摘要:

AbstractThe rate constants for [1O2] [MCLA]and [1O2][NaN3] were measured by quenching the near‐infrared emission (1Δg→3∑g) in steady state with MCLA and NaN3, respectively.1O2was constantly generated by energy transfer to O2from Ar laser‐excited Rose Bengal. The Stern—Volmer plots yielded the second‐order rate constants of 2.94 × 109M−1S−1and 3.83 × 108M−1S−1for quenching1O2with MCLA and NaN3in water at pH 5.4, respectively. The1O2+ MCLA reaction emitted light with maximum at 465 nm at pD 4.5 identical to

ISSN:0884-3996    

DOI:10.1002/bio.1170060203

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractA competitive chemiluminescent immunoassay for quantitation of muramyl tripeptide phosphatidyl‐ethanolamine (MTP‐PE) in plasma has been developed. The assay is based on the use of an acridinium ester‐labelled analogue of muramyl tripeptide and a rabbit antiserum. It includes an overnight incubation and a separation with a second antibody covalently coupled to paramagnetic particles. The sensitivity of detection is 0.012 nmol/l, the assay working range is 0.1‐5 nmol/l, and the inter‐assay CVs are ⩽ 10%. Using up to 6000‐fold sample dilutions, a wide working range (0.1‐30 000 nmol/l) is obtained. Rat plasma samples were collected during and one day after intravenous infusion of MTP‐PE. Following infusion, the concentrations in plasma declined multiphasically. Half‐life time was 0.37 h ± 0.03 (mean ± SD, alpha phase) and 1.76 h ± 0.08 (mean ± SD, beta phase), clearance and volume of distribution were 0.09 ± 0.02 l/h × kg (mean ± SD) and 0.06 ± 0.01 l/kg (mean ± SD) respectively. The use of an acridinium ester as a chemiluminescent (CL) label overcomes the problems associated with re

ISSN:0884-3996    

DOI:10.1002/bio.1170060204

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractSustained generation of reactive oxygen metabolites following respiratory burst activation in neutrophils is a result of continued replenishment of a pool of active NADPH‐oxidase. The sulphydryl‐modifying reagentN‐ethylmaleimide (NEM) has been shown to be without effect on the turnover of activated NADPH‐oxidase but to inhibit the replenishment of active oxidase molecules (Akardet al., 1988). NEM was thus used to determine the rate of deactivation of extracellularly and intracellularly generated chemiluminescence in human neutrophils. We have shown that deactivation is more rapid when activation leads to a release of oxygen metabolites (extracellular chemiluminescence) than when the metabolites are generated intracellularly. The results indicate that the rate of deactivation of NADPH‐oxidase is higher when the oxidase system is localized on the plasma membrane than when it is localized on the phagosomal

ISSN:0884-3996    

DOI:10.1002/bio.1170060205

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractExtraction of whole lobes of normal rat liver with dimethyl sulphoxide (DMSO) under N2gives extracts which contain 5—10 μmol/l·O−2(50‐100 nmol·O−2per 10 ml extract per 4 g liver; 1.25‐2.50 nmol·O−2per millilitre per gram liver). Evidence for ·O−2in the extracts is given by: (1) electron spin resonance signals (ESR), (2) differential pulse polarography (DPP), (3) chemiluminescence (CL), and (4) nitroblue tetrazolium reduction (NBT). All tests yield results identical with those obtained with authentic ·O−2. Extraction of ·O−2is enhanced by tetrabutyl ammonium ion, and is maximal at 1‐3 min. These results raise the possibility that substantial amounts of ·O−2are normally sequestered in prote

ISSN:0884-3996    

DOI:10.1002/bio.1170060206

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractThe reaction rate of ATP‐limited firefly luciferase‐catalysed reactions, is affected by the presence of detergents. Anionic detergents inhibit luciferase activity without causing significant enzyme inactivation during the reaction. Cationic detergents increase reaction rate several‐fold with a sharply defined optimum concentration of detergent for the effect. However, cationic detergents inactivate firefly luciferase during the reaction, resulting in a continuously decreasing reaction rate. Under such conditions, peak light intensity must be used as an indication of initial reaction rate. The inactivation rate increases with increasing detergent concentration. Non‐ionic and zwitterionic detergents increase reaction rate over a broad range of detergent concentrations. Enzyme stability during the reaction is not affected by non‐ionic detergents and only affected by zwitterionic detergents at high detergent concentration. Cyclodextrins, which can increase reaction rates of some chemiluminescent reactions, have little effect on firefly luciferase activity.Assays for ATP using firefly luciferase must be internally standardized by the constant addition technique in which a known amount of ATP is added to the test sample, since external calibration of such assays, by reference to a previously prepared standard curve, can lead to imprecision when detergents ar

ISSN:0884-3996    

DOI:10.1002/bio.1170060207

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

Abstract10‐Methyl‐acridinium‐9‐(N‐sulphonylcarboxamide) salts are prepared via acylation of sulphonamides with acridine‐9‐carboxylic acid chloride and subsequentN‐alkylation with methyl triflate. Substituents on the sulphonamide component were varied to show the effect of steric and electronic factors on the kinetics of light output. The lifetime of the chemiluminescence light output ranged from 1 to 50 seconds for the 15 compounds reported. The long‐term stability of the new compounds was superior to the phenyl e

ISSN:0884-3996    

DOI:10.1002/bio.1170060208

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractLuminol chemiluminescence was used to detect activation of the respiratory burst oxidase in bovine eosinophils and neutrophils. Extracellular and intracellular chemiluminescence were measured by supplementing the medium with horseradish peroxidase and catalase, respectively. Pure bovine eosinophils (>90%), maximally stimulated with 1 nmol/l phorbol 12‐myristate‐13‐acetate (PMA) showed ten times more extracellular luminol‐dependent chemiluminescence (CL) than maximally stimulated pure bovine neutrophils (>96%). Extracellular CL from eosinophils was preferably induced over intracellular CL by both PMA (27‐fold difference) and platelet‐activating factor (PAF) at 2 μmol/l (9‐fold difference), but not by calcium ionophore A23187 (15 μmol/l).Time course information was used in the following experiments to distinguish between the mode of action of various stimulants. A progressively longer lag period was observed in eosinophil suspensions treated with decreasing doses of PMA, whereas platelet‐activating factor induced a dose‐dependent increase in the maximum response with no change in time to peak CL. The time course of extracellular CL was almost identical to intracellular CL for all stimulants tested, providing no evidence to suggest that extracellular CL stems from a different enzyme system than intracellular CL.Eosinophils generated most extracellular CL when stimulated with PMA, whereas neutrophils were most efficiently stimulated with A23187, which induced intracellular CL in eosinophils as well as in neutrophils. This accords with the greater tendency of neutrophils to ingest and kill microorganisms, whereas eosinophils are armed to destroy large ex

ISSN:0884-3996    

DOI:10.1002/bio.1170060209

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractA sensitive method for the analysis of ATP and phosphocreatine (PCr) in single human skeletal muscle fibres is described. Muscle tissue was freeze‐dried and single fibres were dissected free with the aid of low‐power microscopy. The fibres were then extracted in trichloroacetic acid and neutralized with KHCO3. The assay is based on the continuous monitoring of light produced as a result of ATP degradation in the firefly luciferase reaction. PCr is measured as the amount of ATP formed in the creatine kinase reaction. The coefficient of variation was less than 4% for both ATP and PCr determination. The amount of tissue required for the assay is approximately 0.5 μg (dry weight). The assay showed good agreement with spectrophotometric and high‐performance liquid chromatographic (HPLC) measurements made upon extracts of whole muscle

ISSN:0884-3996    

DOI:10.1002/bio.1170060210

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

摘要:

AbstractBovine serum albumin (BSA) affects the amount of light obtained from bacterial luciferase by competing with luciferase for one of the luciferase substrates, the aldehyde. At low aldehyde concentrations BSA behaves as an inhibitor, but at high aldehyde concentrations BSA relieves substrate inhibition. BSA reversibly binds decanal with aKsi= 3.36 μmol/l, approximately half the affinity of luciferase for decanal (KM= 1.5 μmol/l). BSA also increased the rate of intermediate II dark decay. The data suggest that this involves a direct protein‐protein (BSA‐luciferase) intera

ISSN:0884-3996    

DOI:10.1002/bio.1170060211

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY

ISSN:0884-3996    

DOI:10.1002/bio.1170060212

出版商:John Wiley&Sons, Ltd.    

年代:1991

数据来源: WILEY