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101.
In nerve tissue the histochemical nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) reaction is considered a suitable marker for nitric oxide synthase (NOS) activity. We have previously shown that the NOS-specific inhibitorl-nitroarginine (l-NNA) can block NADPH-d staining in intermediolateral (IML) neurons of the rat spinal cord; such a reaction might serve as a control for the presence of a NOS-related catalytic activity, i.e.,l-NNA-dependent NO synthesis in these neurons. However,l-NNA inhibition of neuronal NADPH-d is inconsistent and is therefore disputed by others. This prompted us to reinvestigate the reaction conditions to provide a standardized protocol for inhibition experiments. In IML neurons of formaldehyde-fixed spinal cord tissue, inhibition of NADPH-d reaction was tested by preincubation of frozen sections with the flavin-binder diphenylene iodonium chloride (DPI, 10 M-1 mM) which blocked the NADPH-d reaction in a concentration-dependent way, suggesting an inverse relationship of inhibitor concentration and final reaction product generated. Preincubation with the NOS-specific inhibitorl-NNA in glycine-NaOH buffer (pH 8.5–9.5) but notl-nitroarginine methyl ester (l-NAME) revealed a concentration-dependent blocking effect on neuronal NADPH-d comparable to the effects seen with DPI, suggesting the existence of al-NNA sensitive NADPH-d activity. Blocking withl-NNA (100 M-10 mM) was prevented by excessl-arginine (10–100 mM), suggesting competitive binding sites. NADPH-d staining was not inhibited by 7-nitro indazole, another NOS inhibitor. Thus, in formaldehyde-fixed nervous tissue both DPI andl-NNA inhibit the NOS-associated catalytic NADPH-d activity, thereby preventing NADPH-dependent conversion of nitroblue tetrazolium to formazan.Presented in the Workshop Detection of NO-synthases at the XXXVI Symposium of the Society for Histochemistry on Oxy Radicals, 20–23 September 1994, Heidelberg, Germany 相似文献
102.
Yves Hurtubise Franois Shareck Dieter Kluepfel Rolf Morosoli 《Molecular microbiology》1995,17(2):367-377
Centre de Recherche en Microbiologie Appliquée, Institut Armand-Frappier, Université du Québec, Ville de Laval, Québec H7N 4Z3, Canada. 相似文献
103.
The specificity of thermitase (EC 3.4.21.14), a microbial thermostable serine proteinase fromThermoactinomyces vulgaris, with several oligo- and polypeptide substrates was investigated. Preferred hydrolysis of peptide bonds with a hydrophobic amino acid at the carboxylic site was observed. The proved carboxypeptidolytic splitting of Leu5-enkephalin and bradykinin, as well as the noncleavability of casomorphins by thermitase, can be explained by the position of the glycine and proline residues in these substrates. Major cleavage sites in the oxidized insulin B chain in a 15-min incubation with thermitase at Gln4-His5, Ser9-His10, Leu11-Val12, Leu15-Tyr16 and in the oxidized insulin A chain at Cys SO3H11-Ser12, Leu13-Tyr14, and Leu16-Glu17 were observed. Additional cleavages of the bonds His5-Leu6, Arg22-Gly23, Phe24-Phe25, Phe25-Tyr26, and Tyr26-Thr27 in the oxidized B chain and Cys SO3H6-Cys SO3H7 and Tyr19-Cys SO3H20 in the oxidized A chain in 2-h incubations with thermitase were also noted. Hydrolysis of salmine A I component in a 10-min incubation was observed mainly at four peptide bonds: Arg5-Ser6, Ser6-Ser7, Arg18-Val19, and Gly27-Gly28. The cleavage sites of thermitase in both insulin chains were similar to those reported in the studies of subtilisins. 相似文献
104.
Seven microbial peptide inhibitors—chymostatin, antipain, elastatinal, leupeptin, pepstatin, bestatin, and phosphoramidon—were tested for their efficiency to inhibit thermitase, a thermostable serine protease fromThermoactinomyces vulgaris. Chymostatin and antipain were the most effective inhibitors, with Ki values of 7×10–8
M and 2×10–7
M, respectively. Except for leupeptin, all inhibitors resist hydrolysis by thermitase. Leupeptin, however, is cleaved by thermitase between the two leucylresidues. Further, a close relationship in specificity between thermitase and subtilisin BPN and their distinct discrimination from elastase specificity was demonstrated by using these inhibitors. 相似文献
105.
Schönheit Peter Beimborn Dieter B. Perski Hans-Joachim 《Archives of microbiology》1984,140(2-3):247-251
Cultures of Methanobacterium thermoautotrophicum (Marburg) growing on media low in potassium accumulated the cation up to a maximal concentration gradient ([K+]intracellular/[K+]extracellular) of approximately 50,000-fold. Under these conditions, the membrane potential was determined by measuring the equilibrium distribution of the lipophilic cation (14C) tetraphenylphosphonium (TPP+). This cation was accumulated by the cells 350-to 1,000-fold corresponding to a membrane potential (inside negative) of 170–200 mV. The pH gradient, as measured by equilibrium distribution of the weak acid, benzoic acid, was found to be lower than 0.1 pH units (extracellular pH=6.8). The addition of valinomycin (0.5–1 nmol/mg cells) to the culture reduced the maximal concentration gradient of potassium from 50,000-to approximately 500-fold, without changing the membrane potential. After dissipation of the membrane potential by the addition of 12C-TTP+ (2 mol/mg cells) or tetrachlorosalicylanilide (3 nmol/mg cells), a rapid and complete efflux of potassium was observed.These data indicate that potassium accumulation in the absence of valinomycin is not in equilibrium with the membrane potential. It is concluded that at low extracellular K+ concentrations potassium is not accumulated by M. thermoautotrophicum via an electrogenic uniport mechanism.Non-common abbreviations TPP+
Tetra phenylphosphonium bromide
- DTE
Dithioerythritol
- TCS
3,5,3,4-Tetrachlorosalycylanilide 相似文献
106.
107.
The herbicides amiprophosmethyl (APM) trifluralin, and oryzalin as well as the fungicides methylbenzimidazolyl carbamate (MBC), O-isopropyl N-phenyl carbamate (IPC), and chlorisopropyl N-phenyl carbamate (CIPC), which are known to cause the destruction of microtubules in vivo but do not interfere with tubulin polymerization in vitro, have been examined with respect to their ability to affect Ca2+ transport in isolated cell organelles. In contrast to colchicine which has no effect on Ca2+ transport in isolated mitochondrial and microsomal fractions, all of the substances investigated caused considerable reduction of ca2+ net uptake into mitochondrial but not into microsomal fractions. This reduction has been shown to be due to an increase in passive Ca2+ efflux. These results have been extrapolated to in vivo situations where they are postulated to act by raising cytoplasmic Ca2+ levels.Abbreviations APM
amiprophosmethyl
- CIPC
chlorisopropyl N-phenyl carbamate
- IPC
O-isopropyl N-phenyl carbamate
- MBC
methylbenzimidazolyl carbamate
- Mops
3-(N-Morpholino) propanesulfonic acid
- DMSO
dimethylsulfoxide 相似文献
108.
Analysis of a drosophila tRNA gene cluster 总被引:23,自引:0,他引:23
109.
A study of the subcellular distribution of radioactivity in rat uterus after injection of labelled corticosterone showed that the radioactivity was observed in all fractions from 5 min. to 120 min. A maximum uptake was observed 10 min. after application of the labelled steroid. Competitive uptake of radioactive progesterone and unlabelled corticosterone was assayed 10 min. after injection of the hormone mixture. The ratio between radioactive progesterone and unlabelled corticosterone was 1 : 1 and 1 : 2 (moles:moles), respectively. Compared with control experiments with rats which had received radioactive progesterone alone, the results gave evidence that progesterone found in all subcellular fractions and in the total homogenate was not depressed by unlabelled corticosterone. However, unlabelled progesterone reduced the tritiated progesterone in uterine tissue. This observation demonstrates that the uptake of progesterone by rat uterus is specific. 相似文献
110.
Saccharomyces cerevisiae nuclear membranes were prepared from isolated nuclei by digesting chromatin with deoxyribonuclease and ribonuclease, washing of residual nuclei with 0.5 M MgCl2, and discontinuous gradient centrifugation in buffered Ficoll solutions. Electron microscopic examination of the preparations showed single membrane and double membrane vesicles and membrane sheets. Pores or residual pores were often visible. In double membrane profiles the two unit membranes were often separated by the remains of the perinuclear cistern. The nuclear membrane fragments contained 58% protein, 23.8% phospholipid, 6% sterols, 7.1% neutral acylglycerols, 4.8% RNA, and 0.3% DNA. The phospholipid content of the membrane preparations was influenced by a phospholipase activity with acidic pH optimum. 相似文献