Podcasts about michaelis menten

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.22.550177v1?rss=1 Authors: Knapp, B., Willis, L., Gonzalez, C., Vashistha, H., Touma, J. J., Tikhonov, M., Ram, J., Salman, H., Elias, J. E., Huang, K. C. Abstract: The impact of temperature on growth is typically considered under heat- or cold-shock conditions that elicit specific regulation. In between, cellular growth rate varies according to the Arrhenius law of thermodynamics. Here, we use growth-rate dynamics during transitions between temperatures to discover how this behavior arises and what determines the temperature sensitivity of growth. Using a device that enables single-cell tracking across a wide range of temperatures, we show that bacteria exhibit a highly conserved, slow response to temperatures upshifts with a time scale of ~1.5 doublings at the higher temperature, regardless of initial/final temperature or nutrient source. We rule out transcriptional, translational, and membrane reconfiguration as potential mechanisms. Instead, we demonstrate that an autocatalytic enzyme network incorporating temperature-sensitive Michaelis-Menten kinetics recapitulates all temperature-shift dynamics, reveals that import dictates steady-state Arrhenius growth behavior, and successfully predicts alterations in the upshift response observed under simple-sugar or low-nutrient conditions or in fungi. These findings indicate that metabolome rearrangement dictates how temperature affects microbial growth. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

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Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.18.336891v1?rss=1 Authors: Ueno, H., Kato, M., Minagawa, Y., Hirose, Y., Noji, H. Abstract: Alkaline phosphatase (ALP), a homo-dimeric enzyme has been widely used in various bioassays as disease markers and enzyme probes. Recent advancements of digital bioassay revolutionized ALP-based diagnostic assays as seen in rapid growth of digital ELISA and the emerging multiplex profiling of single-molecule ALP isomers. However, the intrinsic heterogeneity found among ALP molecules hampers the ALP-based quantitative digital bioassays. This study aims quantitative analysis of single-molecule activities of ALP from Escherichia coli and reveals the static heterogeneity in catalytic activity of ALP with two distinct populations: half-active and fully active portions. Digital assays with serial buffer exchange uncovered single-molecule Michaelis-Menten kinetics of ALP; half-active molecules have halved values of the catalytic turnover rate, kcat, and the rate constant of productive binding, kon, of the fully active molecules. These findings suggest that half-active ALP molecules are heterogenic dimers composed of inactive and active monomer units, while fully active ALP molecules comprise two active units. Static heterogeneity was also observed for ALP with other origins: calf intestine or shrimp, showing how the findings can be generalized across species. Cell-free expression of ALP with disulfide bond enhancer and spiked zinc ion resulted in homogenous population of ALP of full activity, revealing that inactive monomer units of ALP are deficient in disulfide bond formation and zinc ion coordination, and also offering the way to prepare homogenous and active populations of ALP for quantitative digital bioassays of ALP. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.07.330233v1?rss=1 Authors: Luong, J. A., Vater, A., Siegel, J. B. Abstract: The relatively small size and scope of most current datasets of biophysical mutation effects in enzymes limit the ability to develop data-driven algorithms enabling accurate generative modeling tools for designing novel enzyme function. Here, the Michaelis-Menten constants (kcat, KM, and kcat/KM) and thermal stability (TM) of five new mutations of {beta}-glucosidase B from Paenibacillus polymyxa (BglB) are characterized. Foldit software was used to create molecular models of the mutants, for which synthetic genes were constructed and the corresponding proteins produced and purified from E. coli. It was found that mutations that disrupted pre-existing hydrogen bonds near the active site had reduced expression in contrast to mutations at the same site that did not affect native hydrogen bonding. This is consistent with previous results showing the relationship between hydrogen bonding and enzyme functionality. These mutants contribute to a growing data set of >100 mutants that have been characterized for expression, kinetic, and thermal properties. Copy rights belong to original authors. Visit the link for more info

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.06.29.177675v1?rss=1 Authors: Shin, M., Copeland, J. M., Venton, B. J. Abstract: Drosophila melanogaster, the fruit fly, is an exquisite model organism to understand neurotransmission. Dopaminergic signaling in the Drosophila mushroom body (MB) is involved in olfactory learning and memory, with different compartments controlling aversive learning (corner) vs appetitive learning (medial tip). Here, the goal was to develop techniques to measure endogenous dopamine in compartments of the MB for the first time. We compared three stimulation methods: acetylcholine (natural stimulus), P2X2 (chemogenetics), and CsChrimson (optogenetics). Evoked dopamine release was measured with fast-scan cyclic voltammetry in isolated adult Drosophila brains. Acetylcholine stimulated the largest dopamine release (0.40 M), followed by P2X2 (0.14 M), and CsChrimson (0.07 M). With the larger acetylcholine and P2X2 stimulations, there were no regional or sex differences in dopamine release. However, with CsChrimson, dopamine release was significantly higher in the corner than the medial tip, and females had more dopamine than males. Michaelis-Menten modeling of the single-light pulse revealed no significant regional differences in Km, but the corner had a significantly lower Vmax (0.12 M/s vs. 0.19 M/s) and higher dopamine release (0.05 M vs. 0.03 M). Optogenetic experiments are challenging because CsChrimson is also sensitive to blue light used to activate green fluorescent protein, and thus, light exposure during brain dissection must be minimized. These experiments expand the toolkit for measuring endogenous dopamine release in Drosophila, introducing chemogenetic and optogenetic experiments for the first time. With a variety of stimulations, different experiments will help improve our understanding of neurochemical signaling in Drosophila. Copy rights belong to original authors. Visit the link for more info

Synes du også, at matematik er svært at forstå - og måske ret kedeligt? Det første er vi på redaktionen enige i; vi har alle kæmpet med det der matematik! Men der er matematik bag nærmest alt fra vejrudsigter, forsøg med at omdanne cellulose til biobenzin, epidemiologi og politik. Så alene af den grund er matematik ikke spor kedeligt! Men for at gå lidt 'let' til det, bruger vi matematisk modellering som gennemgående emne. Her fortæller Lone Simonsen, hvad man gør, når truslen om en pandemi, som f.eks. SARS, rammer verden? Og hvordan man reducerer børnedødelighed?Jesper Hansen fortæller bl.a. om lab on a chip; et slags laboratorium på størrelse med en kuglepen. Det bruger man til at tage blodprøver fra dyr; for at tjekke om de er syge. Ret smart! Natasja Nielsen fortæller om enzymkinetik, som hun har brugt til at prøve at forvandle cellulose om til biobenzin.Du kan også høre om matematikangst, en gymnasielinje på Ordrup Gymnasium hvor man kan læse matematik og musik, Michaelis–Menten ligningen samt om kapillærkraft.Du kan her læse Vagn Lundsgaard Hansen artikel: Din verden er fyldt med matematik. Og hvis du er blevet interesseret i 12-tone musik, så tjek f.eks. Arnold Schoenbergs Piano Concerto, Op. 42 fra 1942. Det kan du læse om her i den engelsksprogede Wikipedia. VI HØRER OGSÅ MEGET GERNE FRA DIG, hvis du har idéer, kommentarer eller andet, du gerne vil dele. Skriv på Facebook eller her på mailen: nalle@polykrom.media. Og vi kommer glad og gerne ud på f.eks. din skole, gymnasie osv. for at fortælle om NATURLIGVIS og/eller naturvidenskabelig formidling :)Værter: Cecilie Magnussen & Nalle Kirkvåg. Sounddesign: Frederik Stilling.

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Prof. Jeff Gore discusses the kinetics of gene expression. Simple input-output relationships and chemical/enzyme kinetics. Response time for stable proteins. Ultrasensitivity: cooperative binding or multimer molecular titration.

This video discusses the importance and utility of enzyme kinetics for drug development and derives the Michaelis-Menten equation for a simple enzyme-substrate system.

Das Ziel dieser Arbeit war es, die konzentrationsabhängige Metabolisierung des tabakspezifischen Nitrosamins 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanon (NNK) in den Mikrosomen von Lunge und Leber des Menschen zu charakterisieren. Es wurden kommerziell erhältliche gepoolte Mikrosomen verwendet um vergleichbare Ergebnisse in den verschiedenen Ansätzen zu erzielen. Die Mikrosomen wurden bei einer Proteinkonzentration von 0,2 mg/mL 20 min mit [5-3H]-NNK inkubiert. Zur Bestimmung der Kinetik kamen je 16 Konzentrationen von 0,006 bis 499 µM zum Einsatz. Die Charakterisierung des NNK-Metabolismus mit spezifischen Hemmstoffen für Cytochrom P450 (CYP) Isoenzyme, α-Naphthoflavon (NF; CYP 1A1/2), 8-Methoxypsoralen (MOP; CYP 2A6/13), Chlorzoxazon (CZ; CYP 2E1) und Troleandomycin (TAO; CYP 3A4/5) alleine und in Kombination aller 4 Stoffe, erfolgte bei 46 nM und 49 µM NNK und Hemmstoffkonzentrationen von 1, 5, 10, 25 und 50 µM. Der Einfluss von Nicotin und seines Hauptmetaboliten Cotinin wurde bei den gleichen NNK-Konzentrationen mit einem 300- bzw. 3000-fachen Überschuss der Alkaloide geprüft. Art und Menge der entstandenen Metaboliten wurde durch Hochdruckflüssigkeitschromatographie (HPLC) mit on-line Radioaktivitätsdetektion ermittelt. Durch Einsatz eines neuen Detektors mit vier hintereinander liegenden Messzellen und integrierten Additionsverfahren gelang es die Nachweisgrenze gegenüber handelsüblichen Detektoren um den Faktor 10 zu senken. Die Zuordnung der Metaboliten erfolgte durch Co-Chromatographie von Referenzsubstanzen, die durch UV-Detektion bei 245 nm bestimmt wurden. In Humanlebermikrosomen wurden 5 NNK-Metaboliten nachgewiesen, das Reduktionsprodukt 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), die Produkte der α-Methylenhydroxylierung von NNK und NNAL, 4-Oxo-4-(3-pyridyl)-butansäure (Ketosäure) und 4-Hydroxy-4-(3-pyridyl)-butansäure (Hydroxysäure), das Produkt der α-Methylhydroxylierung von NNK, 4-Hydroxy-1-(3-pyridyl)-1-butanon (HPB) und das N-Oxidationsprodukt von NNK, das NNK-N-Oxid. Humane Lungenmikrosomen bildeten die gleichen Metaboliten außer HPB. Die Umsätze konnten für alle Metaboliten über den gesamten Konzentrationsbereich von 6 nM bis 500 µM einer Reaktionskinetik nach Michaelis-Menten angepasst werden. Bei Anpassung nur im niedrigen, nanomolaren Bereich ergaben sich für alle Metaboliten außer Ketosäure und HPB km- und Vmax-Werte, die um 2 bis 3 Größenordnungen niedriger lagen als die Werte, die für den gesamten Konzentrationsbereich erhalten wurden. Dabei änderte sich die katalytische Effizienz, der Quotient aus Vmax/km-Werten nur geringfügig. Die Hemmstoffe wirkten in Lebermikrosomen stärker als in Lungenmikrosomen. Bei keinem der Hemmversuche konnte jedoch selbst unter Verwendung der höchsten Konzentration eine vollständige Hemmung der NNK-Verstoffwechslung durch α-Hydroxylierung und N-Oxidation erzielt werden. Die CYP-Inhibitoren hatten erwartungsgemäß nur einen geringen Einfluss auf die NNK-Reduktion zu NNAL. In der Leber wurde die HPB-Bildung am stärksten gehemmt, gefolgt von der NNK-N-Oxidation, der Bildung von Ketosäure und der Bildung von Hydroxysäure. In Lungenmikrosomen war die Hemmung der NNK-N-Oxidation am stärksten ausgeprägt. Die größten Unterschiede zwischen der nano- und der mikromolaren NNK-Konzentration zeigte sich in Lebermikrosomen bei Einsatz von NF mit mäßiger bis starker Hemmung aller Stoffwechselwege bei 46 nM NNK und keiner Hemmung bis z.T. leichter Steigerung des Metabolismus bei 49 µM NNK. Auffällige Unterschiede auch bei TAO in Leber und Lunge und bei CZ in der Leber zeigen, dass sich Versuche mit bisher verwendeten hohen NNK-Konzentrationen nur bedingt auf niedrigere Konzentrationen übertragen lassen. Die in früheren Untersuchungen gezeigte Hemmung des NNK-Stoffwechsels konnte bei der mikromolaren NNK-Konzentration für die α-Hydroxylierung von NNK in Lungen- und Lebermikrosomen bestätigt werden. In der Leber wurde auch die NNK-N-Oxidation deutlich gehemmt. Überraschend war die Hemmung der Reduktion von NNK zu NNAL in Lungen- und Lebermikrosomen. All diese Effekte gingen bei Einsatz der nanomolaren NNK-Konzentration verloren. Es ist deshalb fraglich, ob unter realen Bedingungen bei Rauchern der NNK-Stoffwechsel durch die Tabakalkaloide beeinflusst wird. Die vorliegenden Untersuchungen zeigen, dass die bei Verwendung unrealistisch hoher Konzentrationen von Fremdstoffen in vitro erzielten Ergebnisse nicht ohne weiteres auf die tatsächliche Belastungssituation des Menschen durch Umwelt, Nahrung und Genuss von Tabakwaren zu übertragen sind.

Dense collagen implants were developed which can be easily manufactured by extrusion at room temperature without the need of organic solvents. The physicochemical properties (matrix surface pattern, apparent matrix density, melting temperatures and swelling behavior) of the collagen materials and matrices were investigated. Furthermore, the diffusion coefficients of water inside the collagen devices (5.76*E-02cm²/h) and of various FITC dextrans in solution (e.g. FITC dextran 70: 2.4*E-03cm²/h) were determined by PFG-NMR and FCS, respectively. The developed collagen devices were used to investigate the enzymatic collagen matrix degradation and the release of higher molecular weight drugs, e.g. proteins. Several processes, i.e. diffusion, swelling and erosion, contribute to the overall release profile from collagen devices. Since it was desired to obtain a delivery system which controls release mainly by erosion, insoluble collagen type I materials were used to enhance the resistance against enzymatic attack. Besides this, collagen was physically or chemically cross-linked in some experiments to further restrict collagen digestion and drug delivery. It was shown that model compounds like BSA or FITC dextran 20, 70 and 150, respectively, could be incorporated and that their delivery could be controlled by the used collagen matrix material, e.g. animal source or cross-linking degree, the matrix dimensions (length or diameter of the extrudates), the molecular weight of the incorporated model compound and the drug load. The in vitro release of FITC dextrans and BSA was investigated and delivery of 80% model drug was in the range between 7h and 5d. Comparsion of the in vitro and the in vivo release (monitored in adult domestic pigs) of BSA was made by ESR. Similar results were obtained and it was shown that the mechanism of release changed from mainly diffusion towards erosion control by increasing the degree of matrix cross-linking. The degradation of insoluble collagen type I by bacterial collagenase was studied in detail to gain further insights into the enzymatic hydrolysis of collagen. In contrast to a simple Michaelis-Menten kinetic, adsorption of collagenase onto the substrate surface plays an important role. Based on the obtained in vitro results a mathematical model was developed to describe drug release from collagen matrices undergoing enzymatic degradation. Equations for the collagen degradation and the drug release were implemented, adsorption and diffusion phenomena were incorporated and a mixture of experimentally determined and fitted parameters was used to feed the model. Good correlation between experimental and simulated data was found. Histological evaluations demonstrated that the developed minirods showed good biocompatibility, with only minor inflammation reactions and normal tissue remodeling. This emphasized the assumption that collagen extrudates could be used in vivo without surgical removal after drug depletion.