Podcasts about mg2

PaperPlayer biorxiv biochemistry

Play Episode Listen Later Nov 10, 2020

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.11.09.373902v1?rss=1 Authors: Ladefoged, L. K., Schiott, B., Fedosova, N. U. Abstract: Kinetic properties and crystal structures of the Na+,K+-ATPase in complex with cardiotonic steroids (CTS) revealed significant differences between CTS subfamilies (Laursen et al., 2015): beneficial effects of K+ on bufadienolide binding strongly contrasted with K+/cardenolide antagonism. To solve this riddle we applied docking and molecular dynamics simulations of the complexes involving Na+,K+-ATPase, bufadienolides (bufalin, cinobufagin), and ions (K+, Na+, Mg2+). The results revealed that bufadienolide binding is affected by i) electrostatic attraction of the lactone ring by a cation, and ii) the ability of a cation to stabilize and ''shape'' the site constituted by transmembrane helices of the -subunit (M1-6). The latter effect was due to varying coordination patterns involving amino acid residues from helix bundles M1-4 and M5-10. Substituents on the steroid core of a bufadienolide add to and modify the cation effects. The above rationale is fully consistent with the ion effects on the kinetics of Na+,K+-ATPase/bufadienolide interactions. Copy rights belong to original authors. Visit the link for more info

effects copy binding m1 maleficent cts m5 laursen biorxiv mg2 beneficent na k atpase

Kinetic and thermodynamic analysis defines roles for two metal ions in DNA polymerase specificity and catalysis

Play Episode Listen Later Oct 20, 2020

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.10.20.347138v1?rss=1 Authors: Gong, S., Kirmizialtin, S., Chang, A., Mayfield, J. E., Zhang, Y., Johnson, K. A. Abstract: We examined the roles of Mg2+ ions in DNA polymerization by kinetic analysis of single nucleotide incorporation catalyzed by HIV reverse transcriptase and by molecular dynamics simulation of Mg2+ binding. Binding of the Mg-nucleotide complex induces a conformational change of the enzyme from open to closed states in a process that is independent of free Mg2+ concentration. Subsequently, the second Mg2+ binds weakly to the closed state of the enzyme-DNA-Mg.dNTP complex with an apparent Kd = 3.7 mM and facilitates the catalytic reaction. This weak binding of the catalytic Mg2+ is important to maintain fidelity in that the Mg2+ samples the correctly aligned substrate without perturbing the equilibrium at physiological Mg2+ concentrations. The binding of the catalytic Mg2+ increases nucleotide specificity (kcat/Km) by increasing the rate of the chemistry and decreasing the rate of enzyme opening allowing nucleotide release. Changing the free Mg2+ concentration from 0.25 to 10 mM increased nucleotide specificity (kcat/Km) by 12-fold. Mg2+ binds very weakly to the open state of the enzyme in the absence of nucleotide (Kd ~ 34 mM) and competes with Mg.dNTP. Analysis based on publish crystal structures showed that HIV RT binds only two metal ions during incorporation of a correct base-pair. MD simulations support the kinetic studies suggesting weak binding of the catalytic Mg2+ in open and closed states. They also support the two-metal ion mechanism, although the polymerase may bind a third metal ion in the presence of a mismatched nucleotide. Copy rights belong to original authors. Visit the link for more info

dna md metal hiv roles analysis copy chang kd mm zhang binding defines mg mayfield subsequently km kinetic specificity thermodynamics ions biorxiv catalysis mg2 polymerase

Mg2+-binding shifts the IM30 activity from membrane protection to membrane destabilization

PaperPlayer biorxiv biochemistry

Play Episode Listen Later Sep 26, 2020

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.25.313916v1?rss=1 Authors: Junglas, B., Axt, A., Siebenaller, C., Sonel, H., Hellmann, N., Weber, S. A. L., Schneider, D. Abstract: The inner membrane-associated protein of 30 kDa (IM30) is essential in chloroplasts and cyanobacteria. The spatio-temporal cellular localization of the protein appears to be highly dynamic and triggered by internal as well as external stimuli, mainly light intensity. A soluble fraction of the protein is localized in the cyanobacterial cytoplasm or the chloroplast stroma, respectively. Additionally, the protein attaches to the thylakoid membrane as well as to the chloroplast inner envelope or the cyanobacterial cytoplasmic membrane, respectively, especially under conditions of membrane stress. IM30 is involved in thylakoid membrane biogenesis and/or maintenance, where it either stabilizes membranes and/or triggers membrane-fusion processes. These apparently contradicting processes have to be tightly controlled and separated spatiotemporally in chloroplasts and cyanobacteria. The latter process depends on Mg2+-binding to IM30; yet, it still is unclear how Mg2+-loaded IM30 interacts with membranes and promotes membrane fusion. Here we show that interaction of Mg2+ with IM30 results in increased binding of IM30 to native as well as model membranes. Via Atomic Force Microscopy in liquid, IM30-induced bilayer defects were observed in solid-supported bilayers in presence of Mg2+. The observed interaction of IM30 with membrane surfaces differs dramatically from previously observed membrane-stabilizing, carpet-like structures in the absence of Mg2+. Mg2+-induced alterations of the IM30 structure switches the IM30 activity from a membrane-stabilizing to a membrane-destabilizing function, a crucial step in membrane fusion. Copy rights belong to original authors. Visit the link for more info

activity shifts copy weber schneider binding membrane hellmann axt destabilization biorxiv mg2

Structural determinants of calcium binding beyond the EF-hand binding site: A study of alpha parvalbumins

PaperPlayer biorxiv biochemistry

Play Episode Listen Later Sep 20, 2020

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.18.304063v1?rss=1 Authors: Immadisetty, K., Sun, B., Kekenes-Huskey, P. Abstract: Parvalbumin (PV) is a calcium binding protein expressed in humans, fish and avian species. In these organisms, the calcium (Ca2+) affinities of specific PV isoforms can vary by orders of magnitude. Despite the availability of high resolution structural data for many PV isoforms, the structural bases for how such proteins confer widely-varying divalent Ca2+ affinities and selectivities against common ions like magnesium (Mg2+) has been difficult to rationalize. We therefore conducted molecular simulations of several -pavalbumin (-parvalbumin (PV)) constructs with Ca2+ affinities in the micromolar to nanomolar ranges to identify properties of conformations that contribute to their wide-ranging binding constants and selectivities against Mg2+. Specifically we examined D94S/G98E construct with a reported lower Ca2+ affinity ({approx} -18.2 kcal/mol) relative to the WT ({approx}-22 kcal/mol), an S55D/E59D variant with enhanced affinity ({approx}-24 kcal/mol), and a truncated variant of PV with weak affinity ({approx}-12.6 kcal/mol). We performed molecular dynamics simulations of these constructs and assessed their Ca2+ and Mg2+ binding properties using scores from molecular mechanics generalized Born approximation (MM/GBSA), ion/oxygen coordination patterns and thermodynamics via mean spherical approximation (MSA) theory, as well as via metrics of protein structure and hydration. Our key findings are that although MM/GBSA and MSA scores successfully rank-ordered the variants according to their previously-published affinities and Mg2+ selectivity, importantly, properties of Ca2+ loops in CBPs such as coordination, and charge are alone insufficient to rationalize their binding properties. Rather, Ca2+ affinity and selectivity against 2+ are emergent properties stemming from both local effects within the proteins' ion binding sites as well as non-local contributions from protein folding and solubility. Our findings broaden our understanding of the molecular bases governing PV ion binding that are likely shared by many Ca2+ binding proteins. Copy rights belong to original authors. Visit the link for more info

study sun alpha copy binding structural calcium pv msa wt determinants biorxiv ca2 mg2

Melissa Gonzalez: The Winning Role of Pop-up Shops in Brand Building, Retail + Digital Products

Building A Better Brand

Play Episode Listen Later Sep 17, 2020 56:58

Melissa Gonzalez is a pioneer in bringing the pop-up retail format to the mainstream. She is the founder and CEO of The Lion'esque Group, a firm of award winning retail strategists and pop up architects™ as well as a Principal and Shareholder in MG2, a global architecture firm. Beginning with a deep dive into strategy, Melissa helps clients rethink environments and in-store experiences to convey compelling stories – ones that animate discovery and engagement – attracting new customers, elevating loyalty with existing customers, and building a platform for their future evolution. Some of her impressive clients include brands such as Amazon, COTY, Estee Lauder, M.Gemi, Nordstrom, Purple, Stella & Dot, The RealReal, YUM brands, and others, as well as property groups such as GGP Properties (now Brookfield) and Steiner & Associates. She reflects on her work in her best-selling book, The Pop-Up Paradigm: How Brands Can Build Human Connections in a Digital Age. Melissa is a regular go-to expert in retail and is often featured in Forbes, Fortune, WSJ, Business Insider, Glossy, Digiday, Retail Touchpoints, WWD and on Cheddar and CBS News. She is also a regular contributor for ABC LA Radio, Yahoo Finance and Advertising Week 360. Follow Melissa @Melsstyles and The Lionesque Group @Lionesquegroup Follow Tony Triumph: @tonytriumphofficial (IG), @Tonytriumph10 (Twitter) Co-produced by: Dobi KweMusic by: Yugy

ARL15 modulates magnesium homeostasis through N-glycosylation of CNNMs

Play Episode Listen Later Sep 10, 2020

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.09.09.289835v1?rss=1 Authors: Zolotarov, Y., Ma, C., Gonzalez-Recio, I., Hardy, S., Franken, G., Uetani, N., Latta, F., Kostantin, E., Boulais, J., Thibault, M.-P., Cote, J.-F., Diaz Moreno, I., Diaz Quintana, A., Hoenderop, J. G. J., Martinez-Cruz, L. A., Tremblay, M. L., de Baaij, J. H. F. Abstract: Cyclin M (CNNM1-4) proteins maintain cellular and body magnesium (Mg2+) homeostasis. Using various biochemical approaches, we have identified members of the CNNM family as direct interacting partners of ADP-ribosylation factor-like protein 15 (ARL15), a small GTP-binding protein. ARL15 interacts with CNNMs at their carboxyl-terminal conserved cystathionine-{beta}- synthase (CBS) domains. In silico modeling of the interaction using the reported structures of both CNNM2 and ARL15 supports that the small GTPase specifically binds the CBS1 domain. Immunocytochemical experiments demonstrate that CNNM2 and ARL15 co-localize in the kidney, with both proteins showing subcellular localization in the Golgi-apparatus. Most importantly, we found that ARL15 is required for forming complex N-glycosylation of CNNMs. Overexpression of ARL15 promotes complex N-glycosylation of CNNM3. Mg2+ uptake experiments with a stable isotope demonstrate that there is a significant increase of 25Mg2+ uptake upon knockdown of ARL15 in multiple kidney cancer cell lines. Altogether, our results establish ARL15 as a novel negative regulator of Mg2+ transport by promoting the complex N-glycosylation of CNNMs. Copy rights belong to original authors. Visit the link for more info

cbs copy hardy adp magnesium cote franken thibault tremblay homeostasis latta gtp biorxiv golgi mg2 overexpression glycosylation gtpase

Cryo-EM structure of the MgtE Mg2+ channel pore domain in Mg2+-free conditions reveals cytoplasmic pore opening

Play Episode Listen Later Aug 28, 2020

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.27.270991v1?rss=1 Authors: Jin, F., Sun, M., Fujii, T., Yamada, Y., Wang, J., Maturana, A., Wada, M., Su, S., Ma, J., Takeda, H., Kusakizako, T., Tomita, A., Nakura, Y. N., Liu, K., Uemura, T., Nomura, Y., Nomura, N., Ito, K., Nureki, O., Namba, K., Iwata, S., Yu, Y., Hattori, M. Abstract: MgtE is a Mg2+ channel conserved in organisms ranging from prokaryotes to eukaryotes, including humans, and plays an important role in Mg2+ homeostasis. The previously determined MgtE structures in the Mg2+-bound, closed state and structure-based functional analyses of MgtE revealed that the binding of Mg2+ ions to the MgtE cytoplasmic domain induces channel inactivation to maintain Mg2+ homeostasis. However, due to the lack of a structure of the MgtE channel, including its transmembrane domain in Mg2+-free conditions, the pore-opening mechanism of MgtE has remained unclear. Here, we determined the cryoelectron microscopy (cryo-EM) structure of the MgtE-Fab complex in the absence of Mg2+ ions. The Mg2+-free MgtE transmembrane domain structure and its comparison with the Mg2+-bound, closed-state structure, together with functional analyses, showed the Mg2+-dependent pore opening of MgtE on the cytoplasmic side and revealed the kink motions of the TM2 and TM5 helices at the glycine residues, which are important for channel activity. Overall, our work provides structure-based mechanistic insights into the channel gating of MgtE. Copy rights belong to original authors. Visit the link for more info

sun structure conditions copy wang domain liu yu wada ito takeda nomura yamada iwata pore fujii maturana tomita biorxiv mg2 cryo em cytoplasmic tm2

Synergy of calcium release site determinants in control of calcium release events in cardiac myocytes

PaperPlayer biorxiv bioinformatics

Play Episode Listen Later Aug 27, 2020

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.26.260968v1?rss=1 Authors: Iaparov, B., Zahradnik, I., Moskvin, A. S., Zahradnikova, A. Abstract: Recent data on structure of dyads in cardiac myocytes indicate variable clustering of RyR calcium release channels. The question arises as to how geometric factors of RyR arrangement translate to their role in formation of calcium release events (CRE). Since this question is not experimentally testable in situ, we performed in silico experiments on a large set of calcium release site (CRS) models. The models covered the range of RyR spatial distributions observed in dyads, and included gating of RyRs with open probability dependent on Ca2+ and Mg2+ concentration. The RyR single-channel calcium current, varied in the range of previously reported values, was set constant in the course of CRE simulations. Other known features of dyads were omitted in the model formulation for clarity. CRE simulations initiated by a single random opening of one of the RyRs in a CRS produced spark-like responses with characteristics that varied with RyR vicinity, a newly defined parameter quantifying spatial distribution of RyRs in the CRSs, and with the RyR single-channel calcium current. The CRE characteristics followed the law of mass action with respect to a CRS state variable, defined as a weighed product of RyR vicinity and RyR single-channel calcium current. The results explained the structure-function relations among determinants of cardiac dyads on synergy principles and thus allowed to evolve the concept of CRS as a dynamic unit of cardiac dyad. Copy rights belong to original authors. Visit the link for more info

events copy synergy cardiac cre calcium crs determinants biorxiv ca2 mg2 abstract recent

Carbamazepine and GABA have distinct effects on seizure onset dynamics in mouse brain slices

Play Episode Listen Later Aug 11, 2020

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.08.11.245951v1?rss=1 Authors: Crisp, D., Parent, R., Nakatani, M., Murphy, G. G., Stacey, W. C. Abstract: Optimizing antiepileptic drug therapy is very challenging due to the absence of a reliable method to assess how brain activity changes between seizures. This work uses the Taxonomy of Seizure Dynamics (Saggio et al., 2020) to investigate how anticonvulsants influence seizure onset dynamotypes. The no Mg2+/high K+ mouse brain-slice seizure model (N = 92) was used to generate consistent epileptiform onsets. We compared the onset bifurcations of controls with slices treated with either GABA or carbamazepine. Each anticonvulsant uniquely changed the types of bifurcations in the slices. This experiment provides proof-of-concept evidence that brain states exist on a "map" of seizure dynamics, and that antiepileptic drugs with different mechanisms can change the positioning of the brain states on the map. Copy rights belong to original authors. Visit the link for more info

brain parent effects copy dynamics mouse distinct seizure gaba onset taxonomy slices biorxiv mg2 carbamazepine

46: Accelerated Change within Retail Post Covid 19: Panel Discussion

Influential Visions

Play Episode Listen Later Jun 12, 2020 49:39

Our Expert Panel: - John Ashcroft A specialist in Economics, Strategy, Financial Markets, Creator of The Saturday Economist, NED, Consultant, Advisor, Chair ... Led over 50 corporate finance transactions, worked with First Boston, Bain & Co, Samuel Montagu, Goldman Sachs and many more. Eddie McAtominey: Head of Placecrafting at BECG.COM One of largest environmental communication consultancies in the UK. Eddie is a highly experienced Creative Director with over 25 years of experience across retail and commercial multi-channel brand and Identity development. Melissa Gonzalez is the founder of The Lionesque Group, an award winning firm of experiential retail strategists and pop-up architects™ She is also a Principal and Shareholder in global architecture firm MG2, and author of The Pop-Up Paradigm: Building Human Connections in a Digital Age. For the shownotes visit www.influentialvisions.com/accelerated-change

477: Reinventing Retail

Spark from CBC Radio

Play Episode Listen Later May 28, 2020 54:09

It's been a long time since many of us have stepped inside an actual, physical retail store for anything other than essentials. Across Canada, we're in different stages of reopening in a staggered, uncertain way. Many businesses have shuttered permanently. Some have filed for bankruptcy protection. Others are open, but with restrictions. As e-commerce takes off amid the pandemic, how will smaller vendors compete against the online behemoths? What will the in-store experience look like down the road? Will it change forever? + Craig Patterson is the Founder and Editor-in-Chief of Retail Insider, Canada's leading online retail industry publication. He's also the Director of Applied Research at the School of Retailing at the University of Alberta + Darryl Julott is the senior manager of Digital Main Street, a volunteer-driven group that wants to help independent businesses and artists build online stores free of charge. It's partnered with The City of Toronto and several tech companies to launch the "DMS ShopHERE" initiative. + Melissa Gonzalez is the CEO and founder of retail strategist group The Lion'esque Group, and also a principal and shareholder of global architecture firm MG2.

ceo director university founders canada school toronto chief lion retail reinventing retailing applied research across canada melissa gonzalez mg2 craig patterson retail insider

43: Future of Retail: New Consumer Behaviours in Bricks and Mortar

Influential Visions

Play Episode Listen Later May 22, 2020 36:43

Melissa Gonzalez - CEO of the Lionesque Group and Principal of MG2 and Nat Schooler - discuss: - Online Shopping Contactless Payments Kerbside Pickups BOPIS (Buy online pickup in store) Augmented Reality Fulfilment Centres The Consumer Landscape Here is a link to our website https://www.influentialvisions/future-of-retail-new-consumer-behaviors-in-bricks-and-mortar

principal retail consumer behaviour bricks mortar mg2

Retail Strategist Melissa Gonzalez: Physical Retail Post Covid

Bellwether Culture Podcast

Play Episode Listen Later May 18, 2020 19:16

How retailers need to think through the future of the experiences they will provide. Today's guest is Melissa Gonzalez. She is the CEO & Founder of Lion'esque Group, an award winning firm of experiential retail strategists and pop-up architects™ as well as a Principal and Shareholder in global architecture firm MG2. This conversation took place on April 27, 2020 as part of our Covid Conversations series. We are featuring a conversation daily that we've recently had with over 20 business leaders and influencers, focused on how folks are managing business amidst Covid-19. Our team is putting together a free guide that will highlight the resources and references mentioned across these conversations. DM us on our instagram handle @BellwetherCulture, and we will send you a download link once the series is fully published.

covid-19 ceo founders dm principal retail post covid strategist shareholder covid conversations melissa gonzalez mg2

What's Next In Experiential Retail?

Retail Remix

Play Episode Listen Later Mar 9, 2020 17:39

Melissa Gonzalez, Founder and CEO of the Lion’esque Group, has been one of the driving forces in the era of experiential retail. One of the first experts to delve into the pop-up paradigm (hence her book of the same name), Melissa has worked with countless brands to help them reimagine their brick-and-mortar experiences. Listen in as she shares her take on what’s next in experiential, the technology she’s especially excited about, and how her company’s merger with MG2 will lead to big things for the industry at large.

ceo founders technology lion retail customer experience retailers experiential melissa gonzalez mg2

Novo Toyota Corolla: Análise AO VIVO com o que NINGUÉM comentou sobre os impactos do Lançamento

AutoVideos

Play Episode Listen Later Sep 18, 2019 67:08

O novo Corolla chegou completamente renovado e oferece, pela primeira vez, sistema híbrido que combina três motores, dois elétricos e um a combustão com tecnologia flex. Quais serão os impactos do lançamento no mercado? A 12ª geração do veículo mais vendido do mundo estreia no País com duas opções de motores: 2.0L Dynamic Force flex com transmissão Direct Shift de 10 marchas e injeção direta, e o inédito sistema híbrido que combina três motores, dois elétricos e um a combustão flexfuel. A nova geração do Corolla chega ao mercado brasileiro disponível em três versões diferentes e duas motorizações. O Corolla com motor 2.0L Dynamic Force estará disponível nas versões GLi, XEi e Altis, enquanto a exclusiva versão com sistema híbrido estará disponível apenas na versão topo de linha Altis. Uma das grandes novidades na linha Corolla 2020 são seus novos propulsores. 2.0L Dynamic Force Dual VVT-iE 16V DOHC de ciclo Atkinson Flex, que rende 177 cv de potência a 6.600 rpm, quando abastecido com etanol, e 169 cv, a 6.600 giros, com gasolina. O torque máximo abastecido com etanol ou gasolina é 21,4 kgfm a 4.400 rpm. sistema híbrido da Toyota, nesta nova geração do Corolla, combina um motor a gasolina de 1.8L VVT-i 16V de ciclo Atkinson flex, com 101 cv de potência a 5.200 giros quando abastecido com etanol, e 98 cv também a 5.200 rpm, quando abastecido com gasolina, e 14,5 kgfm de torque a 3.600 rpm (abastecido com etanol ou gasolina). Esse motor funciona em conjunto com dois motores elétricos (MG1 e MG2) de 72 cv de potência e 16,6 kgfm de torque, garantindo aceleração suave e excelente conforto ao rodar em qualquer tipo de condução. Segundo o INMETRO, o Corolla híbrido é capaz de rodar 14,5 km/l na estrada e 16,3 km/l na cidade quando abastecido com gasolina. Com etanol, o modelo roda 9,9 km/l na estrada e 10,9 km/l na cidade. No quesito segurança, desde a versão de entrada GLi, o Corolla já vem dotado de sete airbags (dois frontais, dois laterais, dois de cortina e um de joelho para o motorista), câmera de ré com linhas de distância com projeção na central multimídia, faróis com acendimento automático e com ajuste de altura, controle eletrônico de estabilidade (VSC), controle eletrônico de tração (TRC), sistema de assistência ao arranque em subida (HAC), sistema ISOFIX para fixação de cadeirinhas infantis no banco traseiro Os preços do novo Toyota Corolla 2020 são: GLi 2.0L Dynamic Force Flex R$ 99.990,00 XEi 2.0L Dynamic Force Flex R$ 110.990,00 Altis 2.0L Dynamic Force Flex R$ 124.990,00 Altis Híbrido Flex R$ 124.990,00 E você, acha que o novo Toyota Corolla vai continuar sendo líder de segmento ao revolucionar o mercado com a nova motorização Híbrida? Deixe nos comentários a sua opinião e entre na conversa. Acompanhe o melhor conteúdo automotivo nas nossas redes sociais e site: SITE: https://autovideos.com.br/ FACEBOOK: https://www.facebook.com/AUTOVideosBr... INSTAGRAM: @autovideos_oficial

uma esse segundo toyota quais novo ningu acompanhe gli deixe atkinson impactos ao vivo hac corolla toyota corolla trc vsc altis mg2 inmetro comentou

The Gear Dive S/E: New P790 and MG2 with TaylorMade's Paul Demkowski and Matt Bovee

GolfWRX Radio

Play Episode Listen Later Aug 12, 2019 46:00

In this special edition of THE GEAR DIVE, Johnny Chats with Paul Demkowski and Matt Bovee of TaylorMade on all things P790, UDI, MG2 and so much more.

dive gear taylormade udi bovee mg2

Activity-driven formation and stabilization of functional spine synapses

Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 06/06

Play Episode Listen Later Oct 10, 2015

Physical changes in neuronal connections, dictated by the neuronal network activity, are believed to be essential for learning and memory. Long-term potentiation (LTP) of synaptic transmission has emerged as a model to study activity-driven plasticity. The majority of excitatory contacts between neurons, called synapses, are found on spines, small dendritic protrusions. LTP is known to trigger the formation and stabilization of new dendritic spines in vitro. Similarly, experience-dependent plasticity in vivo is associated with changes in the number and stability of spines. However, to date, the contribution of excitatory synaptogenesis to the enhanced synaptic transmission after LTP remains elusive. Do new spines form functional synapses with the inputs stimulated during LTP induction and thereby follow Hebbian co-activation rules, or do they connect with random partners? Furthermore, at which time-point are de novo spines functionally integrated into the network? I developed an optical approach to stably and exclusively stimulate the axons of a defined channelrhodopsin-2 (ChR2)-transduced subset of CA3 cell in mature hippocampal slice culture over extended periods of time (up to 24h). I continuously monitored synaptic activation and synaptic structure of CA1 cells dendrites using two-photon imaging. To control the dendritic location where LTP and associated spinogenesis were allowed to take place, I globally blocked Na+-dependent action potential firing and directly evoke neurotransmitter release by local light-evoked depolarization of ChR2-expressing presynaptic boutons (in TTX, 4-AP). I induced optical LTP specifically at this location by combining optogenetic activation with chemical pairing (in low [Mg2+]o, high [Ca2+]o, forskolin, and rolipram). Taking advantage of the NMDA-receptor mediated calcium influx during synaptic activation I assessed the formation of functional synapses using the genetically encoded calcium indicator GCaMP6s. I find that optical LTP led to the generation of new spines, decreased the stability of preexisting spines and increased the stability of new spines. Under optical LTP conditions, a fraction of new spines responded to optical presynaptic stimulation within hours after formation. However, the occurrence of the first synaptic calcium response in de novo spines varied considerably, ranging from 8.5 min to 25 h. Most new spines became responsive within 4 h (1.2 ± 0.9 h, mean ± S.D., n = 16 out of 20), whereas the remainder showed their first response only on the second experimental day (18.2 ± 3.7 h). Importantly, new spines generated under optical LTP were more likely to build functional synapses with light-activated, ChR2-expressing axons than spontaneously formed spines (new responsive spines under optical LTP: 64 ± 4 %; control 1: 0%; control 2: 13 ± 4 %; control 3: 11 ± 4 %). Furthermore, new spines that were responsive to optical presynaptic stimulation were less prone to be eliminated after overnight incubation than new spines that failed to respond (% overnight spine survival; 81 ± 3 % new responsive spines; 58 ± 4 % of new unresponsive spines). In summary, the results from my thesis demonstrate that synapses can form rapidly in an input-specific manner.

driven activity formation functional spine stabilization nmda synapses ltp ca2 mg2 ttx ddc:500 ca1 ca3 ddc:570

Drosophila TRPM channel is essential for the control of extracellular magnesium levels

Medizin - Open Access LMU - Teil 17/22

Play Episode Listen Later May 6, 2010

The TRPM group of cation channels plays diverse roles ranging from sensory signaling to Mg2+ homeostasis. In most metazoan organisms the TRPM subfamily is comprised of multiple members, including eight in humans. However, the Drosophila TRPM subfamily is unusual in that it consists of a single member. Currently, the functional requirements for this channel have not been reported. Here, we found that the Drosophila TRPM protein was expressed in the fly counterpart of mammalian kidneys, the Malpighian tubules, which function in the removal of electrolytes and toxic components from the hemolymph. We generated mutations in trpm and found that this resulted in shortening of the Malpighian tubules. In contrast to all other Drosophila trp mutations, loss of trpm was essential for viability, as trpm mutations resulted in pupal lethality. Supplementation of the diet with a high concentration of Mg2+ exacerbated the phenotype, resulting in growth arrest during the larval period. Feeding high Mg2+ also resulted in elevated Mg2+ in the hemolymph, but had relatively little effect on cellular Mg2+. We conclude that loss of Drosophila trpm leads to hypermagnesemia due to a defect in removal of Mg2+ from the hemolymph. These data provide the first evidence for a role for a Drosophila TRP channel in Mg2+ homeostasis, and underscore a broad and evolutionarily conserved role for TRPM channels in Mg2+ homeostasis.

essential levels feeding medizin magnesium supplementation drosophila extracellular mg2

Biophysikalische Untersuchungen zur physiologischen Bedeutung des Prionproteins als Metalloprotein

Medizinische Fakultät - Digitale Hochschulschriften der LMU - Teil 03/19

Play Episode Listen Later Jul 8, 2004

Além da doença de Creutzfeld-Jakob, ìons de cobre tem uma função importante na pathogenese de várias outras doenças neurodegenerativas, por exemplo M. Wilson, M. Alzheimer. Até agora não exisitia nenhuma método para determinar a concentração desde catíon em vivo. Por isso, a minha tése queria achar uma possibilidade para medir cobre por meio de um microscópio ou espetrômetro para seguir a corrente de cobre durante uma depolarisação. O sucesso do uso de corantes fluorescentes para a determinação de concentrações de cálcio nos últimos anos nos encorajou procurar uma substancia que pode detetar especificamente íons de Cu2+. Encontramos que a emissão de TSPP (tetrakis-(4-sulfophenyl)porphine) na região de 645 nm reage muito específico na presença de Cu2+ (KD = 0.43 ± 0.07 µM com pH 7.4). Ela não monstra nenhuma reação com os outros maiores catíons no cérebro, Ca2+ e Mg2+, ao contrário a emissão da maioría das substancias analisadas diminuiu. Também, Zn2+ afeta a fluorescença de TSPP, em outra régião do espétro (605 nm) com uma constante de dissociação KD = 50± 2.5 µM (pH = 7.0). Com TSPP, monstramos primeira vez que existe uma libertação sinaptosomal de cobre depois de depolarisação. Além disso, nós comparamos a quantidade de cobre libertada em camundongos sem (WT) e com (PrP0/0) a deficiência de exprimir o proteina prion (responsável pela doençã de Creutzfeld-Jakob). Achamos a mesma quantidade de vesiculas libertadas em consequência de depolarisação (medido com a corante FM 1-43), mas uma redução significante na quantidade de cobre libertada dos camundongos Prnp0/0. Os resultados foram confirmados de outro método independente, que se chama ICP-MS (inductively-coupled-plasma mass-spectrometry). Com os resultados experimentais, presentamos na tése também uma teoria que pode descrever a função do prion: Assumimos que esta proteína toma o cobre, que sai durante a depolarisação, e leva-o para o interior da célula, não havendo perda. A perda de cobre na ausência do prion seja também uma explicação do processo de CJD.

os alzheimer's disease bedeutung tamb ela mph kd untersuchungen encontramos wt achamos ca2 cjd assumimos mg2 ddc:600 icp ms cu2 zn2

Cloning and characterisation of a Dictyostelium STE20-like protein kinase DST2

Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 01/06

Play Episode Listen Later Dec 2, 2003

In this study a new Dictyostelium STE20-like protein kinase DST2 (Dictyostelium STE20-like kinase 2) was cloned and characterised. STE20 (Sterile 20) kinase was first identified in yeast as a pheromone-induced serine/threonine protein kinase that acts upstream of a MAP kinase cascade. Based on the domain structure, DST2 belongs to the GCK subfamily of STE20-like protein kinases, which include the mammalian STE20-like kinases (MST1/2/3), oxidant stress response kinase SOK-1, and DST1 in Dictyostelium discoideum which phosphorylates severin, a gelsolin-like F-actin fragmenting protein. DST2 was discovered by screening of the D. discoideum cDNA project database using DST1 as query. To confirm the existence of the DST2 gene and its expression, Southern, Northern and Western analyses of DST2 were carried out. It revealed that DST2 may have two copies in the Dictyostelium genome and that DST2 was expressed during all stages of D. discoideum development. In vitro kinase assays with bacterially expressed fusion protein of full length DST2 (aa461), the catalytic domain (aa287) and the regulatory domain (aa174) showed that autophosphorylation of DST2 occurson the regulatory domain and phosphorylates severin in the presence of a Mn2+ or Mg2+. Purified catalytic domain of PKA phosphorylated the regulatory domain of DST2 and caused an increase in the basal autophosphorylation activity of DST2, suggesting that PKA may be a potential upstream kinase of DST2 through the phosphorylation of its regulatory domain. To understand the function of the non-catalytic domain of DST2, three C-terminal truncation constructs (aa1-421, aa1-368 and aa1-326) were used in comparison to full length DST2 in in vitro kinase assays. Deletion of C-terminal regions revealed an inhibitory region amino acids 326-461 of DST2. Gel filtration chromatography showed that DST2 was eluted in a broad peak ranging from approximately 63 kDa to 400 kDa, suggesting that DST2 may exist in vivo as a monomer as well as a high molecular weight complex. The influence of phosphorylated and unphosphorylated severin on F-actin solutions was investigated using falling-ball viscometry and fluorescence spectroscopy. It turned out that phosphorylation by DST2 inhibits the F-actin fragmenting activity of severin, suggesting that DST2 may be directly involved in actin-cytoskeleton rearrangements.

western southern protein northern map gel cloning purified sok deletion kda kinase pka characterisation cdna mg2 ddc:500 dictyostelium ddc:570 gck mn2 ste20

Domänenanalyse des Cyclase-assoziierten Proteins (CAP) und Charakterisierung von Filactin, einem neuartigen actinähnlichen Protein aus Dictyostelium discoideum

Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 01/06

Play Episode Listen Later May 13, 2003

The cyclase-associated protein (CAP) from Dictyostelium discoideum was studied in detail regarding its structure and function relationships. The second part of the thesis describes the characterization of the novel actin-related protein filactin from D. discoideum. CAP homologs are multifunctional proteins: they are involved in signal dependent changes in the actin cytoskeleton, in vesicle transport and cell development. The binding of monomeric actin through the C-terminal domain represents a common feature of all CAPs. From four highly conserved regions in this domain the verprolin homology region of the D. discoideum CAP was analyzed in this work. Loss of this region led to a clear decrease, but not suppression of the actin-sequestering activity. In agreement to these data, stable complexes of the modified CAP-C with G-actin could be identified in chemical crosslinking experiments, and it could be shown that CAP-C is able to dimerize. Considering the high conservation of cyclase-associated proteins and their importance for cell biological processes it is remarkable that no structure of this protein has been elucidated so far. Therefore, in the frame of this thesis the structure of the membrane-associated N-terminal domain of D. discoideum CAP was to be determined in cooperation with the group of T. Holak at the MPI f. Biochemistry (Martinsried). For this purpose numerous constructs from the N-terminal domain had to be cloned and expressed, to be purified and examined for their stability and threedimensional folding. It turned out that the stable core of the aminoterminal domain covers the amino acids 51-226. The structure was determined by nuclear magnetic resonance spectroscopy and X-ray crystallography. Six antiparallel a-helices are connected by loop elements and form a cylindrical core domain which can form a dimer in the crystal, dimerization occurs through Mg2+ ions. The clarification of the N-terminal structure of D. discoideum CAP will now simplify the research on its interaction with phospholipids and CAP binding proteins The second part of this work dealt with the characterization of filactin. The unique 105 kDa protein contains two filamin homologous regions in its N-terminal, and a clearly actin-related domain within the C-terminal part. While in resting cells the endogenous filactin shows cytoplasmic distribution and interaction with protein aggregates as well, the GFP construct of the actin-related domain displays an actin-like behavior during cell movement or phagocytosis. A stimulus-induced colocalization of actin and filactin was observed in experiments with chemotactically stimulated cells. The alignment of its C-terminal amino acid sequence with the structure of muscle actin predicts a globular, actin-related structure containing all residues that are important for ATP/ADP binding.

loss cap einem caps proteins mpi kda actin gfp charakterisierung mg2 ddc:500 dictyostelium ddc:570 atp adp

Glucan synthase of Phytophthora sojae

Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 01/06

Play Episode Listen Later Jun 18, 2002

Glucans, with the (1-3)-b-glucosidic linkage as major feature, are present in most of the higher plants, in many lower plants, as well as in microorganisms (Stone and Clarke, 1992). The synthesis of (1-3)-b-glucan in vivo is catalysed by the enzyme (1-3)-b-glucan synthase (EC 2.4.1.34; UDP-glucose:1,3-b-D-glucan 3-b-D-glucosyl transferase) using UDP-glucose as substrate. The (1-3)-b-glucan synthase was characterised in a number of fungi and plants, but not much work was done with oomycetes (Stone and Clarke, 1992), even though one of the earliest successful in vitro assays for glucan synthase activity was done using Phytophthora cinnamomi (Wang and Bartnicki-Garcia, 1976, Selitrennikoff 1995). In this work, the glucan synthase of the oomycete Phytophthora sojae was characterised, solubilized, and partially purified, and the cDNA for a protein co-purifying with the glucan synthase activity was cloned. The glucan synthase of P. sojae had several features that distinguish it from what is known for glucan synthases from fungi and plants (callose synthases). Its apparent Km value for UDP-glucose was higher than reported for other glucan synthases. The activity was GTP-independent and shown not to be activated by divalent cations like Mg2+ or Ca2+, and shown to be inhibited by some others, like Cu2+ or Zn2+. Some of these properties are shared with the glucan synthase from Achlya ambisexualis (Cabib and Kang, 1987), an organism that belongs to the same kingdom as P. sojae: the Chromista. It was also demonstrated by NMR analysis and enzymatic degradation that the sole product of the CHAPS-solubilized glucan synthase of P. sojae was composed of long linear (1-3)-b-glucan chains. The glucan synthase was purified by product entrapment. Two proteins, with apparent molecular masses of 108 and 50 kDa, were enriched and microsequenced. With the degenerated oligonucleotides derived from the sequenced peptides, PCR experiments were performed using as a template a cDNA library of actively growing P. sojae mycelium. No positive result could be obtained by using the oligonucleotides derived from the 108 kDa protein. In contrast, a full length cDNA (named Ps-P50) was cloned, using the oligonucleotides derived from the 50 kDa protein (P50). The deduced amino acid sequence of Ps-P50 cDNA contains sequence motifs homologous to the peptides sequenced from P50. This cDNA encodes a protein with a molecular mass of 49.991 Da with no homology found in the data bases. Diversity between the PCR product and the cDNA clone, and various different homologous ESTs indicates that Ps-P50 is a member of a gene family.

diversity stone clarke wang kang pcr ec km chaps kda gtp udp nmr p50 ca2 phytophthora cdna mg2 synthase ddc:500 cu2 ddc:570 zn2

Struktur-Funktionsbeziehungen der FhlA- und HydH/G-Regulationssysteme in Escherichia coli

Fakultät für Biologie - Digitale Hochschulschriften der LMU - Teil 01/06

Play Episode Listen Later Mar 28, 2001

Das σ54-abhängige Regulatorprotein FhlA aktiviert die Transkription der für die Bildung eines funktionellen Formiat-Hydrogen-Lyase-Komplexes nötigen Gene. Hierfür weist es eine Reihe von Aktivitäten auf, nämlich ATP-Hydrolyse, Interaktion mit Eσ54 und Stimulierung der Transkription. Die Aktivität von FhlA wird durch Bindung von Formiat und von spezifischen DNA-Sequenzen (UAS) stimuliert und durch HycA negativ beeinflußt. Sequenzähnlichkeiten von FhlA zu anderen Transkriptionsaktivatoren lassen auf einen Aufbau aus drei Domänen schließen. Die in der vorliegenden Arbeit erfolgte Struktur- Funktions-Analyse lieferte wichtige Ergebnisse über die Domänenstruktur und den Wirkungsmechanismus von FhlA. In vivo- und in vitro-Analysen der C-terminalen Hälfte von FhlA (FhlA-C, Aminosäuren 379 bis 693 von FhlA), welche die Domänen B, C und D von FhlA umfaßt, zeigten, daß dieser Teil von FhlA sämtliche für die Transkriptionsaktivierung und ATP-Hydrolyse benötigten Bereiche enthält. Beide Aktivitäten von FhlA-C sind konstitutiv, bedürfen also nicht mehr der Aktivierung durch Formiat-Bindung. Durch die Bindung von DNA konnte die Aktivität von FhlA-C jedoch gesteigert werden, wobei bei spezifischer DNA ein stärkerer Effekt als bei unspezifischer zu beobachten war. Aus den Ergebnissen wurde ein Modell abgeleitet, in welchem die N-terminale Domäne von FhlA im nicht-induzierten Zustand einen hemmenden Einfluß auf die Aktivität des restlichen Proteins hat. Formiat-Bindung an die N-terminale Domäne hebt diese Hemmung auf und bewirkt eine Steigerung der Aktivität von FhlA durch Erhöhung der Affinität für ATP. Auch die HycA-Suszeptibilität konnte der N-terminalen Domäne zugeordnet werden. Darüberhinaus besitzt die N-terminale Domäne eine strukturelle Funktion, da sie für die Oligomerisierung von FhlA verantwortlich ist. Es konnte gezeigt werden, daß die Bindung von ATP an FhlA eine Änderung der Konformation oder des Oligomerisierungszustandes des Proteins zur Folge hat. Eine Studie zur Verbreitung des FhlA-Regulationssystems ergab, daß in mehreren Spezies der Familie Enterobacteriaceae sowohl ein zu FhlA orthologes Protein als auch Homologe zu hyc- oder hyp-Genen existieren. Die Sequenz von fhlA aus S. typhimurium, E. aerogenes und K. oxytoca wurde bestimmt. Sie zeigt über die gesamte Länge große Ähnlichkeit zu FhlA von E. coli, wobei der Grad der Identität der Gene und Proteine den phylogenetischen Verwandtschaftsverhältnissen entspricht. Die FhlA-Orthologe aus E. aerogenes und K. oxytoca können FhlA aus E. coli funktionell ersetzen und zeigen auch in ihrer Aktivität hinsichtlich der Stimulierbarkeit durch Formiat und Anaerobiose große Übereinstimmung. Desweiteren beeinflußt auch HycA von E. coli die Aktivität der orthologen Proteine, was wiederum die nahe Verwandtschaft der Regulationssysteme zeigt. Im Hauptteil dieser Arbeit konnte der Beweis erbracht werden, daß das HydH/G-Zwei- Komponenten-System die Expression von zraP aktiviert, einem stromaufwärts von hydH gelegenen, in divergierender Richtung orientierten Gen. Für gereinigtes HydG wurde durch Gelretardationsexperimente und DNase I Footprinting-Analyse eine Bindestelle identifiziert, die im intergenen Bereich zwischen hydHG und zraP liegt. Diese 55 bp lange Region umfaßt zwei jeweils 17 bp lange Bereiche mit der Sequenz GAGTAAAAATGACTCGC, die als inverted repeat angeordnet sind. Diese Bindestelle wirkt als UAS in beide Richtungen. Als auslösender Stimulus für die Expressionsaktivierung von zraP durch HydH/G konnte eine Zn2+-Konzentration im Medium von mehr als 0,5 mM identifiziert werden. Pb2+ war zu einem gewissen Grad in der Lage, Zn2+ in der Funktion als Induktor zu ersetzen, jedoch erfolgte keine Aktivierung durch Ni2+, Co2+, Mn2+, Fe2+, Mg2+, Cu2+, Cd2+ oder Hg2+. Gleichzeitig ist HydH/G einer positiven Autoregulation unterworfen, die durch die gleichen Metalle induziert wird. Sowohl für die durch HydH/G induzierte Expression von zraP als auch von hydHG konnte eine Abhängigkeit von σ54 gezeigt werden. Der Einfluß von HydH/G auf die Regulation der Gene für die Hydrogenase 3 tritt entgegen früherer Postulate nur bei Überproduktion des Regulators auf und ist auf "cross-talk" zurückzuführen. Zweidimensionale Gelelektrophorese erbrachte Hinweise auf weitere durch HydG in ihrer zellulären Konzentration beeinflußte Proteine. Obwohl zraP im Zusammenhang mit Zink- Toleranz identifiziert worden war, zeigten die Ergebnisse keinen Einfluß von HydH/G oder ZraP auf die Zink-Toleranz der Zellen.

dna medium arbeit dar durch diese dom region co2 lage protein bereich expression reihe identit grad zusammenhang ergebnisse richtung stimulus mm zustand struktur aktivit funktion bereiche hinweise regulators atp abh beweis ergebnissen konzentration sowohl bindung analysen des weiteren proteins erh interaktion verbreitung richtungen zellen steigerung hierf proteine aktivierung uas verwandtschaft eine studie genen affinit aminos einflu metalle sequenz transkription hemmung im hauptteil autoregulation cd2 escherichia coli die aktivit postulate mg2 ddc:500 pb2 der einflu cu2 ddc:570 mn2 induktor oligomerisierung stimulierbarkeit zn2 die sequenz ni2 bindestelle atp hydrolyse

Another member of the cyclic nucleotide-gated channel family, expressed in testis, kidney, and heart.

Chemie und Pharmazie - Open Access LMU - Teil 02/02

Play Episode Listen Later Jan 1, 1994

Cyclic nucleotide-gated cation channels are essential in visual and olfactory signal transduction. An additional member of the cGMP-gated channel family, termed CNG-3, has been cloned from bovine kidney. Its deduced amino acid sequence is 60% and 62% identical with the CNG-channel proteins from bovine rod outer segment and bovine olfactory epithelium, respectively. Northern analysis and sequences amplified by the PCR showed that the CNG-3 mRNA is present in testis, kidney, and heart. Calcium permeated the expressed channel in the presence of extracellular Mg2+ and Na+ at membrane potentials from -100 to +45 mV. It is likely that CNG-3 protein is responsible for cGMP-induced Ca2+ entry in cells other than sensory cells.

family member northern mrna pcr kidney calcium mv expressed gated cyclic cng cgmp ca2 mg2 nucleotide chemie und pharmazie

Millimolar concentrations of free magnesium enhance exocytosis from permeabilized rat pheochromocytoma (PC 12) cells

Medizin - Open Access LMU - Teil 07/22

Play Episode Listen Later Jan 1, 1989

The role of Mg2+ during the final steps of exocytosis was investigated using rat pheochromocytoma cells (PC12) permeabilized with bacterial pore forming toxins. Concentrations of free Mg2+ between 0.2 and 2 mM slightly lowered the basal but greatly enhanced the [3H]dopamine release elicited by 8 μM free Ca2+. Maximal effects were obtained at approximately 1 mM free Mg2+. At higher concentrations Mg2+ was less potent. Similar effects of Mg2+ were obtained in cells permeabilized either for small molecules (by α-toxin) or for large ones (by streptolysin O). It is concluded that millimolar concentrations of cytoplasmic Mg2+ play an important role in Ca2+ triggered exocytosis.

Further Characterization of Dopamine Release by Permeabilized PC 12 Cells

Medizin - Open Access LMU - Teil 06/22

Play Episode Listen Later Jan 1, 1987

Rat pheochromocytoma cells (PC 12) permeabilized with staphylococcal α-toxin release [3H]dopamine after addition of micromolar Ca2+. This does not require additional Mg2+-ATP (in contrast to bovine adrenal medullary chromaffin cells). We also observed Ca2+-dependent [3H]-dopamine release from digitonin-permeabilized PC 12 cells. Permeabilization with α-toxin or digitonin and stimulation of the cells were done consecutively to wash out endogenous Mg2+-ATP. During permeabilization, ATP was removed effectively from the cytoplasm by both agents but the cells released [3H]dopamine in response to micromolar Ca2+ alone. Replacement by chloride of glutamate, which could sustain mitochondrial ATP production in permeabilized cells, does not significantly alter catecholamine release induced by Ca2+. However, Mg2+ without ATP augments the Ca2+-induced release. The release was unaltered by thiol-, hydroxyl-, or calmodulin-interfering substances. Thus Mg2+-ATP, calmodulin, or proteins containing -SH or -OH groups are not necessary for exocytosis in permeabilized PC 12 cells.

pc sh rat replacement dopamine medizin cells atp characterization 3h ca2 mg2 mg2 atp

Evidence for a magnesium-insensitive membrane resistance increase during NMDA-induced depolarizations in rat neocortical neurons in vitro

Medizin - Open Access LMU - Teil 05/22

Play Episode Listen Later Jan 1, 1987

The responses of rat neocortical neurons in vitro to iontophoretically applied N-methyl-d-aspartate (NMDA) were investigated by means of intracellular recording in the presence and absence of extracellular magnesium ions (Mg2+). At Mg2+-concentrations of 1.3 mM the neurons responded with a depolarization accompanied by an increase in membrane resistance. Upon removal of Mg2+ the NMDA-induced depolarization was markedly potentiated. However, even in neurons recorded from slices which were incubated in a Mg2+-free solution for 3–7 h, the NMDA response was still associated with a resistance increase, suggesting that the voltage-dependence of the NMDA-activated conductance is not exclusively determined by Mg2+.

resistance mm medizin magnesium induced neurons membrane vitro insensitive nmda mg2

Ca2+ binding to Chromaffin Vesicle Matrix Proteins

Medizin - Open Access LMU - Teil 05/22

Play Episode Listen Later Jan 1, 1986

Recently we found that Ca2+ within chromaffin vesicles is largely bound [Bulenda, D., & Gratzl, M. (1985) Biochemistry 24, 7760-77651. In order to explore the nature of these bonds, we analyzed the binding of Ca2+ to the vesicle matrix proteins as well as to ATP, the main nucleotide present in these vesicles. The dissociation constant at pH 7 is 50 pM (number of binding sites, n = 180 nmol/mg of protein) for Ca2+-protein bonds and 15 pM (n = 0.8 pmol/pmoi) for Ca2+-ATP bonds. When the pH is decreased to more physiological values (pH 6), the number of binding sites remains the same. However, the affinity of Ca2+ for the proteins decreases much less than its affinity for ATP (dissociation constant of 90 vs. 70 pM). At pH 6 monovalent cations (30-50 mM) as well as Mg2+ (0.1-0.5 mM), which are also present within chromaffin vesicles, do not affect the number of binding sites for Ca2+ but cause a decrease in the affinity of Ca2+ for both proteins and ATP. For Ca2+ binding to ATP in the presence of 0.5 mM Mg2+ we found a dissociation constant of 340 pM and after addition of 35 mM K+ a dissociation constant of 170 pM. Ca2+ binding to the chromaffin vesicle matrix proteins in the presence of 0.5 mM Mg2+ is characterized by a Kd of 240 pM and after addition of 15 mM Na' by a Kd of 340 pM. The similar affinity of Ca2+ for protein and ATP, especially at pH 6, in media of increased ionic strength and after addition of Mg2+, points to the possibility that the intravesicular medium determines whether Ca2+ is preferentially bound to ATP or the chromaffin vesicle matrix proteins. Purified chromogranin A, after sodium dodecyl sulfate- polyacrylamide gel electrophoresis, stains with a carbocyanine dye ("Stains-all") and, following blotting onto nitrocellulose, binds to 45Ca2+. A spectrophotometric analysis of dye binding to chromaffin vesicle matrix proteins revealed a strong absorption band at 615 nm for the dye-protein complex. Since the observed spectral changes were unaffected by the presence of Ca2+ (100 pM free), the sites interacting with the dye and Ca2+ must be regarded as different.

matrix kd mm medizin binding atp biochemistry proteins stains purified mmk ca2 mg2 vesicle gratzl

Na+/Ca2+ exchange in coated microvesicles

Medizin - Open Access LMU - Teil 05/22

Play Episode Listen Later Jan 1, 1986

Coated microvesicles isolated from bovine neurohypophyses could be loaded with Ca2+ in two different ways, either by incubation in the presence of ATP or by imposition of an outwardly directed Na+ gradient. Na+, but not K+, was able to release Ca2+ accumulated by the coated microvesicles. These results suggest the existence of an ATP-dependent Ca2+-transport system as well as of a Na+/Ca2+ carrier in the membrane of coated microvesicles similar to that present in the membranes of secretory vesicles from the neurohypophysis. A kinetic analysis of transport indicates that the apparent Km for free Ca2+ of the ATP-dependent uptake was 0.8 microM. The average Vmax. was 2 nmol of Ca2+/5 min per mg of protein. The total capacity of microvesicles for Ca2+ uptake was 3.7 nmol/mg of protein. Both nifedipine (10 microM) and NH4Cl (50 mM) inhibited Ca2+ uptake. The ATPase activity in purified coated-microvesicles fractions from brain and neurohypophysis was characterized. Micromolar concentrations of Ca2+ in the presence of millimolar concentrations of Mg2+ did not change enzyme activity. Ionophores increasing the proton permeability across membranes activated the ATPase activity in preparations of coated microvesicles from brain as well as from the neurohypophysis. Thus the enzyme exhibits properties of a proton-transporting ATPase. This enzyme seems to be linked to the ion accumulation by coated microvesicles, although the precise coupling of the proton transport to Ca2+ and Na+ fluxes remains to be determined.

exchange mm medizin atp km coated ca2 vmax atpase mg2 microm na ca2

Phosphoproteins and protein-kinase activity in isolated envelopes of pea (Pisum sativum L.) chloroplasts

Biologie - Open Access LMU - Teil 01/02

Play Episode Listen Later Jan 1, 1985

A protein kinase was found in envelope membranes of purified pea (Pisum sativum L.) chloroplasts. Separation of the two envelope membranes showed that most of the enzyme activity was localized in the outer envelope. The kinase was activated by Mg2+ and inhibited by ADP and pyrophosphate. It showed no response to changes in pH in the physiological range (pH 7-8) or conventional protein substrates. Up to ten phosphorylated proteins could be detected in the envelope-membrane fraction. The molecular weights of these proteins, as determined by polyacrylamide-gel electrophoresis were: two proteins higher than 145 kDa, 97, 86, 62, 55, 46, 34 and 14 kDa. The 86-kDa band being the most pronounced. Experiments with separated inner and outer envelopes showed that most labeled proteins are also localized in the outer-envelope fraction. The results indicate a major function of the outer envelope in the communication between the chloroplast and the parent cell.

experiments separation activity protein isolated adp biologie envelopes kda kinase mg2 chloroplasts pisum

Matrix-free calcium in isolated chromaffin vesicles

Play Episode Listen Later Jan 1, 1985

Isolated secretory vesicles from bovine adrenal medulla contain 80 nmol of Ca2+ and 25 nmol of Mg2+ per milligram of protein. As determined with a Ca2+-selective electrode, a further accumulation of about 160 nmol of Ca2+/mg of protein can be attained upon addition of the Ca2+ ionophore A23187. During this process protons are released from the vesicles, in exchange for Ca2+ ions, as indicated by the decrease of the pH in the incubation medium or the release of 9-aminoacridine previously taken up by the vesicles. Intravesicular Mg2+ is not released from the vesicles by A23 187, as determined by atomic emission spectroscopy. In the presence of N H Q , which causes the collapse of the secretory vesicle transmembrane proton gradient (ApH), Ca2+ uptake decreases. Under these conditions A23 187-mediated influx of Ca2+ and efflux of H+ cease at Ca2+ concentrations of about 4 pM. Below this concentration Ca2+ is even released from the vesicles. At the Ca2+ concentration at which no net flux of ions occurs the intravesicular matrix free Ca2+ equals the extravesicular free Ca2+. In the absence of NH4C1 we determined an intravesicular pH of 6.2. Under these conditions the Ca2+ influx ceases around 0.15 pM. From this value and the known pH across the vesicular membrane an intravesicular matrix free Ca2+ concentration of about 24 pM was calculated. This is within the same order of magnitude as the concentration of free Ca2+ in the vesicles determined in the presence of NH4C1. Calculation of the total Ca2+ present in the secretory vesicles gives an apparent intravesicular Ca2+ concentration of 40 mM, which is a factor of lo4 higher than the free intravesicular concentration of Ca2+. It can be concluded, therefore, that the concentration gradient of free Ca2+ across the secretory vesicle membrane in the intact chromaffin cells is probably small, which implies that less energy is required to accumulate and maintain Ca2+ within the vesicles than was previously anticipated.

matrix mm medizin isolated calcium calculation aph ca2 a23 mg2 vesicles

Calcium/sodium exchange in purified secretory vesicles from bovine neurohypophyses

Play Episode Listen Later Jan 1, 1983

Purified secretory vesicles isolated from bovine neurohypophyses take up Na+ under the same circumstances where an efflux of Ca2+ takes place, suggesting a Na+/Ca2+ exchange. Potassium cannot substitute for Na+ in this process. Also, a Ca2+/Ca2+ exchange can occur. Inhibiting the latter process by Mg2+ allowed to estimate an apparent KM of 0.7 μM free Ca2+ and a maximal uptake of 1.5 nmol × mg protein−1 × min−1 Ca2+ in exchange for Na+. The vesicles did not contain plasma membrane marker (Na+/K+ ATPase) as shown by distribution analyses on the density gradients on which they were purified. Similarly, distribution studies also showed that no other ATPase activity could be detected in the purified vesicle fraction. It is concluded that a Na+/Ca2+ exchange is operating across the secretory vesicle membrane and that it is not directly dependent on ATP hydrolysis.

exchange medizin atp km calcium sodium potassium purified bovine inhibiting ca2 atpase mg2 vesicles secretory na k atpase na ca2

3',5'-Cyclic Adenosine Monophosphate- and Ca2+-Calmodulin-Dependent Endogenous Protein Phosphorylation Activity in Membranes of the Bovine Chromaffin Secretory Vesicles: Identification of Two Phosphorylated Components as Tyrosine Hydroxylase and Protein K

Play Episode Listen Later Jan 1, 1983

Abstract: Membranes of the secretory vesicles from bovine adrenal medulla were investigated for the presence of the endogenous protein phosphorylation activity. Seven phosphoprotein bands in the molecular weight range of 250,000 to 30,000 were observed by means of the sodium dodecyl sulphate electrophoresis and autoradiography. On the basis of the criteria of molecular weight, selective stimulation of the phosphorylation by cyclic AMP (as compared with cyclic GMP) and immunoprecipitation by specific antibodies, band 5 (molecular weight 60,300) was found to represent the phosphorylated form of the secretory vesicle-bound tyrosine hydroxylase. The electrophoretic mobility, the stimulatory and inhibitory effects of cyclic AMP in presence of Mg2+ and Zn,2+ respectively, and immunoreactivity toward antibodies showed band 6 to contain two forms of the regulatory subunits of the type II cyclic AMP-dependent protein kinase, distinguishable by their molecular weights (56,000 and 52,000, respectively). Phosphorylation of band 7 (molecular weight 29,800) was stimulated about 2 to 3 times by Ca2+ and calmodulin in the concentration range of both agents believed to occur in the secretory tissues under physiological conditions.

activity protein medizin components identification dependent zn bovine gmp membranes cyclic endogenous adenosine tyrosine ca2 phosphorylation mg2 vesicles secretory calmodulin

Effects of Monovalent and Divalent Cations on Ca2+ Fluxes Across Chromaffin Secretory Membrane Vesicles

Biologie - Open Access LMU - Teil 01/02

Play Episode Listen Later Jan 1, 1983

Abstract: Bovine chromaffin secretory vesicle ghosts loaded with Na+ were found to take up Ca2+ when incubated in K+ media or in sucrose media containing micromolar concentrations of free Ca2+. Li+- or choline+loaded ghosts did not take up Ca2+. The Ca2+ accumulated by Na+-loaded ghosts could be released by the Ca2+ ionophore A23187, but not by EGTA. Ca2+ uptake was inhibited by external Sr2+, Na +, Li +, or choline +. All the 45Ca2+ accumulated by Na+-dependent Ca2+ uptake could be released by external Na +, indicating that both Ca2+ influx and efflux occur in a Na+-dependent manner. Na + -dependent Ca2+ uptake and release were only slightly inhibited by Mg2+. In the presence of the Na+ ionophore Monensin the Ca2+ uptake by Na +-loaded ghosts was reduced. Ca2+ sequestered by the Na+-dependent mechanism could also be released by external Ca2+ or Sr2+ but not by Mg2+, indicating the presence of a Ca2+/Ca2+ exchange activity in secretory membrane vesicles. This Ca2+/Ca2+ exchange system is inhibited by Mg2+, but not by Sr2+. The Na + -dependent Ca2+ uptake system in the presence of Mg2+ is a saturable process with an apparent Km of 0.28 μM and a Vmax= 14.5 nmol min−1 mg protein−1. Ruthenium red inhibited neither the Na+/Ca2+ nor the Ca2+/Ca2+ exchange, even at high concentrations.

effects li medizin km membrane ca2 vmax mg2 vesicles secretory ruthenium sr2 monensin na ca2

Phosphorylation of chloroplast ribulose bisphosphate carboxylase/oxygenase small subunit by an envelope-bound protein kinase in situ

Play Episode Listen Later Jan 1, 1983

A new protein kinase of the cAMP independent type was found to be bound to the outer envelope membrane of spinach chloroplasts. While stimulated by Mg2+ and inhibited by ADP, the enzyme showed no response to conventional protein substrates and was essentially independent of pH in the physiological (pH 7 to 8) range. The new protein kinase phosphorylated the mature form of the small subunit of ribulose 1,5- bisphosphate carboxylase/oxygenase and, to a lesser extent, an unidentified 24-kDa polypeptide, both of which were bound to the outer envelope membrane. The results suggest that phosphorylation of cytoplasmically synthesized protein constituents of chloroplasts is involved in their transport through the chloroplast envelope membrane barrier.

camp bound protein adp situ biologie envelope kda kinase phosphorylation mg2 subunit chloroplast

Uptake of Ca2+ by isolated secretory vesicles from adrenal medulla

Play Episode Listen Later Jan 1, 1982

Intact secretory vesicles isolated from bovine adrenal medulla contain 94 nmol Na+ per mg of protein, and Ca2+ influx into the vesicles is inhibited by increasing concentrations of extravesicular Na+ (but not of K+, Li+ or choline+) or by addition of the Na+ ionophore monensin. Thus Ca2+ influx is determined by the Na+ gradient across the vesicular membrane. Half maximal inhibition of Ca2+ influx occurs with 34 mM Na+ extravesicularly. The fact that Ca2+ can also be released from the vesicles by inversion of the Na+ gradient provides direct evidence that an Na+-Ca2+ exchange may operate. According to an analysis of the inhibition of Ca2+ uptake by Na+ in a Hill plot 2 Na+ would be exchanged for 1 Ca2+. Ca2+ influx into the vesicles increases with temperature (energy of activation: 16 kcal/mol), can be observed already with 10−7 M free Ca2+ and increases up to 10−4 M Ca2+. Ca2+ influx is not affected by Mg2+ but Sr2+ is inhibitory. Since the process is only slightly influenced by the pH of the incubation medium and is insensitive to Mg2+-ATP or inhibitors of the proton translocating Mg2+-ATPase the electrochemical proton gradient across the vesicular membrane does not affect directly the Ca2+ influx into the secretory vesicles. Ca2+ uptake is insensitive to ruthenium red and oligomycin.

li medizin isolated adrenal intact uptake medulla ca2 mg2 vesicles secretory sr2 na ca2 mg2 atp

Ca2+ uptake to purified secretory vesicles from bovine neurohypophyses

Play Episode Listen Later Jan 1, 1982

Purified secretory vesicles isolated from bovine neurohypophyses were found to take up Ca2+ when incubated at 30°C in media containing 10−7 to 10−4 M free Ca2+. At 10−4 free Ca2+ 19 nmol/mg protein were taken up within 30 min. The initial uptake at this Ca2+ concentration was about 2 nmol/mg protein per min. The uptake of Ca2+ to secretory vesicles was not affected by ATP, oligomycin, ruthenium red, trifluoperazine, Mg2+ or K+, but was inhibited by Na+ and Sr2+. From these characteristics it can be concluded that the uptake system does not utilize directly ATP (as the Ca2+-ATPases known to be present in the cell membrane and the endoplasmic reticulum) and is different from the mitochondrial Ca2+ uptake system driven by respiration and/or ATP hydrolysis. However, Ca2+-Na+ exchange may well operate: In experiments using different concentrations of Na+ we found half-maximal inhibition of Ca2+ uptake with 33.3 mM Na+. An analysis of the data in a Hill plot indicated that at least 2 Na+ would be exchanged for 1 Ca2+. Also, it was found that Ca2+ previously taken up could be released again by external Na+ but not by K+.

medizin atp purified bovine uptake ca2 mg2 vesicles secretory sr2

Latent acetylcholinesterase in secretory vesicles isolated from adrenal medulla

Play Episode Listen Later Jan 1, 1981

A new procedure is described for the preparation of highly purified and stable secretory vesicles from adrenal medulla. Two forms of acetylcholinesterase, a membrane bound form as well as a soluble form, were found within these vesicles. The secretory vesicles, isolated by differential centrifugation, were further purified on a continuous isotonic Percoll™ gradient. In this way, secretory vesicles were separated from mitochondrial, microsomal and cell membrane contamination. The secretory vesicles recovered from the gradient contained an average of 2.26 μmol adrenalin/mg protein. On incubation for 30 min at 37°C in media differing in ionic strength, pH, Mg2+ and Ca2+ concentration, the vesicles released less than 20% of total adrenalin. Acetylcholinesterase could hardly be detected in the secretory vesicle fraction when assayed in isotonic media. However, in hypotonic media (

medizin isolated adrenal latent medulla ca2 mg2 vesicles secretory triton x

Synaptic modulation of calcium-dependent potassium conductance in myenteric neurones in the guinea-pig.