Luciferase inactivation

Chemical modifications of the aC106 (not shown in Figure 136.1) result in luciferase inactivation. This residue is beheved to be at or near the aldehyde inhibitor site but is not essential to the binding of FMNHj or decanaP or catalysis. ... 

Mutations of five conserved histidine residues (at Positions 44, 45, 82, 224, and 285) on the a were shown to result in markedly different degrees of luciferase inactivation. Residues aH44 and aH45 are particularly critical their mutations can individually result in four to six orders of magnitude of activity reduction. Although aH44 and aH45 are adjacent residues, they are associated with different functions. 

Purification of luciferase. Cypridina luciferase is more stable than many other luciferases, except that this enzyme is rapidly inactivated at acidity below pH 5.0. The dried specimens that have been stored for over 50 years at room temperature (sometimes exceeding 30°C) still possess strong luciferase activity that can be extracted and purified. Preparations of highly purified luciferase have been obtained by various methods (McElroy and Chase, 1951 Shimomura et al., 1961,1969 Tsuji and Sowinski, 1961 Stone, 1968 Tsuji etal., 1974 Thompson et al., 1989) the purification methods employed include... 

Heat stability The Oplophorus luminescence system is more thermostable than several other known bioluminescence systems the most stable system presently known is that of Periphylla (Section 4.5). The luminescence of the Oplophorus system is optimum at about 40°C in reference to light intensity (Fig. 3.3.3 Shimomura et al., 1978). The quantum yield of coelenterazine is nearly constant from 0°C to 20°C, decreasing slightly while the temperature is increased up to 50°C (Fig. 3.3.3) at temperatures above 50°C, the inactivation of luciferase becomes too rapid to obtain reliable data of quantum yield. In contrast, in the bioluminescence systems of Cypridina, Latia, Chaetopterus, luminous bacteria and aequorin, the relative quantum yields decrease steeply when the temperature is raised, and become almost zero at a temperature near 40-50°C (Shimomura et al., 1978). 

Stability. Luciferase L (32 kDa) is highly resistant to heat and its activity is little affected by boiling it for 1 min (Fig. 4.5.4) however, it is unstable in low ionic-strength solutions if some BSA is not included in the solution. Luciferases A, B and C are also highly resistant to inactivation their luminescence activities are only slightly diminished... 

Purification of Pholas luciferase (Michelson, 1978). Acetone powder of the light organs is extracted with 10 mM Tris-HCl buffer, pH 7.5, and the luciferase extracted is chromatographed on a column of DEAE Sephadex A-50 (elution by NaCl gradient from 0.1 M to 0.6 M). Two peaks of proteins are eluted, first luciferase, followed by a stable complex of luciferase and inactivated pholasin. The fractions of each peak are combined, and centrifuged in 40% cesium chloride... 

Luciferase is irreversibly inactivated by H2O2, and strongly inhibited by KCN, diethyldithiocarbamate and o-phenanthroline. Electron paramagnetic resonance spectroscopy (EPR) of luciferase showed a... 

Effect of pH. The light emission from most bioluminescence systems is affected by the pH of the medium, and some luciferases and photoproteins can be made inactive at certain pH ranges without resulting in permanent inactivation. For example, the luminescence of euphausiids can be quenched at pH 6, the luminescence of aequorin can be suppressed at pH 4.2-4A, and the luciferase of the decapod shrimp Oplophorus becomes inactive at about pH 4. In the case of Cypridina luminescence, however, the acidification of an extract to below pH 5 results in an irreversible inactivation of the luciferase. 

If a trace activity is indicated by the luminescence intensity measurement, the following two methods can be used to determine whether the light emission is due to the luciferase or it is an artifact (1) Measure the luminescence intensity with a buffer that contains 1 mM EDTA (add luciferase to this buffer and wait 1 min before mixing with luciferin). If the luminescence was caused totally by luciferase, the light intensity will be decreased to about 20% by EDTA (see Section 3.1.7). (2) Inactivate luciferase by acidifying the sample to pH about 2.0, followed by neutralization with NaHCC>3. Inactivated luciferase should not show any luciferase activity. 

Bacterial bioluminescence, 30-46 factors required, 31 general scheme, 32 in vivo luminescence, 41 luminescence reaction, 37, 38 Bacterial luciferase, 33-35, 343 assay, 39 cloning, 34 crystal structure, 34 extraction and purification, 34 inactivation, 34, 35 molecular weight, 34 properties, 34 storage, 35 subunits, 34... 

Permeable Cell Assay for high-throughput measurement of cellular ATP synthetic activity (4). In this assay, osmotic shock and Triton X-100 treatment made bacterial cells permeable for ATP Discharged ATP reacted with external luciferase and is detected as bioluminescence. An increased bioluminescence is observed with permeable cells, whereas it is not observed with standard ATP solution and heat-inactivated permeable cells. The cellular ATP synthetic activity is calculated from the slope of increasing bioluminescence. Permeable Cell Assay is simple and rapid with a small amount of cell culmre for quantification of ATP synthesis. 

Wfe have recently carried out experiments with cell-based and animal models of inflammatory diseases. The experiments have revealed that human polymorphonuclear cells once activated with phorbol esters to cause an inflammatory response produce copious amounts of chlorinated polyphenol species. Rats treated with lipopolysaccharide (LPS) in an in vivo model of inflammatory disease heavily nitrate polyphenols in tissues (e.g., lung and liver) where inflammatory cell invasion occurs. These modified polyphenols are better antioxidants than their parent compound. Using a luciferase reporter gene assay in COS cells, both chloro- and nitrogenistein were shown to have 1-2 orders lower estrogen receptor activation than genistein itself. In summary, metabolism of polyphenols is rampant, but not always inactivating. 

CAT assay. An enzyme assay. CAT stands for chloramphenicol acetyl transferase, a bacterial enzyme which inactivates chloramphenicol by acety-lating it. CAT assays are often performed to test the function of a promoter. The gene coding for CAT is linked onto a promoter (transcription control region) from another gene, and the construct is transfected into cultured cells. Largely supplanted by the reporter gene luciferase. 

Enhancer-dependent expression is observed following transplantation of an injected pronucleus into a two-cell blastomere (Henery et al., 1995) (Figure 4). The first mitosis—and concomitant loss of the plasmid from the injected pronucleus—was circumvented by the transplantation procedure and hence cannot account for the enhancer requirement observed in the two-cell embryo. The luciferase reporter gene is driven by the / promoter, which has two Spl binding sites (termed the proximal and distal sites)—a CAAT box binding site that binds CTF and a TATA box that binds TBP. Analysis of the expression of a set of linker scanning mutations that inactivated each of these sites without altering the distances between each of the promoter elements revealed that each mutation had the same relative effect on tk... 

The main conclusion from this study is that both PP and LM firefly luciferases have similar kinetic mechanisms characterized by significant inactivation of the enzyme induced by its interaction with the substrates. The difference in kinetic properties for both enzymes is mainly in the reaction rates for formation and dissociation of the luciferin-luciferase comlex. The addition of pyrophosphate to the reaction mixture increases the reaction yield due to enhanced regeneration of active enzyme from the enzyme-product complex. 

Incubation of luciferase with the not reduced activated FMN derivative results in irreversible inactivation of the enzyme. The activated FMN derivative, obviously, modifies no more one functional groups of luciferase, because the coefficient characterizing a number of modified protein residues was determined as 1.05. The addition in an incubation mixture the second substrate of luciferase - tetradecanal before adding the activated FMN derivative, does not protect the luciferase from inactivation. The addition of the protector,of SH-groups - DTT result in essential inhibition decreasing. The activated FMN derivative reacts most likely with SH-groups of luciferases, which, as is known, are good nucleophile. "... 

FIGURE 7.11 Heat inactivation of some enzymes, expressed as activity divided by initial activity, (a) A bacterial protease as a function of heating time at various heating temperatures (°C, indicated), (b) Luciferase, same variables, (c) A bacterial protease heated during 30 min as a function of heating temperature. 

The incubation of luciferase with nonreduced activated FMN also results in irreversible inactivation of the enzyme (Fig.l). 

Fig. 1. The second order rate constant for inactivation of the luciferase by nonreduced activated flavin mononucleotide.

The addition of the second substrate of luciferase - tetradecanal - to the mixture before addition of the activated FMN derivate did not protect the luciferase from inactivation. The addition of the protector of SH-groups - DTT (8 mM) resulted in the essential removal of inhibition. The inhibition was about 20% within 45 min with the nonreduced form of FMN and was completely removed by incubation with the photoreduced form of activated FMN derivate in the presence of the protectors in the reaction mixture. The activated FMN derivative should react with SH-groups of luciferase which are known to be good nucleophiles.4 However, the presence of DTT in the reaction mixture, or its direct co-addition with the flavin does not turn the photoreduced activated flavin a substrate of the reaction. 

In this study the site-directed mutagenesis of Cys62 and 146 to Ser in L. mingrelica luciferase was performed. The effect of these mutations on thermal inactivation of the enzyme in the absence and in presence of dithiothreitol (DDT) and on the activity of L. mingrelica luciferase was studied. 

Irreversible inactivation of luciferase. Aliquots of luciferase solution (10"8, 10 7 and 10 6 mol/L), 30 pL, in the working buffer were incubated at 37°C in the presence and in absence of 12 mmol/L DTT. At certain time intervals the microtubes were taken out of the thermostat and immediately chilled on ice for 10 min before activity measurements. The residual activity was measured at 22°C. Kinetic constants and statistics were calculated using Origin 6.0 software. 

Irreversible inactivation of L. mingrelica luciferase at 37°C. Thermal inactivation of the WT luciferase and its mutants was investigated at 37°C at various enzyme concentrations (10 6-10"8 mol/L). Two-exponential time-curves of thermoinactivation corresponding to the fast and the slow phases of inactivation were observed. The fast hi) and slow (k2) inactivation rate constants of the luciferase were shown to be dependent on the enzyme concentration. This phenomenon is typical for oligomeric enzyme when monomers are less stable than oligomers.4,5 At the concentration 10 6 mol/L, the rate constants kt and k2 of WT and mutant enzymes were similar. At 1 O 7 mol/L enzyme and lower, the ki and k2 values increased both for the WT and mutant luciferases, but constants ki and k2 of mutant forms were four to six times lower that those of the WT (Table 1). Thus, mutations significantly increased the stability of luciferase at both stages of inactivation. 

Table 1. Kinetic rate constants of thermal inactivation for the WT firefly luciferase Luciola mingrelica and its mutant forms at 37°C at the enzyme concentration 106 - 108mol/L. Conditions 0.05 mol/L Tris-acetate buffer,...

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