Purification and characterization of parvalbumins, the major allergens in red stingray (Dasyatis akajei)

Fish has received increasing attention because it induces IgE-mediated food allergy. Parvalbumin (PV) represents the major allergen of fish, and IgE cross-reactivity to PV in various teleost fish species has been shown, while little information is available about allergens in elasmobranch fish. In this study, two PV isoforms (named as PV-I and PV-II) from red stingray (Dasyatis akajei) were purified to homogeneity by a series of procedures including ammonium sulfate precipitation and column chromatographies of DEAE-Sepharose and Sephacryl S-200. Purified PVs revealed a single band on tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular masses of PV-I and PV-II were 12.29 and 11.95 kDa, respectively, as determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Western blot using antifrog PV monoclonal antibody (PARV-19) showed positive reactions to the two proteins, confirming that they were PVs, although their immunological reactivities were weaker than those of PV from silver carp. The N-terminal amino acid sequence of PV-I was determined, and comparison with PVs from other fish species showed low homology between teleost and elasmobranch fish. The isoelectric points of PV-I and PV-II were 5.4 and 5.0, respectively, as determined by two-dimensional electrophoresis (2-DE), suggesting that both isoforms belong to the α-group. IgE immunoblotting analysis showed that sera from fish-allergic patients reacted to both PV-I and PV-II from red stingray. Thermal stability revealed that PV-I easily formed oligomers than PV-II, which might contribute to the maintenance of its allerginicity during heat processing.     

Isolation and characterization of aminopeptidase (Jc-peptidase) from Japanese cedar pollen (Cryptomeria japonica)

An aminopeptidase, Jc-peptidase, was purified from Japanese cedar pollen by seven steps, including precipitation with ammonium sulfate, ion-exchange chromatography, gel filtration, hydrophobic interaction chromatography on phenyl-agarose, and high-performance liquid chromatography. Purified Jc-peptidease has a molecular weight of 42 kDa and hydrolyzes the synthetic substrates of L-phenylalanyl-4-methylcoumaryl-7-amide (Phe-MCA) with Km = 5 x 10(-5) M, Tyr-MCA with Km = 7 x 10(-4) M, Leu-MCA with Km = 1 x 10(-3) M, and Met-MCA with Km = 1 x 10(-3) M. Other MCA analogues such as Arg-MCA or Glu-MCA failed to serve as its substrates. The activity was inhibited in the presence of phebestin, [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-L-valyl]-L-phenylalanine, with Ki = 4.7 x 10(-5) M, or bestatin, [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]-L-leucine, with Ki = 1.1 x 10(-4) M. According to amino acid sequence analysis, the N-terminal amino group seems to be blocked. The physiological function of the aminopeptidase (Jc-peptidase) has not been clarified in vivo.     

Purification, characterization, and cDNA cloning of a myofibril-bound serine proteinase from the skeletal muscle of crucian carp (Carassius auratus)

A myofibril-bound serine proteinase (MBSP) was highly purified from the skeletal muscle of crucian carp (Carasius auratus) by acidic treatment of myofibril solution and chromatographies on Q-Sepharose and benzamidine-Sepharose 6B. MBSP revealed a main protein band of approximately 28 kDa on SDS-polyacrylamide gel electrophoresis (PAGE) and was particularly inhibited by serine proteinase inhibitors. Substrate-specificity analysis revealed that the enzyme specifically cleaved at the carboxyl side of arginine and lysine residues, suggesting the characteristics of a trypsin-type serine proteinase. MBSP gene was cloned on the basis of the N-terminal sequence and the conserved active site peptide of serine proteinases together with 5'-rapid amplification of cDNA ends (5'-RACE) and 3'-RACE. The coding region gave an amino acid sequence of 242 residues including the initiation methionine and a signal peptide of 20 residues. Amino acid residues of His60, Asp106, and Ser196 consisting of the catalytic triad of serine proteinases were conserved in the sequence. Crucian carp MBSP shared relatively high identities with other serine proteinases, especially in well-conserved regions.     

Invertase inhibitors from sweet potato (Ipomoea batatas): purification and biochemical characterization

Two proteinaceous invertase inhibitors, designated ITI-L and ITI-R, were purified to electrophoretic homogeneity. ITI-L was purified from acetone powder of sweet potato leaves through sequential steps entailing buffer extraction, acid treatment, DEAE-Sephacel ion-exchange chromatography, and Sephacryl S-100 gel filtration. ITI-R was purified from sweet potato tuberous roots by sequentially applying buffer extraction, Con A-Sepharose affinity chromatography, DEAE-Sephacel ion-exchange chromatography, Sephacryl S-200, and Superose 12 gel filtration. The optimal pHs for interaction between ITI-L and ITI-R and acid invertase from sweet potato leaves were 5.5 and 5.0, respectively. The molecular masses of ITI-L and ITI-R were 10 and 22 kDa, respectively, as estimated by both gel filtration and SDS-PAGE. Both inhibitors were thermostable (90% of the activity remained after incubation at 100 degrees C for 20 min), and Western blotting showed them to be immunologically related.     

Purification and characterization of calpastatin from grass prawn muscle (Penaeus monodon)

Calpastatin, a specific calpain inhibitor was purified to electrophoretical homogeneity from grass prawn (Penaeus monodon) muscle by 100 degrees C heat-treatment, DEAE-Sephacel, and Q-Sepharose chromatographs. No significant change in the inhibitory activity of crude calpastatin was observed even after 20 min incubation at 100 degrees C, pH 7.0. The purified prawn calpastatin had a molecular weight (M(r)) of 80 and 88.7 kDa determined by SDS-PAGE and Sephacryl S-200 HR gel filtration, respectively. According to the active site titration, the purified calpastatin revealed four beef mu-calpain and two beef m-calpain binding domains, respectively. It was stable during 1 h of incubation at 30 degrees C under pH 4.5-10.0 and shown to be a highly specific inhibitor for calpain.     

Classification of gilthead sea bream (Sparus aurata) from 1H NMR lipid profiling combined with principal component and linear discriminant analysis

The combination of (1)H NMR fingerprinting of lipids from gilthead sea bream (Sparus aurata) with nonsupervised and supervised multivariate analysis was applied to differentiate wild and farmed fish and to classify farmed specimen according to their areas of production belonging to the Mediterranean basin. Principal component analysis (PCA) applied on processed (1)H NMR profiles made a clear distinction between wild and farmed samples. Linear discriminant analysis (LDA) allowed classification of samples according to the geographic origin, as well as for the wild and farmed status using both PCA scores and NMR data as variables. Variable selection for LDA was achieved with forward selection (stepwise) with a predefined 5% error level. The methods allowed the classification of 100% of the samples according to their wild and farmed status and 85-97% to geographic origin. Probabilistic neural network (PNN) analyses provided complementary means for the successful discrimination among classes investigated.     

The 26S proteasome in garlic (Allium sativum): purification and partial characterization

The 26S proteasome (multicatalytic protease complex, MPC) was purified from fresh garlic cloves (Allium sativum) to near homogeneity by ion exchange chromatography on DEAE-sephacel, gel filtration on Sepharose-4B, and glycerol density gradient centrifugation. Two alpha-type (20S proteasome "catalytic core") subunits were identified by the direct sequencing of peptide fragments (mass fingerprint analysis, Mass Spectrometry Lab, Stanford University) or the sequencing of a cloned cDNA generated using a garlic cDNA library as the template; these subunits were found to have a high homology to those from other plants. Polyacrylamide gel electrophoresis under denaturing conditions separated the garlic MPC into multiple polypeptides having molecular masses in the range of 21-35 (components of the 20S catalytic core) and 55-100 kDa (components of the 19S regulatory units). The banding pattern of the garlic MCP is similar to that of spinach and rat liver with minor differences in some components; however, polyclonal antibodies against mammalian proteasomes failed to significantly stain the enzyme from garlic. This is the first work to identify the garlic proteasome.     

Partial purification and kinetic characterization of a carotenoid cleavage enzyme from quince fruit (Cydonia oblonga)

For the first time, a cytosolic carotenoid cleavage enzyme isolated from quince (Cydonia oblonga) fruit is described. The enzyme was partially purified by using centrifugation, acetone precipitation, ultrafiltration (300 kD, 50 kD), isoelectric focusing (pH 3-10), and sodium dodecyl sulfate polyacrylamide gel electrophoresis (7.5%). In this way, an enzymatically active protein fraction was obtained that contained three similar proteins, all exhibiting molecular weights in the range of 20 kD. Using beta-carotene as substrate, the enzyme activity was detected spectrophotometrically at a wavelength of 505 nm. The time constant of the reaction was 8.2 min, the Michaelis constant (K(m)) was 11.0 micromol x L(-1), and the maximum velocity (v(max)) was 0.083 micromol x L(-1) x min(-1) x mg(protein)(-1). The optimum temperature was above 50 degrees C.     

Partial purification,characterization, and thermal and high-pressure inactivation of pectin methylesterase from carrots (Daucus carrota L.)

Pectin methylesterase (PME) from carrots (Daucus carrota L.) was extracted and purified by affinity chromatography on a CNBr-Sepharose 4B-PME inhibitor column. A single protein and PME activity peak was obtained. A biochemical characterization in terms of molar mass (MM), isoelectric points (pI), and kinetic parameters of carrot PME was performed. In a second step, the thermal and high-pressure stability of the enzyme was studied. Isothermal and combined isothermal-isobaric inactivation of purified carrot PME could be described by a fractional-conversion model.     

Purification and characterization of cathepsin L from the muscle of silver carp (Hypophthalmichthys molitrix)

Cathepsin L in silver carp musle was purified to 48.4-fold by acid-heat treatment and ammonium sulfate fractionation, followed by a series of chromatographic separations. The molecular mass of the purified enzyme was 30 kDa determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme was activated by dithiothreitol and cysteine while it was substantially inhibited by E-64 and insensitive to PMSF and pepstatin A, suggesting that the purified enzyme belongs to a family of cysteine proteinase. Consistent with this conclusion, Zn2+, Cu2+, Co2+, Ni2+, and Fe2+ could strongly inhibit the activity of this enzyme. The optimal pH and temperature were 5.0 and 55 degrees C, respectively. The enzyme catalyzed the hydrolysis of Z-Phe-Arg-MCA with a parameter of K(m) (8.27 microM) and K(cat) (28.7 s(-1)) but hardly hydrolyzed Z-Arg-Arg-MCA, Arg-MCA, and Boc-Val-Leu-Lys-MCA. The microstructure analysis by scanning electron microscopy showed that this proteinase is capable of destroying the network structure of silver carp surimi gels. The enzyme exhibited a higher hydrolytic activity on surimi protein at 65 degrees C than at 40 degrees C.     

Latent polyphenol oxidases from sago log (Metroxylon sagu): partial purification, activation, and some properties

Latent polyphenol oxidase (LPPO), an enzyme responsible for the browning reaction of sago starches during processing and storage, was investigated. The enzyme was effectively extracted and partially purified from the pith using combinations of nonionic detergents. With Triton X-114 and a temperature-induced phase partitioning method, the enzyme showed a recovery of 70% and purification of 4. 1-fold. Native PAGE analysis of the partially purified LPPO revealed three activity bands when stained with catechol and two bands with pyrogallol. The molecular masses of the enzymes were estimated by SDS-PAGE to be 37, 45, and 53 kDa. The enzyme showed optimum pH values of 4.5 with 4-methylcatechol as a substrate and 7.5 with pyrogallol. The LPPO was highly reactive toward diphenols and triphenols. The activity of the enzyme was greatly enhanced in the presence of trypsin, SDS, ethanol, and linoleic acid.     

Chemical and sensory characterization of DOC red wines from Marche (Italy) related to vintage and grape cultivars

Monomeric phenols, color and copigmentation parameters, pigments with different chemical structure, tannin, glucose, fructose, glycerol, ethanol, and organic acids were determined in DOC red wines from Marche (Italy), obtained during three different vintages ranging from 1996 to 2000. The intensity of the bitter and astringent tastes of the wines was determined with panel tastings. Lacrima di Morro and Vernaccia di Serrapetrona (obtained from local cultivars) were different from Rosso Piceno, Rosso Piceno Superiore, and Rosso Conero (produced from different percentages of Sangiovese and Montepulciano). Vernaccia, a red, sweet, "spumante" wine, was an outlier. Lacrima showed a low tannin content, a high content of small pigments and phenols, and a high ratio of copigmented color, which persisted after 3 years of aging. The chemical determinations accounted for a high percentage of variability of measured panel astringency, copigmented color, and measured wine absorbance at 520 nm. It was not possible to create a predictive model for bitterness.     

Partial purification, characterization, and histochemical localization of fully latent desert truffle (Terfezia claveryi Chatin) polyphenol oxidase

In the present paper, a fully latent polyphenol oxidase (PPO) from desert truffle (Terfezia claveryi Chatin) ascocarps is described for the first time. The enzyme was partially purified by using phase partitioning in Triton X-114 (TX-114). The achieved purification was 2-fold from a crude extract, with a 66% recovery of activity. The interfering lipids were reduced to 13% of the original content. In addition, the purification gave rise to a reduction of phenolic compounds to only 37.5%, thus avoiding the postpurification tanning of the enzyme. Latent PPO was activated by the anionic surfactant sodium dodecyl sulfate (SDS) or by incubation with trypsin. The amount of SDS necessary to obtain a maximum activation was dependent on the nature of the substrate. The use of SDS also permitted the histochemical localization of the latent enzyme within the ascocarp. Terfezia polyphenol oxidase was kinetically characterized using two phenolic substrates (L-DOPA and tert-butylcatechol). The latter substrate presented inhibition at high substrate concentration with a K(si) of 6.3 mM. Different inhibiting agents (kojic and cinnamic acid, mimosine and tropolone) were also studied, tropolone being the most effective.     

Purification and characterization of gelatinase-like proteinases from the dark muscle of common carp (Cyprinus carpio)

Gelatinolytic proteinases from common carp dark muscle were purified by 30-60% ammonium sulfate fractionation and a combination of chromatographic steps including ion exchange on DEAE-Sephacel, gel filtration on Sephacryl S-200, ion exchange on High-Q, and affinity on gelatin-Sepharose. The molecular masses of these proteinases as estimated by SDS-PAGE were 75, 67, and 64 kDa under nonreducing conditions. The enzymes revealed high activity at a slightly alkaline pH range, and their activities were investigated using gelatin as substrate. Metalloproteinase inhibitors, EDTA, EGTA, and 1,10-phenanthroline, almost completely suppressed the gelatinolytic activity, whereas other proteinase inhibitors did not show any inhibitory effect. Divalent metal ion Ca (2+) is essential for the gelatinolytic activity. Furthermore, these gelatinolytic proteinases hydrolyze native type I collagen effectively even at 4 degrees C, strongly suggesting their involvement in the texture softening of fish muscle during the post-mortem stage.     

Isolation,purification, and biochemical characterization of a novel water soluble protein from Inca peanut (Plukenetia volubilis L.)

A water soluble storage albumin from Inca peanut (IPA) accounted for approximately 25% (w/w) of defatted seed flour weight, representing 31% of the total seed protein. IPA is a 3S storage protein composed of two glycosylated polypeptides, with estimated molecular weights (MW) of 32800 and 34800 Da, respectively. IPA has an estimated sugar content of 4.8% +/- 0.92% (n = 6). IPA is a basic protein (pI of approximately 9.4) and contains all of the essential amino acids in adequate amounts when compared to the FAO/WHO recommended pattern for a human adult. The tryptophan content of IPA is unusually high (44 mg/g of protein), whereas the phenylalanine content is low (9 mg/g of protein). IPA is a highly digestible protein in vitro.     

Protein from red cabbage (Brassica oleracea) seeds with antifungal, antibacterial, and anticancer activities

A 30 kDa antifungal protein was purified from red cabbage ( Brassica oleracea ) seeds. It exhibited a molecular mass and N-terminal amino acid sequence disinct from those of previously isolated Brassica antifungal proteins. The protocol used entailed ion exchange chromatography on Q-Sepharose and SP-Sepharose followed by fast protein liquid chromatography on Mono S. The protein hindered mycelial growth in Mycosphaerella arachidicola (with an IC50=5 μM), Setospaeria turcica, and Bipolaris maydis. It also inhibited the yeast Candida albicans with an IC50=96 μM. It exerted its antifungal action by permeabilizing the fungal membrane as evidenced by staining with Sytox green. The antifungal activity was stable from pH 3 to 11 and from 0 to 65 °C. It manifested antibacterial activity against Pseudomonas aeruginosa (IC50=53 μM). Furthermore, after 48 h of culture, it suppressed proliferation of nasopharyngeal cancer and hepatoma cells with IC50=50 and 90 μM, respectively.     

Fatty acid distribution in muscle,liver, and gonads of rays (Dasyatis marmorata,Rhinobatos cemiculus,and Rhinoptera marginata) from the East Tropical Atlantic Ocean

If a great number of rays are fished in the Tropical East Atlantic Ocean for their caudal fins, only a small amount of ray flesh is processed. Among them, three species of rays, Dasyatis marmorata, Rhinobatos cemiculus, and Rhinoptera marginata, from the Mauritanian coast have been investigated for the fatty acid composition of their lipids. Gas chromatography and gas chromatography-mass spectrometry allowed identification of 50 molecules from muscles, livers, and gonads of these fishes. Principal component analysis, starting from >50 samples, reveals significant differences in various fatty acid distributions, related to the species and sex of the sampled fish. Some of them are preferentially present in one sex or in both species, whereas the occurrence of others characterizes the male and female of one or two species. The results show that rays are potential resources of polyunsaturated fatty acids (PUFA) and should be used in the diet of local populations. The lipidic fractions contained a high amount of PUFA (up to 30% of the total), mainly composed of docosa-4,7,10,13,16,19-hexaenoic acid, eicosa-5,8,11,14-tetraenoic acid, and eicosa-5,8,11,14,17-pentaenoic acid.     

Rare earth and other trace and major elemental distribution in a pedogenic calcrete profile (Slimene,NE Tunisia)

A calcrete profile developed on the top of a calcareous consolidated dune located in a coastal area of NE Tunisia (semi-arid climate) was studied with the aim to investigate the behavior of the chemical elements (rare earth elements—REE—and other trace and major elements) during the processes associated with calcrete formation, particularly dissolution and precipitation of carbonates in the vadose zone. The profile shows a vertical sequence, with clear zonations from the surface downwards: a reddish soil at the land surface, a nodular horizon, a laminar-structured level, and the consolidated old dune. Chemical and mineralogical analyses of samples from all levels were performed by neutron activation analysis and X-ray diffraction (bulk samples and < 2 μm fraction). Detailed studies of iron speciation and iron minerals were done by Mössbauer spectroscopy. Microfauna, mineralogical and chemical variations in the Slimene weathering profile point to a long term aerial exposure of the old dune in a semi-arid carbonated environment leading to the development of a pedogenic calcrete profile. The mineralogical variations with depth indicate carbonate dissolution at the surface and downward leaching of calcium. Secondary carbonates overgrow primary ones and precipitate as coating or concretions below. Minerals found in the upper levels correspond to original materials of the old dune and also weathering phases and atmospheric inputs. Phyllosilicates decrease while calcite and K-feldspars increase with depth. Kaolinite was the only clay mineral found in the old dune. Illite and chlorite were also found in all samples with calcrete. Authigenic smectite formation occurs above the laminar-structured calcrete due to restricted drainage conditions. The most significant chemical variations associated with the calcrete formation are (i) enrichment of Co, U, Br, and REE in calcrete; (ii) depletion of middle REE in the upper levels, particularly Eu, and enrichment of middle REE and heavy REE in calcrete; and (iii) Hf, Zr, Cr, Th, Cs, Ta, Ga, Rb, and K appear to be retained in the upper levels, where calcite has been dissolved. Here iron is more reduced. Fe³⁺ occurs in iron oxides (goethite and hematite), and clay minerals. The ratio goethite/hematite appears to increase down the profile; and Fe²⁺ is mainly present in clay minerals.     

Forest fragmentation and the distribution, abundance and conservation of the Tana river red colobus (Procolobus rufomitratus)

Evidence suggests that habitat heterogeneity, disturbance, disease, predation and food resources influence primate population abundance. However, few studies have focused on what habitat factors determine the probability that a primate species will occur in a habitat fragment. We analysed forest habitat and colobus population attributes to determine factors that influenced occurrence and abundance of the critically endangered Tana River red colobus. The probability of colobus inhabiting forests was positively related to the relative amount of habitat edge and canopy tree species composition. Most of the variance in the density of colobus monkeys was explained by basal area of food trees (per ha), basal area per food tree, density of food trees, and basal area per tree for all trees. We conclude that effective conservation of primates in fragmented habitats may require strategies that consider factors that influence both the occurrence of a species and its relative abundance.