N-terminal Amino Acid Sequence Analysis Research Articles

Purification and characterization of a serine protease from the fruit of Ficus carica cultivar Masui Dauphine.

Ficus carica produces, in addition to the cysteine protease ficin, a serine protease (FSP). Here, we purified FSP to homogeneity from the fruit of F. carica cultivar Masui Dauphine. An 81-fold enrichment in specific activity of FSP with 2.1% recovery was attained. Three protein bands (70, 62, and 60kDa) were identified on SDS-PAGE. Each band was identified as a subtilisin-like protease (661 amino acids) by trypsin digestion and LC-MS/MS analysis, and the partial N-terminal amino-acid sequence analysis. Gelatin zymography revealed that the active FSP exists as a dimer. The optimum hydrolysis pH of FSP was 7.5, and the pHs at which the enzyme retained its initial activity by 70% in 24h were 8.0-11.0. The optimum hydrolysis temperature of FSP was 50-60ºC, and the temperature required to reduce the initial activity by 50% in 15min was 70ºC. These results will inform the industrial use of FSP.

A heterodimeric hyaluronate lyase secreted by the activated sludge bacterium Haliscomenobacter hydrossis.

Haliscomenobacter hydrossis is a filamentous bacterium common in activated sludge. The bacterium was found to utilize hyaluronic acid, and hyaluronate lyase activity was detected in its culture. However, no hyaluronate lyase gene was found in the genome, suggesting the bacterium secretes a novel hyaluronate lyase. The purified enzyme exhibited two bands on SDS-PAGE and a single peak on gel filtration chromatography, suggesting a heterodimeric composition. N-terminal amino acid sequence and mass spectrometric analyses suggested that the subunits are molybdopterin-binding and [2Fe-2S]-binding subunits of a xanthine oxidase family protein. The presence of the cofactors was confirmed using spectrometric analysis. Oxidase activity was not detected, revealing that the enzyme is not an oxidase but a hyaluronate lyase. Nuclear magnetic resonance analysis of the enzymatic digest revealed that the enzyme breaks hyaluronic acid to 3-(4-deoxy-β-d-gluc-4-enuronosyl)-N-acetyl-d-glucosamine. As hyaluronate lyases (EC 4.2.2.1) are monomeric or trimeric, the enzyme is the first heterodimeric hyaluronate lyase.

Family-level diversity of extracellular proteases of sedimentary bacteria from the South China Sea

Protease-producing bacteria and their extracellular proteases are key players in degrading organic nitrogen to drive marine nitrogen cycling and yet knowledge on both of them is still very limited. This study screened protease-producing bacteria from the South China Sea sediments and analyzed the diversity of their extracellular proteases at the family level through N-terminal amino acid sequencing. Results of the 16S rRNA gene sequence analysis showed that all screened protease-producing bacteria belonged to the class Gammaproteobacteria and most of them were affiliated with different genera within the orders Alteromonadales and Vibrionales. The N-terminal amino acid sequence analysis for fourteen extracellular proteases from fourteen screened bacterial strains revealed that all these proteases belonged to the M4 family of metalloproteases or the S8 family of serine proteases. This study presents new details on taxa of marine sedimentary protease-producing bacteria and types of their extracellular proteases, which will help to comprehensively understand the process and mechanism of the microbial enzymatic degradation of marine sedimentary organic nitrogen.

Isolation and partial structural characterization of new Kunitz-type trypsin inhibitors from the pike cestode Triaenophorus nodulosus

The inhibitors produced by the parasitic worms successfully protect them from the host’s proteases and are supposed to underlie the host-parasite specificity. Our previous study has shown that the extracts from the pike tapeworm Triaenophorus nodulosus inhibit host proteinases and commercial trypsin. We aimed to isolate and identify the components responsible for trypsin inactivation. After a two-step separation the molecular masses were measured by SE-HPLC. The sample proved to contain four fractions represented by polypeptides (1–45 kDa) and low-molecular hydrophobic compounds. According to SDS-PAGE analysis, the major polypeptides in the fractions displaying the highest inhibition had masses of 14.4 kDa. The study culminated in partial N-terminal amino acid sequence analysis with a further search for homology. The research revealed two novel Kunitz-type proteins potentially responsible for the inhibitory capacity of the tapeworms against trypsin. Our findings extend the list of cestodes relying on Kunitz-type proteins in the host-parasite molecular cross-talk.

Purification and characterization of \u03b2-secretase inhibitory peptide from sea hare (Aplysia kurodai) by enzymatic hydrolysis

Amyloid plaque, also called senile plaque, the product of aggregation of β-amyloid peptides (Aβ), is observed in brains of the patients with Alzheimer’s disease (AD) and is one of the key factors in etiology of the disease. In this study, hydrolysates obtained from the sea hare (Aplysia kurodai) were investigated for β-secretase inhibitory peptide. The sea hare’s muscle protein was hydrolyzed using six enzymes in a batch reactor. Trypsin hydrolysate had highest β-secretase inhibitory activity compared to the other hydrolysates. β-secretase inhibitory peptide was separated using Sephadex G-25 column chromatography and high-performance liquid chromatography on a C18 column. β-secretase inhibitory peptide was identified as eight amino acid residues of Val-Ala-Ala-Leu-Met-Leu-Phe-Asn by N-terminal amino acid sequence analysis. IC50 value of purified β-secretase inhibitory peptide was 74.25 μM, and Lineweaver−Burk plots suggested that the peptide purified from sea hare muscle protein acts as a competitive inhibitor against β-secretase. Results of this study suggest that peptides derived from sea hare muscle may be beneficial as anti-dementia compounds in functional foods or as pharmaceuticals.

Construction and Characterization of Cell-Penetrating Peptide-Fused Fibroblast Growth Factor and Vascular Endothelial Growth Factor for an Enhanced Percutaneous Delivery System.

The exogenous administration of growth factors has been examined for treating wounds and such factors serve as cosmetic agents for skin regeneration. However, the topical application of growth factors is hampered by their limited percutaneous absorption. In this study, we genetically modified basic fibroblast growth factor (bFGF) and vascular endothelial growth factor-A (VEGF-A) using a cell-penetrating peptide to facilitate their permeation into skin and to avoid multiple steps in the chemical or physical modification of biomaterials. Low-molecular-weight protamine (LMWP)-fused bFGF and VEGF-A (LMWP-bFGF and LMWP-VEGF-A) were designed by serial polymerase chain reaction (PCR)-mediated addition of the LMWP codons onto the bFGF and VEGF-A genes and then produced in Escherichia coli. The purified LMWP-bFGF and LMWP-VEGF-A represented >90% of the total proteins, as determined by SDS-PAGE and densitometric quantification, and their actual molecular weights, determined by mass spectroscopy, were 19,026 and 15,715 Da, respectively, corresponding to the theoretical values. N-terminal amino acid sequencing analyses confirmed the N-terminal conjugation of LMWP to bFGF and VEGF-A. Immunoblotting analyses using antibodies against bFGF and VEGF-A, together with the proliferative effects of LMWP-bFGF and LMWP- VEGF-A on human keratinocytes and fibroblasts, demonstrated that the original biological activity of each growth factor was not altered by LMWP conjugation. In addition, the LMWP conjugation did not induce further cytotoxic effects on the skin cells, while the cell membrane-penetrating activities of LMWP-bFGF and LMWP-VEGF-A were significantly enhanced compared with the respective unconjugated growth factors. These results suggest that LMWP-bFGF and LMWP-VEGF-A can be used as effective topical therapeutic or cosmetic agents for skin regeneration and anti-aging treatments.

A novel epididymal quiescence factor inhibits sperm motility by modulating NOS activity and intracellular NO-cGMP pathway.

Mature and potentially motile spermatozoa stored in cauda epididymis in an inactive state for approximately 30 days; however, during ejaculation they regain motility. To understand the actual molecular mechanism of the sperm quiescence during caudal stay, a proteinaceous quiescence factor (QF) has been purified from caprine epididymal plasma to apparent homogeneity. In the present study complete purification, detailed characterization as well as mechanistic pathway of QF has been described. QF is purified to 215-fold with 45% activity recovery. It is a 59 kDa monomeric protein with isoelectric point 5.8 and optimally active at pH 7.5. Circular dichroism spectroscopy and atomic force microscopy study confirm its α-helical secondary structure and globular tertiary conformation. QF is a thermo-stable protein as higher temperature does not alter its helical structure. N-terminal amino acid sequencing and MALDI analysis of QF did not find 100% similarity with any available protein of the database, proved its novelty. QF at 2 μM dose inhibits sperm progressive forward motility within 10 min. This motility inhibitory activity of QF is mediated by reducing NOS enzyme activity and subsequently decreasing the intracellular NO and cGMP concentration. It does not modulate intracellular Ca++ and cAMP concentration. QF has no adverse effect on DNA integrity and morphology of spermatozoa. Motility inhibitory action of QF is reversible. Thus, the role of QF in maintaining energy saving quiescence state of mature cauda spermatozoa and its reactive nitrogen species reducing activity may lead to a new direction for storage of spermatozoa and idiopathic male infertility.

Characterization of porcine sapelovirus isolated from Japanese swine with PLC/PRF/5 cells.

Porcine sapelovirus (PSV) is a causative agent of neurological disorders, fertility disorders and dermal lesions of swine. In this study, we isolated two PSV strains, Jpsv477 and Jpsv1315, from swine faecal specimens using a PLC/PRF/5 cell culture system. The PSV infection of PLC/PRF/5 cells induced a cytopathic effect (CPE). Two types of virus particles with identical diameter (~35nm) but different densities (1.300 and 1.285g/cm3 ) were observed in the cell culture supernatants. Analysis of the entire genome sequence of Jpsv477 and Jpsv1315 revealed that both strains possess 7,558 nucleotides and the poly (A) tail and have a typical PSV genome organization consisting of a 5' terminal untranslated region (5'UTR), a large open reading frame (ORF), and a 3' terminal untranslated region (3'UTR). The ORF encodes a single polyprotein that is subsequently processed into a leader protein (L), four structural proteins (VP1, VP2, VP3 and VP4) and seven functional proteins (2A, 2B, 2C, 3A, 3B, 3C and 3D). The structural proteins VP1, VP2, VP3 and VP4 have molecular masses of ~35, ~26, ~25 and ~6kDa. The N-terminal amino acid sequence analysis of VP1, VP2, VP3 and VP4 confirmed that the cleavage sites between VP4 and VP2, VP2 and VP3, and VP3 and VP1 are K/A, Q/G and Q/G, respectively. We further confirmed that HepG2/C3A, Vero E6 and primary green monkey kidney cells (PGMKC) were also susceptible to PSV infection. The stability assay demonstrated that PSV was inactivated by heating at 60°C for 10min or 65°C for 5min. The virus also lost infectivity by incubation with 62.5ppm of NaClO for 30min.

Small leucine-rich repeat proteoglycans associated with mature insoluble elastin serve as binding sites for galectins.

We previously reported that galectin-9 (Gal-9), an immunomodulatory animal lectin, could bind to insoluble collagen preparations and exerted direct cytocidal effects on immune cells. In the present study, we found that mature insoluble elastin is capable of binding Gal-9 and other members of the human galectin family. Lectin blot analysis of a series of commercial water-soluble elastin preparations, PES-(A) ~ PES-(E), revealed that only PES-(E) contained substances recognized by Gal-9. Gal-9-interacting substances in PES-(E) were affinity-purified, digested with trypsin and then analyzed by reversed-phase HPLC. Peptide fragments derived from five members of the small leucine-rich repeat proteoglycan family, versican, lumican, osteoglycin/mimecan, prolargin, and fibromodulin, were identified by N-terminal amino acid sequence analysis. The results indicate that Gal-9 and possibly other galectins recognize glycans attached to small leucine-rich repeat proteoglycans associated with insoluble elastin and also indicate the possibility that mature insoluble elastin serves as an extracellular reservoir for galectins.

High-level expression and purification of a molluskan endoglucanase from Ampullaria crossean in Pichia pastoris

EG27I is an endogenous glucanase belonging to glycoside hydrolase family (GHF) 45 from the mollusk Ampullaria crossean. In this study, the mature EG27I peptide gene fused to the HFBII secretion signal of Trichoderma reesei was expressed under the GAP promoter of Pichia pastoris in SMD1163 strain. A bioactive EG27I with a molecular weight of 27 kDa was successfully expressed and secreted into our culture medium. The respective final OD600 and hydrolytic activity were 333 and 1.28 U/mL when high-cell-density fermentation of the recombinant P. pastoris was performed in a 7.5 L fermenter through a fed-batch strategy for 132 h. The recombinant protein concentration of the fermentation supernatant was 47.7 mg/L. EG27I was consecutively purified from the fermentation supernatant through ultrafiltration, cation exchange, and hydrophobic interaction. The specific activity of the recombinant EG27I was 26.8 U/mg, and the optimal pH and temperature of the enzyme were 5 and 50 °C, respectively. The half-life of the enzyme activity at 100 °C could reach 40 min. The N-terminal amino acid sequence analysis of the purified recombinant protein confirmed that the amino terminal sequence was consistent with the natural structure. The high quantity and purity of the EG27I provide a basis for future structural and functional studies.

Chemical and structural characterization of \u03b1-N-acetylgalactosaminidase I and II from starfish, asterina amurensis

BackgroundThe marine invertebrate starfish was found to contain a novel α-N-acetylgalactosaminidase, α-GalNAcase II, which catalyzes removal of terminal α-N-acetylgalactosamine (α-GalNAc), in addition to a typical α-N-acetylgalactosaminidase, α-GalNAcase I, which catalyzes removal of terminal α-N-acetylgalactosamine (α-GalNAc) and, to a lesser extent, galactose. The interrelationship between α-GalNAcase I and α-GalNAcase II and the molecular basis of their differences in substrate specificity remain unknown.ResultsChemical and structural comparisons between α-GalNAcase I and II using immunostaining, N-terminal amino acid sequencing and peptide analysis showed high homology to each other and also to other glycoside hydrolase family (GHF) 27 members. The amino acid sequence of peptides showed conserved residues at the active site as seen in typical α-GalNAcase. Some substitutions of conserved amino acid residues were found in α-GalNAcase II that were located near catalytic site. Among them G171 and A173, in place of C171 and W173, respectively in α-GalNAcase were identified to be responsible for lacking intrinsic α-galactosidase activity of α-GalNAcase II. Chemical modifications supported the presence of serine, aspartate and tryptophan as active site residues. Two tryptophan residues (W16 and W173) were involved in α-galactosidase activity, and one (W16) of them was involved in α-GalNAcase activity.ConclusionsThe results suggested that α-GalNAcase I and II are closely related with respect to primary and higher order structure and that their structural differences are responsible for difference in substrate specificities.

Purification and partial characterization of M1-UVs300, a novel bacteriocin produced by Lactobacillus plantarum isolated from fermented sausage

A novel bacteriocin-M1-UVs300, which was produced by Lactobacillus plantarum M1-UVs300, was purified and characterized. Bacteriocin-M1-UVs300 was purified sequentially by an aqueous two-phase system (ATPS) and a Sephadex G-50 gel chromatography assay, combined with reverse phase high-performance liquid chromatography (RE-HPLC). The purification resulted in a 3221.63 IU ml−1 titer with a 20.41- fold purification of the original activity. According to a Tricine-SDS-PAGE analysis, the molecular weight of the bacteriocin-M1-UVs300 was approximately 3.4 kDa. A quantitative analysis of the secondary structure of bacteriocin-M1-UVs300 results showed that it had a major β-sheet content of 52.43%, α-helix of 16.17%, β-turn of 15.27%, and a random coil of 16.12%. A partial sequence, GAKSKYGNVG-, was obtained by N-terminal amino acid sequence analysis. The antimicrobial spectrum of bacteriocin-M1-UVs300 exhibited activity against Gram-positive bacteria and Gram-negative bacteria. In addition, it was relatively heat-resistant, active over a range of pH 2–8, and sensitive to proteolytic enzymes, but it was not sensitive to α-amylase. The additives significantly increased the activity of bacteriocin-M1-UVs300 significantly. These findings indicated that bacteriocin-M1-UVs300, is a novel bacteriocin with a broad inhibitory spectrum, and that it had the potential to act as a natural preservative in the food industry.

SAXS and other spectroscopic analysis of 12S cruciferin isolated from the seeds of Brassica nigra

Oilseeds of the plant family Brassicaceae are important for providing both lipid and protein contents to human nutrition. Cruciferins (12S globulins) are seed storage proteins, which are getting attention due to their allergenic and pathogenicity related nature. This study describes the purification and characterization of a trimeric (∼190 kDa) cruciferin protein from the seeds of Brassica nigra (L.). Cruciferin was first partially purified by ammonium sulfate precipitation (30% saturation constant) and further purified by size exclusion chromatography. The N-terminal amino-acid sequence analysis showed 82% sequence homology with cruciferin from Arabidopsis thaliana. The 50–55 kDa monomeric cruciferin produced multiple bands of two major molecular weight ranges (α-polypeptides of 28–32 kDa and β-polypeptides of 17–20 kDa) under reduced conditions of SDS-PAGE. The 2D gel electrophoretic analysis showed the further separation of the bands into their isoforms with major pI ranges between 5.7 and 8.0 (α-polypeptides) and 5.5–8.5 (β-polypeptides). The Dynamic Light Scattering (DLS) showed the monodisperse nature of the cruciferin with hydrodynamic radius of 5.8 ± 0.1 nm confirming the trimeric nature of the protein. The Circular Dichroism (CD) spectra showed both α-helices and β-sheets in the native conformation of the trimeric protein. The pure cruciferin protein (40 mg/ml) was successfully crystallized; however, the crystals diffracted only to low resolution data (8 Å). Small-angle x-ray scattering (SAXS) was applied to gain insights into the three-dimensional structure in solution. SAXS showed that the radius of gyration is 4.24 ± 0.25 nm and confirmed the nearly globular shape. The SAXS based ab initio dummy model of B. nigra cruciferin was compared with 11S globulins (PDB ID: 3KGL) of B. napus which further confirmed a highly similar molecular weight and globular shape indicating a conserved trimerization of B. nigra cruciferin. The comparison of the scattering patterns of both proteins showed a minimized χ2-value of 1.337 confirming a similar molecular structure. This is the first report describing the purification and characterization of a cruciferin protein from seeds of B. nigra.

Proteins causing membrane fouling in membrane bioreactors.

In this study, the details of proteins causing membrane fouling in membrane bioreactors (MBRs) treating real municipal wastewater were investigated. Two separate pilot-scale MBRs were continuously operated under significantly different operating conditions; one MBR was a submerged type whereas the other was a side-stream type. The submerged and side-stream MBRs were operated for 20 and 10 days, respectively. At the end of continuous operation, the foulants were extracted from the fouled membranes. The proteins contained in the extracted foulants were enriched by using the combination of crude concentration with an ultrafiltration membrane and trichloroacetic acid precipitation, and then separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The N-terminal amino acid sequencing analysis of the proteins which formed intensive spots on the 2D-PAGE gels allowed us to partially identify one protein (OmpA family protein originated from genus Brevundimonas or Riemerella anatipestifer) from the foulant obtained from the submerged MBR, and two proteins (OprD and OprF originated from genus Pseudomonas) from that obtained from the side-stream MBR. Despite the significant difference in operating conditions of the two MBRs, all proteins identified in this study belong to β-barrel protein. These findings strongly suggest the importance of β-barrel proteins in developing membrane fouling in MBRs.

Isolation of an Adoxophyes orana granulovirus (AdorGV) occlusion body morphology mutant: biological activity, genome sequence and relationship to other isolates of AdorGV.

A granulovirus (GV) producing occlusion bodies (OBs) with an unusual appearance was isolated from Adoxophyes spp. larvae in the field. Ultrastructural observations revealed that its OBs were significantly larger and cuboidal in shape, rather than the standard ovo-cylindrical shape typical of GVs. N-terminal amino acid sequence analysis of the OB matrix protein from this virus suggested that this new isolate was a variant of Adoxophyes orana granulovirus (AdorGV). Bioassays of this GV (termed AdorGV-M) and an English isolate of AdorGV (termed AdorGV-E) indicated that the two isolates were equally pathogenic against larvae of Adoxophyes honmai. However, AdorGV-M retained more infectivity towards larvae after irradiation with UV light than did AdorGV-E. Sequencing and analysis of the AdorGV-M genome revealed little sequence divergence between this isolate and AdorGV-E. Comparison of selected genes among the two AdorGV isolates and other Japanese AdorGV isolates revealed differences that may account for the unusual OB morphology of AdorGV-M.

A C-type lectin isolated from the skin of Japanese bullhead shark (Heterodontus japonicus) binds a remarkably broad range of sugars and induces blood coagulation.

The aim of this study was to determine the physiological role of skin lectins of the Japanese bullhead shark (Heterodontus japonicus). A skin extract was subjected to affinity chromatography using seven different sugars as ligands. Molecular mass and N-terminal amino acid sequence analyses indicated elution of the same protein by each of the seven respective cognate ligands from sugar affinity columns. The predicted amino acid sequence encoded by the cDNA of this protein [designated as H. japonicus C-type-lectin (HjCL)] identified it as a novel fish subgroup VII C-type lectin evolutionarily related to snake venom lectins. HjCL was predicted to bind to mannose because of the presence of a Glu-Pro-Asn (EPN) motif; however, haemagglutination inhibition assays and glycoconjugate microarray analysis demonstrated its binding to numerous structurally diverse sugars. Competitive sugar-binding assays using affinity chromatography indicated that HjCL bound multiple sugars via a common carbohydrate-recognition domain. The mRNA encoding HjCL was specifically detected in the skin, and immunohistochemical analysis detected its expression in uncharacterized large cells in the epidermis. HjCL agglutinated the bacterial pathogen Edwardsiella tarda and promoted immediate clotting of shark blood, indicating that HjCL is involved in host defence on the skin surface especially when the shark is injured and bleeds.

Purification and identification of a novel antifungal protein secreted by Penicillium citrinum from the Southwest Indian Ocean.

A novel antifungal protein produced by the fungal strain Penicillium citrinum W1, which was isolated from a Southwest Indian Ocean sediment sample, was purified and characterized. The culture supernatant of P. citrinum W1 inhibited the mycelial growth of some plant pathogenic fungi. After saturation of P. citrinum W1 culture supernatants with ammonium sulfate and ion-exchange chromatography, an antifungal protein (PcPAF) was purified. The N-terminal amino acid sequence analysis showed that PcPAF might be an unknown antifungal protein. PcPAF displayed antifungal activity against Trichoderma viride, Fusarium oxysporum, Paecilomyces variotii, and Alternaria longipes at minimum inhibitory concentrations of 1.52, 6.08, 3.04, and 6.08 µg/disc, respectively. PcPAF possessed high thermostability and had a certain extent of protease and metal ion resistance. The results suggested that PcPAF may represent a novel antifungal protein with potential application in controlling plant pathogenic fungal infection.

Molybdenum-containing membrane-bound formate dehydrogenase isolated from Citrobacter sp. S-77 having high stability against oxygen, pH, and temperature

Membrane-bound formate dehydrogenase (FDH) was purified to homogeneity from a facultative anaerobic bacterium Citrobacter sp. S-77. The FDH from Citrobacter sp. S-77 (FDHS77) was a monomer with molecular mass of approximately 150kDa. On SDS-PAGE, the purified FDHS77 showed as three different protein bands with molecular mass of approximately 95, 87, and 32kDa, respectively. Based on the N-terminal amino acid sequence analysis, the sequence alignments observed for the 87kDa protein band were identical to that of the large subunit of 95kDa, indicating that the purified FDHS77 consisted of two subunits; a 95kDa large subunit and a 32kDa small subunit. The purified FDHS77 in this purification did not contain a heme b subunit, but the FDHS77 showed significant activity for formate oxidation, determined by the Vmax of 30.4U/mg using benzyl viologen as an electron acceptor. The EPR and ICP-MS spectra indicate that the FDHS77 is a molybdenum-containing enzyme, displaying a remarkable O2-stability along with thermostability and pH resistance. This is the first report of the purification and characterization of a FDH from Citrobacter species.

Characterization of α-1,3-glucanase isozyme from Paenibacillus glycanilyticus FH11 in a new subgroup of family 87 α-1,3-glucanase

Two α-1,3-glucanase isozymes, designated as α-1,3-glucanase 1 (Agl-FH1) and α-1,3-glucanase 2 (Agl-FH2), were purified from the culture medium of Paenibacillus glycanilyticus FH11. Agl-FH1 and Agl-FH2 exhibited similar characteristics such as optimal pH, pH stability, optimal temperature, thermostability, and molecular masses on SDS-PAGE. However, their hydrolysis products of α-1,3-glucan varied somewhat. Agl-FH1 hydrolyzed α-1,3-glucan into a mixture of maltotriose and maltotetraose, and maltotetraose was the major hydrolysis product of Agl-FH2. N-terminal amino acid sequence analysis and LC-MS/MS analysis of trypsin digested fragments revealed several differences between the amino acid sequences of Agl-FH1 and Agl-FH2. Genes of Agl-FH1 and Agl-FH2 were subcloned into an expression plasmid, and both enzymes were successfully expressed in Escherichia coli. The recombinant Agl-FH1 and Agl-FH2 exhibited the same enzymatic properties as those of each wild-type enzyme, and both of the recombinants showed the activity on the protoplast formation of Schizophyllum commune mycelia. A great diversity was detected in the C-terminal region of family 87 α-1,3-glucanases. Compared with Agl-FH2 which is highly sequence-related to the known α-1,3-glucanases, the C-terminal region of Agl-FH1 has only slight similarity to them (approximately 20% identity). Our analysis revealed that Agl-FH1 was the first member of a new subgroup of family 87 α-1,3-glucanases.

Identification of perfluorooctane sulfonate binding protein in the plasma of tiger pufferfish Takifugu rubripes

It is well known that perfluorooctane sulfonate (PFOS) preferentially accumulates in the plasma of wildlife and humans. Although earlier studies have suggested that this was due to binding of PFOS to a plasma protein, definite characterization of the protein in in vivo exposure studies was not conducted thus far. In this study, we conducted both in vitro and in vivo experiments to identify PFOS binding protein in the plasma of fish. For the in vivo studies, PFOS was administered intraperitoneally to tiger pufferfish, Takifugu rubripes, and the plasma was separated by ammonium sulfate fractionation. High concentrations of PFOS were found in the 65–70 percent ammonium sulfate fraction (190ng/mL). After SDS-PAGE and N-terminal amino acid sequence analysis, the PFOS-binding protein was identified as an apolipoprotein A–I, which was confirmed on the basis of a significant correlation to the PFOS concentration in each fraction. The plasma samples fractionated by ammonium sulfate from untreated pufferfish were subjected to PFOS binding assay by the equilibrium dialysis method. The results further confirmed that the 60–65 percent ammonium sulfate fraction showed a high PFOS-binding ratio, similar to that found from in vivo studies. We demonstrated that PFOS is likely bound to an apolipoprotein A–I in the plasma of tiger pufferfish in in vivo and in vitro studies.