Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.13.12.7 (firefly luciferase)
 2,792 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The continuous bioluminescent assay of ATP has been adapted to the study of Mg2+-dependent ATPases, including the (Na+; K+) pump, in amphibian tissues. A discrete bioluminescent assay procedure for ATPase has also been developed. Components of the firefly luciferase assay reagent modify the observed ATPase activity but this can be circumvented by performing discrete instead of continuous measurements of enzyme activity. In assays with commercial ATPase preparations the continuous bioluminescent assay procedure gave ATPase activities 2.2-fold lower than obtained with the discrete procedure. In Xenopus oocyte or egg homogenates, in contrast, the total ATPase activity measured is stimulated eight times by the luciferase reagent, mainly through an unexplained activation of a Mg2+-independent ATPase. In other tissues, such as Xenopus brain homogenates, both the continuous and discrete monitoring procedures are equally suitable for the determination of ATPase activity.
 ...
PMID:Bioluminescent assay of ATPase activity in embryonic material using firefly luciferase. 285 Jul 21

(1) The kinetics of the release of the mitochondrial inhibitor protein (IF1) is studied in bovine heart submitochondrial vesicles supplemented with 125I-labelled IF1, using a method for rapidly 'freezing' the state of F1-IF1 interaction. It is shown that generation of a protonmotive force leads to release of IF1 from F1 into solution, following an exponential process. (2) In one set of experiments the rate of IF1 release, in IF1 supplemented vesicles generating a protonmotive force, is correlated with the induction of ATP hydrolytic capacity. It is found that, even under different metabolic states (phosphorylating and non-phosphorylating conditions), both processes follow the same time-course (half-time of around 40 s) and that there is a direct correlation between induced ATPase capacity and IF1 released. This finding rules out the possibility of a non-inhibitory binding site for IF1 on the membrane. (3) In a second set of experiments, also using IF1 supplemented vesicles, the induction of the ATP hydrolytic capacity after energisation is correlated with the induction of the ATP synthetic capacity. Initial rates of both processes are monitored using firefly luciferase, keeping the assay systems as similar as possible. It is shown that the induction of each capacity follows an exponential time-course, with a half-time of around 40 s. This is in good agreement with the half-times obtained for the induction of ATP hydrolytic capacity and the rate of IF1 release, using the quench-stop method. (4) If the induction of ATP hydrolytic and synthetic capacities is followed in untreated submitochondrial vesicles, i.e., vesicles not supplemented with IF1, the extent and time-course of the change in both hydrolytic and synthetic capacities remain correlated, but the half-time of the transient falls to around 10 s. It is suggested that the length of the transient, observed in IF1 supplemented vesicles, results from partial loss of coupling during repeated centrifugations. (5) These results demonstrate that energy-dependent release of IF1 from F1 into solution results in a concomitant increase in both ATP synthetic and hydrolytic capacities of the ATP synthase complex, and that the time-course of this process is sensitive to the degree of coupling of the vesicles.
 ...
PMID:Kinetics of the release of the mitochondrial inhibitor protein. Correlation with synthesis and hydrolysis of ATP. 289 52

We have reconstituted an ATP-dependent protein folding machinery using purified yeast cytosolic proteins. The S. cerevisiae Hsp70 Ssa1p and the DnaJ homolog Ydj1p refolded denatured firefly luciferase. In E. coli, efficient refolding of luciferase requires the Hsp70 DnaK and two modulators, DnaJ and GrpE, that synergistically stimulate its ATPase activity. Exchanging DnaJ homologs between the S. cerevisiae and E. coli systems revealed that their ability to stimulate Hsp70 ATPase activity was conserved. In contrast, GrpE further stimulated only DnaK's ATPase activity. Efficient refolding of luciferase by Ssa1p and DnaJ, but not by DnaK and Ydj1p, suggests that a compatible Hsp70/DnaJ homolog pair can act as a protein folding machinery.
 ...
PMID:Conserved ATPase and luciferase refolding activities between bacteria and yeast Hsp70 chaperones and modulators. 763 93

The Hsp70 family of molecular chaperones has an essential role in the synthesis, folding and translocation of the nascent peptide chain. While the general features of these activities are well documented, less is understood about the regulation of these activities. The ATPase rate is stimulated by non-native proteins, furthermore, interaction with ATP leads to the release of protein substrate concurrent with a conformational change in Hsp70. One interpretation of these data is that the two domains of Hsp70 interact. In the process of mapping the carboxyl-terminal boundary of the substrate binding domain for human Hsp70, we identified a regulatory motif, EEVD, which is conserved at the extreme carboxyl terminus among nearly all cloned cytosolic eukaryotic Hsp70s. Deletion or mutation of EEVD affects the ATPase activity, the ability to interact with substrates, and interferes with the ability of the mutant Hsp70 to interact with HDJ-1 in the refolding of denatured firefly luciferase. Examination of the biophysical properties of the mutant Hsp70s reveals a change in the overall shape and conformation of the protein consistent with reduced interactions between the two domains. These data suggest that the EEVD motif is involved in the intramolecular regulation of Hsp70 function and intermolecular interactions with HDJ-1.
 ...
PMID:Identification of a regulatory motif in Hsp70 that affects ATPase activity, substrate binding and interaction with HDJ-1. 777 86

Thyroid hormone (T3) stimulates Na,K-ATPase activity and alpha and beta subunit mRNA abundances in myocardial cells in vivo and in vitro. In this study, we used transient transfection and nuclear run-on assays to determine whether T3 regulates the transcription rate of the Na,K-ATPase alpha 2 subunit gene. Primary cultures of neonatal rat cardiac myocytes were incubated with 100 nM T3 for 1, 3, and 6 d, and alpha 2 mRNA levels were measured by Northern blot hybridization analysis. There was no change in the abundance of alpha 2 mRNA by 1 d of T3 treatment, whereas a two- and threefold increase in alpha 2 mRNA was evident when cells were exposed to T3 for 3 and 6 d, respectively. A portion of the rat alpha 2 gene containing 1700 base pairs (bp) of 5'-flanking DNA sequence was isolated and fused to the firefly luciferase gene. Transient transfection experiments utilizing this chimeric gene showed no T3 trans-activation of reporter gene activity either in the absence or presence of cotransfected beta 1 or alpha 1 isoforms of rat T3 receptor (T3R). In contrast, cotransfection of T3R facilitated a strong stimulation of luciferase activity driven by a construct containing a single copy of a palindromic T3 response element (TRE). Nuclear run-on analysis indicated that the rate of transcription of the endogenous alpha 2 gene was enhanced 1.2-fold at 3 d of T3 treatment, and was not regulated at either 1 or 6 d. These results indicate that the T3-dependent increase in alpha 2 mRNA content at 6 d is mediated at a post-transcriptional level. Unexpectedly, we observed a T3-dependent three-to sixfold repression of alpha 2/luciferase expression in cardiac myocytes cotransfected with T3R. Deletion analysis of the 5' end of the alpha 2 gene revealed a negative TRE between nucleotides -354 and -100.
 ...
PMID:Thyroid hormone regulation of Na,K-ATPase alpha 2 gene expression in cardiac myocytes. 780 25

Incubation of mammalian cells in medium containing low K+ is often associated with an increase in Na,K-ATPase activity and content. In the present studies, we investigated the effect of low K+ on the regulation of Na,K-ATPase alpha 1 and beta 1 gene expression in primary cultures of neonatal rat cardiac myocytes. Northern blot hybridization analysis indicated that exposure of cardiocytes to a medium containing 0.3 mmol/L K+ for 72 h resulted in 1.8- and 3.3-fold increases in the abundance of alpha 1 and beta 1 mRNAs, respectively, compared with control cells exposed to 5.4 mmol/L K+. To investigate the molecular mechanism of the low K effect on beta 1 mRNA content, we constructed chimeric genes with a full-length and deleted portion of the 5' end of the rat beta 1 gene linked to the firefly luciferase gene. Transient transfection experiments using these constructs indicated that beta 1 gene sequences between -102 and +151 base pairs (bp) are required for low K(+)-induced trans-activation of reporter gene expression. Taken together, these results indicate that low K+ induction of beta 1 mRNA abundance in neonatal rat cardiac myocytes is mediated by regulatory DNA sequences in close proximity to the site of transcription initiation of the beta 1 gene.
 ...
PMID:Low external K+ regulates Na,K-ATPase alpha 1 and beta 1 gene expression in rat cardiac myocytes. 813 18

The human Na,K-ATPase beta 1 subunit gene promoter activity is stimulated by thyroid hormone (T3) in the human intestinal Caco-2 cells. To identify potential cis-acting transcriptional regulatory elements involved in this process, chimeric plasmids containing varying lengths of the 5' flanking region of the human beta 1 Na,K-ATPase gene linked to the firefly luciferase reporter gene were introduced into Caco-2 cells by transient transfection. Analysis of T3-regulated luciferase activity of cells carrying these plasmids, and subsequent use of site-directed mutagenesis revealed that a region from -459 to -438 (relative to the transcriptional start site) is required for the induction of the beta 1 Na,K-ATPase gene by T3. An oligonucleotide containing this sequence from -465 to -433 confers T3 responsiveness to a heterologous promoter. Gel mobility shift assays showed specific binding of nuclear proteins of Caco-2 cells to this region and immunoreactive T3 receptor was identified in one of these complexes. These data demonstrate that there is a cis-acting thyroid hormone responsive element in the 5' flanking region of the human beta 1 Na,K-ATPase gene and induction of transcription of this gene by T3 involves specific binding of the thyroid hormone receptor to the TRE located at position -459 to -438.
 ...
PMID:Identification of a functional thyroid hormone response element in the upstream flanking region of the human Na,K-ATPase beta 1 gene. 839 3

We have purified to apparent homogeneity a 66-kDa protein from rabbit reticulocyte lysate which is associated with hsp 70. Our characterization of this 66-kDa protein demonstrates that its physiological role is to promote the recycling of hsp 70 by catalyzing the dissociation of hsp 70-bound ADP in exchange for ATP. We have therefore termed the 66-kDa protein RF-hsp 70, a recycling factor for hsp 70. RF-hsp 70 promotes stoichiometric binding of ATP to hsp 70, and it increases about 5-fold the rate of dissociation of hsp 70.ADP in the presence of ATP. This process represents adenine nucleotide exchange, since dissociation of ADP does not occur unless ATP is added; dATP, GTP, and ITP cannot substitute for ATP. The mechanism of action of RF-hsp 70 is to lower the KD of hsp 70 for ATP about 6-7-fold to a value that is close to the KDof hsp 70 for ADP. RF-hsp 70 also stimulates the ATPase activity of hsp 70, including the 42-kDa amino-terminal portion of hsp 70 generated by chymotrypsin, demonstrating that RF-hsp 70 interacts with that part of hsp 70 known to contain the ATP/ADP binding site. Confirming its recycling function, RF-hsp 70 stimulates about 7-10-fold the ability of hsp 70 to reactivate heat-denatured firefly luciferase. In addition, RF-hsp 70 acts catalytically to recycle hsp 70, since, at 0.2 times the molar concentration of hsp 70, RF-hsp 70 increases the rate of renaturation of luciferase by hsp 70 about 3-4-fold. The action of RF-hsp 70 is also partially species-specific since it is most effective with rabbit reticulocyte hsp 70, less effective with bovine brain hsp 70, even less effective with human hsp 70, and ineffective with broad bean hsp 70.
 ...
PMID:Purification and characterization of a 66-kDa protein from rabbit reticulocyte lysate which promotes the recycling of hsp 70. 866 20

The effects of the human DnaJ homolog, Hsp40, on the ATPase and chaperone functions of the constitutively expressed Hsp70 homolog, Hsc70, were analyzed. Hsp40 stimulates the hydrolysis of ATP by Hsc70, causing a approximately 7-fold increase in its steady-state ATPase activity. In contrast to the prokaryotic Hsp70 system, ATP-hydrolysis and not the release of bound ADP is the rate-limiting step in the overall ATPase cycle of mammalian Hsc70. The ability to activate the Hsc70 ATPase is partially preserved in a deletion mutant containing the J-domain and the G/F region of Hsp40 but not in a deletion mutant that contains the J-domain alone. As a result of its ATPase stimulating activity, addition of Hsp40 allows Hsc70 to bind peptide in the presence of ATP, whereas in the absence of Hsp40, peptide is efficiently released upon ATP binding to Hsc70. The functional cooperation of Hsp40 with Hsc70 is essential to ensure the ATP hydrolysis-dependent binding of aggregation-sensitive denatured polypeptides, such as thermally denatured firefly luciferase and chemically denatured rhodanese. Binding of these proteins results in the formation of ternary complexes of Hsc70, Hsp40, and substrates. Hsc70 and Hsp40 cooperate with further factors in protein renaturation, as demonstrated by the finding that luciferase, thermally denatured in the presence of Hsc70, Hsp40, and ATP, refolds upon addition of rabbit reticulocyte cytosol. Our results indicate that Hsp40 has a critical regulatory function in the Hsc70 ATPase cycle that is required for the efficient loading of peptide substrate onto Hsc70.
 ...
PMID:Regulation of the heat-shock protein 70 reaction cycle by the mammalian DnaJ homolog, Hsp40. 870 58

The DnaK/DnaJ/GrpE heat shock proteins of Escherichia coli constitute the prototype DnaK chaperone machine. Various studies have shown that these three proteins work synergistically in a diverse array of biological functions, including protein folding and disaggregation, proteolysis, and transport across biological membranes. We have overexpressed and purified the mitochondrial Saccharomyces cerevisiae DnaJ homologue, Mdj1pDelta55, which lacks the mitochondrial presequence, and studied its biochemical properties in well defined in vitro systems. We find that Mdj1pDelta55 interacts with DnaK as judged both by an enzyme-linked immunosorbent assay, as well as stimulation of DnaK's weak ATPase activity in the presence of GrpE. In addition, Mdj1pDelta55 not only interacts with denatured firefly luciferase on its own, but also enables DnaK to bind to it in an ATP-dependent mode. Using co-immunoprecipitation assays we can demonstrate the presence of a stable Mdj1pDelta55-luciferase-DnaK complex. However, in contrast to DnaJ, Mdj1pDelta55 does not appear to interact well with certain seemingly folded proteins, such as the sigma32 heat shock transcription factor or the lambdaP DNA replication protein. Finally, Mdj1pDelta55 can substitute perfectly well for DnaJ in the refolding of denatured firefly luciferase by the DnaK chaperone machine. These studies demonstrate that Mdj1pDelta55 has conserved most of DnaJ's known biological properties, thus supporting an analogous functional role in yeast mitochondria.
 ...
PMID:Purification and biochemical properties of Saccharomyces cerevisiae Mdj1p, the mitochondrial DnaJ homologue. 935 16


1 2 3 4 Next >>