scholarly journals Regulatory effects of ATP and luciferin on firefly luciferase activity

1995 ◽  
Vol 305 (3) ◽  
pp. 929-933 ◽  
Author(s):  
N Lembert ◽  
L Å Idahl
Keyword(s):  
Steady State ◽  
Active Site ◽  
Luciferase Activity ◽  
Firefly Luciferase ◽  
Kinetic Properties ◽  
Allosteric Site ◽  
Experimental Conditions ◽  
Regulatory Effects ◽  
High Concentrations ◽  
Firefly Luciferase Activity

ATP and luciferin are not only substrates of firefly luciferase, but can, in addition, modulate its activity. High concentrations of luciferin induce a conformational change of the enzyme that temporarily reduces the catalytic rate. Re-activation takes approx. 20 min and is independent of variation in the concentration of enzyme or ATP, but lengthens with increasing luciferin concentration. High concentrations of albumin reduce this luciferin effect. The kinetic properties of firefly luciferase determined from initial rates and at steady state after 1 min of catalysis have been analysed according to Michaelis-Menten kinetics. There is only one active site for each of the substrates. At steady state the Km and Vmax. values for both substrates are reduced in an uncompetitive manner. Hyperbolic Lineweaver-Burk plots indicate an activation by ATP probably by binding to an allosteric site. A model is presented which incorporates luciferin induced de- and re-activation effects. Experimental conditions to avoid the regulatory effects of substrates during ATP monitoring are proposed.

scholarly journals Re-examination of the roles of PEP and Mg2+ in the reaction catalysed by the phosphorylated and non-phosphorylated forms of phosphoenolpyruvate carboxylase from leaves of Zea mays

1998 ◽  
Vol 332 (3) ◽  
pp. 633-642 ◽  
Author(s):  
Alejandro TOVAR-MÉNDEZ ◽  
Rogelio RODRÍGUEZ-SOTRES ◽  
Dulce M. LÓPEZ-VALENTÍN ◽  
Rosario A. MUÑOZ-CLARES
Keyword(s):  
Active Site ◽  
Phosphoenolpyruvate Carboxylase ◽  
Metal Ion ◽  
Dissociation Constants ◽  
Physiological Role ◽  
Free Enzyme ◽  
Kinetic Properties ◽  
Allosteric Site ◽  
Physiological Concentration

To study the effects of phosphoenolpyruvate (PEP) and Mg2+ on the activity of the non-phosphorylated and phosphorylated forms of phosphoenolpyruvate carboxylase (PEPC) from Zea maysleaves, steady-state measurements have been carried out with the free forms of PEP (fPEP) and Mg2+ (fMg2+), both in a near-physiological concentration range. At pH 7.3, in the absence of activators, the initial velocity data obtained with both forms of the enzyme are consistent with the exclusive binding of MgPEP to the active site and of fPEP to an activating allosteric site. At pH 8.3, and in the presence of saturating concentrations of glucose 6-phosphate (Glc6P) or Gly, the free species also combined with the active site in the free enzyme, but with dissociation constants at least 35-fold that estimated for MgPEP. The latter dissociation constant was lowered to the same extent by saturating Glc6P and Gly, to approx. one-tenth and one-sixteenth in the non-phosphorylated and phosphorylated enzymes respectively. When Glc6P is present, fPEP binds to the active site in the free enzyme better than fMg2+, whereas the metal ion binds better in the presence of Gly. Saturation of the enzyme with Glc6P abolished the activation by fPEP, consistent with a common binding site, whereas saturation with Gly increased the affinity of the allosteric site for fPEP. Under all the conditions tested, our results suggest that fPEP is not able to combine with the allosteric site in the free enzyme, i.e. it cannot combine until after MgPEP, fPEP or fMg2+ are bound at the active site. The physiological role of Mg2+ in the regulation of the enzyme is only that of a substrate, mainly as part of the MgPEP complex. The kinetic properties of maize leaf PEPC reported here are consistent with the enzyme being well below saturation under the physiological concentrations of fMg2+ and PEP, particularly during the dark period; it is therefore suggested that the basal PEPC activity in vivois very low, but highly responsive to even small changes in the intracellular concentration of its substrate and effectors.

Opiate Analgesics' Dual Role in Firefly Luciferase Activity

Biochemistry ◽  
1998 ◽  
Vol 37 (13) ◽  
pp. 4451-4458 ◽  
Author(s):  
T. Sudhaharan ◽  
A. Ram Reddy
Keyword(s):  
Luciferase Activity ◽  
Firefly Luciferase ◽  
Dual Role ◽  
Opiate Analgesics ◽  
Firefly Luciferase Activity

Plasmalemma and Tonoplast Influxes of Potassium in Barley Roots, and Interpretation of the Absorption Isotherm at High Concentrations

1975 ◽  
Vol 2 (2) ◽  
pp. 177 ◽  
Author(s):  
JB Robinson ◽  
GG Laties
Keyword(s):  
Steady State ◽  
High Concentration ◽  
Experimental Conditions ◽  
Apparent Increase ◽  
Absorption Isotherm ◽  
Low Salt ◽  
High Concentrations ◽  
High Concentration Range ◽  
Kinetics Of ◽  
Potassium Exchange

Estimates of plasmalemma influx and steady-state vacuolar influx of potassium in low-salt barley roots have been obtained in the concentration range 10-80 mM by the use of controlled loading and washing times. Both fluxes are reduced by preloading the tissue in solutions containing potassium. When the experimental temperature is increased from 20 to 30°C, an apparent increase in the steady-state vacuolar influx occurs; separation of this flux from the apparent plasmalemma influx is not possible. The data support the hypothesis that the kinetics of potassium exchange in barley root tissue may be confounded by both loading time and temperature, and thus interpretation of the influx isotherm in the high concentration range is difficult. Where experiments are carried out at 20-25°C the steady- state vacuolar influx is measured unless closely defined experimental conditions obtain. At higher temperatures the plasmalemma influx may be measured.

Another Look at Thrombin-GPIba Interaction in Solution Phaseα

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1646-1646
Author(s):  
Subramanian Yegneswaran ◽  
James R. Roberts ◽  
Richard A. McClintock ◽  
Zaverio M. Ruggeri
Keyword(s):  
Steady State ◽  
Active Site ◽  
Binding Capacity ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Solution Phase ◽  
Complete Agreement ◽  
Experimental Conditions ◽  
Platelet Surface ◽  
Covalently Linked

Abstract Glycoprotein (GP) Ib in the GPIb-IX-V receptor complex is the most abundant binding site for thrombin on the platelet surface. Virtually the entire thrombin binding capacity of GPIb has been shown to reside on the N-terminal region of the GPIba subunit of GPIb. Recently, Celikel et al and Dumas et al independently solved the structure of the thrombin-GPIba complex. Although comparable N-terminal fragments, comprising residues 1–290 of GPIba, were used for crystallization in both studies, significant differences existed between the two structures. Thus, it is still unclear how GPIb interacts with thrombin. In this study we have examined the interaction of GPIba with thrombin in solution phase. Human a-thrombin was labeled active site-specifically with either dansyl (D) dye via a Glu-Gly-Arg (EGR) linker to yield DEGR-thrombin or with a fluorescein or 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid dye (IAEDANS) via a Phe-Pro-Arg tether to yield Fluorescein-thrombin and AEDANS-thrombin, respectively. When DEGR-thrombin (initially 100 nM) was titrated with human glycocalicin, the N-terminal fragment of GPIbα compring ~400 residues, the steady state anisotropy of DEGR-thrombin decreased by ~ 22% before reaching a plateau at ~ 100 nM protein suggesting an interaction between Glycocalicin and DEGR-thrombin. A ~ 10% increase in anisotropy of the dansyl moiety was observed when a recombinant wild-type fragment of GPIba (residues 1–290) containing the three sulfated-tyrosines at positions 276, 277 and 279 was titrated into DEGR-thrombin. However, this change in anisotropy was not observed when either a mutant with tyrosine 276 mutated to phenylalanine (Y276F) or a Y279F mutant (named analogously) were titrated into DEGR-thrombin. To examine if dimerization of GPIba was important for thrombin interaction, a construct was made such that residues 1–288 of GPIba were covalently linked through a C-terminal extended sequence containing 4 Cys residues, and expressed as dimer (C65 +). When C65+ was titrated into DEGR-thrombin, the anisotropy of the dansyl probe increased by ~ 29% before reaching a plateau at 130 nM C65+, suggesting that thrombin can bind dimeric GPIba. To elucidate the stoicheometry of the thrombin-GPIba complex, resonance energy transfer (RET) experiments were performed between AEDANS donor-labeled thrombin and Fluorescein acceptor labeled thrombin. The AEDANS-thrombin and Fl-thrombin were mixed in equimolar ratios and then titrated with increasing amounts of GPIba. No change in donor intensity was observed suggestive of the absence of a AEDANS-thrombin- GPIba- Fl-thrombin complex. In conclusion, our data suggests that the GPIba interaction with thrombin can be observed in solution phase using steady state fluorescence by appropriately active site-labeled thrombin. Tyrosine sulfation at positions 276 and 279 are critical for this interaction. This observation is in complete agreement with both crystal structures where the contact site with exosite II of thrombin seems to be mediated by residues 275–279 of GPIba. However, using RET, we could not find a thrombin-GPIba-thrombin complex in solution under the present experimental conditions.

Distinct functional roles of the two terminal halves of eukaryotic phosphofructokinase

2012 ◽  
Vol 445 (2) ◽  
pp. 213-218 ◽  
Author(s):  
Oscar H. Martínez-Costa ◽  
Valentina Sánchez ◽  
Antonio Lázaro ◽  
Eloy D. Hernández ◽  
Keith Tornheim ◽  
...  
Keyword(s):  
Active Site ◽  
Kinetic Properties ◽  
Wild Type ◽  
Allosteric Site ◽  
Functional Roles ◽  
Allosteric Interactions ◽  
C Terminus ◽  
N Terminus ◽  
Metabolite Binding

Eukaryotic PFK (phosphofructokinase), a key regulatory enzyme in glycolysis, has homologous N- and C-terminal domains thought to result from duplication, fusion and divergence of an ancestral prokaryotic gene. It has been suggested that both the active site and the Fru-2,6-P2 (fructose 2,6-bisphosphate) allosteric site are formed by opposing N- and C-termini of subunits orientated antiparallel in a dimer. In contrast, we show in the present study that in fact the N-terminal halves form the active site, since expression of the N-terminal half of the enzymes from Dictyostelium discoideum and human muscle in PFK-deficient yeast restored growth on glucose. However, the N-terminus alone was not stable in vitro. The C-terminus is not catalytic, but is needed for stability of the enzyme, as is the connecting peptide that normally joins the two domains (here included in the N-terminus). Co-expression of homologous, but not heterologous, N- and C-termini yielded stable fully active enzymes in vitro with sizes and kinetic properties similar to those of the wild-type tetrameric enzymes. This indicates that the separately translated domains can fold sufficiently well to bind to each other, that such binding of complementary domains is stable and that the alignment is sufficiently accurate and tight as to preserve metabolite binding sites and allosteric interactions.

Enhancement of firefly luciferase activity by cytidine nucleotides

1992 ◽  
Vol 204 (2) ◽  
pp. 283-291 ◽  
Author(s):  
Sharon R. Ford ◽  
Marliese S. Hall ◽  
Franklin R. Leach
Keyword(s):  
Luciferase Activity ◽  
Firefly Luciferase ◽  
Firefly Luciferase Activity
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