The complete amino acid sequence of growth hormone and partial amino acid sequence of prolactin and somatolactin from sea perch (Lateolabrax japonicus)

Growth hormone (GH), prolactin (PRL) and somatolactin (SL) were purified simultaneously under alkaline condition (pH 9.0) from pituitary glands of sea perch (Lateolabrax japonicas) by a two-step procedure involving gel filtration on Sephadex G-100 and reverse-phase high-performance liquid chromatography (rpHPLC). At each step of purification, fractions were monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and by immunoblotting with chum salmon GH. PRL and SL antisera. The yields of sea perch GH, PRL and SL were 4.2, 1.0 and 0.28 mg/g wet tissue, respectively. The molecular weights of 19,200 and 20,370 Da were estimated by SDS-PAGE for sea perch GH and PRL, respectively. Two forms of sea perch SL were found: one (28,400 Da) is probably glycosylated, while the other one (23,200 Da) is believed to be deglycosylated. GH bioactivity was examined by an in vivo assay. Intraperitoneal injection of sea perch GH at a dose of 0.01 and 0.1 mug/g body weight at 7-day intervals resulted in a significant increase in body weight and length of juvenile rainbow trout. The complete sea-perch GH amino acid sequence of 187 residues was determined by sequencing fragments cleaved by chemicals and enzymes. Alignment of sea-perch GH with those of other fish GHs revealed that sea-perch GH is most similar to advanced marine fish, such as tuna, gilthead sea bream, yellowfin porgy, red sea bream, bonito and yellow tail with 98.4, 96.2%, 95.7%, 95.2%, 94.1% and 91% sequence identity, respectively. Sea-perch GH has low identity to Atlantic cod (76.5%), hardtail (73.3%), flounder (68.4%), chum salmon (66.3%), carp (54%) and blue shark (38%). Partial amino-acid sequences of 127 of sea-perch PRL and the N-terminal of 16 amino-acid sequence of sea-perch SL have been determined. The data show that sea-perch PRL has a slightly higher sequence identity with tilapia PRL( 73.2%) than with chum salmon PRL(70%) in this 127 amino-acid sequence. (C) 2001 Elsevier Science B.V. All rights reserved.

Application of 2-(11H-benzo[a] carbazol-11-yl) Ethyl Carbonochloridate as a Pro-column Derivatization Reagent of Amino Acid by High Performance Liquid Chromatography with Fluorescence Detection

On a reversed phase Hypersil BDS C-18 (200 mm x 4. 6 mm, 5 mu m) column, 20 amino acids, which were derivatized using 2-(11H-benzo [a] carbazol-11-yl) ethyl carbonochloridate (BCEC-Cl) as pre-column derivatization reagent, were separated in conjunction with a gradient elution. Optimum derivatization was obtained by reacting of amino acids with BCEC-Cl at room temperature for 5 min in the presence of sodium borate catalyst in acetonitrile solvent. The fluorescence excitation and emission wavelengths were 279 nm and 380 nm respectively. The identification of amino acid derivatives from hydrolyzed bovine serum albumin and bee pollen was carried out by post-column mass spectrometry with electrospray ion source in positive ion mode. Linear correlation coefficients of the amino acid derivatives were > 0.9990, and detection limits (at signal to noise of 3:1) were 1.49 - 19.74 fmol for the labeled amino acids.

Simultaneous determination of monoamine and amino acid neurotransmitters in rat endbrain tissues by pre-column derivatization with high-performance liquid chromatographic fluorescence detection and mass spectrometric identification

A sensitive and efficient method for simultaneous determination of glutamic acid (Glu), gamma-amino-butyric acid (GABA), dopamine (DA), 5-hydroxytryptamine (5-HT) and 5-hydroxyindole acetic acid (5-HIAA) in rat endbrains was developed by high-performance liquid chromatography (HPLC) with fluorescence detection and on-line mass spectrometric identification following derivatization with 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC). Different parameters which influenced derivatization and separation were optimized. The complete separation of five neurotransmitter (NT) derivatives was performed on a reversed-phase Hypersil BDS-C-18 column with a gradient elution. The rapid structure identification of five neurotransmitter derivatives was carried out by on-line mass spectrometry with electrospray ionization (ESI) source in positive ion mode, and the BCEOC-labeled derivatives were characterized by easy-to-interpret mass spectra. Stability of derivatives, repeatability, precision and accuracy were evaluated and the results were excellent for efficient HPLC analysis. The quantitative linear range of five neurotransmitters were 2.441-2 x 10(4) nM, and limits of detection were in the range of 0.398-1.258 nM (S/N = 3:1). The changes of their concentrations in endbrains of three rat groups were also studied using this HPLC fluorescence detection method. The results indicated that exhausting exercise could obviously influence the concentrations of neurotransmitters in rat endbrains. The established method exhibited excellent validity, high sensitivity and convenience, and provided a new technique for simultaneous analysis of monoamine and amino acid neurotransmitters in rat brain. (C) 2008 Elsevier B.V. All rights reserved.

Chiral separation of enantiomers of amino acid derivatives by high-performance liquid chromatography on a norvancomycin-bonded chiral stationary phase

A novel norvancomycin-bonded chiral stationary phase (NVC-CSP) was synthesized by using the chiral selector of norvancomycin. The chiral separation of enantiomers of several dansyl-amino acids by high-performance liquid chromatography (HPLC) in the reversed-phase mode is described. The effects of some parameters, such as organic modifier concentration, column temperature, pH and flow rate of the mobile phase, on the retention and enantioselectivity were investigated. The study showed that ionic, as well as hydrophobic interactions were engaged between the analyte and macrocycle in this chromatographic system. Increasing pH of buffers usually improved the chiral resolution for dansyl-alpha-amino-n-butyric acid (Dns-But), dansyl-methionine (Dns-Met) and dansyl-threonine (Dns-Thr), but not for dansyl-glutamic acid (Dns-Glu) which contains two carboxylic groups in its molecular structure. The natural logarithms of selectivity factors (In alpha) of all the investigated compounds depended linearly on the reciprocal of temperature (1/T), most processes of enantioseparation were controlled enthalpically. Interestingly, the process of enantioseparation for dansyl-threonine was enthalpy-controlled at pH of 3.5, while at pH of 7.0, it was entropy-controlled according to thermodynamic parameters Delta(R,S)DeltaHdegrees and Delta(R,S)DeltaSdegrees afforded by Van't Hoff plots. In order to get baseline separation for all the solutes researched, norvancomycin was also used as a chiral mobile phase additive. In combination with the NVC-CSP remarkable increases in enanselectivity were observed for all the compounds, as the result of a "synergistic" effect. (C) 2003 Elsevier B.V. All rights reserved.

A novel heme-containing protein with anti-HIV-1 activity from skin secretions of Bufo andrewsi

A novel protein, named BAS-AH, was purified and characterized from the skin of the toad Bufo andrewsi. BAS-AH is a single chain protein and the apparent molecular weight is about 63 kDa as judged by SDS-PAGE. BAS-AH was determined to bind heme (0.89 mol heme/mol protein) as determined by pyridine haemochrome analysis. Fifty percentage cytotoxic concentration (CC50) of BAS-AH on C8166 cells was 9.5 mu M. However, at concentrations that showed little effect oil cell viability, BAS-AH displayed dose dependent inhibition oil HIV-1 infection and replication. The antiviral selectivity indexes corresponding to the measurements of syncytium formation and HIV-1 p24 (CC50/EC50) were 14.4 and 11.4, respectively, corresponding to the . BAS-AH also showed an inhibitory effect on the activity of recombinant HIV-1 reverse transcriptase (IC50 = 1.32 mu M). The N-terminal sequence of BAS-AH was determined to be NAKXKADVIGKISILLGQDNLSNIVAM, which exhibited little identity with other known anti-HIV-1 proteins. BAS-AH is devoid of antibacterial, protcolytic, trypsin inhibitory activity, (L)-amino acid oxidase activity and catalase activity. (c) 2005 Elsevier Ltd. All rights reserved.

Biochemical and biological properties of Trimeresurus jerdonii venom and characterization of a platelet aggregation-inhibiting acidic phospholipase A(2)

Several biochemical and biological activities such as phospholipase A(2), arginine esterase, proteolytic, L-amino acid oxidase, 5'nucleotidase, acetylcholinesterase, thrombin-like, anticoagulant, and hemorrhagic activities were determined for whole desiccated venom of Trimeresurus jerdonii. An acidic phospholipase (named TJ-PLA(2)) was purified by anionic exchange chromatography, gel filtration, and reverse phase HPLC. TJ-PLA(2) had a molecular weight of 16,000 and a pI of 4.8. TJ-PLA(2) was non-lethal to mice up to an i.p. dose of 15 mg/kg body weight and lacked neurotoxicity and myotoxicity. It induced edema in the footpads of mice. The purified enzyme inhibited ADP- and collagen-induced human platelet aggregation in a manner which was both dose- and time-dependent.

Purification and cloning of cysteine-rich proteins from Trimeresurus jerdonii and Naja atra venoms

Three 26 kDa proteins, named as TJ-CRVP, NA-CRVP1 and NA-CRVP2, were isolated from the venoms of Trimeresurus jerdonii and Naja atra, respectively. The N-terminal sequences of TJ-CRVP and NA-CRVPs were determined. These components were devoid of the enzymatic activities tested, such as phospholipase A(2), arginine esterase, proteolysis, L-amino acid oxidase, 5' nucleotidase, acetylcholinesterase. Furthermore, these three components did not have the following biological activities: coagulant and anticoagulant activities, lethal activity, myotoxicity, hemorrhagic activity, platelet aggregation and platelet aggregation-inhibiting activities. These proteins are named as cysteine-rich venom protein (CRVP) because their sequences showed high level of similarity with mammalian cysteine-rich secretory protein (CRISP) family. Recently, some CRISP-like proteins were also isolated from several different snake venoms, including Agkistrodon blomhoffi, Trimeresurus flavoviridis, Lanticauda semifascita and king cobra. We presumed that CRVP might be a common component in snake venoms. Of particular interest, phylogenetic analysis and sequence alignment showed that NA-CRVP1 and ophanin, both from elapid snakes, share higher similarity with CRVPs from Viperidae snakes. (C) 2003 Elsevier Ltd. All rights reserved.

固斑蝰蛇毒C-型凝集素样蛋白Dabocetin及L-氨基酸氧化酶DRS-LAO的研究

本论文研究了从圆斑蛙蛇泰国亚种（Daboia russellii siamensis）蛇毒中纯化的C一型凝集素样蛋白Dabocetin和L一氨基酸氧化酶DRS一AO的理化性质、生物学活性和分子克隆。Dabocetin是分子量约为28扔。的异二聚体蛋白，它由分子量约为15.0kDa和14．5kDa的两个同源亚基以和p共价结合形成。N－末端氨基酸序列比较显示，Dabocetin与目前已知的蛇毒c一型凝集素样蛋白有很高的同源性。即使在终浓度达50．0。叫而时，Dabocetin也不能直接诱导血小板聚集。此外，在终浓度为40.00μg/ml时，Dabocetin几乎不能抑制由AdP，TMVA和stejnulxin诱导的血小板聚集。但是，Dabocetin呈剂量依赖地抑制瑞斯托霉素诱导的血小板凝集，其半数抑制率ICS。值为10.80ug/ml。流式细胞仪分析表明，Dabocetin显著抑制单克隆抗体522与GPIba的结合，提示Dabocetin很可能是一个GPIb结合蛋白。从圆斑蛙蛇的毒腺中克隆到了7个编码不同蛇毒C一型凝集素样蛋白亚基的七DNA（命名为DRs一1至DRs一7）。其中，DRsLS编码Dabocetin的a亚基，DRS一6编码Dabocetin的p亚基。DRs一1和DRS一2很可能是圆斑蛙蛇毒腺中表达的X因子激活剂的两条轻链LCZ和LCI的山NA。DRS一3，DRS毛4和DRSL7可能是圆斑蛙蛇毒腺中表达的C一型凝集素样蛋白p亚基的。NA。DRsLAO是一个新的L一氨基酸氧化酶，比活力为1．98U加噶。十二烷基磺酸钠一聚丙烯酞氨凝胶电泳（SDs-PAGE）分析显示，该酶在还原和非还原条件下均呈现一条蛋白带，表观分子量约为58kDa。N-末端氨基酸序列比较显示，DRS一AO与目前已知的蛇毒L一氨基酸氧化酶有很高的同源性。该酶的最适底物为L一亮氨酸，最适pH为8．8。DRs一Ao呈剂量依赖地抑制扔P和仆IvA诱导的血小板聚集，其半数抑制率ICS。值分别为32.8μg/ml和32.3μg/ml。DRS-LAO对金黄色葡萄球菌（灯Cc25923）和耐甲氧西林金黄色葡萄球菌有较强的抗菌作用。DRs一AO对金黄色葡萄球菌必Tcc25923）的最低抑菌浓度卿C）和最低杀菌浓度耐甲氧西林金黄色葡萄球菌的孤CS。和呱Cg。值分别为18．。林留时和36.0μg/ml；DRSLAo对耐甲氧西林金黄色葡萄球菌的MBC50和MBCg。值分别为36.0μg/ml和72.0μg/ml。通过对DRS一AO的分子克隆，得到了编码DRS-AO的部分cDNA序列。

Mass spectrometry study on snake venoms and enzymes

In this paper, three kinds of snake venoms and lour kinds of enzymes (phospholipase A(2), fibrinolytic enzyme, arginine esterase and L-amino acid oxidase) isolated from the snake venom were analyzed. As the snake venom was different, the MALDI/TOF/MS showed difference, The MALDI/TOF/MS determination results could be affected Ly the concentrations of snake venom enzymes, And the mechanisms of desorption and ionization was also given in this study, By using MALDI/TOF/MS we obtained the accurate molecular weights and homogeneities of the enzymes. The apparent characteristics of the positive MALDI/TOF/MS of enzymes composed by two subunits were also given out, The results showed that MALDI/TOF/MS is an effective analytic method for discovering new components from snake venom complexes. And it is reliable to use this method to determine the molecular weights and purifies of protein molecules.

白眉蝮蛇蛇毒L-氨基酸氧化酶的纯化与表征

用离子交换和凝胶过滤层析技术从长白山白眉蝮蛇蛇毒(A gkistrodon blom hoffii ussurensis)中分离得到了L氨基酸氧化酶(L-amino acid oxidase)，经SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)和基体辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)鉴定，发现L-氨基酸氧化酶由两个不相等的亚基组成，一个亚基的分子量约为36000，另一个亚基分子量约为57000．以L-亮氨酸作为底物进行L-氨基酸氧化酶活力测定时发现，其反应的适宜PH值为45.5和89，紫外吸收光谱明显表现出核黄素蛋白的特征

同步荧光法研究白眉蝮蛇蛇毒酶发色团的微环境

利用同步荧光技术对长白山白眉蝮蛇蛇毒中纯化得到的4种酶：磷脂酶A2(phospholipase A2,PLA2)、精氨酸酯酶(arginine esterase,AEase)、纤溶酶(fibrinolytic enzyme,FEase)和L-氨基酸氧化酶(L-amino acid oxidase,L-a a oxidase)进行了研究． 结果表明：当Δλ＝20nm时， 酶的荧光主要由酪氨酸(Tyr)残基所贡献； 当Δλ≥75nm时，酶的荧光主要由色氨酸(Try)所贡献．而且，长白山白眉蝮蛇蛇毒4种酶的Tyr和Trp残基所处的微环境并不相同.

Secondary structural analysis of the mRNA regions encoding the hemagglutinin cleavage site basic amino acids of the avian influenza virus H5N1 subtype samples

Here we report the codon bias and the mRNA secondary structural features of the hemagglutinin (HA) cleavage site basic amino acid regions of avian influenza virus H5N1 subtypes. We have developed a dynamic extended folding strategy to predict RNA secondar

Effect of amino acids on cryopreservation of cynomolgus monkey (Macaca fascicularis) sperm

The effects of three amino acids (proline, glutamine, and glycine) added to the freezing medium Tes-Tris-egg yolk (TTE) for cryopreservation of cynomolgus monkey (Macaca fascicularis) spermatozoa were studied. This is the first report on the effects of amino acids on nonhuman primate sperm cryopreservation. The addition of 5 mM proline, 10 mM glutamine, and 10 or 20 mM glycine each significantly improved post-thaw sperm motility and membrane and acrosome integrity compared with the control (TTE alone). However, a significant decrease in motility and membrane/acrosome integrity was observed when amino acid concentrations increased to 60 mM for proline and glutamine, and 80 mM for glycine. The results suggest that adding a limited amount of amino acids to the freezing media is beneficial for freezing cynomolgus monkey sperm.