Purification, characterization and functional expression of a new peptide with an analgesic effect from Chinese scorpion Buthus martensii Karsch (BmK AGP-SYPU1)

In this study, a new peptide named BmK AGP‐SYPU1 with an analgesic effect was purified from the venom of Chinese scorpion Buthus martensi Karsch (BmK) through a four‐step chromatographic process. The mouse twisting test was used to identify the target peptides in every separation step. The purified BmK AGP‐SYPU1 was further qualified by RP‐HPLC and HPCE. The molecular mass determined by the MALDI‐4800‐TOF/TOF MS for BmK AGP‐SYPU1 was 7544 Da. Its primary structure of the N‐terminal was obtained using Edman degradation. The gene sequence of BmK AGP‐SYPU1 was cloned from the cDNA pool and genomic of scorpion glands, respectively, and then expressed in Escherichia coli. The sequence determination showed that BmK AGP‐SYPU1 was composed of 66 amino acid residues with a new primary structure. The metal chelating affinity column and cation exchange chromatography were used to purify the recombinant BmK AGP‐SYPU1. Consequently, the native and recombinant BmK AGP‐SYPU1 showed similar analgesic effects on mice as assayed using a mouse twisting model. These results suggested that BmK AGP‐SYPU1 is a new analgesic component found in the Chinese scorpion Buthus martensi Karsch. Copyright © 2010 John Wiley & Sons, Ltd.     

Structure and function of ferritin

Ferritin is the major iron storage protein of mammals and consists of up to 4500 atoms of ferric iron surrounded by a shell of protein subunits. The protein component, apoferritin, consists of 24 identical polypeptide chains each of molecular weight 18500. The function of ferritin is to store iron in a soluble form from which it can be readily mobilized. Recent results concerning the structure of the protein are reported, and progress in the elucidation of the mechanisms whereby iron is introduced into apoferritin and released from ferritin is reviewed.     

Structure and biosynthesis of the jamaicamides, new mixed polyketide-peptide neurotoxins from the marine cyanobacterium Lyngbya majuscula

A screening program for bioactive compounds from marine cyanobacteria led to the isolation of jamaicamides A-C. Jamaicamide A is a novel and highly functionalized lipopeptide containing an alkynyl bromide, vinyl chloride, beta-methoxy eneone system, and pyrrolinone ring. The jamaicamides show sodium channelblocking activity and fish toxicity. Precursor feeding to jamaicamide-producing cultures mapped out the series of acetate and amino acid residues and helped develop an effective cloning strategy for the biosynthetic gene cluster. The 58 kbp gene cluster is composed of 17 open reading frames that show an exact colinearity with their expected utilization. A novel cassette of genes appears to form a pendent carbon atom possessing the vinyl chloride functionality; at its core this contains an HMG-CoA synthase-like motif, giving insight into the mechanism by which this functional group is created.     

马氏钳蝎蝎毒短肽BmK622的分离纯化和一级结构测定

应用凝胶过滤、离子交换和HPLC反相色谱法从马氏蝎粗毒中分离纯化得到蝎毒 多肽BmK622．联合运用串联质谱法和Edman降解法，鉴定了KmK622N端19个残基的序 列，经过数据库检索，发现数据库中用cDNA克隆方法鉴定了序列的马氏钳蝎蝎毒短 肽BmTX3一级序列N端19个残基与BmK622已测定的N端19个残基序列完全相同， BmK622的分子量测定和氨基酸组成分折的结果表明，BmK22与BmTX3分子量相同、氨 基酸组成一致，从而BmK22的一级结构为：GFLID VKCFA SSECW TACKK VTGSG QGKCQ NNQCR CY．     

Structure and function of vanadium haloperoxidases

A quantum mechanics/molecular mechanics study of the resting state of the vanadium dependent chloroperoxidase from fungi Curvularia inaequalis and of the early intermediates of the halide oxidation is reported. The investigation of different protonation states indicates that the enzyme likely consists of an anionic H2VO4- vanadate moiety where one hydroxo group is in axial position. The calculations suggest that the hydrogen peroxide binding may not involve an initial protonation of the vanadate cofactor. A low free energy reactive path is found where the hydrogen peroxide directly attacks the axial hydroxo group, resulting in the formation of an hydrogen peroxide intermediate. This intermediate is promptly protonated to yield a peroxo species. The free energy barrier for the formation of the peroxo species does not depend significantly upon the protonation state of the cofactor. The most likely protonation states of the peroxo cofactor are neutral forms HVO2(O2) with a hydroxo group either H-bonded to Ser402 or coordinated to Arg360. The peroxo cofactor is also coordinated to an axial water molecule, which could be important for the stability of the peroxovanadate/His496 adduct. Our calculations strongly suggest that the halide oxidation may take place with the preliminary formation of a peroxovanadate/halogen adduct. Subsequently, the halogen reacts with the peroxo moiety yielding a hypohalogen vanadate. The most reactive protonation state of peroxovanadate is the neutral HVO2(O2) with the hydroxo group H-bonded to Ser402. The important role of Lys353 in determining the catalytic activity is also confirmed.     

Structure and function of noncanonical nucleobases

DNA and RNA contain, next to the four canonical nucleobases, a number of modified nucleosides that extend their chemical information content. RNA is particularly rich in modifications, which is obviously an adaptation to their highly complex and variable functions. In fact, the modified nucleosides and their chemical structures establish a second layer of information which is of central importance to the function of the RNA molecules. Also the chemical diversity of DNA is greater than originally thought. Next to the four canonical bases, the DNA of higher organisms contains a total of four epigenetic bases: m(5) dC, hm(5) dC, f(5) dC und ca(5) dC. While all cells of an organism contain the same genetic material, their vastly different function and properties inside complex higher organisms require the controlled silencing and activation of cell-type specific genes. The regulation of the underlying silencing and activation process requires an additional layer of epigenetic information, which is clearly linked to increased chemical diversity. This diversity is provided by the modified non-canonical nucleosides in both DNA and RNA.     

东亚马氏钳蝎哺乳动物类神经毒素III的快原子轰击酶谱分析

应用快原子轰击酶谱分析检验了东亚马氏钳蝎哺乳动物类神经毒素III的氨基酸序列,对前人用Edman降解法测定的序列作了两处修正:(1)第60位氨基酸残基是脯氨酸而不是色氨酸;(2)C-端氨基酸是组氨酸而不是甘氨酸。分别用羧肽酶B降解和部分胰蛋白酶酶解后的质谱数据进一步证实了以上的修正。在分析中采用了化学修饰方法以减少表面抑制效应,从而得到完整的快原子轰击酶谱。     

Ion-spray mass spectrometry of marine neurotoxins

Ion-spray mass spectrometry was investigated for the analysis of three marine neurotoxins: domoic acid, saxitoxin and tetrodotoxin. All three compounds gave positive-ion spectra with abundant ions of protonated molecules and no significant fragmentation. Domoic acid gave a negative-ion spectrum with a strong [M-H]- ion. Tandem mass spectrometry provided useful fragment-ion spectra for all compounds. Detection limits for flow injection analyses with selected-ion monitoring were determined to be 30 pg for saxitoxin, 100 pg for domoic acid and 200 pg for tetrodotoxin. Combining liquid chromatography with ion-spray mass spectrometry allowed the determination of domoic acid and some of its isomers in toxic shellfish tissue extracts.     

Purification, Characterization and 1H NMR Resonance Assignment of an a－Like Neurotoxin BmK 16 from the Venom of Chinese Scor－pion Buthus martensii Karsch

A natural scorpion toxin BmK 16 was purified for the first time from the venom of the Chinese scorpion Buthus martensii Karsch (BmK) by using combined gel-filtration, ion exchange and reversed phase chromatography. The sequence of the N-terminal 8 amino acid residues was determined by Edman degradation. Using the N-terminal sequence as a tag, the database searching revealed a hit in the scorpion cDNA Bank. The sequence for N-terminal 8 amino acid residues, molecular weight and amino acid compositions of BmK 16 were identical with the calculated values according to the first 64 residues‘ se-quence of the precursor peptide alpha-neurotoxin TX16 derived from the sequence of the cDNA AF156597 (EMBL). The se-quence-specific resonance assignment of BmK 16 was achieved and the intact sequence of BmK 16 was determined as follow-ings: VRDAY IAKPH NCVYE CARNE YCNDL CTKNG AKSGY CQWVG KYGNG CWCKE LPDNV PIRVP GKCH. Furthermore, the results from the sequence homology analysis and the toxicity assays indicated that BmK 16 was an α-likescorpion neurotoxin.     

Structure and function of bacterial initiation factors

Bacteria require three initiation factors, IF1, IF2 and IF3, to start protein synthesis. In the last few years the elucidation of both structural and mechanistic aspects pertaining to these proteins has made substantial progress. In this article we outline the translation initiation process in bacteria and review these recent developments giving a summary of the main features of the structure and function of the initiation factors.     

Structure and function of annexin V-decorsin fusion

Decorsin is an antagonist of fibrinogen receptor on platelets and Annexin V is able to recognize stimulated platelets. Two recombinant proteins, Annexin V plus Decorsin (AnnV-D39) and Annexin V plus the C terminal 27 amino acids variant of Decorsin (AnnV-D27), were constructed, expressed, and purified. Platelet Aggregation assay results appear that AnnV-D39 shows good anti-platelet aggregation activity, but AnnV-D27 no such activities in any platelet aggregation assay test. And computational simulations reveal that AnnV-D39 showed good anti-platelet aggregation activity as a new antagonist of fibrinogen receptor, while Annv-D27 needs re-modification. Despite the AnnV_D39 fusion is more than decorsin, the former maintains the binding sites of decorsin interacton with its receptor, which contains Asp10, Arg28-Asp33, and Tyr37-Glu39. And the addition of Annexin V could not influence the interaction between its decorsin part with its receptor GPIIb–IIIa due to the linkage peptide (GGGGSGGGGS). Although the AnnV_D27 fusion is similar to the AnnV_D39 fusion, there are differences between them, where the former is in shortage of the linkage peptide and the N-terminal segment of decorsin whose one residue (Asp10) contribution to its interaction with GPIIb–IIIa. Meanwhile, these complex models suggest that decorsin plays a role in antiplatelet aggregation not only by its RGD motif interaction with its GPIIb–IIIa receptor, but also by other residues, especially Asp10 of its N-terminal segment and Tyr37-Glu39 of its C-terminal segment. On the other hand, the linkage peptide acts as avoidance of influence and blockage between domains of fusion. This is the cause that AnnV-D39 shows good anti-platelet aggregation activity.     

Chiral CE separation of dopamine-derived neurotoxins.

An enantiomeric separation of dopamine-derived neurotoxins by capillary electrophoresis has been developed. Tetrahydroisoquinoline (TIQ), dopamine (DA), (R/S)-1-benzyl-TIQ (BTIQ), (R/S)-6,7-dihydroxy-1-methyl-TIQ (salsolinol, Sal), and (R/S)-6,7-dihydroxy-1, 2-dimethyl-TIQ (N-methyl-salsolinol, NMSal) were studied as model compounds. The CE running buffer (50 mM phosphate buffer at pH 3.0) contained 1.5 M urea and 12 mM beta-CD as a chiral selector. During separation, the (R)-enantiomers formed more stable inclusion complexes with beta-CD, and thus had a longer migration time than their optical antipodes. It was noticed that the recovery rates of these TIQ derivatives were very poor (< 15%) during protein precipitation, a procedure widely used for cleaning up biological samples. The recovery was significantly improved by pre-mixing the sample with a surfactant (e.g., sodium hexanesulfonate or Triton X-100) to reduce the co-precipitation. The present method in combination with electrospray ionization tandem mass spectrometry (ESI-MS/MS) was applied to study samples obtained from in vitro incubation of two catecholamines, dopamine and epinine, with aldehydes forming neurotoxins including (S)- and (R)-NMSal enantiomers. The later is known to induce Parkinsonism in rats.     

Structure and function of window glass and Pyrex

Window glass is a ternary mixture, while Pyrex (after window glass, the most common form of commercial glass) is a quaternary. Here, we investigate the chemical, physical, and mathematical factors that determine the compositions of these optimized glasses. Previously, we succeeded in deriving exactly the composition of window glass (sodium calcium silicate) without adjustable parameters. Borosilicates are a much more challenging problem, and Pyrex (sodium aluminum borosilicate) requires a different approach. Our analysis shows that mean-field (or global) models (networks without significant clustering) are sufficient for window glass and probably most other commercial silicate glasses. However, it appears that the most important property of pyrex, its ability to resist mechanical shocks, requires a cluster model (large medium range order). We propose such a model, and argue that it also follows from hierarchical principles. Our model is strongly supported by specific experiments, and we suggest further experiments to test the principles underlying commercial glasses.     

Dinoflagellate neurotoxins related to saxitoxin: Structure and latent activity of toxins B1 and B21

Toxins B1 and B2 from cultured dinoflagellates of the genus Protogonyaulax are shown to be the carbamoyl-N-sulfo derivatives of saxitoxin and neosaxitoxin, the structures confirmed by synthesis from the corresponding decarbamoyl toxins.     

Purification and characterization of an analgesic peptide from Buthus martensii Karsch

The scorpion Buthus martensii Karsch (BmK) has been one of the indispensable materials in Chinese traditional medicine for thousands of years. In this work, an analgesic peptide was purified through four continuous chromatographic steps. The mouse twisting test was used to identify the target peptides in every separation step. The molecular weight, isoelectric point, and N-terminal residues of the purified peptide were determined. Based on the N-terminal sequence, the cDNA was also cloned by rapid amplification of cDNA ends from the cDNA pool of scorpion glands. This peptide was identical to BmK AS, an agonist of rabbit skeletal muscle ryanodine receptors. Preliminary pharmacodynamics revealed the following: the dose-effect curve plotted by the mouse twisting test showed an ED(50) of 1.42 mg/kg; and the time-effect curves plotted by a hot plate procedure showed a similar effect to the painkiller morphine. We report a purification procedure that yields substantial amounts of natural BmK AS having high activity. BmK AS has the potential to become a new analgesic medicine.     

Structure and function of cis-prenyl chain elongating enzymes

All carbon skeletons of isoprenoids, whose chain lengths vary widely from geranyl diphosphate (C10) to natural rubber (C>10,000), are synthesized by sequential condensation of isopentenyl diphosphate with an allylic diphosphate through catalytic functions of a group of enzymes commonly called "prenyltransferases." Prenyltransferases are classified into two major groups, trans- or (E)-prenyltransferases and cis- or (Z)-prenyltransferases, according to the geometry of the prenyl chain units in the products. From the year 1987, many genes encoding trans-prenyltransferases were cloned and clearly characterized. In contrast, the structure and detailed mechanism of cis-prenyltransferase was completely unknown until the identification of a gene encoding the undecaprenyl diphosphate (UPP) synthase from Micrococcus luteus B-P 26 in 1998. Not only the primary but also the tertiary structure of the UPP synthase is quite different from that of the trans-prenyltransferases. Multiple alignment of the primary structures of cis-prenyltransferases identified from various organisms reveals five highly conserved regions. Site-directed mutagenesis of the conserved amino acid residues in UPP synthases based on the crystal structure has elucidated the basic catalytic mechanisms. Moreover, comparison of the structures of short-, medium-, and long-chain cis-prenyltransferases reveals important amino acid residues for product chain length determination, which enabled us to understand the regulation mechanism of the ultimate chain length among cis-prenyltransferases.     

Structure of ascorbic acid and its biological function

Summary Only LAMMA spectra of negative ions of ascorbic acid and isoascorbic acid exhibit a deprotonated peak. In the case of the radicals Na-ascorbate and K-isoascorbate neither the positive nor the negative ion spectra show a similar peak. Only a peak at m_r/z=41 can be detected in the negative ion spectra of both of the radicals. Positive ion spectra exhibit peaks at m_r/z =63 (for Na-ASC) and at 95 (for K-Iso-ASC) in addition to the Na⁺ and K⁺ peaks in the corresponding radicals. The peaks at 41, 63, and 95 might represent Na and K complexes engulfed in the cyclic structure of the side chain. From the results obtained one can be certain, that both of the radicals are electroneutral.

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Struktur von Ascorbinsäure und ihre biologische Funktion: IV. Untersuchung der Struktur durch laser-induzierte Massenspektrometrie

Zusammenfassung Nur die LAMMA-Spektren der negativen Ionen von Ascorbinsäure und Isoascorbinsäure haben einen deprotonierten Peak. Bei den Radikalen Na-Ascorbat und K-Isoascorbat ist dieser weder bei den positiven noch bei den negativen Ionen-Spektren vorhanden. Bei beiden Radikalen kann nur bei m_r/z=41 ein Peak im negativen Ionen-Spektrum nachgewiesen werden. Die positiven Ionen-Spektren haben neben den Na⁺- und K⁺-Peaks solche bei m_r/z=63 (für Na-ASC) und 95 (für K-Iso-ASC). Die Peaks bei 41,63 und 95 stellen wahrscheinlich Na- und K-Komplexe dar, die in der cyclischen Struktur der Seitenkette eingebettet sind. Auf jeden Fall kann aus den erhaltenen Ergebnissen geschlossen werden, daß beide Radikale elektroneutral sind.