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)

Exogenous and endogenously generated reduced pyridine nucleotides caused marked stimulation of O(2) uptake when added to treponemal cell-free extracts, which indicated that terminal electron transport was coupled to the consumption of O(2). Oxidation of reduced nicotinamide adenine dinucleotide (NADH) was shown to correlate stoichiometrically with O(2) reduction, suggesting that NADH was being oxidized through a mainstream respiratory chain dehydrogenase. Oxygen evolution in treponemal extracts was observed after the completion of O(2) uptake which was stimulated by exogenous NADH and endogenously generated reduced NAD phosphate. Oxygen evolution was inhibited by both cyanide and pyruvate, which was consistent with O(2) release from H(2)O(2) by catalase. The addition of exogenous H(2)O(2) to treponemal extracts caused rapid O(2) evolution characteristic of a catalase reaction. A spectrophotometric assay was used to measure ATP formation in T. pallidum cell-free extracts that were stimulated with NADH. P/O ratios from 0.5 to 1.1 were calculated from the amounts of ATP formed versus NADH oxidized. Phosphorylating activity was dependent on P(i) concentration and was sensitive to cyanide, N, N'-dicyclohexylcarbodiimide, and carbonyl cyanide m-chlorophenyl hydrazone. Adenine nucleotide pools of T. pallidum were measured by the firefly luciferin-luciferase assay. Shifts in adenine nucleotide levels upon the addition of NADH to cell-free extracts were impossible to evaluate due to the presence of NAD(+) nucleosidase. However, when whole cells, previously incubated under an atmosphere of 95% N(2)-5% CO(2), were sparged with air, ATP and ADP levels increased, while AMP levels decreased. The shift was attributed to both oxidative phosphorylation and to the presence of an adenylate kinase activity. T. pallidum was also found to possess an Mg(2+) - and Ca(2+) -stimulated ATPase activity which was sensitive to N, N' -dicyclohexylcarbodiimide. These data indicated a capability for oxidative phosphorylation by T. pallidum.
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PMID:Respiration and oxidative phosphorylation in Treponema pallidum. 2 9

1. Purified luciferase and luciferin were used to study the time course of phosphorylation in submitochondrial particles. The light emitted was detected by a single-photon counter, using a multichannel analyser, and the results were analysed by an 'on-line' digital computer. 2. Using NADH as substrate, phosphorylation showed, in general, four phases. These were (i) a period of increasing rate ('lag'); (ii) a period of constant (positive) rate; (iii) a period of zero net rate (plateau), when the phosphorylation potential was maintained at its equilibrium value, and (iv) a period of negative rate (atp hydrolysis) after all the oxygen has been consumed. 3. The lag phase, several seconds in length, was a function of the inhibitor protein content of the particles. It was decreased in particles treated to remove the inhibitor protein, either by prior energisation of the particles with NADH, or by addition of aurovertin, which competes with the inhibitor protein for the ATPase. It was concluded that the ATPase inhibitor inhibits both ATP synthesis and hydrolysis by the ATPase. 4. The rate constant for the release of the inhibitor protein from the energised membrane was determined from the time course of ATP production during the lag phase. The activation energy of this process was measured from the temperature dependence of the lag, and was shown to be 13.3 kcal/mol, lower than the activation energy of ATP synthesis or NADH oxidation. 5. The rate constant for inhibitor release was dependent on 'energisation' of the membrane, being lower in the presence of uncouplers. However, it was possible to decrease the rate constant considerably with agents that collapsed the membrane potential without uncoupling the membrane. It was concluded that the inhibitor protein responded to the membrane potential component of the energisation. 6. A kinetic model for energy-dependent dissociation of the ATPase-inhibitor complex is proposed.
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PMID:The ATPase inhibitor protein in oxidative phosphorylation. The rate-limiting factor to phosphorylation in submitochondrial particles. 22 34

The plasmid origin of DNA replication of Epstein-Barr virus, oriP, is replicated once per cell division, employing cellular replication machinery and only one viral protein. To understand how replication from this origin is initiated and regulated, we purified this viral protein, EBNA1. EBNA1 was expressed in CV-1p cells by using an infectious simian virus 40 vector containing the EBNA1 gene. It was purified in two chromatographic steps to apparent homogeneity. The purified protein is capable of supporting transcription of the luciferase gene from a reporter plasmid carrying the FR enhancer element to which EBNA1 binds. EBNA1 does not have oriP-dependent ATPase activity, indicating that it does not carry out an energy-dependent step in the initiation of DNA replication. However, EBNA1 does mediate an association between the two elements of oriP. We measured this association by binding one of the elements, the enhancer element, to a solid matrix and measuring retention by this element of the other one, the initiator element, in the presence of EBNA1. This retention is specific for DNA fragments containing EBNA1-binding sites. EBNA1 thus can link the two elements of the origin, providing a locally high concentration of EBNA1 at the site of initiation of DNA replication. We propose that this association is important either (i) to affect DNA structure to allow a cellular helicase to initiate DNA strand separation or (ii) to bind replication proteins to bring them to the origin of replication.
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PMID:EBNA1 can link the enhancer element to the initiator element of the Epstein-Barr virus plasmid origin of DNA replication. 130 58

Synthesis and activity of the enzymatic equivalent of the sodium pump, Na,K-ATPase, are regulated by thyroid hormone in responsive tissues. The purpose of this study was to determine whether triiodothyronine (T3) regulates the level of the messenger RNA (mRNA) coding for Na,K-ATPase alpha- and beta-subunits in the heart. The expression of Na,K-ATPase mRNAs in in vitro myocardial cells was directly assayed by Northern and slot blot hybridization using Na,K-ATPase alpha- and beta-isoform-specific cDNA probes. Exposure of cultured neonatal rat cardiocytes to 10(-8) M T3 resulted in 1) threefold to fourfold increase in alpha 1- and beta 1-mRNA accumulation, with a maximum elevation at 48 hours, 2) sevenfold increase in alpha 2-mRNA accumulation with a peak elevation at 72 hours, and 3) transient threefold increase in alpha 3-mRNA within the first 24 hours followed by a deinduction thereafter. The increase in alpha 1-mRNA accumulation by T3 occurred over the physiological T3 concentration range with an EC50 of 5 x 10(-10) M. This was associated with a twofold increase in alpha 1-subunit protein accumulation and an increase in Na,K-ATPase transport activity. The half-life of alpha 1-mRNA analyzed by actinomycin D chase was less than 3 hours and was not affected by T3. Transfection experiments with the luciferase reporter gene revealed that thyroid hormone response sequences are located within the 5'-flanking regions of each alpha-isoform gene.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Regulation of Na,K-ATPase gene expression by thyroid hormone in rat cardiocytes. 133 Mar 58

1. The ATP concentration of intact, cold-tolerant (ground squirrel) red cells and cold-sensitive (guinea-pig and human) red cells was monitored by use of the firefly tail, luciferin-luciferase assay. ATP kinetics of the pump in intact red blood cells was investigated by altering cell [ATP] by progressive depletion of ATP in the presence of 2-deoxy-D-glucose and then by measurement of ouabain-sensitive K+ influx at each level of [ATP] at various temperatures between 37 and 5 degrees C. Na(+)-K(+)-ATPase activity of broken membranes was also determined in parallel experiments using ouabain-sensitive release of 32P from [gamma-32P]ATP as a measure of activity. 2. Without depletion, there is no immediate decrease in [ATP] of intact cold-sensitive cells at low temperature (5 degrees C) at times when there are marked differences in the activities of the Na(+)-K+ pump of cold-tolerant and cold-sensitive cells. 3. At 37 degrees C Na(+)-K(+)-ATPase of all three species exhibited two components of ATP dependence at 37 degrees C, one with high velocity, low affinity, the other with low velocity, high affinity. Affinities of both components rose with cooling. 4. A similar, two component pattern was observed in intact guinea-pig and human red cells at 37 degrees C, except that the segment corresponding to the high affinity component had an apparent Km (Michaelis-Menten constant) 3- to 4-fold higher than that of the broken membrane preparation. 5. Cooling intact guinea-pig and human red cells decreased the apparent affinity of the high velocity, low affinity component for ATP, so that at 20 degrees C the value of Km approached or exceeded the levels of physiological ATP concentration. Below 20 degrees C only one component with values corresponding to that of the low velocity, high affinity component could be observed. 6. In intact ground squirrel cells only the low affinity, high velocity component was apparent between 37 and 5 degrees C. Its affinity for ATP rose with cooling between 37 and 5 degrees C.
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PMID:ATP dependence of Na(+)-K+ pump of cold-sensitive and cold-tolerant mammalian red blood cells. 133 4

A high molecular weight (HMW) fraction of the 150,000 g supernatant of rat brain homogenates contains protein-tRNA complexes which are able to incorporate [3H]Arg and [3H]Lys into tRNA. The aminoacylation of tRNA(Arg) was found to be dependent on ATP and inhibited by RNase. Conversely, the aminoacylation of tRNA(Lys) did not require exogenous ATP and was resistant to RNase and ATPase. In HMW fractions of regenerating rat sciatic nerves, the charging of both tRNA(Arg) and tRNA(Lys) was resistant to RNase and ATPase and did not require exogenous ATP. Because sciatic nerves are rich in axoplasm and tRNAs are known to be present in axons, we tested the hypothesis that degradative enzyme-resistant, ATP-tRNA complexes were of axonal origin. In HMW fractions from rat liver (containing no axons), both tRNA(Arg) and tRNA(Lys) were sensitive to RNase and required exogenous ATP for charging. But, in similar fractions of axoplasm obtained from the giant axon of squid, both tRNAs were insensitive to RNase and ATPase and did not require exogenous ATP for charging. These results suggest that tRNAs in axons are present in protected HMW complexes and contain endogenous stores of ATP. The presence of ATP in the HMW complexes was demonstrated by the luciferase-luciferin assay for ATP. The nature of the protection of tRNAs from RNases was examined by dissociating proteins from HMW complexes by boiling, treating with proteinase K, or overhomogenizing the tissue. These procedures failed to render brain tRNA(Lys) susceptible to RNase. But phenol-extracted, ethanol-precipitated brain tRNA(Lys) was sensitive to RNase, suggesting that the protection of tRNA(Lys) may be by a protease- and heat-resistant polypeptide or by a nonproteinaceous mechanism.
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PMID:Evidence that axonal tRNAs are resistant to RNase and ATPase and can be aminoacylated in the absence of exogenous ATP. 153 73

2,6-diisopropylphenol (propofol), a general intravenous anesthetic, inhibits the glutamate-dependent Ca2+ entry in rat synaptosomes with an approximate IC50 of 3.0 x 10(-5) M. Propofol, at concentrations above 10(-6) M, also inhibits the ATP-dependent uptake of glutamate in the presence of Ca2+, with an approximate IC50 of 3.5 x 10(-5) M, while it only has a slight inhibitory effect on the release of glutamate. The ouabain-insensitive synaptosomal ATPase is strongly inhibited by propofol, with an IC50 of about 2.5 x 10(-6) M, at concentrations which do not affect the luciferase system.
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PMID:2,6-diisopropylphenol, a general anesthetic, inhibits glutamate action on rat synaptosomes. 168 36

Cellular cystine loading with cystine dimethyl ester inhibits volume absorption, transepithelial potential difference, glucose transport, and bicarbonate transport in proximal convoluted tubules perfused in vitro. This study examined the roles of ATP and NaK ATPase in this in vitro model of the Fanconi syndrome of cystinosis. Intracellular ATP was measured using the luciferin-luciferase assay. Intracellular ATP was reduced by 60% in proximal convoluted tubules incubated with 0.5 mM cystine dimethyl ester for 15 min at 37 degrees C (P less than 0.001). Incubation of cystine loaded tubules with 1 mM exogenous ATP increased intracellular ATP to levels not significantly different than that of controls. On the other hand, Vmax NaK ATPase activity was unchanged even though the incubation times and the concentration of cystine dimethyl ester were doubled to 30 min and 1 mM, respectively. In proximal convoluted tubules perfused in vitro, 0.5 mM cystine dimethyl ester resulted in an 89% inhibition in volume absorption (0.81 +/- 0.14 to 0.09 +/- 0.09 nl/mm.min), while there was only a 45% inhibition in volume absorption (P less than 0.01) due to cellular cystine loading in the presence of 1 mM lumen and bath ATP (0.94 +/- 0.05 to 0.52 +/- 0.11 nl/mm.min). These data demonstrate that proximal tubule cellular cystine loading decreases cellular ATP concentration, but does not directly inhibit NaK ATPase activity. The inhibition in transport and decrease in intracellular ATP due to cellular cystine loading was ameliorated by exogenous ATP. These data are consistent with cellular ATP depletion playing a major role in the inhibition of proximal tubule transport due to intracellular cystine loading.
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PMID:Role of adenosine triphosphate (ATP) and NaK ATPase in the inhibition of proximal tubule transport with intracellular cystine loading. 184 41

Turtle bladder mitochondria-rich (MR) cells secrete H+ by an ATP-dependent process. MR cells also secrete HCO3- by an energy-requiring, Cl- -dependent process that may depend on a serosal H+-ATPase. To determine whether HCO3- is linked to an H+-ATPase, O2 consumption was assessed in MR and granular (G) cells exposed to H+ and HCO3- transport inhibitors alone and also with an H+-ATPase inhibitor. MR and G cells were separated by Ficoll density-gradient centrifugation and treated with ouabain and 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS) to maximally inhibit Na+ and luminal H+ transport. O2 consumption was measured before and after cells were additionally treated with 10 microM N-ethylmaleimide (NEM), an inhibitor of nonmitochondrial H+-ATPase. O2 consumption of MR cells fell after treatment with NEM (delta = 8.64 +/- 2.35 microliters O2.h-1.mg protein-1, P less than 0.025, n = 5). There was no significant difference in G cells similarly treated (delta = 1.61 +/- 0.62 microliters O2.h-1.mg protein-1, P greater than 0.05, n = 5). Because luminal H+ secretion is nearly abolished after treatment with SITS, the decline in O2 consumption of 44.4 +/- 7.11% after addition of NEM is probably due to inhibition of other non-mitochondrial H+-ATPases. In the intact bladder, HCO3- secretion was reduced by 35.1% after serosal application of NEM. Furthermore, in SITS-treated MR and G cells, ATP levels as measured by the luciferin-luciferase assay method were not appreciably different in the presence or absence of NEM.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:HCO3- secretion in mitochondria-rich cells is linked to an H+-ATPase. 252 69

In the present study the effect of phosphorylation of skeletal muscle myosin light chains on the interaction between myosin and actin has been investigated. The actomyosin ATPase activities were determined for synthetic actomyosins formed from either phosphorylated or non-phosphorylated myosin and pure actin, with the help of the luciferin-luciferase system. The contractile properties of our preparations were simultaneously studied by the superprecipitation model. Phosphorylated form of myosin had lower actin-activated ATPase activity at particular conditions studied. In agreement with this, superprecipitation of phosphorylated actomyosin was delayed. On the basis of these results one can expect that phosphorylation of myosin light chains modulates contractile properties of intact skeletal muscle.
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PMID:Effects of skeletal muscle myosin light chain phosphorylation on synthetic actomyosin ATPase activity and superprecipitation. 253 28


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