical procedure based on manual dynamic headspace solid-phase microextraction (HS-SPME) method and the conventional extraction method by liquid–liquid extraction (LLE), were compared for their effectiveness in the extraction and quantification of volatile compounds from commercial whiskey samples. Seven extraction solvents covering a wide range of polarities and two SPME fibres coatings, has been evaluated. The highest amounts extracted, were achieved using dichloromethane (CH2Cl2) by LLE method (LLECH2Cl2)(LLECH2Cl2) and using a CAR/PDMS fibre (SPMECAR/PDMS) in HS-SPME. Each method was used to determine the responses of 25 analytes from whiskeys and calibration standards, in order to provide sensitivity comparisons between the two methods. Calibration curves were established in a synthetic whiskey and linear correlation coefficient (r ) were greater than 0.9929 for LLECH2Cl2LLECH2Cl2 and 0.9935 for SPMECAR/PDMS, for all target compounds. Recoveries greater than 80% were achieved. For most compounds, precision (expressed by relative standard deviation, R.S.D.) are very good, with R.S.D. values lower than 14.78% for HS-SPME method and than 19.42% for LLE method. The detection limits ranged from 0.13 to 19.03 μg L−1 for SPME procedure and from 0.50 to 12.48 μg L−1 for LLE. A tentative study to estimate the contribution of a specific compound to the aroma of a whiskey, on the basis of their odour activity values (OAV) was made. Ethyl octanoate followed by isoamyl acetate and isobutyl alcohol, were found the most potent odour-active compounds.

A new and improved strategy combining a dispersive-solid phase extraction-based multiclass method with ultra high pressure liquid chromatography for analysis of low molecular weight polyphenols in vegetables

This paper reports on the development and optimization of a modified Quick, Easy, Cheap Effective, Rugged and Safe (QuEChERS) based extraction technique coupled with a clean-up dispersive-solid phase extraction (dSPE) as a new, reliable and powerful strategy to enhance the extraction efficiency of free low molecular-weight polyphenols in selected species of dietary vegetables. The process involves two simple steps. First, the homogenized samples are extracted and partitioned using an organic solvent and salt solution. Then, the supernatant is further extracted and cleaned using a dSPE technique. Final clear extracts of vegetables were concentrated under vacuum to near dryness and taken up into initial mobile phase (0.1% formic acid and 20% methanol). The separation and quantification of free low molecular weight polyphenols from the vegetable extracts was achieved by ultrahigh pressure liquid chromatography (UHPLC) equipped with a phodiode array (PDA) detection system and a Trifunctional High Strength Silica capillary analytical column (HSS T3), specially designed for polar compounds. The performance of the method was assessed by studying the selectivity, linear dynamic range, the limit of detection (LOD) and limit of quantification (LOQ), precision, trueness, and matrix effects. The validation parameters of the method showed satisfactory figures of merit. Good linearity (View the MathML sourceRvalues2>0.954; (+)-catechin in carrot samples) was achieved at the studied concentration range. Reproducibility was better than 3%. Consistent recoveries of polyphenols ranging from 78.4 to 99.9% were observed when all target vegetable samples were spiked at two concentration levels, with relative standard deviations (RSDs, n = 5) lower than 2.9%. The LODs and the LOQs ranged from 0.005 μg mL−1 (trans-resveratrol, carrot) to 0.62 μg mL−1 (syringic acid, garlic) and from 0.016 μg mL−1 (trans-resveratrol, carrot) to 0.87 μg mL−1 ((+)-catechin, carrot) depending on the compound. The method was applied for studying the occurrence of free low molecular weight polyphenols in eight selected dietary vegetables (broccoli, tomato, carrot, garlic, onion, red pepper, green pepper and beetroot), providing a valuable and promising tool for food quality evaluation.

Solid phase microextraction as a reliable alternative to conventional extraction techniques to evaluate the pattern of hydrolytically released components in Vitis vinifera L. grapes

In present research, headspace solid-phase microextraction (HS-SPME) followed by gas chromatography–mass spectrometry (GC–qMS), was evaluated as a reliable and improved alternative to the commonly used liquid–liquid extraction (LLE) technique for the establishment of the pattern of hydrolytically released components of 7 Vitis vinifera L. grape varieties, commonly used to produce the world-famous Madeira wine. Since there is no data available on their glycosidic fractions, at a first step, two hydrolyse procedures, acid and enzymatic, were carried out using Boal grapes as matrix. Several parameters susceptible of influencing the hydrolytic process were studied. The best results, expressed as GC peak area, number of identified components and reproducibility, were obtained using ProZym M with b-glucosidase activity at 35 °C for 42 h. For the extraction of hydrolytically released components, HS-SPME technique was evaluated as a reliable and improved alternative to the conventional extraction technique, LLE (ethyl acetate). HS-SPME using DVB/CAR/PDMS as coating fiber displayed an extraction capacity two fold higher than LLE (ethyl acetate). The hydrolyzed fraction was mainly characterized by the occurrence of aliphatic and aromatic alcohols, followed by acids, esters, carbonyl compounds, terpenoids, and volatile phenols. Concerning to terpenoids its contribution to the total hydrolyzed fraction is highest for Malvasia Cândida (23%) and Malvasia Roxa (13%), and their presence according previous studies, even at low concentration, is important from a sensorial point of view (can impart floral notes to the wines), due to their low odor threshold (μg/L). According to the obtained data by principal component analysis (PCA), the sensorial properties of Madeira wines produced by Malvasia Cândida and Malvasia Roxa could be improved by hydrolysis procedure, since their hydrolyzed fraction is mainly characterized by terpenoids (e.g. linalool, geraniol) which are responsible for floral notes. Bual and Sercial grapes are characterized by aromatic alcohols (e.g. benzyl alcohol, 2-phenylethyl alcohol), so an improvement in sensorial characteristics (citrus, sweet and floral odors) of the corresponding wines, as result of hydrolytic process, is expected.

Potentialities of two solventless extraction approaches—Stir bar sorptive extraction and headspace solid-phase microextraction for determination of higher alcohol acetates, isoamyl esters and ethyl esters in wines

A stir bar sorptive extraction with liquid desorption followed by large volume injection coupled to gas chromatography–quadrupole mass spectrometry (SBSE-LD/LVI-GC–qMS) was evaluated for the simultaneous determination of higher alcohol acetates (HAA), isoamyl esters (IsoE) and ethyl esters (EE) of fatty acids. The method performance was assessed and compared with other solventless technique, the solid-phase microextraction (SPME) in headspace mode (HS). For both techniques, influential experimental parameters were optimised to provide sensitive and robust methods. The SBSE-LD/LVI methodology was previously optimised in terms of extraction time, influence of ethanol in the matrix, liquid desorption (LD) conditions and instrumental settings. Higher extraction efficiency was obtained using 60 min of extraction time, 10% ethanol content, n-pentane as desorption solvent, 15 min for the back-extraction period, 10 mL min−1 for the solvent vent flow rate and 10 °C for the inlet temperature. For HS-SPME, the fibre coated with 50/30 μm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) afforded highest extraction efficiency, providing the best sensitivity for the target volatiles, particularly when the samples were extracted at 25 °C for 60 min under continuous stirring in the presence of sodium chloride (10% (w/v)). Both methodologies showed good linearity over the concentration range tested, with correlation coefficients higher than 0.984 for HS-SPME and 0.982 for SBES-LD approach, for all analytes. A good reproducibility was attained and low detection limits were achieved using both SBSE-LD (0.03–28.96 μg L−1) and HS-SPME (0.02–20.29 μg L−1) methodologies. The quantification limits for SBSE-LD approach ranging from 0.11 to 96.56 μg L−and from 0.06 to 67.63 μg L−1 for HS-SPME. Using the HS-SPME approach an average recovery of about 70% was obtained whilst by using SBSE-LD obtained average recovery were close to 80%. The analytical and procedural advantages and disadvantages of these two methods have been compared. Both analytical methods were used to determine the HAA, IsoE and EE fatty acids content in “Terras Madeirenses” table wines. A total of 16 esters were identified and quantified from the wine extracts by HS-SPME whereas by SBSE-LD technique were found 25 esters which include 2 higher alcohol acetates, 4 isoamyl esters and 19 ethyl esters of fatty acids. Generally SBSE-LD provided higher sensitivity with decreased analysis time.

Optimisation of stir bar sorptive extraction and liquid desorption combined with large volume injection-gas chromatography–quadrupole mass spectrometry for the determination of volatile compounds in wines

Stir bar sorptive extraction and liquid desorption followed by large volume injection coupled to gas chromatography–quadrupole mass spectrometry (SBSE–LD/LVI-GC–qMS) had been applied for the determination of volatiles in wines. The methodology was optimised in terms of extraction time and influence of ethanol in the matrix; LD conditions, and instrumental settings. The optimisation was carried out by using 10 standards representative of the main chemical families of wine, i.e. guaiazulene, E,E-farnesol, β-ionone, geranylacetone, ethyl decanoate, β-citronellol, 2-phenylethanol, linalool, hexyl acetate and hexanol. The methodology shows good linearity over the concentration range tested, with correlation coefficients higher than 0.9821, a good reproducibility was attained (8.9–17.8%), and low detection limits were achieved for nine volatile compounds (0.05–9.09 μg L−1), with the exception of 2-phenylethanol due to low recovery by SBSE. The analytical ability of the SBSE–LD/LVI-GC–qMS methodology was tested in real matrices, such as sparkling and table wines using analytical curves prepared by using the 10 standards where each one was applied to quantify the structurally related compounds. This methodology allowed, in a single run, the quantification of 67 wine volatiles at levels lower than their respective olfactory thresholds. The proposed methodology demonstrated to be easy to work-up, reliable, sensitive and with low sample requirement to monitor the volatile fraction of wine.

Atividades Biológicas De Xilana De Sabugo De Milho

The corn cob is an agricultural by-product still little used, this in part due to the low knowledge of the biotechnological potential of their molecules. Xylan from corn cobs (XSM) is a polysaccharide present in greater quantity in the structure of plant and its biotechnology potential is little known. This study aimed to the extraction, chemical characterization and evaluation of biological activities of xylan from corn cobs. To this end, corncobs were cleaned, cut, dried and crushed, resulting in flour. This was subjected to a methodology that combines the use of alkaline conditions with waves of ultrasound. After methanol precipitation, centrifugation and drying was obtained a yield of 40% (g/g flour). Chemical analysis indicated a high percentage of polysaccharides in the sample (60%) and low contamination by protein (0.4%) and phenolic compounds (> 0.01%). Analysis of monosaccharide composition indicated the presence of xylose:glucose:arabinose:galactose:mannose:glucuronic acid in a molar ratio 50:20:15:10:2.5:2.5. The presence of xylan in the sample was confirmed by nuclear magnetic resonance (¹H and ¹³C) and infrared spectroscopy (IR). Tests were conducted to evaluate the antioxidant potential of XSM. This showed a total antioxidant capacity of 48.45 EAA/g sample. However, did not show scavenging activity of superoxide and hydroxyl radical and also reducing power. But, showing a high capacity chelating iron ions with 70% with about 2 mg/mL. The ability to XSM to influence cell proliferation in culture was also evaluated. This polymer did not influence the proliferation of normal fibroblast cells (3T3), however, decreased the rate of proliferation of tumor cells (HeLa) in a dose-dependent, reaching an inhibition of about 50% with a concentration around 2 mg/mL. Analyzing proteins related to cell death, by immunoblotting, XSM increases the amount of Bax, Bcl-2 decrease, increase cytochrome c and AIF, and reduce pro-caspase-3, indicating the induction of cell death induced apoptosis dependent and independent of caspase. XSM did not show anticoagulant activity in the PT test. However, the test of activated partial thromboplastin time (aPTT), XSM increased clotting time at about 5 times with 600 μg of sample compared with the negative control. The presence of sulfate on the XSM was discarded by agarose gel electrophoresis and IR. After carboxyl-reduction of XSM the anticoagulant activity decreased dramatically. The data of this study demonstrate that XSM has potential as antioxidant, antiproliferative and anticoagulant compound. Future studies to characterize these activities of XSM will help to increase knowledge about this molecule extracted from corn and allow their use in functional foods, pharmaceuticals and chemical industries.

Síntese enzimática de ésteres de açúcar: surfactantes e polímeros como novos materiais ambientalmente seguros

Sugar esters are substances which possess surfactant, antifungical and bactericidal actions and can be obtained through two renewable sources of raw materials: sugars and vegetable oils. Their excellent biodegradability, allied to lhe fact that they are non toxic, insipid, inodorous, biocompatible, no-ionic, digestible and because they can resist to adverse conditions of temperature, pH and salinity, explain lhe crescent use of these substances in several sections of lhe industry. The objective of this thesis was to synthesize and characterize surfactants and polymers containing sugar branched in their structures, through enzymatic transesterification of vinyl esters and sugars, using alkaline protease from Bacillus subtilis as catalyst, in organic medium (DMF).Three types of sugars were used: L-arabinose, D-glucose and sucrose and two types of vinyl esters: vinyl laurate and vinyl adipate. Aiming to reach high conversions from substrates to products for a possible future large scale industrial production, a serie of variables was optimized, through Design of Experiments (DOE), using Response Surface Methodology (RSM).The investigated variables were: (1) enzyme concentration; (2) molar reason of substrates; (3) water/solvent rale; (4) temperature and (5) time. We obtained six distinct sugar esters: 5-0-lauroyl L-arabinose, 6-0-lauroyl D-glucose, 1'-O-lauroyl sucrose, 5-0-vinyladipoyl L-arabinose, 6-0-vinyladipoyl D-glucose and 1 '-O-vinyladipoyl sucrose, being lhe last three polymerizable. The progress of lhe reaction was monitored by HPLC analysis, through lhe decrease of sugar concentration in comparison to lhe blank. Qualitative analysis by TLC confirmed lhe formation of lhe products. In lhe purification step, two methodologies were adopted: (1) chromatographic column and (2) extraction with hot acetone. The acylation position and lhe chemical structure were determined by 13C-RMN. The polymerization of lhe three vinyl sugar esters was possible, through chemical catalysis, using H2O2 and K2S2O8 as initiators, at 60°C, for 24 hours. IR spectra of lhe monomers and respective polymers were compared revealing lhe disappearance of lhe vinyl group in lhe polymer spectra. The molar weights of lhe polymers were determined by GPC and presented lhe following results: poly (5-0-vinyladipoyl L-arabinose): Mw = 7.2 X 104; PD = 2.48; poly (6-0-vinyladipoyl D-glucose): Mw = 2.7 X 103; PD = 1.75 and poly (1'-O-vinyladipoyl sucrose): Mw = 4.2 X 104; PD = 6.57. The six sugar esters were submitted to superficial tension tests for determination of the critical micelle concentrations (CMC), which varied from 122 to 167 ppm. Finally, a study of applicability of these sugar esters, as lubricants for completion fluids of petroleum wells was' accomplished through comparative analysis of lhe efficiency of these sugar esters, in relation to three commercial lubricants. The products synthesized in this thesis presented equivalent or superior action to lhe tested commercial products

Extração de boro de água produzida sintética por sistema microemulsionado

Boron is a semi-metal present in certain types of soils and natural waters. It is essential to the healthy development of plants and non-toxic to humans, depending on its concentration. It is used in various industries and it s present in water production coming from oil production. More specifically in Rio Grande do Norte, one of the largest oil producers on shore of Brazil, the relationship water/oil in some fields becomes more than 90%. The most common destination of this produced water is disposal in open sea after processing to meet the legal specification. In this context, this research proposes to study the extraction of boron in water produced by microemulsion systems for industrial utilization. It was taken into account the efficiency of extraction of boron related to surfactant (DDA and OCS, both characterized by FT-IR), cosurfactant (butanol and isoamyl alcohol), organic phase (kerosene and heptanes) and aqueous phase (solution of boron 3.6 ppm in alkaline pH). The ratio cosurfactant/ surfactant used was four and the percentage of organic phases for all points of study was set at 5%. It was chosen points with the highest percentage of aqueous phase. Each system was designed for three points of different compositions in relation to the constituents of a pseudoternary diagram. These points were chosen according to studies of phase behavior in pseudoternary diagrams made in previous studies. For this research, points were chosen in the Winsor II region. The excess aqueous solution obtained in these systems was separated and analyzed by ICP OES. For the data set obtained, the better efficiency in the extraction of boron was obtained using the system with DAC, isoamyl alcohol and heptanes, which extracted 49% in a single step. OCS was not viable to the extraction of boron by microemulsion system in the conditions defined in this study

Comparison of DNA-extraction methods and Selective Enrichment broths on the detection of Salmonella Typhimurium in swine feces by polymerase chain reaction (PCR)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Culture of osteogenic cells from human alveolar bone: A useful source of alkaline phosphatase

The aim of this study was to obtain membrane-bound alkaline phosphatase from osteoblastic-like cells of human alveolar bone. Cells were obtained by enzymatic digestion and maintained in primary culture in osteogenic medium until subconfluence. First passage cells were cultured in the same medium and at 7, 14, and 21 days, total protein content, collagen content, and alkaline phosphatase activity were evaluated. Bone-like nodule formation was evaluated at 21 days. Cells in primary culture at day 14 were washed with Tris-HCl buffer, and used to extract the membrane-bound alkaline phosphatase. Cells expressed osteoblastic phenotype. The apparent optimum pH for PNPP hydrolysis by the enzyme was pH 10.0. This enzyme also hydrolyzes ATP, ADP, fructose-1-phosphate, fructose-6-phosphate, pyrophosphate and beta-glycerophosphate. PNPPase activity was reduced by typical inhibitors of alkaline phosphatase. SDS-PAGE of membrane fraction showed a single band with activity of similar to 120 kDa that could be solubilized by phospholipase C or Polidocanol. (c) 2007 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved.

Allosteric modulation of pyrophosphatase activity of rat osseous plate alkaline phosphatase by magnesium ions

Pyrophosphatase activity of rat osseous plate alkaline phosphatase was studied at different concentrations of calcium and magnesium ions. with the aim of characterizing the modulation of enzyme activity by these metals. In the absence of metal ions, the enzyme hydrolysed pyrophosphate following Michaelian kinetics with a specific activity of 36.7 U/mg and K-0.5 = 88 mu M. In the presence of low concentrations (0.1 mM) of magnesium (or calcium) ions, the enzyme also exhibited Michaclian kinetics for the hydrolysis of pyrophosphate, but a significant increase in specific activity (123 U/mg) was observed. K-m values remained almost unchanged. Quite different behavior occurred in the presence of 2 mM magnesium (or calcium) ions. In addition to low-affinity sites (K-0.5 = 40 and 90 mu M, for magnesium and calcium, respectively), high-affinity sites were also observed with K-0.5 values 100-fold lower. The high-affinity sites observed in the presence of calcium ions represented about 10% of those observed for magnesium ions. This was correlated with the fact that only magnesium ions triggered conformational changes yielding a fully active enzyme. These results suggested that the enzyme could hydrolyse pyrophosphate, even at physiological concentrations (4 mu M), since magnesium concentrations are high enough to trigger conformational changes increasing the enzyme activity. A model, suggesting the involvement of magnesium ions in the hydrolysis of pyrophosphate by rat osseous plate alkaline phosphatase is proposed. (C) 1998 Published by Elsevier B.V. Ltd. All rights reserved.

Membrane-bound alkaline phosphatase from ectopic mineralization and rat bone marrow cell culture

Cells from rat bone marrow exhibit the proliferation-differentiation sequence of osteoblasts, form mineralized extracellular matrix in vitro and release alkaline phosphatase into the medium. Membrane-bound alkaline phosphatase was obtained by method that is easy to reproduce, simpler and fast when compared with the method used to obtain the enzyme from rat osseous plate. The membrane-bound alkaline phosphatase from cultures of rat bone marrow cells has a MWr of about 120 kDa and specific PNPP activity of 1200 U/tng. The ecto-enzyme is anchored to the plasma membrane by the GPI anchor and can be released by PIPLC (selective treatment) or polidocanol (0.2 mg/mL protein and 1% (w/v) detergent). The apparent optimum pH for PNPP hydrolysis by the enzyme was pH 10. This fraction hydrolyzes ATP (240 U/mg), ADP (350 U/ mg), glucose 1-phosphate (1100 U/mg), glucose 6-phosphate (340 Wing), fructose 6-phosphate (460 U/mg), pyrophosphate (330 U/mg) and (3glycerophosphate (600 U/mg). Cooperative effects were observed for the hydrolysis of PPi and beta-glycerophosphate. PNPPase activity was inhibited by 0.1 mM vanadate (46%), 0.1 mM ZnCl2 (68%), 1 mM levamisole (66%), 1 mM arsenate (44%), 10 mM phosphate (21%) and 1 mM theophylline (72%). We report the biochemical characterization of membrane-bound alkaline phosphatase obtained from rat bone marrow cells cultures, using a method that is simple, rapid and easy to reproduce. Its properties are compared with those of rat osseous plate enzyme and revealed that the alkaline phosphatase obtained has some kinetics and structural behaviors with higher levels of enzymatic activity, facilitating the comprehension of the mineralization process and its function. (c) 2006 Elsevier B.V. All rights reserved.

Inorganic pyrophosphate-phosphohydrolytic activity associated with rat osseous plate alkaline phosphatase

Purified membrane-bound alkaline phosphatase from rat osseous plate hydrolyzed pyrophosphate in the presence of magnesium ions, with a specific activity of 92.7 U/mg. Optimal apparent pH for pyrophosphatase activity was 8.0 and it remained unchanged on increasing the pyrophosphate concentration. In the absence of magnesium ions the enzyme had a K-m = 88 mu M and V = 36.7 U/mg for pyrophosphate and no inhibition by excess substrate was observed. Pyrophosphatase activity was rapidly destroyed at temperatures above 40 degrees C, but magnesium ions apparently protected the enzyme against danaturation. Sodium metavanadate (Ki = 1.0 mM) was a competitive inhibitor of pyrophosphatase activity, while levamisole (Ki = 8.2 mM) and theophylline (Ki = 7.4 mM) were uncompetitive inhibitors. Magnesium ions (K-0.5 = 1.7 mu M) stimulated pyrophosphatase activity, while cobalt (Ki = 48.5 mu M) and zinc (Ki = 22.0 mu M) ions were non-competitive inhibitors. Manganese and calcium ions had no effect on pyrophosphatase activity. The M-w of the pyrophosphatase: protein was 130 kDa by gel filtration, but a value of 65 kDa was obtained by dissociative gel electrophoresis, suggesting that it was a dimer of apparently identical subunits. These results suggested that pyrophosphatase activity stems from the membrane-bound osseous plate alkaline phosphatase and not from a different protein.

Construction of an alkaline phosphatase-liposome system: a tool for biomineralization study

Alkaline phosphatase is required for the mineralization of bone and cartilage. This enzyme is localized in the matrix vesicle, which plays a role key in calcifying cartilage. In this paper. we standardize a method for construction an alkaline phosphatase liposome system to mimic matrix vesicles and examine a some kinetic behavior of the incorporated enzyme. Polidocanol-solubilized alkaline phosphatase, free of detergent, was incorporated into liposomes constituted from dimyristoylphosphatidylcholine (DMPC), dilaurilphosphatidylcholine (DLPC) or dipalmitoylphosphatidylcholine (DPPC). This process was time-dependent and >95% of the enzyme was incorporated into the liposome after 4 h of incubation at 25 degreesC. Although, incorporation was more rapid when vesicles constituted from DPPC were used, the incorporation was more efficient using vesicles constituted from DMPC. The 395 nm diameter of the alkaline phosphatase-liposome system was relatively homogeneous and more stable when stored at 4 degreesC.Alkaline phosphatase was completely released from liposome system only using purified phosphatidylinositol-specific phospholipase C (PIPLC). These experiments confirm that the interaction between alkaline phosphatase and lipid bilayer of liposome is via GPI anchor of the enzyme, alone. An important point shown is that an enzyme bound to liposome does not lose the ability to hydrolyze ATP, pyrophosphate and p-nitrophenyl phosphate (PNPP), but a liposome environment affects its kinetic properties, specifically for pyrophosphate.The standardization of such system allows the study of the effect of phospholipids and the enzyme in in vitro and in vivo mineralization, since it reproduces many essential features of the matrix vesicle. (C) 2002 Elsevier B.V. Ltd. All rights reserved.

Erythrocyte ghost cell-alkaline phosphatase: construction and characterization of a vesicular system for use in biomineralization studies

Alkaline phosphatase is required for the mineralization of bone and cartilage. This enzyme is localized in the matrix vesicle, which plays a role key in calcifying cartilage. In this paper we standardize a method to construction a resealed ghost cell-alkaline phosphatase system to mimic matrix vesicles and examine the kinetic behavior of the incorporated enzyme. Polidocanol-solubilized alkaline phosphatase, free of detergent, was incorporated into resealed ghost cells. This process was time-dependent and practically 50% of the enzyme was incorporated into the vesicles in 40 h of incubation, at 25 degreesC. Alkaline phosphatase-ghost cell systems were relatively homogeneous with diameters of about 300 nm and were more stable when stored at -20 degreesC.Alkaline phosphatase was completely released from the resealed ghost cell-system using only phospholipase C. These experiments confirm that the interaction between alkaline phosphatase and the lipid bilayer of resealed ghost cell is exclusively via glycosylphosphatidylinositol (GPI) anchor of the enzyme.An important point shown is that an enzyme bound to resealed ghost cell does not lose the ability to hydrolyze ATP, pyrophosphate and p-nitrophenyl phosphate (PNPP), but the presence of a ghost membrane, as a support of the enzyme, affects its kinetic properties. Moreover, calcium ions stimulate and phosphate ions inhibit the PNPPase activity of alkaline phosphatase present in resealed ghost cells. (C) 2002 Elsevier B.V. B.V. All rights reserved.