Diffusional increase and decrease in half-maximal-activity substrate concentrations with two-substrate enzymic reactions.
Ontology highlight
ABSTRACT:
SUBMITTER: Engasser JM
PROVIDER: S-EPMC1185780 | biostudies-other | 1978 Jul
REPOSITORIES: biostudies-other
Similar Datasets
Project description:BackgroundSnake envenoming is a major clinical problem in Sri Lanka, with an estimated 40,000 bites annually. Antivenom is only available from India and there is a high rate of systemic hypersensitivity reactions. This study aimed to investigate whether the rate of infusion of antivenom reduced the frequency of severe systemic hypersensitivity reactions.Methods and findingsThis was a randomized comparison trial of two infusion rates of antivenom for treatment of non-pregnant adult patients (>14 y) with snake envenoming in Sri Lanka. Snake identification was by patient or hospital examination of dead snakes when available and confirmed by enzyme-immunoassay for Russell's viper envenoming. Patients were blindly allocated in a 11 randomisation schedule to receive antivenom either as a 20 minute infusion (rapid) or a two hour infusion (slow). The primary outcome was the proportion with severe systemic hypersensitivity reactions (grade 3 by Brown grading system) within 4 hours of commencement of antivenom. Secondary outcomes included the proportion with mild/moderate hypersensitivity reactions and repeat antivenom doses. Of 1004 patients with suspected snakebites, 247 patients received antivenom. 49 patients were excluded or not recruited leaving 104 patients allocated to the rapid antivenom infusion and 94 to the slow antivenom infusion. The median actual duration of antivenom infusion in the rapid group was 20 min (Interquartile range[IQR]:20-25 min) versus 120 min (IQR:75-120 min) in the slow group. There was no difference in severe systemic hypersensitivity reactions between those given rapid and slow infusions (32% vs. 35%; difference 3%; 95%CI:-10% to +17%;p = 0.65). The frequency of mild/moderate reactions was also similar. Similar numbers of patients in each arm received further doses of antivenom (30/104 vs. 23/94).ConclusionsA slower infusion rate would not reduce the rate of severe systemic hypersensitivity reactions from current high rates. More effort should be put into developing better quality antivenoms.Trial registrationwww.slctr.lk SLCTR/2007/005.
| S-EPMC3377702 | biostudies-literature
Project description:Insights into the pathogenesis of migraine with aura may be gained from a study of human Ca(V)2.1 channels containing mutations linked to familial hemiplegic migraine (FHM). Here, we extend the previous single-channel analysis to human Ca(V)2.1 channels containing mutation V1457L. This mutation increased the channel open probability by shifting its activation to more negative voltages and reduced both the unitary conductance and the density of functional channels in the membrane. To investigate the possibility of changes in Ca(V)2.1 function common to all FHM mutations, we calculated the product of single-channel current and open probability as a measure of Ca(2+) influx through single Ca(V)2.1 channels. All five FHM mutants analyzed showed a single-channel Ca(2+) influx larger than wild type in a broad voltage range around the threshold of activation. We also expressed the FHM mutants in cerebellar granule cells from Ca(V)2.1alpha(1)-/- mice rather than HEK293 cells. The FHM mutations invariably led to a decrease of the maximal Ca(V)2.1 current density in neurons. Current densities were similar to wild type at lower voltages because of the negatively shifted activation of FHM mutants. Our data show that mutational changes of functional channel densities can be different in different cell types, and they uncover two functional effects common to all FHM mutations analyzed: increase of single-channel Ca(2+) influx and decrease of maximal Ca(V)2.1 current density in neurons. We discuss the relevance of these findings for the pathogenesis of migraine with aura.
| S-EPMC130625 | biostudies-literature
Project description:A graphical procedure is presented for distinguishing between possible mechanisms of one substrate-one product enzymic reactions.
| S-EPMC1173774 | biostudies-other
Project description:Steady-state kinetic equations for isotope exchange are derived for a number of one substrate-one product enzymic mechanisms in which two molecules of substrate or product can be combined with an enzyme molecule at the one time (e.g. allosteric mechanisms). The usual assumption, that the radioactive material is distributed among the substrate and product components according to a first-order law, is not valid. One can recognize whether isotope-exchange kinetics of an enzyme reaction follows first-order behaviour by using various initial concentrations of the labelled substance added to a mixture.
| S-EPMC1165905 | biostudies-other
Project description:BackgroundIt has been suggested that intraoperative electroencephalographic (EEG) burst suppression (BSupp) may be associated with post-operative neurocognitive disorders in the elderly, and EEG-guided anaesthesia may help to reduce BSupp. Despite of this suggestion, a standard treatment does not exist, as we have yet to fully understand the phenomenon and its underlying pathomechanism. This study was designed to address two underlying phenomena-cerebral hypoperfusion and individual anaesthetic overdose.ObjectivesWe aimed to demonstrate that targeted anaesthetic interventions-treating intraoperative hypotension and/or reducing the anaesthetic concentration-reduce BSupp.MethodsWe randomly assigned patients to receive EEG-based interventions during anaesthesia or EEG-blinded standard anaesthesia. If BSupp was detected, defined as burst suppression ratio (BSR) > 0, the primary intervention aimed to adjust the mean arterial blood pressure to patient baseline (MAP intervention) followed by reduction of anaesthetic concentration (MAC intervention).ResultsEEG-based intervention significantly reduced total cumulative BSR, BSR duration, and maximum BSR. MAP intervention caused a significant MAP increase at the end of a BSR > 0 episode compared to the control group. Coincidentally, the maximum BSR decreased significantly; in 55% of all MAP interventions, the BSR decreased to 0% without any further action. In the remaining events, additional MAC intervention was required.ConclusionOur results show that targeted interventions (MAC/MAP) reduce total cumulative amount, duration, and maximum BSR > 0 in the elderly undergoing general anaesthesia. Haemodynamic intervention already interrupted or reduced BSupp, strengthening the current reflections that hypotension-induced cerebral hypoperfusion may be seen as potential pathomechanism of intraoperative BSupp.Clinical trial registrationNCT03775356 [ClinicalTrials.gov], DRKS00015839 [German Clinical Trials Register (Deutsches Register klinischer Studien, DRKS)].
| S-EPMC8931826 | biostudies-literature
Project description:Salutaridine reductase (SalR, EC 1.1.1.248) catalyzes the stereospecific reduction of salutaridine to 7(S)-salutaridinol in the biosynthesis of morphine. It belongs to a new, plant-specific class of short-chain dehydrogenases, which are characterized by their monomeric nature and increased length compared with related enzymes. Homology modeling and substrate docking suggested that additional amino acids form a novel alpha-helical element, which is involved in substrate binding. Site-directed mutagenesis and subsequent studies on enzyme kinetics revealed the importance of three residues in this element for substrate binding. Further replacement of eight additional residues led to the characterization of the entire substrate binding pocket. In addition, a specific role in salutaridine binding by either hydrogen bond formation or hydrophobic interactions was assigned to each amino acid. Substrate docking also revealed an alternative mode for salutaridine binding, which could explain the strong substrate inhibition of SalR. An alternate arrangement of salutaridine in the enzyme was corroborated by the effect of various amino acid substitutions on substrate inhibition. In most cases, the complete removal of substrate inhibition was accompanied by a substantial loss in enzyme activity. However, some mutations greatly reduced substrate inhibition while maintaining or even increasing the maximal velocity. Based on these results, a double mutant of SalR was created that exhibited the complete absence of substrate inhibition and higher activity compared with wild-type SalR.
| S-EPMC2785364 | biostudies-literature
Project description:Some bacterial genomes contain an incomplete set of genes encoding phosphoribosyl isomerases, raising the question of whether there exists broadened substrate specificity for the missing gene products. To investigate the underlying molecular principles of this hypothesis, we have determined the crystal structure of the bifunctional enzyme PriA from Streptomyces coelicolor at 1.8 A resolution. It consists of a (betaalpha)(8)-barrel fold that is assembled by two symmetric (betaalpha)(4) half-barrels. The structure shows how its active site may catalyse the isomerization reactions of two different substrates, and we provide a plausible model of how the smaller of the two substrates could be bound in two different orientations. Our findings expand the half-barrel ancestor concept by demonstrating that symmetry-related half-barrels could provide a smart solution to cope with dual substrate specificity. The data may help to unravel molecular rationales regarding how organisms with miniature genomes can keep central biological pathways functional.
| S-EPMC1299240 | biostudies-literature
Project description:ObjectiveCortisol clearance is reduced in sepsis and may contribute to the development of impaired adrenocortical function that is thought to contribute to the pathophysiology of critical illness-related corticosteroid insufficiency. We sought to assess adrenocortical function using computer-assisted numerical modeling methodology to characterize and compare maximal cortisol secretion rate and free cortisol half-life in septic shock, sepsis, and healthy control subjects.DesignPost hoc analysis of previously published total cortisol, free cortisol, corticosteroid-binding globulin, and albumin concentration data.SettingSingle academic medical center.PatientsSubjects included septic shock (n = 45), sepsis (n = 25), and healthy controls (n = 10).InterventionsI.v. cosyntropin (250 μg).Measurements and main resultsSolutions for maximal cortisol secretion rate and free cortisol half-life were obtained by least squares solution of simultaneous, nonlinear differential equations that account for free cortisol appearance and elimination as well as reversible binding to corticosteroid-binding globulin and albumin. Maximal cortisol secretion rate was significantly greater in septic shock (0.83 nM/s [0.44, 1.58 nM/s] reported as median [lower quartile, upper quartile]) compared with sepsis (0.51 nM/s [0.36, 0.62 nM/s]; p = 0.007) and controls (0.49 nM/s [0.42, 0.62 nM/s]; p = 0.04). The variance of maximal cortisol secretion rate in septic shock was also greater than that of sepsis or control groups (F test, p < 0.001). Free cortisol half-life was significantly increased in septic shock (4.6 min [2.2, 6.3 min]) and sepsis (3.0 min [2.3, 4.8 min] when compared with controls (2.0 min [1.2, 2.6 min]) (both p < 0.004).ConclusionsResults obtained by numerical modeling are consistent with comparable measures obtained by the gold standard stable isotope dilution method. Septic shock is associated with generally not only higher levels but also greater variance of maximal cortisol secretion rate when compared with control and sepsis groups. Additional studies would be needed to determine whether assessment of cortisol kinetic parameters such as maximal cortisol secretion rate and free cortisol half-life is useful in the diagnosis or management of critical illness-related corticosteroid insufficiency.
| S-EPMC8259018 | biostudies-literature
Project description:Citrate synthase (CS) performs two half-reactions: the mechanistically intriguing condensation of acetyl-CoA with oxaloacetate (OAA) to form citryl-CoA and the subsequent, slower hydrolysis of citryl-CoA that generally dominates steady-state kinetics. The condensation reaction requires the abstraction of a proton from the methyl carbon of acetyl-CoA to generate a reactive enolate intermediate. The carbanion of that intermediate then attacks the OAA carbonyl to furnish citryl-CoA, the initial product. Using stopped-flow and steady-state fluorescence methods, kinetic substrate isotope effects, and mutagenesis of active site residues, we show that all of the processes that occur in the condensation half-reaction performed by Thermoplasma acidophilum citrate synthase (TpCS) with the natural thioester substrate, acetyl-CoA, also occur with the ketone inhibitor dethiaacetyl-CoA. Free energy profiles demonstrate that the nonhydrolyzable product of the condensation reaction, dethiacitryl-CoA, forms a particularly stable complex with TpCS but not pig heart CS.
| S-EPMC2765034 | biostudies-literature
Project description:Background50% of leprosy patients suffer from episodes of Type 1/ reversal reactions (RR) and Type 2/ Erythema Nodosum Leprosum (ENL) reactions which lead to morbidity and nerve damage. CD4+ subsets of Th17 cells and CD25+FOXP3+ regulatory T cells (Tregs) have been shown to play a major role in disease associated immunopathology and in stable leprosy as reported by us and others. The aim of our study was to analyze their role in leprosy reactions.Methodology and principle findingsQuantitative reverse transcribed PCR (qPCR), flowcytometry and ELISA were used to respectively investigate gene expression, cell phenotypes and supernatant levels of cytokines in antigen stimulated PBMC cultures in patients with stable disease and those undergoing leprosy reactions. Both types of reactions are associated with significant increase of Th17 cells and associated cytokines IL-17A, IL-17F, IL-21, IL-23 and chemokines CCL20, CCL22 as compared to matching stable forms of leprosy. Concurrently patients in reactions show reduction in FOXP3+ Treg cells as well as reduction in TGF-β and increase in IL-6. Moreover, expression of many T cell markers, cytokines, chemokines and signaling factors were observed to be increased in RR as compared to ENL reaction patients.ConclusionsPatients with leprosy reactions show an imbalance in Th17 and Treg populations. The reduction in Treg suppressor activity is associated withhigherTh17cell activity. The combined effect of reduced TGF-β and enhanced IL-6, IL-21 cytokines influence the balance between Th17 or Treg cells in leprosy reactions as reported in the murine models and autoimmune diseases. The increase in Th17 cell associated cytokines may contribute to lesional inflammation.
| S-EPMC4818038 | biostudies-literature