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1.
Numerous investigations have been carried out on the spectral distribution of the light of different species of fireflies. Here we record the emission spectrum of the Indian species of the firefly Luciola praeusta Kiesenwetter 1874 (Coleoptera : Lampyridae : Luciolinae) on a color film. Green and red color-sectors, with an intense yellow one in between, appear in this spectrum. Intensity profile of this spectrum reveals a hitherto undetected strong narrow yellow line, which lies within the full-width-at-half maximum (FWHM) of the intensity profile. The spectrum recorded in a high-resolution spectrometer confirms the presence of this sharp intense line. This finding lends support to an earlier drawn analogy between the in vivo emission of the firefly and laser light. 相似文献
2.
Ultrasensitive bioluminescence immunoassays for the determination of peptides and proteins (illustrated with human urinary kallikrein, bradykinin and the determination of human urinary kallikrein antibody titres) have been developed. The usable ranges of the standard curves are from 5 pg to 5000 pg per litre. The relative intra-assay coefficients of variation of the tests were between 2% and 6%, and the inter-assay coefficients of variation between 4% and 12%. 相似文献
3.
A modified purification method for bacterial luciferases and NAD(P)H:FMN oxidoreductases is described which uses FMN-Sepharose alone or coupled to DEAE ion exchange chromatography for the simultaneous purification of luciferase and the various oxidoreductases from Vibrio harveyi, a bright mutant of Vibrio harveyi, Vibrio fischeri, and Photobacterium phosphoreum. This purification method is compared with DEAE-Sepharose CI 6B fractionations from these organisms. Both methods allow the separation of oxidoreductases specific for either NADH or NADPH. The use of FMN-Sepharose coupled to DEAE-Sepharose fractionation allows the isolation of highly purified enzymes. Lacking interfering factors, these are very suitable for various analytical applications based on bacterial bioluminescence enzymes. The partially purified enzymes from the affinity column have higher specific activities than those obtained using DEAE-Sepharose. 相似文献
4.
Osamu Shimomura 《Luminescence》1995,10(2):91-101
Bioluminescence of euphausiids takes place when a fluorescent tetrapyrrole F and a highly unstable protein P react in the presence of oxygen. A previous study on the euphausiid Meganyctiphanes norvegica indicated that F acts as a catalyst and P is consumed in the luminescence reaction, differing from the luminescence system of dinoflagellates in which a tetrapyrrole luciferin, nearly identical to F, is enzymatically oxidized in the presence of dinoflagellate luciferase. In the present study, P was extracted from Euphausia pacifica as well as from M. norvegica, then purified separately by affinity chromatography on a column of biliverdin–Sepharose 4B, completing the whole process in less than 5h. The samples of P obtained from both species had a molecular weight of 600,000, a purity of about 80%, and a specific activity 50–100 times greater than that previously found. The activity of P rapidly decreased in solutions, even at 0°C, and the inactivation of P derived from M. norvegica was more than four times faster than that derived from E. pacifica. The kinetics of the luminescence reaction was investigated with F and P whose concentrations were systematically varied. The reaction was characteristically slow and involved two different reaction rates; the turnover number at 0°C was 30/h for the initial 20 min and 20/h after the initial 1 h. The total light emitted in a 50-h period indicated that the bioluminescence quantum yield of F was about 0.6 at 0°C, and P recycled many times in the luminescence reaction. Thus, the present results conclusively show that F is a luciferin and P is a luciferase of an unusually slow-working type, contrary to early report. 相似文献
5.
The use of 6-(N-acetyl-L -phenylalanyl)-aminoluciferin as a novel substrate for α-chymotrypsin has been demonstrated. The kinetic parameters determined are KM = 0.38mmol/L, kcat = 6.5 s?1 and kcat/kM = 17,100 (L/mols). The test principle of the coupled assay is the release of aminoluciferin by enzymatic cleavage of 6-(N-acetyl-L -phenylalanyl)-aminoluciferin. Aminoluciferin is oxidized, with light emission, by firefly luciferase (Photinus pyralis) and can be quantified in a luminometric assay. The detection limit for chymotrypsin was found to be 0.3 ng per assay. 6-(N-acetyl-L -phenylalanyl)-aminoluciferin has been synthesized as an example for a new class of highly sensitive substrates. By modification of the peptide residue these new substrates may be suitable for ultrasensitive detection of different proteinases. 相似文献
6.
固定化虫荧光素酶光纤传感器 总被引:1,自引:0,他引:1
固定化虫荧光素酶光纤传感器蔡谨,王顺光,杨歧生,吉鑫松(浙江大学化工系生化教研室,杭州310027;中国科学院上海生物化学研究所,200031)关键词虫荧光素酶,ATP,固定化酶,光纤生物传感器ATP是生物体内极为重要的能量物质。如何准确快速地定量A... 相似文献
7.
Abstract The firefly luciferase gene, luc , was demonstrated to hold promise as a specific marker for monitoring of genetically modified bacteria in the environment. PCR amplification and bioluminescence procedures were modified and compared for environmental monitoring of luc -tagged bacteria, using Escherichia coli as a model. The methods were used to track luc -tagged bacterial cells added to intact sediment core microcosms. Detection limits for the luc -tagged cells were the following, expressed as cells per 0.5 g of sediment: 102 , by PCR amplification; 103 , by whole cell luminescence; and 103 −104 , by measurement of luminescence in cell extracts. 相似文献
8.
Common structural features of the luxF protein and the subunits of bacterial luciferase: evidence for a (beta alpha)8 fold in luciferase.
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S. A. Moore M. N. James 《Protein science : a publication of the Protein Society》1994,3(11):1914-1926
The amino acid sequence identity and potential structural similarity between the subunits of bacterial luciferase and the recently determined structure of the luxF molecule are examined. The unique beta/alpha barrel fold found in luxF appears to be conserved in part in the luciferase subunits. From secondary structural predictions of both luciferase subunits, and from structural comparisons between the protein product of the luxF gene, NFP, and glycolate oxidase, we propose that it is feasible for both luciferase subunits to adopt a (beta alpha)8 barrel fold with at least 2 excursions from the (beta alpha)8 topology. Amino acids conserved between NFP and the luciferase subunits cluster together in 3 distinct "pockets" of NFP, which are located at hydrophobic interfaces between the beta-strands and alpha-helices. Several tight turns joining the C-termini of beta-strands and the N-termini of alpha-helices are found as key components of these conserved regions. Helix start and end points are easily demarcated in the luciferase subunit protein sequences; the N-cap residues are the most strongly conserved structural features. A partial model of the luciferase beta subunit from Photobacterium leiognathi has been built based on our crystallographically determined structure of luxF at 1.6 A resolution. 相似文献
9.
A novel method is described for the on-line determination of viable cell number. It has been tested in fermentations of Escherichia coli. The cells are transfected with the gene for firefly luciferase and fed low levels of luciferin in the medium. The reaction requires ATP, so the nonviable cells cannot produce light. Thus, light production is linear with viable cell density from innoculation through most of exponential growth. The light emitted by these cells is then conducted from the reaction vessel to the light detection equipment by an optical fiber. With the equipment described below, as few as a 10(6) cells/mL, or an OD(600) of 0.004, are easily detectable and concentrations greater than 10(10) cells/mL are well within range. The data are collected by a computer, so adaptation to on-line control applications is straightforward. During lag phase, this method is much more accurate then optical density measurements. At the end of exponential growth, rapid changes in light production mark carbon source depletion and the onset of cell lysis. A simple model accounts for the luciferin used during the fermentation and corrects the light detected to the proper cell density. (c) 1993 John Wiley & Sons, Inc. 相似文献
10.
《Journal of molecular biology》2023,435(13):168129
Mitochondrial protein import is critical for organelle biogenesis, bioenergetic function, and health. The mechanism of which is poorly understood, particularly of the mammalian system. To address this problem we have established an assay to quantitatively monitor mitochondrial import inside mammalian cells. The reporter is based on a split luciferase, whereby the large fragment is segregated in the mitochondrial matrix and the small complementary fragment is fused to the C-terminus of a purified recombinant precursor protein destined for import. Following import the complementary fragments combine to form an active luciferase–providing a sensitive, accurate and continuous measure of protein import. This advance allows detailed mechanistic examination of the transport process in live cells, including the analysis of import breakdown associated with disease, and high-throughput drug screening. Furthermore, the set-up has the potential to be adapted for the analysis of alternative protein transport systems within different cell types, and multicellular model organisms. 相似文献