Reaction mechanism of surfactant‐sensitized chemiluminescence of bis(2,4,6‐trichlorophyenyl) oxalate and hydrogen peroxide induced by gold nanoparticles

The chemiluminescence (CL) of bis(2,4,6‐trichlorophyenyl) oxalate with hydrogen peroxide in the present of cationic surfactant and gold nanoparticles was studied. The CL emission was obviously enhanced in the presence of surfactant at a suitable concentration, with a synergetic catalysis effect exhibited. Different sizes of gold nanoparticles (15 and 50 nm) showed different effects on CL intensity. Mechanisms of the CL reaction and sensitization effect are discussed. Copyright © 2008 John Wiley & Sons, Ltd.     

Determination of immunoreactive endothelin in medium from cultured endothelial cells and human plasma

We have developed a sensitive and selective radioimmunoassay for porcine/human endothelin (ET1). The assay has a detection limit of 0.62 pg/tube and exhibits no cross-reactivity to atrial natriuretic peptide, arginine vasopressin, or angiotensin II. Procedures were developed for extraction of endothelin from human plasma samples and samples of buffer from endothelial cell incubations using C18 Sep-Pak extraction cartridges. The mean recovery following extraction was approximately 80%. Both bovine and porcine aortic endothelial cells were found to produce immunoreactive endothelin (IR-ET) with porcine cells producing 4.7 +/- 1.1 ng of IR-ET/mg cell protein after 6 hours. Human plasma samples were extracted, assayed and found to contain a mean concentration of 2.0 +/- 0.4 pg/ml of IR-ET.     

Enzymes of vitamin B6 degradation. Purification and properties of two N-acetylamidohydrolases

alpha-(N-Acetylaminomethylene)succinic acid hydrolase (Compound A hydrolase, EC 3.5.1-) and alpha-hydroxymethyl-alpha'-(N-acetylaminomethylene)succinic acid hydrolase (Compound B hydrolase, EC 3.5.1-) were purified to homogeneity from Pseudomonas MA-1 and Arthrobacter Cr-7, respectively. The two inducible enzymes catalyze Reactions 1 and 2, respectively, which release the first generally useful anabolic intermediates during growth of these organisms with (formula; see text) pyridoxine as a sole source of carbon and nitrogen. Compound A hydrolase is a monomeric protein of Mr 32,500 with aspartic acid as its NH2-terminal residue. Compound B hydrolase (Mr congruent to 205,000) is a multimer containing probably six identical subunits with glycine as the NH2 terminus. The two enzymes have quite different amino acid analyses, although both are high in Asx and Glx, lack tryptophan, and show similar stabilities to pH and temperature. Compound A hydrolase has a pI of 4.4, a Km of 3.3 microM, and a Vmax of 3.1 mumol X min-1 X mg-1 at pH 6.5 and 25 degrees C; no analogue substrates were found. Compound B hydrolase has a pI of 4.2, a Km of 25 microM, and a Vmax of 3.8 mumol X min-1 X mg-1 at 25 degrees C and pH 7.0; it also hydrolyzes Compound A slowly. Both enzymes are inhibited competitively by di- and tricarboxylic acids, itaconic acid being among the most effective. Sulfite inhibits both enzymes by a time-dependent mechanism not yet understood. The two amidases appear to differ greatly in architecture despite the similarity in properties and in the overall reactions they catalyze.     

Novel extracellular enzymes (ligninases) of Phanerochaete chrysosporium

Abstract 3 New spectrophotometric enzyme assays were developed for the study of microbial lignin-degrading enzymes. The conversion of 2-methoxy-3-phenylbenzoic acid to 2-hydroxy-3-phenylbenzoic acid led to the discovery of an extracellular, aromatic methyl ether demethylase produced by the white-rot fungus Phanerochaete chrysosporium . The conversion of methyl 2-hydroxy-3-phenylbenzoate to 2-hydroxy-3-phenylbenzoic acid allowed the identification of an extracellular, aromatic methyl ester esterase produced by this fungus. The Phanerochaete sp. also excreted an enzyme complex that oxidized 4-(4-hydroxy-3-methoxyphenyl)-3-buten-2-one, probably to aliphatic products. All 3 novel enzyme activities were produced together with, and probably comprise a part of, the Phanerochaete ligninolytic enzyme complex. Unlike previously known ligninases, these enzymes did not oxidize 3,4-dimethoxybenzyl alcohol. All 3 were H₂O₂-dependent and were activated by Mn²⁺ ions.     

Histidine decarboxylase of Lactobacillus 30a. Sequences of the overlapping peptides, the complete alpha chain, and prohistidine decarboxylase

The complete amino acid sequence of the alpha chain of histidine decarboxylase of Lactobacillus 30a has been established by isolation and analysis of the eight methionine-containing tryptic peptides of this chain. These peptides provide the overlaps required to order all nine peptides derived by complete cyanogen bromide cleavage of the alpha chain (Huynh, Q.K., Vaaler, G.L., Recsei, P.A., and Snell, E.E. (1984) J. Biol. Chem. 259, 2826-2832). Ordering of six of the latter peptides was confirmed by isolation and analysis of four peptides derived by incomplete cyanogen bromide cleavage. The alpha chain is composed of 226 residues and has a molecular weight of 24,892 calculated from the sequence. These results and the previously determined sequence of the beta chain (Vaaler, G.L., Recsei, P.A., Fox, J.L., and Snell, E.E. (1982) J. Biol. Chem. 257, 12770-12774) establish the complete amino acid sequence of the enzyme and of the pi chain of prohistidine decarboxylase. The latter is composed of 307 amino acids and has a calculated molecular weight of 33,731. Four segments of the pi chain sequence are repeated. The bond between Ser-81 and Ser-82 that is cleaved during proenzyme activation is in an uncharged portion of the sequence that is rich in serine and threonine residues and is predicted to be part of a beta sheet structure.     

类脂过氧化对竹红菌甲素引起膜蛋白光敏交联的影响

本文研究了丙二醛与红细胞膜温育后所形成的交联。当丙二醛浓度在5×10~-4mol/L以上时,膜蛋白发生交联,并且在460nm处有荧光特征峰出现。而甲素光敏所致膜蛋白的交联,在460nm处没有荧光峰,光敏所产生的内源丙二醛量很少,不足以引起红细胞膜反应形成交联。我们还研究了BHT和Vit E两种抗氧化剂对甲素光敏作用的影响。BHT能抑制类脂过氧化,不能抑制膜蛋白巯基的变化和膜蛋白的交联。而Vit E仅不能抑制膜蛋白的交联。以上结果均说明甲素光敏所致膜类脂过氧化是不参与膜蛋白交联的。     

EPR and M?ssbauer studies of protocatechuate 4,5-dioxygenase. Characterization of a new Fe2+ environment

Protocatechuate 4,5-dioxygenase from Pseudomonas testosteroni has been purified to homogeneity and crystallized. The iron containing, extradiol dioxygenase is shown to be composed of two subunit types (alpha, Mr = 17,700 and beta, Mr = 33,800) in a 1:1 ratio; such a composition has not been observed for other extradiol dioxygenases. The 4.2 K M?ssbauer spectrum of native protocatechuate 4,5-dioxygenase prepared from cells grown in 57Fe-enriched media consists of a doublet with quadrupole splitting, delta EQ = 2.22 mm/s, and isomer shift delta Fe = 1.28 mm/s, demonstrating a high spin Fe2+ site. These parameters, and the temperature dependence of delta EQ, are unique among enzymes but are strikingly similar to those reported for the reaction center of the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26, suggesting very similar ligand environments. The Fe2+ of protocatechuate 4,5-dioxygenase can be oxidized, for instance by H2O2, to yield high spin Fe3+ with EPR g values around g = 6 (and g = 4.3). In the oxidized state, protocatechuate 4,5-dioxygenase is inactive; the iron, however, can be rereduced by ascorbate to yield active enzyme. Our data suggest that protocatechuate binds to Fe2+; the spectra indicate that the ligand binding is heterogenous. The M?ssbauer spectra observed here are fundamentally different from those reported earlier (Zabinski, R., Münck, E., Champion, P., and Wood, J. M. (1972) Biochemistry 11, 3212-3219). The spectra of the earlier (reconstituted) preparations, which had substantially lower specific activities, probably reflect adventitiously bound Fe3+. We discuss here how adventitiously bound iron can be identified and removed. The Fe2+ which is present in native protocatechuate 4,5-dioxygenase and its complexes with substrates and inhibitors reacts quantitatively with nitric oxide to produce a species with electronic spin S = 3/2. The EPR and M?ssbauer spectra of these complexes compare favorably with EDTA . Fe(II) . NO. We have studied the latter complex extensively and have analyzed the M?ssbauer spectra with an S = 3/2 spin Hamiltonian. EPR spectra show that protocatechuate 4,5-dioxygenase-NO complexes with substrates or inhibitors are heterogeneous and consist of several well defined subspecies. The data show that NO, and presumably also O2, has access to the active site Fe2+ in the enzyme-substrate complex. The use of EPR-detectable NO complexes as a rapid and sensitive tool for the study of the EPR silent active site iron of extradiol dioxygenases is discussed.     

Studies of the ferredoxin from Thermus thermophilus

The soluble ferredoxin from Thermus thermophilus was examined by M?ssbauer and EPR spectroscopies and by reductive titrations. These studies demonstrate the presence of one 3Fe center, responsible for the characteristic g = 2.02 EPR signal in the oxidized protein, and one [4Fe-4S] center which is responsible for the rhombic EPR spectrum of the fully reduced protein. These assignments should replace those made by Ohnishi et al. (Ohnishi, T., Blum, H., Sato, S., Nakazawa, K., Hon-nami, K., and Oshima, T. (1980) J. Biol. Chem. 255, 345-348) prior to the discovery of the 3Fe clusters. The amino acid composition was determined and is discussed with reference to recent structural studies of 7Fe ferredoxins.     

Doxorubicin affects tau protein metabolism in human neuroblastoma cells

The microtubule associated protein called tau, found primarily in neurons, was detected in a human neuroblastoma cell line, LAN-5. Cells treated with retinoic acid (2.0×10^–5M) differentiate and acquire processes similar to neurons. Differentiated and logarithmically growing undifferentiated cells were exposed to varying doses of doxorubicin (an anthracycline chemotherapeutic antibiotic). While doxorubicin was lethal to many undifferentiated dividing cells, it was not as damaging to differentiated cells. After 2 to 4 days of doxorubicin treatment, the cells were harvested, the protein concentration determined and SDS-PAGE performed. Proteins were blotted onto nitrocellulose paper and immunostained with either a rabbit antiserum or mouse monoclonal antibody to tau. Undifferentiated LAN-5 cells treated with 4.0×10^–8M doxorubicin for 4 days and cells treated with 8.0×10^–8M doxorubicin for 2 days displayed a distinct lower band (just below the 50kd marker) that was either absent or very faint in untreated controls.Special issue dedicated to Dr. Paola S. Timiras.