Primary structure of 6.5k-arginine/glutamate-rich polypeptide from the seeds of sponge gourd (Luffa cylindrica)

The amino acid sequence of 6.5k-arginine/glutamate rich polypeptide (6.5k-AGRP) from the seeds of sponge gourd (Luffa cylindrica) has been determined. The 6.5k-AGRP consists of a 47-residue polypeptide chain containing two disulfide bonds, and a molecular mass calculated to be 5695 Da, which fully coincides with a value of [M+H]+ = m/zeta 5693.39 obtained by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). The mass spectrometric evidence indicated that 6.5k-AGRP is also present partially truncated at the C-terminus. In our preparations, approximately half of the polypeptide molecules have the C-terminal sequence Arg-Arg-Glu-Val-Asp; the other half lack Val-Asp and end with the glutamic acid, making a total of 45 residues in the polypeptide chain. The two disulfide bonds connect Cys12 to Cys33 and Cys16 to Cys29. Comparison of the amino acid sequence of 6.5k-AGRP with those of the other known proteins included in the PIR protein sequence database showed that it is related to the amino acid sequence of the N-terminal region encoded by the first exon of the cocoa (Theobroma cacao) and cotton seeds vicilin genes, sharing a characteristic two Cys-Xaa-Xaa-Xaa-Cys motif.     

Novel gangliosides containing the sialyl-Le(a) structure from a human rectal adenocarcinoma

Oligosaccharides were released from the gangliosides of a human rectal adenocarcinoma with endoglycoceramidase (Rhodococcus sp. G-74-2) and then purified by affinity chromatography on a column of an immobilized monoclonal antibody, MSW 113. Structural studies, involving 600-MHz 1H NMR spectrometry, indicated the structures of these compounds to be as follows. [formula: see text] Three of these oligosaccharides, 2, 3, and 4, are novel as to ganglioside sugar chains and contain both lacto series types 1 and 2 chains. Oligosaccharides 3 and 4 are unique in that they contain the sialyl-Le(a)-X structure in linear and branched structures. Gangliosides with the oligosaccharide structures presented above might be new potential tumor markers.     

The structure of an insect parvovirus (Galleria mellonella densovirus) at 3.7 A resolution

BACKGROUND: Parvoviruses infect vertebrates, insects and crustaceans. Many arthropod parvoviruses (densoviruses) are highly pathogenic and kill approximately 90% of the host larvae within days, making them potentially effective as selective pesticides. Improved understanding of densoviral structure and function is therefore desirable. There are four different initiation sites for translation of the densovirus capsid protein mRNA, giving rise to the viral proteins VP1 to VP4. Sixty copies of the common, C-terminal domain make up the ordered part of the icosahedral capsid. RESULTS: The Galleria mellonella densovirus (GMDNV) capsid protein consists of a core beta-barrel motif, similar to that found in many other viral capsid proteins. The structure most closely resembles that of the vertebrate parvoviruses, but it has diverged beyond recognition in many of the long loop regions that constitute the surface features and intersubunit contacts. The N termini of twofold-related subunits have swapped their positions relative to those of the vertebrate parvoviruses. Unlike in the vertebrate parvoviruses, in GmDNV there is no continuous electron density in the channels running along the fivefold axes of the virus. Electron density corresponding to some of the single-stranded DNA genome is visible in the crystal structure, but it is not as well defined as in the vertebrate parvoviruses. CONCLUSIONS: The sequence of the glycine-rich motif, which occupies each of the channels along the fivefold axes in vertebrate viruses, is conserved between mammalian and insect parvoviruses. This motif may serve to externalize the N-terminal region of the single VP1 subunit per particle. The domain swapping of the N termini between insect and vertebrate parvoviruses may have the effect of increasing capsid stability in GmDNV.     

Primary structure of hemocyanin from Palinurus vulgaris

The primary structure of hemocyanin from the spiny lobster Palinurus vulgaris was determined using a mixture of at least four slightly different subunits. Heterogeneities were observed in 32 (5%) of the positions. The amino acid sequence differs at about 20% of the positions from that of subunit a of Panulirus interruptus hemocyanin.     

Glucose oxidase from Penicillium amagasakiense. Primary structure and comparison with other glucose-methanol-choline (GMC) oxidoreductases

The complete amino acid sequence of glucose oxidase from Penicillium amagasakiense was determined by Edman degradation and mass spectrometry of peptide fragments derived from three different specific proteolytic digests and a cyanogen bromide cleavage. The complete sequence of each monomer comprises 587 amino acid residues, contains three cysteine residues, and seven potential N-glycosylation sites, of which at least five were confirmed to be glycosylated. Glucose oxidase from P. amagasakiense shows a high degree of identity (66%) and 79% similarity to glucose oxidase from Aspergillus niger, and is a member of the glucose-methanol-choline (GMC) oxidoreductase family. The tertiary structures of glucose oxidase from A. niger and cholesterol oxidase from Brevibacterium sterolicum were superimposed to provide a template for the sequence comparison of members of the GMC family. The general topology of the GMC oxidoreductases is conserved, with the exception of the presence of an active site lid in cholesterol oxidase and the insertion of additional structural elements in the substrate-binding domain of alcohol oxidase. The overall structure can be divided into five distinct sequence regions: FAD-binding domain, extended FAD-binding domain, flavin attachment loop and intermediate region, FAD covering lid, and substrate-binding domain. The FAD-binding and the extended FAD-binding domains are composed of several separate sequence regions. The other three regions each comprise a single contiguous sequence. Four major consensus patterns have been identified, including the nucleotide-binding consensus sequence close to their N-termini. The functions of the two motifs recently selected by the Genetics Computer Group, Madison, Wisconsin, as additional signature patterns of the GMC oxidoreductases are discussed. The other consensus patterns belong to either the FAD-binding or the extended FAD-binding domain. In addition, the roles of conserved residues are discussed wherever possible.     

Primary structure of trypsin inhibitors from Sicyos australis

Three trypsin inhibitors from Sicyos australis, have been isolated, purified and sequenced. Following protein extraction with ammonium sulphate, the mixture of inhibitors was separated from other proteins by trypsin-affinity chromatography. Subsequent purification of the individual inhibitors was accomplished by reversed-phase HPLC. The primary structures of each inhibitor were elucidated by a combination of protein sequencing and electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS-MS) on both the untreated and the reduced and S-carboxymethylated inhibitors. All three inhibitors show extensive sequence similarity with inhibitors from cultivated Cucurbitaceae species, although there are a number of novel residues present. One of the inhibitors has a blocked N-terminus (pyroglutamic acid) and the use of MS-MS was crucial to the elucidation of its primary structure. ESI-MS was further used to characterize the non-covalent complex between one of the inhibitors and trypsin.     

Primary structure of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens

The amino acid sequence of the p-hydroxybenzoate hydroxylase (4-hydroxybenzoate,NADPH:oxygen oxidoreductase (3-hydroxylating), EC 1.14.13.2) monomer from Pseudomonas fluorescens has been determined. The sequence was elucidated by a combination of the results from an X-ray crystallographic study at 0.25 nm resolution (Wierenga, R.K., de Jong, R.J., Kalk, K.H., Hol, W.G.J. and Drenth, J. (1979) J. Mol. Biol. 131, 55-73) and from protein sequence analysis. The polypeptide chain of the monomer contains 394 amino acids and has a molecular weight of 44 299.     

Crystal structure of the IIB subunit of a fructose permease (IIBLev) from Bacillus subtilis

The bacterial phosphoenolpyruvate-dependent phosphotransferase system (PTS) mediates both the uptake of carbohydrates across the cytoplasmic membrane and their phosphorylation. During this process, a phosphoryl group is transferred from phosphoenolpyruvate via the general PTS proteins enzyme I, HPr and the sugar-specific components IIA, IIB to the transported sugar. The crystal structure of the IIB subunit of a fructose transporter from Bacillus subtilis (IIBLev) was solved by MIRAS to a resolution of 2.9 A. IIBLev comprises 163 amino acid residues that are folded into an open, mainly parallel beta-sheet with helices packed on either face. The phosphorylation site (His15) is located on the first loop (1/A) at one of the topological switch-points of the fold. Despite different global folds, IIBLev and HPr have very similar active-site loop conformations with the active-site histidine residues located close to the N terminus of the first helix. This resemblance may be of functional importance, since both proteins exchange a phosphoryl group with the same IIA subunit. The structural basis of phosphoryl transfer from HPr to IIAMan to IIBMan was investigated by modeling of the respective transition state complexes using the known HPr and IIAMan structures and a homology model of IIBMan that was derived from the IIBLev structure. All three proteins contain a helix that appears to be suitable for stabilization of the phospho-histidine by dipole and H-bonding interactions. Smooth phosphoryl transfer from one N-cap position to the other appears feasible with a minimized transition state energy due to simultaneous interactions with the donor and the acceptor helix.     

Primary structure of apolipophorin-III from the greater wax moth, Galleria mellonella

BACKGROUND: Neurobiological research has implicated the dopamine and serotonin systems in the pathogenesis of autism. Open-label reports suggest that the serotonin2A-dopamine D2 antagonist risperidone may be safe and effective in reducing the interfering symptoms of patients with autism. METHODS: Thirty-one adults (age [mean+/-SD], 28.1+/-7.3 years) with autistic disorder (n=17) or pervasive developmental disorder not otherwise specified (n=14) participated in a 12-week double-blind, placebo-controlled trial of risperidone. Patients treated with placebo subsequently received a 12-week open-label trial of risperidone. RESULTS: For persons completing the study, 8 (57%) of 14 patients treated with risperidone were categorized as responders (daily dose [mean+/-SD], 2.9+/-1.4 mg) compared with none of 16 in the placebo group (P<.002). Risperidone was superior to placebo in reducing repetitive behavior (P<.001), aggression (P<.001), anxiety or nervousness (P<.02), depression (P<.03), irritability (P<.01), and the overall behavioral symptoms of autism (P<.02). Objective, measurable change in social behavior and language did not occur. Nine (60%) of 15 patients who received treatment with open-label risperidone following the double-blind placebo phase responded. Other than mild, transient sedation, risperidone was well tolerated, with no evidence of extrapyramidal effects, cardiac events, or seizures. CONCLUSION: Risperidone is more effective than placebo in the short-term treatment of symptoms of autism in adults.     

Primary structure and characterization of an exopolygalacturonase from Aspergillus tubingensis

From the culture fluid of the hyphal fungus Aspergillus tubingensis, an exopolygalacturonase with a molecular mass of 78 kDa, an isoelectric point in the pH-range 3.7-4.4 and a pH optimum of 4.2 was purified. The enzyme has been characterized as an exopolygalacturonase [poly(1,4-alpha-D-galacturonide)galacturonohydrolase] that cleaves monomer units from the non-reducing end of the substrate molecule. K(m) and Vmax for polygalacturonic acid hydrolysis were 3.2 mg ml-1 and 3.1 mg ml-1 and 255 U mg-1 and 262 U mg-1 for the wild-type and recombinant enzymes, respectively. The kinetic data of exopolygalacturonase on oligogalacturonates of different degree of polymerization (2-7) were interpreted in terms of a subsite model to obtain more insight into catalysis and substrate binding. On oligogalacturonates of different degrees of polymerization (2-7), the Michaelis constant (K(m)) decreased with increasing chain length (n). The Vmax value increased with chain length up to n = 4, then reached a plateau value. The enzyme was competitively inhibited by galacturonic acid (Ki = 0.3 mM) as well as by reduced digalacturonate (Ki = 0.4 mM). The exopolygalacturonase gene (pgaX) was cloned by reverse genetics and shows only 13% overall amino acid sequence identity with A. niger endopolygalacturonases. The exopolygalacturonase is most related to plant polygalacturonases. Only four small stretches of amino acids are conserved between all known endogalacturonases and exopolygalacturonases. Expression of the pgaX gene is inducible with galacturonic acid and is subject to catabolite repression. A fusion between the promoter of the A. niger glycolytic gene encoding pyruvate kinase and the pgaX-coding region was used to achieve high level production of exopolygalacturonase under conditions where no endopolygalacturonases were produced.     

Primary structure and crystallization of orotate phosphoribosyltransferase from Salmonella typhimurium

Orotate phosphoribosyltransferase (OPRTase; EC 2.4.2.10) catalyzes phosphoribosyl group transfer between alpha-D-5-phosphoribosyl-1-pyrophosphate and orotate to form orotidine-5'-monophosphate and pyrophosphate, the nucleotide-forming step in pyrimidine biosynthesis. It is one of ten PRTases that perform vital roles in de novo and salvage pathways for purine, pyrimidine and pyridine nucleotides. Although the PRTases are important drug targets, they are poorly understood mechanistically, and no three-dimensional structures exist. Here, we report the complete sequence of the Salmonella typhimurium pyrE gene and the deduced sequence of the OPRTase gene product. OPRTase forms tetragonal crystals from polyethylene glycol solutions; these crystals diffract to better than 2 A resolution, and are stable to radiation damage. The space group is P4(1)2(1)2 (or P4(3)2(1)2) with unit cell dimensions of a = b = 48.5 A, c = 210.5 A, and alpha = beta = gamma = 90 degrees. A crystalline form of the selenomethionine derivative of the protein is also reported.     

A two-dimensional NMR study of Co(II)7 rabbit liver metallothionein

The 600-MHz 1H-NMR NOESY spectra on Co(II)7-reconstituted metallothionein (Co7MT), exhibiting hyperfine signals in the range 350 ppm to -50 ppm, with nuclear relaxation times of the order of a few milliseconds, have been measured and several interproton connectivities have been detected. To our knowledge, this is the largest spectral window ever reported for a two-dimensional 1H-NMR spectrum in the case of a paramagnetic metalloprotein. No scalar connectivities could be detected. The hyperfine-shifted signals belong to the cysteine-ligand protons of the Co4S11 cluster of Co7MT. Together with results from one-dimensional NOE experiments, the two-dimensional experiments allowed us to proceed with the pairwise assignment of the isotropically shifted signals of the C beta H2 groups of the metal-coordinated cysteines. With the aid of computer-graphics inspection of the four-metal-cluster domain, based on the NMR solution structure of Cd7MT, it is possible to purpose sequence-specific assignments of a few hyperfine-shifted 1H-NMR signals. In particular, a tentative assignment is given for the six signals whose shifts exhibit an antiCurie temperature dependence. The assignment relies on the theoretical model that qualitatively rationalizes the isotropic-shift pattern and its temperature dependence. Inferences on the solution structure of the Co4S11 cluster are drawn.     

Primary structure of two-chain botrocetin, a von Willebrand factor modulator purified from the venom of Bothrops jararaca

The complete amino acid sequence and location of the disulfide bonds of two-chain botrocetin, which promotes platelet agglutination in the presence of von Willebrand factor, from venom of the snake Bothrops jararaca are presented. Sequences of the alpha and beta subunits were determined by analysis of peptides generated by digestion of the S-pyridylethylated protein with Achromobacter protease I or alpha-chymotrypsin and by chemical cleavage with cyanogen bromide or 2-(2'-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine. Two-chain botrocetin is a heterodimer composed of the alpha subunit (consisting of 133 amino acid residues) and the beta subunit (consisting of 125 amino acid residues) held together by a disulfide bond. Seven disulfide bonds link half-cystine residues 2 to 13, 30 to 128, and 103 to 120 of the alpha subunit; 2 to 13, 30 to 121, and 98 to 113 of the beta subunit; and 80 of the alpha subunit to 75 of the beta subunit. In terms of amino acid sequence and disulfide bond location, two-chain botrocetin is homologous to echinoidin (a sea urchin lectin) and other C-type (Ca(2+)-dependent) lectins.     

栗山天牛提取物主要营养成分分析与评价

采用现代分析技术对栗山天牛提取物的主要营养成分进行了分析.结果显示:栗山天牛提取物的蛋白质、氨基酸、糖类质量分数分别为37.28%、37.25%、8.61%;含有7种人体必需氨基酸,其必需氨基酸占总氨基酸的40.97%,必需氨基酸与非必需氨基酸的比值为0.693 9,第一限制性氨基酸为含硫氨基酸,即蛋氨酸和胱氨酸;还含有Ca、Mg、Fe、Mn、Zn、Cu、Mo、Ni、Se、Cr等多种矿物质与微量元素.