黑蕊虎耳草中岩白菜素没食子酸酯类及其对丙型肝炎丝氨酸蛋白酶的抑制作用

研究了黑蕊虎耳草(Saxifraga melanocentra)中岩白菜素衍生物的化学和生物活性,从其地上部分分离纯化得到一个新的岩白菜素没食子酸酯,主要通过1维和2维核磁共振波谱鉴定结构为11-氧-(4-氧甲基没食子酰)岩白菜素[11-O-(4-O-methylgalloyl) bergenin(1)] ,该化合物对丙型肝炎丝氨酸蛋白酶(HCVNS3serine protease)具有抑制活性,其IC50为0.32 mg/mL。     

Purification and Characterization of an Alkaline Protease from the Marine Yeast <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> for Bioactive Peptide Production from Different Sources

The extracellular alkaline protease in the supernatant of cell culture of the marine yeast Aureobasidium pullulans 10 was purified to homogeneity with a 2.1-fold increase in specific protease activity as compared to that in the supernatant by ammonium sulfate fractionation, gel filtration chromatography (Sephadex™ G-75), and anion-exchange chromatography (DEAE Sepharose Fast Flow). According to the sodium dodecyl sulfate-polyacrylamide gel electrophoresis data, the molecular mass of the purified enzyme was estimated to be 32.0 kDa. The optimal pH and temperature of the purified enzyme were 9.0 and 45°C, respectively. The enzyme was activated by Cu²⁺ (at a concentration of 1.0 mM) and Mn²⁺ and inhibited by Hg²⁺, Fe²⁺, Fe³⁺, Zn²⁺, and Co²⁺. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride, but weakly inhibited by EDTA, 1–10-phenanthroline, and iodoacetic acid. The K _m and V _max values of the purified enzyme for casein were 0.25 mg/ml and 0.0286 μmol/min/mg of protein, respectively. After digestion of shrimp protein, spirulina (Arthospira platensis) protein, proteins of marine yeast strains N3C (Yarrowia lipolytica) and YA03a (Hanseniaspora uvarum), milk protein, and casein with the purified alkaline protease, angiotensin I converting enzyme (ACE) inhibitory activities of the resulting peptides reached 85.3%, 12.1%, 29.8%, 22.8%, 14.1%, and 15.5%, respectively, while the antioxidant activities of these were 52.1%. 54.6%, 25.1%, 35%, 12.5%, and 24.2%, respectively, indicating that ACE inhibitory activity of the resulting peptides from the shrimp protein and antioxidant activity of those produced from the spirulina protein were the highest, respectively. These results suggest that the bioactive peptides produced by digestion of the shrimp protein with the purified alkaline protease have potential applications in the food and pharmaceutical industries.     

Efficient identification of side-chain patterns using a multidimensional index tree

Convergent evolution often produces similar functional sites in nonhomologous proteins. The identification of these sites can make it possible to infer function from structure, to pinpoint the location of a functional site, to identify enzymes with similar enzymatic mechanisms, or to discover putative functional sites. In this article, a novel method is presented that (a) queries a database of protein structures for the occurrence of a given side chain pattern and (b) identifies interesting side-chain patterns in a given structure. For efficiency and to make a robust statistical evaluation of the significance of a similarity possible, patterns of three residues (or triads) are considered. Each triad is encoded as a high-dimensional vector and stored in an SR (Sphere/Rectangle) tree, an efficient multidimensional index tree. Identifying similar triads can then be reformulated as identifying neighboring vectors. The method deals with many features that otherwise complicate the identification of meaningful patterns: shifted backbone positions, conservative substitutions, various atom label ambiguities and mirror imaged geometries. The combined treatment of these features leads to the identification of previously unidentified patterns. In particular, the identification of mirror imaged side-chain patterns is unique to the here-described method. Interesting triads in a given structure can be identified by extracting all triads and comparing them with a database of triads involved in ligand binding. The approach was tested by an all-against-all comparison of unique representatives of all SCOP superfamilies. New findings include mirror imaged metal binding and active sites, and a putative active site in bacterial luciferase.     

Cloning and over-expression of an alkaline protease from Bacillus licheniformis

The alkaline protease gene, apr, from Bacillus licheniformis 2709 was cloned into a Bacillus shuttle expression vector, pHL, to yield the recombinant plasmid pHL-apr. The pHL-apr was expressed in Bacillus subtilis WB600, yielding a high expression strain BW-016. The amount of alkaline protease produced in the recombinant increased by 65% relative to the original strain. SDS-PAGE analysis indicated a Mr of 30.5 kDa. The amino acid sequence deduced from the DNA sequence analysis revealed a 98% identity to that of Bacillus licheniformis 6816.     

Resonant mirror biosensor detection method based on yessotoxin-phosphodiesterase interactions

Yessotoxin (YTX) is a generic name for a group of lipophilic compounds recently discovered and chemically characterized. Association measurements were done in a resonant mirror biosensor. The instrument detects changes in the refractive index and/or thickness occurring within a few hundred nanometers form the sensor surface where a molecule is attached. We used aminosilane surfaces where phosphodiesterase 3',5'-cyclic-nucleotide-specific from bovine brain (PDEs) was immobilized. Over this immobilized ligand different amounts of YTX were added and typical association curve profiles were observed. These association curves fit a pseudo-first-order kinetic equation where the apparent association rate constant (k(on)) can be calculated. The value of this constant increases with YTX concentration. From the representation of k(on) versus YTX concentration we obtained the association rate constant (k(ass)) 248+/-40 M(-1)s(-1) and the dissociation rate constant (k(diss)) 9.36 x 10(-4)+/-1.72 x 10(-4)s(-1). From these values the kinetic equilibrium dissociation constant (K(D)) for YTX-PDEs association can be calculated. The value of this last constant is 3.74 x 10(-6)+/-8.25 x 10(-8)M YTX. The PDE-YTX association was used as a method suitable for determination of the toxin concentration in a shellfish sample. The assay had sufficient sensitivity and can be used on simple shellfish extracts.     

Crystal structure of human factor VIIa/tissue factor in complex with peptide mimetic inhibitor

The 3D structure of human factor VIIa/soluble tissue factor in complex with a peptide mimetic inhibitor, propylsulfonamide-D-Thr-Met-p-aminobenzamidine, is determined by X-ray crystallography. As compared with the interactions between thrombin and thrombin inhibitors, the interactions at S2 and S3 sites characteristic of factor VIIa and factor VIIa inhibitors are revealed. The S2 site has a small pocket, which is filled by the hydrophobic methionine side chain in P2. The small S3 site fits the small size residue, D-threonine in P3. The structural data and SAR data of the peptide mimetic inhibitor show that these interactions in the S2 and S3 sites play an important role for the improvement of selectivity versus thrombin. The results will provide valuable information for the structure-based drug design of specific inhibitors for FVIIa/TF.     

Fluorescent Location of Human Colonic Tumour Cells by Means of an Enzyme-Inhibitor Complex

Abstract

We studied the enzymic status of the tumour cell surface protease, guanidinobenzoatase (GB) in frozen sections of a human colonic tumour grown in nude mice and also in human colons. Active enzyme was demonstrated by the binding of a synthetic fluorescent probe for the active centre of guanidinobenzoatase (GB). It was observed that tissue derived inhibitors of GB blocked the binding of this fluorescent probe and that enzyme inhibitor complex formation could be controlled by lowering the pH of the medium with lactic acid. The presence of an inhibitor of GB in the mouse tumour extract was taken advantage of by making two fluorescent derivatives of this inhibitor; both of which located GB on colonic tumour cells in frozen sections of human colon.     

A 54 kDa cysteine protease purified from the crude extract of Neodiplostomum seoulense adult worms

As a preliminary study for the explanation of pathobiology of Neodiplostomum seoulense infection, a 54 kDa protease was purified from the crude extract of adult worms by sequential chromatographic methods. The crude extract was subjected to DEAE-Sepharose Fast Flow column, and protein was eluted using 25 mM Tris-HCl (pH 7.4) containing 0.05, 0.1, 0.2 and 0.4 M NaCl in stepwise elution. The 0.2 M NaCl fraction was further purified by Q-Sepharose chromatography and protein was eluted using 20 mM sodium acetate (pH 6.4) containing 0.05, 0.1, 0.2 and 0.3 M NaCl, respectively. The 0.1M NaCl fraction showed a single protein band on SDS-PAGE carried out on a 7.5-15% gradient gel. The proteolytic activities of the purified enzyme were specifically inhibited by L-trans-epoxy-succinylleucylamide (4-guanidino) butane (E-64) and iodoacetic acid. The enzyme, cysteine protease, showed the maximum proteolytic activity at pH 6.0 in 0.1 M buffer, and degraded extracellular matrix proteins such as collagen and fibronectin with different activities. It is suggested that the cysteine protease may play a role in the nutrient uptake of N. seoulense from the host intestine.     

Letter to the Editor: 1H, 15N and 13C NMR Resonance Assignments of Staphostatin A, a Specific Staphylococcus Aureus Cysteine Proteinase Inhibitor

The increasing antibiotic resistance of an important human pathogen Staphylococcus aureus calls for the development of new therapeutic strategies. Staphylococcal cysteine proteases have been suggested as targets for such therapies. The recent discovery of staphostatins, specific protein inhibitors of these enzymes, gives prospects for the design and production of synthetic, low molecular weight analogs which might become drugs. We have decided to structurally characterize staphostatin A, a representative inhibitor of staphylococcal cysteine proteases, and to assess its binding mode to the target protease with the view of clarifying the specificity determinants. Here we report the (1)H, (15)N and (13)C NMR resonance assignments of staphostatin A.     

Partial purification and characterization of an extracellular protease from<Emphasis Type="Italic">Xenorhabdus nematophilus</Emphasis>, a symbiotic bacterium isolated from an entomopathogenic nematode,<Emphasis Type="Italic">Steinernema glaseri</Emphasis>

Entomopathogenic nematodes are used for insect control. Herein, an extracellular protease was partially purified from a culture supernatant ofXenorhabdus nematophilus, a symbiotic bacterium of an entomopathogenic nematode,Steinernema glaseri, using precipitation with 80% v/v isopropyl alcohol followed by gel permeation chromatography with a packed Sephacryl S-300 HR media. The partially purified protease exhibited maximal activity at pH 7 in the presence of 1 mM CaCl₂. The protease was identified as a metallo-protease based on the inhibition of its activity by the metal chelating agent, EDTA.