Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.14.14.3 (luciferase)
 38,195 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Peroxisome, an ubiquitous subcellular organelle in eukaryotes, functions in many crucial pathways in metabolisms such as catabolism by beta-oxidation of very long chain fatty acids, biosynthesis of etherglycerolipids, and metabolism of cholesterol. To address the question how peroxisomes are assembled in eukaryotic cells, we discuss here two topics undertaken in our laboratory. Peroxisomes are formed by posttranslational assembly mechanism; peroxisomal proteins are synthesized on free polysomes in the cytosol, mostly at their final sizes. This implies that topogenic signal(s) for import of newly synthesized polypeptides into peroxisomes reside in the internal sequence of proteins. Peroxisome-targeting signal has been noted in vivo and in vitro for enzymes such as luciferase and acyl-CoA oxidase (AOX). The topogenic signal resides at the extreme C-terminus and comprises tripeptide-Ser-Lys-Leu-COOH (SKL). Further experiments have strongly suggested that the SKL motif, Ser/Ala-Lys/Arg/His-Leu-COOH commonly found at C-termini of many peroxisomal proteins, functions as a peroxisome-targeting signal. Among several human genetic peroxisomal disorders, cerebrohepatorenal syndrome (Zellweger syndrome) is a typical, severe disease with absence of peroxisome, where a peroxisome assembly is likely to be defective. We isolated three mutants (Z24, Z65, and ZP92), recessive to wild-type cell and mutually complementary, of Chinese hamster ovary (CHO) cells that resemble the fibroblasts from Zellweger patients. To investigate molecular mechanism of peroxisome assembly and primary defects of human peroxisome-deficient disorders, we searched for the genes encoding factors that complement dysfunctions of CHO cell mutants. The mutants transfected with a pcD2-rat liver cDNA library were selected in the presence of G418.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[Biogenesis of peroxisome--targeting signal and peroxisome assembly factor]. 156 55

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)
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PMID:Functional consequences of site-directed mutation of conserved histidyl residues of the bacterial luciferase alpha subunit. 195 63

Flavin-dependent external monooxygenases and oxidases could catalyze the same flavin oxidation reaction involving distinct mechanisms. To gain insights into enzyme structure-function relationship, site-directed mutagenesis was carried out for Vibrio harveyi luciferase, a monooxygenase. The substitution of the alpha subunit cysteine 106 by alanine shows unambiguously that the alphaCys106 is not essential to catalysis. The corresponding substitution by valine resulted in a substantial reduction of the bioluminescence activity correlatable with the induction of a new flavin oxidation activity typical for oxidases. These findings indicate that mutation of a single noncatalytic residue at the active center of a flavoenzyme could transform one enzyme type to another, thus highlighting the subtlety of enzyme active site structure in relation to catalysis and the versatility of enzyme evolution.
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PMID:Elicitation of an oxidase activity in bacterial luciferase by site-directed mutation of a noncatalytic residue. 230 67

It has been appreciated for many years that the luciferase from the luminous marine bacterium Vibrio harveyi has a highly reactive cysteinyl residue which is protected from alkylation by binding of flavin. Alkylation of the reactive thiol, which resides in a hydrophobic pocket, leads to inactivation of the enzyme. To determine conclusively whether the reactive thiol is required for the catalytic mechanism, we have constructed a mutant by oligonucleotide directed site-specific mutagenesis in which the reactive cysteinyl residue, which resides at position 106 of the alpha subunit, has been replaced with a seryl residue. The resulting alpha 106Ser luciferase retains full activity in the bioluminescence reaction, although the mutant enzyme has a ca 100-fold increase in the FMNH2 dissociation constant. The alpha 106Ser luciferase is still inactivated by N-ethylmaleimide, albeit at about 1/10 the rate of the wild-type (alpha 106Cys) enzyme, demonstrating the existence of a second, less reactive, cysteinyl residue that was obscured in the wild-type enzyme by the highly reactive cysteinyl residue at position alpha 106. An alpha 106Ala variant luciferase was also active, but the alpha 106Val mutant enzyme was about 50-fold less active than the wild type. All three variants (Ser, Ala and Val) appeared to have somewhat reduced affinities for the aldehyde substrate, the valine mutant being the most affected. It is interesting to note that the alpha 106 mutant luciferases are much less subject to aldehyde substrate inhibition than is the wild-type V. harveyi luciferase, suggesting that the molecular mechanism of aldehyde substrate inhibition involves the Cys at alpha 106.
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PMID:Site-directed mutagenesis of bacterial luciferase: analysis of the 'essential' thiol. 267 23

Numerous luciferase structural gene mutants of Vibrio harveyi have been generated by random mutagenesis and phenotypically characterized [Cline, T.W., & Hastings, J.W. (1972) Biochemistry 11, 3359-3370]. All mutants selected by Cline and Hastings for altered kinetics in the bioluminescence reaction had lesions in the alpha subunit. One of these mutants, AK-20, has normal or slightly enhanced thermal stability and enhanced FMNH2 binding affinity but a much-reduced quantum yield of bioluminescence and dramatically altered stability of the aldehyde-C4a-peroxydihydroflavin-luciferase intermediate (IIA), with a different aldehyde chain length dependence from that of the wild-type luciferase. To better understand the structural aspects of the aldehyde binding site in bacterial luciferase, we have cloned the luxAB genes from the V. harveyi mutant AK-20, determined the nucleotide sequence of the entire luxA gene, and determined the mutation to be TCT----TTT, resulting in a change of serine----phenylalanine at position 227 of the alpha subunit. To confirm that this alteration caused the altered kinetic properties of AK-20, we reverted the AK-20 luxA gene by oligonucleotide-directed site-specific mutagenesis to the wild-type sequence and found that the resulting enzyme is indistinguishable from the wild-type luciferase with respect to quantum yield, FMNH2 binding affinity, and intermediate IIA decay rates with 1-octanal, 1-decanal, and 1-dodecanal. To investigate the cause of the AK-20 phenotype, i.e., whether the phenotype is due to loss of the seryl residue or to the properties of the phenylalanyl residue, we have constructed mutants with alanine, tyrosine, and tryptophan at alpha 227.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Random and site-directed mutagenesis of bacterial luciferase: investigation of the aldehyde binding site. 273 Aug 82

31P NMR of living 32D cl23 cells and 1H NMR of cell extracts were used to study the metabolic effects of interleukin 3 (IL3). When IL3 was removed from 32D cl23 for 9-10 hours 31P spectra showed a decrease in sugar phosphate, gamma ATP/ADP, alpha ATP/ADP/NAD, and beta ATP resonances which declined progressively over a time period of up to 16 hours. By comparison, ATP measurements using the luciferin/luciferase method resulted in the decline of ATP levels from 12 hours in the absence of IL3. At this time, viability of the cells was unaffected. For 1H NMR experiments cells were grown in the presence and absence of IL3 for 4 and 24 hours, after which acid cell extracts were prepared. These spectra revealed a four-fold decrease in lactate 4 hours post-IL3 removal. Alanine levels were unchanged but glycine was elevated 1.5-fold whilst various other amino acids were elevated slightly. After 24 hours without IL3, only 22% of cells were viable which was reflected in a general decline of most resonance intensities. These findings suggest that IL3 exerts its effect primarily on glucose metabolism and has a delayed secondary effect on maintenance of ATP levels in the cell. We have demonstrated the applicability of high resolution 1H and 31P NMR to the study of cellular metabolism in hemopoietic cells.
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PMID:Metabolic effects of interleukin 3 on 32D cl23 cells analyzed by NMR. 350 Jan 78

Human immunodeficiency virus type 1 Vpr is a virion-associated, regulatory protein that is required for efficient viral replication in monocytes/macrophages. The protein is believed to act in conjunction with the Gag matrix protein to allow import of the viral preintegration complex in nondividing cells. In cells, Vpr localizes to the nucleus. Recently, we showed that Vpr prevents the activation of p34cdc2-cyclin B. This results in arrest of Vpr-expressing cells in the G2/M phase of the cell cycle. Here, we use a panel of expression vectors encoding Vpr molecules mutated in the amino-terminal alpha-helical region, the central hydrophobic region, or the carboxy-terminal basic region to define the functional domains of the protein. The results showed cell cycle arrest was largely controlled by the carboxy-terminal basic domain of the protein. In contrast, the amino-terminal alpha-helical region of Vpr was required for nuclear localization and packaging into virions. The carboxy terminus appeared to be unnecessary for nuclear localization. In the alpha-helical region, mutation of Ala-30 to Pro resulted in a protein that localized to the cytoplasm. Surprisingly, fusion of Vpr to luciferase resulted in a molecule that failed to localize to the nucleus. In addition, we show that simian immunodeficiency virus Vpr, but not Vpx, induces G2 arrest. We speculate that Vpr has two sites for interaction with cellular factors: one in the alpha-helical region that specifies nuclear localization and one in the carboxy-terminal domain that is required for Cdc2 inhibition.
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PMID:Mutational analysis of cell cycle arrest, nuclear localization and virion packaging of human immunodeficiency virus type 1 Vpr. 749 3

Alanine/glyoxylate aminotransferase 1 (AGT) is peroxisomal in most normal humans, but in some patients with the hereditary disease primary hyperoxaluria type 1 (PH1), AGT is mislocalized to the mitochondria. In an attempt to identify the sequences in AGT that mediate its targeting to peroxisomes, and to determine the mechanism by which AGT is mistargeted in PH1, we have studied the intracellular compartmentalization of various normal and mutant AGT polypeptides in normal human fibroblasts and cell lines with selective deficiencies of peroxisomal protein import, using immunofluorescence microscopy after intranuclear microinjection of AGT expression plasmids. The results show that AGT is imported into peroxisomes via the peroxisomal targeting sequence type 1 (PTS1) translocation pathway. Although the COOH-terminal KKL of human AGT was shown to be necessary for its peroxisomal import, this tripeptide was unable to direct the peroxisomal import of the bona fide peroxisomal protein firefly luciferase or the reporter protein bacterial chloramphenicol acetyltransferase. An ill-defined region immediately upstream of the COOH-terminal KKL was also found to be necessary for the peroxisomal import of AGT, but again this region was found to be insufficient to direct the peroxisomal import of chloramphenicol acetyltransferase. Substitution of the COOH-terminal KKL of human AGT by the COOH-terminal tripeptides found in the AGTs of other mammalian species (SQL, NKL), the prototypical PTS1 (SKL), or the glycosomal PTS1 (SSL) also allowed peroxisomal targeting, showing that the allowable PTS1 motif in AGT is considerably more degenerate than, or at least very different from, that acceptable in luciferase. AGT possessing the two amino acid substitutions responsible for its mistargeting in PH1 (i.e., Pro11-->Leu and Gly170-->Arg) was targeted mainly to the mitochondria. However, AGTs possessing each amino acid substitution on its own were targeted normally to the peroxisomes. This suggests that Gly170-->Arg-mediated increased functional efficiency of the otherwise weak mitochondrial targeting sequence (generated by the Pro11-->Leu polymorphism) is not due to interference with the peroxisomal targeting or import of AGT.
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PMID:Mammalian alanine/glyoxylate aminotransferase 1 is imported into peroxisomes via the PTS1 translocation pathway. Increased degeneracy and context specificity of the mammalian PTS1 motif and implications for the peroxisome-to-mitochondrion mistargeting of AGT in primary hyperoxaluria type 1. 755 90

The pathogenesis of the eosinophilia myalgia syndrome (EMS) remains unclear. Several abnormal constituents have been found in the L-tryptophan lots responsible for the illness, particularly, 1,1-ethylidenebis[L-tryptophan], also called peak E or EBT, and 3-phenylamino-alanine or peak 5. However, the role of these contaminants in the pathogenesis of EMS and in the development of fibrosis is unknown. We now report that peak E, a dimer of L-tryptophan, is a potent stimulus for human dermal fibroblast DNA and collagen synthesis. Peak E (0.1-1.0 microM) increased DNA synthesis up to four-fold (P = 0.0001) in a dose-dependent manner (r = 0.987). When added to monolayer cultures for 2 to 24 h, peak E (0.5 to 100 microM) caused a progressive, more than threefold increase in alpha 1(I) procollagen mRNA levels and collagenous protein. No increase in procollagen mRNA levels was found after the addition of another major L-tryptophan contaminant, peak 5, or with L-tryptophan itself. Transient transfection with a 2.5-kb alpha 1(I) procollagen promoter-luciferase construct showed that peak E causes a twofold upregulation of promoter activity (P = 0.022). Contraction of collagen gels, consisting of human dermal fibroblasts incorporated into a type I collagen lattice, was enhanced two-fold by exposure to peak E (P = 0.001). We conclude that a major constituent of contaminated batches of L-tryptophan, peak E, is a potent stimulus for fibroblast activation and collagen synthesis. This stimulatory action of peak E may provide a direct mechanism for the development of fibrosis in EMS.
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PMID:Enhanced collagen synthesis and transcription by peak E, a contaminant of L-tryptophan preparations associated with the eosinophilia myalgia syndrome epidemic. 759 96

Stimulation of alpha 1-adrenergic receptors in neonatal ventricular cardiomyocytes induces hypertrophic changes including activation of the atrial natriuretic factor (ANF) gene. This receptor couples to Gq to activate phospholipase C (PLC) and protein kinase C, which have been implicated as mediators of the hypertrophic response. To directly determine whether receptor coupling to Gq/PLC is sufficient to induce ANF expression, we expressed wild-type and chimeric muscarinic cholinergic receptors (mAChRs) with altered G-protein coupling properties in cardiac myocytes and examined their ability to activate an ANF promoter/luciferase reporter gene. The cholinergic agonist carbachol failed to induce transcriptional activation of the ANF reporter gene through endogenous Gi-linked M2mAChRs or in cells transfected with M2mAChRs. In contrast, in cells transfected with M1mAChRs, which effectively couple to Gq/PLC, carbachol increased ANF reporter gene expression 10-fold and also increased ANF protein, as determined by immunofluorescence. Carbachol-mediated ANF gene expression was inhibited by the mAChR antagonist pirenzepine with a Ki value characteristic of an M1mAChR. Studies using chimeric M1- and M2mAChRs demonstrated that the N-terminal 21 amino acids of the third intracellular loop of the M1mAChR were required for receptor coupling to ANF gene expression. This region, previously shown to specify receptor coupling to Gq/PLC, also conferred partial activity to a chimeric M2 receptor. We further demonstrated that M1mAChR coupling to ANF gene expression was Ras-dependent since co-expression of dominant-interfering Ala-15 Ras inhibited M1mAChR-induced ANF expression by 60%. In contrast, ANF expression induced by the chimeric M2 receptor was not blocked by dominant-interfering Ras. We suggest that receptor coupling to Gq/PLC is sufficient to induce ANF expression and that a Ras-dependent pathway contributes additional signals required for maximal M1mAChR-mediated ANF gene expression.
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PMID:M1 muscarinic receptors heterologously expressed in cardiac myocytes mediate Ras-dependent changes in gene expression. 772 39


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