Degradation of luteinizing hormone-releasing hormone by serum and plasma in vitro

Luteinizing hormone-releasing hormone (LH-RH) is degraded in vitro by serum and plasma from several species (human, rat, guinea-pig and cattle). Separation of the degradation products by high-performance liquid chromatography (HPLC) followed by amino acid analysis and radioimmunoassay showed that the main sites of cleavage are the Trp₃-Ser₄ and Tyr₅-Gly₆ bonds. Two peptidases are responsible since the cleavage at Trp₃-Ser₄ can be selectively inhibited by EDTA. In human plasma, the peptidase responsible for Trp₃-Ser₄ hydrolyssishas a K_m of 2.9 · 10^?4 M and V of 30 nmol/h per ml plasma. The half-life in vitro of LH-RH in serum and plasma from various species ranges from 3 h (guinea-pig) to 9.8 h (human). The peptidase cleaving LH-RH at Tyr₅-Gly₆ is present as an impurity in some commercial bovine serum and plasma albumins. Such contamination may have important practical implications for work involving peptide assays where albumins are used as carrier proteins.     

Interaction of synthetic Alzheimerβ-protein-derived analogs with aqueous aluminum: A low-field27Al NMR investigation

Synthetic peptides corresponding to the soluble Alzheimer-protein, i.e., 1–40 and 6-25, were utilized to investigate the association of aluminum using low-field²⁷Al nuclear magnetic resonance (NMR) spectroscopy and reversed-phase high-performance liquid chromatography (RP-HPLC). Addition of 1-40 or 6-25 to aqueous Al³⁺ gives rise to a²⁷Al NMR signal corresponding to the association of Al³⁺ with the peptides; this effect is not easily reversed by EDTA. Based on the relative intensity of the Al³⁺-peptide signal between pH 4 and 6, there are at least 4 Al³⁺ ions associated with each peptide molecule. Microheterogeneity is observed with RP-HPLC on incubating solutions of Al³⁺ with 1-40 and 6-25. The²⁷Al NMR spectra of chromatographically pure fractions of 1-40 and 6-25 indicate that the peptide-associated Al³⁺ is released below pH 3.5. We propose that soluble 1-40 provides an anchor for Al³⁺ to bind, eventually leading to an increased deposition of amyloid in the Alzheimer brain.     

Differential mutagenicity of reaction products of various pyrazolones with nitrite

Four pyrazolones in frequent use, i.e. antipyrine (AP), aminopyrine (AMP), sulpyrine (SP) and isopropylantipyrine (IPA), were compared for their reactivity with nitrite and for the in vitro mutagenicity of their reaction products by Ames' reversion test. In various acidic solutions at 37°C, AP, AMP and SP were found to react easily with nitrite and yield various products including dimethylnitrosamine (DMNA) and 4-nitrosoantipyrine (4-NAP) in the cases of AMP and AP, respectively. When tested with Salmonella typhimurium TA100 and TA98 after lyophilization, the reaction products of AP (AP-N) were found to be mutagenic in both strains, while products of AMP and SP (AMP-N and SP-N) were mutagenic only in TA100. The presence of unknown ultimate mutagens, other than DMNA and 4-NAP, were evidenced in AP-N, AMP-N and SP-N. Incubation with S-9 mixture did not affect the mutagenicity of SP-N and decreased that of AP-N and AMP-N.In clear contrast to AP, AMP and SP, it was found that IPA remained essentially intact upon reaction with nitrite. No mutagenicity was detected with the reaction mixture (IPA-N) in either strain.     

Reversed-phase high-performance liquid chromatography of chlorophylls and carotenoids

Reversed-phase high-performance liquid chromatography with octadecyl- or octylsilylated silica gel as the stationary phase provides a powerful tool in the analysis of chloroplast pigments from higher plants and green algae. Chromatographic columns packed with 10 μm chemically bonded silica gel particles allow the simultaneous separation of chlorophylls a and b, chlorophyll isomers, pheophytins a and b, α-carotene, β-carotene, lutein, violaxanthin, lutein-5,6-epoxide, antheraxanthin, neoxanthin and several minor carotenoids from a single sample within a short analysis time. The quantitative analysis requires a minimum of 1–5 pmol for carotenoids and 5–10 pmol for chlorophylls. Pigment degradation products, formed on polar stationary phases, are not found in reversed-phase high-performance liquid chromatography due to the weak hydrophobic forces on which the separation mechanism is based. The production of altered pigments however, either induced by various treatments or generated during the isolation, can be monitored as the reversed-phase system is selective enough to separate cis-isomers and oxidation products from their parent compounds. The reproducibility of the individual retention time for each pigment is better than ±1.5% which facilitates the identification of unknown pigments. The method is applied to the analysis of the pigment composition of Chlorella fusca, spinach (Spinacia oleracea) chloroplasts, and to the rapid determination of the ratio of chlorophyll a to chlorophyll b.     

不同贮藏条件对沙田柚果实苦味物质含量的影响

沙田柚原产广西,营养丰富且适合高血压和糖尿病等患者食用,近年来在沙田柚产业中存在品质下降、有苦味、异味生成等问题。为了研究不同贮藏条件下沙田柚苦味物质的含量变化,该文利用高效液相色谱仪,对室温包薄膜袋(A)、室温不包薄膜袋(B)和4℃低温不包薄膜袋(C)三种不同贮藏条件下,沙田柚外果皮、中果皮、囊衣、汁胞和种子中柚皮苷、柠檬苦素和诺米林在贮藏过程中的含量变化进行了测定。结果表明：沙田柚果实苦味物质以柚皮苷为主,主要分布在中果皮和囊衣中,柠檬苦素主要分布在种子和外果皮中,诺米林主要分布在种子中。各处理果实在贮藏过程中苦味物质含量变化不同,其中柚皮苷含量略有上升,柠檬苦素和诺米林含量整体呈下降趋势,略有波动,而汁胞中柠檬苦素和诺米林含量先上升再下降。三种不同贮藏条件相比,4℃低温不包薄膜袋贮藏的果实汁胞中柚皮苷和柠檬苦素的含量最高,在贮藏第30天,A、B、C 三种不同贮藏条件果实汁胞中柚皮苷含量分别为(0.139±0.006)、(0.190±0.009)和(0.194±0.019) mg?g-1,柠檬苦素含量分别为(47.28±1.91)、(33.64±1.90)和(84.19±5.56)μg?g-1。与此相反,外果皮、中果皮、囊衣和种子中柚皮苷、柠檬苦素和诺米林含量最低。该研究结果为采后沙田柚贮藏保鲜方式的选择提供了理论指导。     

Colorimetric detection of bisphenol A based on unmodified aptamer and cationic polymer aggregated gold nanoparticles

In this study, a colorimetric method was exploited to detect bisphenol A (BPA) based on BPA-specific aptamer and cationic polymer-induced aggregation of gold nanoparticles (AuNPs). The principle of this assay is very classical. The aggregation of AuNPs was induced by the concentration of cationic polymer, which is controlled by specific recognition of aptamer with BPA and the reaction of aptamer and cationic polymer forming “duplex” structure. This method enables colorimetric detection of BPA with selectivity and a detection limit of 1.50 nM. In addition, this colorimetric method was successfully used to determine spiked BPA in tap water and river water samples.     

Structural characterization of a rhamnose-binding glycoprotein (lectin) from Spanish mackerel (Scomberomorous niphonius) eggs

A rhamnose-binding glycoprotein (lectin), named SML, was isolated from the eggs of Spanish mackerel (Scomberomorous niphonius) by affinity and ion-exchange chromatographies. SML was composed of a non-covalently linked homodimer. The SML subunit was composed of 201 amino acid residues with two tandemly repeated domains, and contained 8 half-Cys residues in each domain, which is highly homologous to the N-terminal lectin domain of calcium-independent α-latrotoxin receptor in mammalian brains. Each domain has the same disulfide bonding pattern; Cys10–Cys40, Cys20–Cys99, Cys54–Cys86 and Cys67–Cys73 were located in the N-terminal domain, and Cys108–Cys138, Cys117–Cys195, Cys152–Cys182 and Cys163–Cys169 were in the C-terminal domain. SML was N-glycosylated at Asn168 in the C-terminal domain. The structure of the sugar chain was determined to be NeuAc-Galβ1-4GlcNAcβ1-2Manα1-6-(NeuAc-Galβ1-4GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc-Asn.     

Toward protein structure in situ: comparison of two bifunctional rhodamine adducts of troponin C

As part of a program to develop methods for determining protein structure in situ, sTnC was labeled with a bifunctional rhodamine (BR or BSR), cross-linking residues 56 and 63 of its C-helix. NMR spectroscopy of the N-terminal domain of BSR-labeled sTnC in complex with Ca(2+) and the troponin I switch peptide (residues 115-131) showed that BSR labeling does not significantly affect the secondary structure of the protein or its dynamics in solution. BR-labeling was previously shown to have no effect on the solution structure of this complex. Isometric force generation in isolated demembranated fibers from rabbit psoas muscle into which BR- or BSR-labeled sTnC had been exchanged showed reduced Ca(2+)-sensitivity, and this effect was larger with the BSR label. The orientation of rhodamine dipoles with respect to the fiber axis was determined by polarized fluorescence. The mean orientations of the BR and BSR dipoles were almost identical in relaxed muscle, suggesting that both probes accurately report the orientation of the C-helix to which they are attached. The BSR dipole had smaller orientational dispersion, consistent with less flexible linkers between the rhodamine dipole and cysteine-reactive groups.     

Tryptophan 334 oxidation in bovine cytochrome c oxidase subunit I involves free radical migration

A single tryptophan (W(334(I))) within the mitochondrial-encoded core subunits of cytochrome c oxidase (CcO) is selectively oxidized when hydrogen peroxide reacts with the binuclear center. W(334(I)) is converted to hydroxytryptophan as identified by reversed-phase HPLC-electrospray ionization tandem mass spectrometry analysis of peptides derived from the three SDS-PAGE purified subunits. Total sequence coverage of subunits I, II and III was limited to 84%, 66% and 54%, respectively. W(334(I)) is located on the surface of CcO at the membrane interface. Two other surface tryptophans within nuclear-encoded subunits, W(48(IV)) and W(19(VIIc)), are also oxidized when hydrogen peroxide reacts with the binuclear center (Musatov et al. (2004) Biochemistry 43, 1003-1009). Two aromatic-rich networks of amino acids were identified that link the binuclear center to the three oxidized tryptophans. We propose the following mechanism to explain these results. Electron transfer through the aromatic networks moves the free radicals generated at the binuclear center to the surface-exposed tryptophans, where they produce hydroxytryptophan.     

Isolation and characterization of oxygen-evolving thylakoid membranes and Photosystem II particles from a marine diatom Chaetoceros gracilis

Thylakoid membranes retaining high oxygen-evolving activity (about 250 μmol O₂/mg Chl/h) were prepared from a marine centric diatom, Chaetoceros gracilis, after disruption of the cells by freeze-thawing. We also succeeded in purification of Photosystem II (PSII) particles by differential centrifugation of the thylakoid membranes after treatment with 1% Triton X-100. The diatom PSII particles showed an oxygen-evolving activity of 850 and 1045 μmol O₂/mg Chl/h in the absence and presence of CaCl₂, respectively. The PSII particles contained fucoxanthin chlorophyll a/c-binding proteins in addition to main intrinsic proteins of CP47, CP43, D2, D1, cytochrome b559, and the antenna size was estimated to be 229 Chl a per 2 molecules of pheophytin. Five extrinsic proteins were stoichiometrically released from the diatom PSII particles by alkaline Tris-treatment. Among these five extrinsic proteins, four proteins were red algal-type extrinsic proteins, namely, PsbO, PsbQ', PsbV and PsbU, whereas the other one was a novel, hypothetical protein. This is the first report on isolation and characterization of diatom PSII particles that are highly active in oxygen evolution and retain the full set of extrinsic proteins including an unknown protein.