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PubMed2005

Random mutagenesis of bacterial luciferase: critical role of Glu175 in the control of luminescence decay.

Bacterial luciferases (LuxAB) can be readily classed as slow or fast decay luciferases based on their rates of luminescence decay in a single turnover assay. Luciferases from Vibrio harveyi and Xenorhabdus (Photorhabdus) luminescens have slow decay rates, and those from the Photobacterium genus, such as Photobacterium fisheri, P. phosphoreum and P. leiognathi, have rapid decay rates. By substitution of a 67-amino-acid stretch of P. phosphoreum LuxA in the central region of the LuxA subunit, the 'slow' X. luminescens luciferase was converted into a chimaeric luciferase with a significantly more rapid decay rate [Valkova, Szittner and Meighen (1999) Biochemistry 38, 13820-13828]. To understand better the role of specific residues in the classification of luciferases as slow and fast decay, we have conducted random mutagenesis on this region. One of the mutants generated by a single mutation on LuxA at position 175 [E175G (Glu175-->Gly)] resulted in the 'slow decay' X. luminescens luciferase being converted into a luciferase with a significantly more rapid decay rate. These results indicate the importance of Glu175 in LuxA as a critical residue for differentiating between 'slow' and 'fast' luciferases and show that this distinction is primarily due to differences in aldehyde affinity and in the decomposition of the luciferase-flavin-oxygen intermediate.

Aldehydes↗

PubMed2003

Nitric oxide donors inhibit luciferase expression in a promoter-independent fashion.

Nitric oxide (NO) is an important molecule with diverse bio-messenger functions including regulation of gene expression. Transcriptional studies using sensitive luciferase reporter systems have suggested that NO inhibits the promoter activity of a variety of genes. Here we report that NO donors (sodium nitroprusside, 2',2'-(hydroxynitrosohydrazono)bis-ethanimine, and (+/-)-(E)-4-ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexen-1-yl-nicotinamide) decrease luciferase activity in a promoter-independent fashion in both viral and eukaryotic promoters, with a reduction to nearly 50% in the presence of 100 microm NO donor. Addition of an SV40 enhancer downstream of the luciferase coding region shifted NO donor inhibition to the right, with inhibition at approximately 300 microm. In contrast, when studied in a chloramphenicol acetyltransferase reporter, two promoters indicating inhibition by NO were unaffected. The decrease in luciferase activity was not caused by NO suppression of the luciferase enzyme. Real-time PCR data showed that luciferase mRNA half-life decreased by nearly half in the presence of NO donor (from 75 to 45 min). The SV40 enhancer prolonged luciferase mRNA half-life and somewhat blunted the NO effect. Our data suggest that exogenous NO inhibits luciferase activity in a dose-dependent manner through decreasing luciferase mRNA stability. Thus, the use of luciferase reporter systems to study transcriptional regulation by NO should be attempted with caution.

Animals↗

PubMed1991

Firefly luciferase as a marker for herpesvirus (pseudorabies virus) replication in vitro and in vivo.

Insertion of reporter genes into complex viral genomes and monitoring virus replication by detecting the corresponding protein products is increasingly used in pathogenesis studies. We present here the isolation and characterization of a recombinant neurotropic alpha-herpesvirus, pseudorabies virus (PrV), which stably carries the gene encoding firefly luciferase. To express the enzyme the complete open reading frame for luciferase was fused to the promoter and first seven codons of the non-essential glycoprotein gX gene of PrV. A recombinant PrV carrying the luciferase gene inserted into the gX gene and exhibiting strong luciferase activity after infection of cultured cells was further characterized. Kinetic analyses showed that luciferase activity was detectable as early as 90 min after infection. Luciferase expression could be monitored in cell extracts in a luminometer. For facilitating plaque isolation of luciferase recombinant viruses it was also visualized in situ on sensitive film. Kinetic experiments in mice proved the suitability of luciferase as an excellent marker for following herpesvirus spread in the animal. By way of luciferase detection we show that PrV invasion of the central nervous system after intranasal infection of mice occurred independently of replication in non-neural tissues such as lung or thymus. Furthermore, comparison of isogenic luciferase recombinant PrV strains carrying intact or deleted glycoprotein gI genes showed differences in the organotropism between these two viruses.

Animals↗

PubMed2004

cDNA cloning and characterization of a secreted luciferase from the luminous Japanese ostracod, Cypridina noctiluca.

A secreted luciferase from the marine ostracod, Vargula hilgendorfii, is a useful tool for gene expression assays in living mammalian cells. We have cloned the cDNA of a new secreted luciferase from the ostracod Cypridina noctiluca, which inhabits the coast of Japan. C. noctiluca luciferase consists of 553 amino acid residues with a molecular mass of 61,415 Da, as deduced from the nucleotide sequence. The homologies of nucleotide and amino acid sequences with V. hilgendorfii luciferase are 79.2% and 83.1%, respectively. C. noctiluca luciferase can expressed in and secreted from cultured mammalian cells. The characteristic properties of expressed C. noctiluca luciferase are similar to those of V. hilgendorfii luciferase. However, the activity of C. noctiluca luciferase in culture medium is much higher than that of V. hilgendorfii luciferase, suggesting that C. noctiluca luciferase is a highly potent reporter enzyme for real-time and continuous monitoring of gene expression in living cells.

Amino Acid Sequence↗

PubMed2003

Characteristics and application of established luciferase hepatoma cell line that responds to dioxin-like chemicals.

AIM: To establish a luciferase reporter cell line that responds dioxin-like chemicals (DLCs) and on this basis to evaluate its characteristics and application in the determination of DLCs. METHODS: A recombinant luciferase reporter plasmid was constructed by inserting dioxin-responsive element (DREs) and MMTV promoter segments into the pGL(3)-promoter plasmid immediately upstream of the luciferase gene, which was structurally demonstrated by fragment mapping analysis in gel electrophoresis and transfected into the human hepatoma cell line HepG(2), both transiently and stably, to identify the inducible expression of luciferase by 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD). The time course, responsive period, sensitivity, structure-inducibility and dose-effect relationships of inducible luciferase expression to DLCs was dynamically observed in HepG(2) cells stably transfected by the recombinant vector (HepG(2)-Luc) and compared with that assayed by ethoxyresorufin-O-deethylase (EROD) in non-transfected HepG(2) cells (HepG(2)-wt). RESULTS: The inducible luciferase expression of HepG(2)-Luc cells was noted in a time-, dose-, and AhR-dependent manner, which peaked at 4 h and then decreased to a stable level at 14 h after TCDD treatment. The responsiveness of HepG(2)-Luc cells to TCDD induction was decreased with culture time and became undetectable at 10th month of HepG(2)-Luc cell formation. The fact that luciferase activity induced by 3, 3', 4, 4'-PCB in HepG(2)-Luc cells was much less than that induced by TCDD suggests a structure-inducibility relationship existing among DLCs. Within the concentrations from 3.5 x 10(-12) to 5 x 10(-9) mol/L, significant correlations between TCDD doses and EROD activities were observed in both HepG(2)-luc and HepG(2)-wt cells. The correlation between TCDD doses from 1.1 x 10(-13) to 1 x 10(-8) mol/L and luciferase activities was also found to be significant in HepG(2)-luc cells (r=0.997, P<0.001), but not in their HepG(2)-wt counterparts. For the comparison of the enzyme responsiveness between cell lines to TCDD, the luciferase sensitivity and reproducibility in HepG(2)-luc cells were both better than that of EROD in HepG(2)-wt cells, the former was at 1.1 x 10(-13) mol/L and 3.5 x 10(-12) mol/L, and the coefficients of variation (CV) of the latter was 15-30 % and 22-38 %, respectively. CONCLUSION: The luciferase expression of HepG(2)-luc cells established in the present study could sensitively respond to the DLCs stimulation and might be a prospective tool for the determination of DLCs.

Carcinoma, Hepatocellular↗

PubMed2003

Hoechst 33342 alters luciferase gene expression in transfected BC3H-1 myocytes.

BACKGROUND: Hoechst 33342 and Hoechst 33258 bind to the minor groove of DNA. Hoechst 33342 induces apoptosis in a variety of cell types by a mechanism that is associated with disruption of the formation of the TATA box-binding protein/DNA complex. OBJECTIVE: To further investigate the role of Hoechst 33342 in gene regulation using BC3H-1 myocytes transfected with 4 different pGL3 luciferase reporter vectors constructed with or without the SV40 promoter and/or enhancer regions or with 2 synthetic Renilla luciferase vectors (phRL-null and phRL-TK). METHODS: Luciferase messenger RNA content was measured by reverse transcriptase-polymerase chain reaction, and luciferase activity was measured by luminometry. The ability of transcription factors in nuclei prepared from BC3H-1 myocytes to bind to a [32P]-labeled 24-base pair oligonucleotide containing the TATA box-binding element was determined by a gel mobility shift assay. RESULTS: In vivo, 4.4 and 8.9 microM of Hoechst 33342 (sublethal doses) increased luciferase enzyme activity in cells transfected with each of the 4 pGL3 luciferase reporter vectors and both of the Renilla luciferase vectors. Hoechst 33258 had no effect on luciferase enzyme activity. In vitro, Hoechst 33342 increased transcription factor binding to the 24-mer oligonucleotide containing the TATA box-binding element, which would be favorable to increased RNA polymerase II efficiency. CONCLUSION: Hoechst 33342 stimulates luciferase activity by a pathway that is independent of the integrity of the promoters in the luciferase gene expression vectors used (pGL3 basic, pGL3 control, pGL3 enhancer, and pGL3 promoter vectors, phRL-null, or phRL-TK).

Animals↗

PubMedDate not supplied

[The effect of Clp proteins on DnaK-dependent refolding of bacterial luciferases].

A study was made of the refolding of bacterial luciferases of Vibrio fischeri, V. harveyi, Photobacterium phosphoreum, and Photorhabdus luminescens. By reaction rate, luciferases were divided into two groups. The reaction rate constants of fast luciferases of V. fischeri and Ph. phosphoreum were about tenfold higher than those of slow luciferases of Ph. luminescens and V. harveyi. The order of increasing luciferase thermostability was Ph. phosphoreum, V. fischeri, V. harveyi, and Ph. luminescens. The refolding of thermoinactivated luciferases completely depended on the active DnaK-DnaJ-GrpE chaperone system. Thermolabile fast luciferases of V. fischeri and Ph. phosphoreum showed highly efficient rapid refolding. Slower and less efficient refolding was characteristic of thermostable slow luciferases of V. harveyi and Ph. luminescens. Chaperones of the Clp family were tested for effect on the efficiency of DnaK-dependent refolding of bacterial luciferases in Escherichia coli cells. The rate and extent of refolding were considerably lower in the clpB mutant than in wild-type cells. In E. coli cells with mutant clpA, clpP, of clpX showed a substantially lower luciferase refolding after heat shock.

Adenosine Triphosphatases↗

PubMed1998

Quantitative analysis of luciferase activity of viral and hybrid promoters in bovine preimplantation embryos.

This study was conducted to investigate quantitatively the luciferase activity of gene constructs with viral and hybrid enhancers and promoters in bovine preimplantation embryos by using firefly luciferase reporter genes. In Experiment I, to examine the stability of the luciferase, bioluminescence intensity of bovine embryos injected with the luciferase gene driven by the SV40 early promoter and enhancer (SVEluc) was measured with a luminometer at 2 days after microinjection. The results indicated that the bioluminescence could be analysed at any time within 30 min because the luciferase activity was constant during the measurement period from 5 to 30 min. In Experiment II, the luciferase expression of fertilized oocytes injected with four gene constructs (TKEluc, TK6WEluc, SVEluc, and Miwluc) was analysed by using a photon imaging system at 2 or 6 days following microinjection. The results from Experiment II indicated that the reporter gene governed by the Miw promoter (RSV LTR and chicken beta-actin promoter) was expressed more intensively in bovine morulae and blastocysts than three other gene constructs. In Experiment III, the effect of SV40 enhancer was investigated when fused downstream to the luciferase cDNA of the Miwluc vector. The results showed that SV40 enhancer further activated the luciferase activity of the Miw promoter in bovine preimplantation embryos. It was concluded, therefore, that the Miw promoter together with the SV40 enhancer would confer the strongest expression of the firefly luciferase reporter gene among the gene constructs tested in preimplantation bovine embryos.

Animals↗

PubMed2003

One-step measurement of firefly luciferase activity in yeast.

Firefly luciferase is often used as a sensitive genetic reporter in various cell types. The pitfall in yeast, however, has been the need to break down the rigid cells in order to measure the enzyme activity. In this study we have removed the peroxisomal targeting codons from the Photinus pyralis luciferase gene (luc) and shown that in the yeast Saccharomyces cerevisiae this modified luciferase gives high levels of light emission that is easy to measure from intact living cells. Furthermore, cells with the modified luciferase grew essentially faster than those with the wild-type luciferase, indicating that peroxisomal targeting of a foreign enzyme puts some constraints to cellular viability. As a model system we used two different reporter constructs. In the first, expression of the luciferase gene is under control of CUP1-promoter, a well known yeast promoter that is inducible by copper ions. In the second, luciferase activity is dependent on activation of the human oestrogen receptor and its interaction with oestrogen-responsive elements incorporated in a yeast promoter. The luciferase activity measurement could be done on a 96-well plate by simple addition of the substrate, D-luciferin, at a moderately acidic pH of 5.0. The ease of use of the non-peroxisomal luciferase makes it an interesting alternative for reporter genes that are conventionally used in yeast, such as lacZ.

Copper↗

PubMed1999

Relationship between the conserved alpha subunit arginine 107 and effects of phosphate on the activity and stability of Vibrio harveyi luciferase.

The Arg107 of the alpha subunit is a conserved residue for all known bacterial luciferases. The phosphate moiety of the reduced flavin mononucleotide (FMNH(2)) side chain has been hypothesized to be anchored at this site (A. J. Fisher, F. M. Raushel, T. O. Baldwin, and I. Rayment Biochemistry 34, 6581-6586, 1995). Mutations of alphaArg107 of the Vibrio harveyi luciferase to alanine, serine, and glutamate were carried out to test such a hypothesis. These variants were characterized and compared with the wild-type luciferase with respect to their K(m) for decanal, FMNH(2), and reduced riboflavin in both low- (0.01 or 0.05 M) and high- (0.3 M) phosphate buffers at pH 7.0. Results are consistent with the hypothesized binding of the FMNH(2) phosphate group by alphaArg107. Moreover, the alphaArg107 residue was apparently important in the expression of the luciferase maximal activity and aldehyde binding. Phosphate ion is also known to have other effects on luciferase stability. We compared the three luciferase variants with the native enzyme with respect to the decay rate of the FMN 4a-hydroperoxide intermediate II, and rates of inactivation by trypsin digestion, modification by N-ethylmaleimide, and heat treatment in low- and high-phosphate buffers. On the basis of patterns of the phosphate effects, alphaArg107 appeared to be important to the enhancement of luciferase stability against trypsin proteolysis at high phosphate but was not involved in regulating the intermediate II decay or sensitivity to N-ethylmaleimide modification. Differential effects of mutations on luciferase thermal stability were observed. It is uncertain whether alphaArg107 is involved in the enhanced thermal stability of the native luciferase in high phosphate buffer.

Alanine↗

PubMed2000

Use of a dual firefly and Renilla luciferase reporter gene assay to simultaneously determine drug selectivity at human corticotrophin releasing hormone 1 and 2 receptors.

The aim of this study was to investigate and validate the use of a dual glow-signal luciferase reporter gene assay to simultaneously evaluate drug activity at two different seven-transmembrane receptor subtypes. Stable cell lines (CHO) transfected with either human corticotrophin releasing hormone 1 (hCRH1) receptors and a firefly luciferase reporter gene or hCRH2 and a Renilla luciferase reporter gene were created to provide different luciferase readouts for CRH1 and CRH2 receptors, respectively. Cells were combined for stimulation and measurement of luciferase luminescence in a 96-well plate format assay. The nonselective CRH agonists rat/human CRH and sauvagine caused concentration-dependent increases in luminescence via activation of CRH1 (firefly luciferase; pEC50 = 8.40 +/- 0.06 and 8.39 +/- 0.08, respectively, n = 8) and CRH2 (Renilla luciferase; pEC50 = 8.89 +/- 0.14 and 8.92 +/- 0.13, respectively, n = 8) receptors. The nonselective CRH antagonist astressin blocked these agonist-induced increases in luciferase at both CRH1 and CRH2 receptors. The selective CRH1 antagonist CP154,526 blocked r/hCRH- and sauvagine-induced increases in luciferase at CRH1 receptors only. These data report the expected pharmacology for CRH1 and CRH2 receptors. This assay enabled two receptor subtypes to be studied simultaneously in the same 96-well plate and generated robust data with low variability. It has the potential advantage of significant time and cost savings, with application to both basic research and compound screening.

Animals↗

PubMed1998

Demonstration of the specificity of poliovirus encapsidation using a novel replicon which encodes enzymatically active firefly luciferase.

The specificity of poliovirus encapsidation has been studied using a novel chimeric genome in which the gene encoding firefly luciferase has been substituted for the VP2-VP3-VP1 genes of the poliovirus capsid (P1) gene. Transfection of RNA transcribed in vitro from this genome resulted in a VP4-luciferase fusion protein which retained luciferase enzyme activity. Since the detection of enzyme activity was dependent upon replication of the transfected RNA genome, we refer to these genomes as replicons. The replicon encoding luciferase was encapsidated upon transfection of the genomic RNA into cells previously infected with a recombinant vaccinia virus, VV-P1, which encodes the poliovirus type 1 capsid proteins (P1). Infection of cells with each serial passage, followed by analysis of luciferase enzyme activity, revealed that encapsidated replicons could be detected at the first passage with VV-P1. Amplification of the titer of encapsidated replicons occurred upon serial passage with VV-P1, as evidenced by the high expression levels of luciferase enzyme activity following infection. Serial passage of the luciferase replicons with poliovirus type 1, 2, or 3 resulted in the trans encapsidation into the type 1, 2, or 3 capsids, respectively. In contrast, serial passage with bovine enterovirus, Coxsackievirus A21 or B3, or enterovirus 70 did not result in trans encapsidation, even though co-infection of cells with the replicon and different enteroviruses resulted in high-level expression of luciferase. The results of this study highlight the specificity of poliovirus encapsidation and point to the use of encapsidated replicons encoding luciferase as a reagent for dissecting elements of replication and encapsidation.

Animals↗

PubMed1990

The in vivo pattern of firefly luciferase expression in transgenic plants.

Expression of the firefly luciferase gene in transgenic plants produces light emission patterns when the plants are supplied with luciferin. We explored whether in in vivo pattern of light emission truly reveals the pattern of luciferase gene expression or whether it reflects other parameters such as the availability of the substrate, luciferin, or the tissue-specific distribution of organelles in which luciferase was localized. The tissue-specific distribution of luciferase activity and the in vivo pattern of light were examined when the luciferase gene was driven by different promoters and when luciferase was was redirected from the peroxisome, where it is normally targeted, to the chloroplast compartment. It was found that the distribution of luciferase activity closely correlated with the tissue-specific pattern of luciferase mRNA. However, the in vivo light pattern appeared to reflect not only tissue-specific distribution of luciferase activity, but also the pattern of luciferin uptake.

Animals↗

PubMed1992

Use of bacterial and firefly luciferases as reporter genes in DEAE-dextran-mediated transfection of mammalian cells.

The aim of this study was to compare three different luciferase genes by placing them in a single reporter vector and expressing them in the same mammalian cell type. The luciferase genes investigated were the luc genes from the fireflies Photinus pyralis (PP) and Luciola mingrelica (LM) and the lux AB5 gene, a translational fusion of the two subunits of the bacterial luciferase from Vibrio harveyi (VH). The chloramphenicol acetyltransferase (CAT) gene was also included in this study for comparison. The performances of the assay methods of the corresponding enzymes were evaluated using reference materials and the results of the expressed enzymes following transfection were calculated using calibration curves. All of the bioluminescent assays possess high reproducibility both within and between the batches (less than 15%). The comparison of the assay methods shows that firefly luciferases have the highest detection sensitivity (0.05 and 0.08 amol for PP and LM, respectively) whereas the VH bacterial luciferase has 5 amol and CAT 100 amol. On the other hand, the transfection of the various plasmids shows that the content of the expressed enzyme within the cells is much higher for CAT than for the other luciferase genes. VH luciferase is expressed at very low levels in mammalian cells due to the relatively high temperature of growing of the mammalian cells that seems to impair the correct folding of the active enzyme. PP and LM luciferases are both expressed at picomolar level but usually 10 to 70 times less in content with respect to CAT within the transfected cells. On the basis of these results the overall improvement in sensitivity related to the use of firefly luciferases as reporter genes in mammalian cells is about 30 to 50 times with respect to that of CAT.

Animals↗

PubMed1998

Expressed luciferase viability assay (ELVA) for the measurement of cell growth and viability.

An expressed luciferase viability assay (ELVA) has been developed for cell viability and cell number based on detecting the expression of luciferase transfected into the cells. Stable transfectants were produced that expressed luciferase constitutively. Like many endogenous enzymes, luciferase is rapidly degraded following cell death, so that the enzyme can be used as a measure of cell viability. A modified luciferase assay was used in which the reagents were added directly to the cells in a microplate. The main advantages compared to other cell viability assays are the wide dynamic range, high sensitivity, low background, and the absence of any requirement to wash or harvest the cells. Stable transfectants of three factor-dependent cell lines (B13, Ba/F3 and CTLL) were produced and used in cytokine assays. Three strategies of selection after electroporation were tested: (1) using a plasmid containing both the genes encoding firefly luciferase and a selectable marker (neo), (2) cotransfection of a plasmid containing luciferase and a plasmid containing a selectable marker (puromycin resistance), and (3) cotransfection of a plasmid containing luciferase and a plasmid containing the human IL-5Ralpha-chain, and selecting in IL-5. This latter strategy produces an IL-5 responsive cell line expressing luciferase in a single step without the need for antibiotic selection.

Animals↗

PubMed1999

Improved assay sensitivity of an engineered secreted Renilla luciferase.

We have previously reported the construction of a functional Renilla luciferase enzyme secreted by mammalian cells when fused to the signal peptide of human interleukin-2. The presence of three predicted cysteine residues in the amino acid sequence of Renilla luciferase suggested that its secreted form could contain oxidized sulfhydryls, which might impair enzyme activity. In this work, four secreted Renilla luciferase mutants were constructed to investigate this possibility: three luciferase mutants in which a different cysteine residue was replaced by an alanine residue, and one luciferase mutant in which all three cysteine residues were replaced by alanine residues. Simian cells were transfected with the genes encoding these mutant luciferases, as well as with the original gene construct, and cell culture media were assayed for bioluminescence activity. Only media containing a mutated luciferase with a cysteine to alanine substitution at position 152 in the preprotein showed a marked increase in bioluminescence activity when compared to media containing the original secreted Renilla luciferase. This increase in light emission was due in part to enhanced stability of the mutant enzyme. This new enzyme represents a significant improvement in the sensitivity of the secreted Renilla luciferase assay for monitoring gene expression.

Animals↗

PubMed1991

Functional consequences of site-directed mutation of conserved histidyl residues of the bacterial luciferase alpha subunit.

The available sequences for the different bacterial luciferases reveal five conserved histidyl residues at positions 44, 45, 82, 224, and 285 of the alpha subunit. Ten variants of Vibrio harveyi luciferase were obtained by selective site-directed mutations of these five histidines. The essentiality of alpha His44 and alpha His45 was indicated by 4-7 orders of magnitude of bioluminescence activity reductions resulting from the substitution of either histidine by alanine (alpha H44A or alpha H45A), aspartate (alpha H44D or alpha H45D), or lysine (alpha H45K). Moreover, alpha H44A and alpha H45A were distinct from the native luciferase in thermal stabilities. Mutations at the other three positions also resulted in activity reductions ranging from a fewfold to 3 orders of magnitude. Despite these widely different bioluminescence light outputs, mutated luciferases exhibited, in nonturnover in vitro assays, light emission decay rates mostly similar to that of the native luciferase using octanal, decanal, or dodecanal as a substrate. This is attributed to a similarity in the catalytic rate constants of the light-emitting pathway for the native and mutated luciferases, but various mutated luciferases suffer in different degrees from competing dark reaction(s). In accord with this interpretation, the bioluminescence activities of mutated luciferases showed a general parallel with the relative stabilities of their 4a-hydroperoxyflavin intermediate species. Furthermore, the drastically reduced bioluminescence activities for luciferases with the alpha His44 or alpha His45 substituted by aspartate, alanine, or lysine were accompanied by little or no activities for consuming the aldehyde substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1996

Effect of geldanamycin on the kinetics of chaperone-mediated renaturation of firefly luciferase in rabbit reticulocyte lysate.

Renaturation of thermally denatured firefly luciferase in rabbit reticulocyte lysate (RRL) requires hsp90, hsc70, and other as yet unidentified RRL components [Schumacher, R.J., et al. (1994) J. Biol. Chem. 269, 9493-9499]. Benzoquinonoid ansamycins (BAs) have recently been shown to specifically bind hsp90 and inhibit its function. In this report, we present data that indicate BAs are specific inhibitors of hsp90 function. The effects of the BA geldanamycin (GA) on the kinetics of the luciferase renaturation in RRL were examined to gain insight into the mechanism by which GA inhibits the function of the hsp90 chaperone machinery. Chaperone-mediated renaturation of luciferase obeyed Michaelis-Menten kinetics. The GA inhibited luciferase renaturation uncompetitively with respect to ATP concentration and noncompetitively with respect to luciferase concentration, indicating that GA binds after the binding of ATP and that it binds to both the hsp90 chaperone machine/ATP complex and the hsp90 chaperone machine/ATP/luciferase complex. GA markedly decreased the Kapp of the hsp90 chaperone machine for ATP, suggesting that GA increases the binding affinity of the hsp90 chaperone machinery for ATP or it slows the rate of ATP hydrolysis. Consistent with the notion that GA specifically binds hsp90 and inhibits its function, addition of hsp90, but not hsc70, p60, or p23, reversed GA-induced inhibition of luciferase renaturation in RRL. Hsp90, hsc70, and the hsp cohorts p60, p48, and p23 were coimmunoprecipitated with luciferase from RRL. GA increased the steady-state levels of luciferase associated with hsp90/hsp70 chaperone machine complexes that contain p60 and blocked the association of the hsp90 cohort p23 with chaperone-bound luciferase. The data suggest that the function of the hsp90 chaperone machinery is not specific to its previously described interaction with steroid hormone receptors, and that it carries out some more generalized function in vivo.

Animals↗