The collisional limit: an important consideration for membrane‐associated enzymes and receptors

Abbott, Alan J.; Nelsestuen, Gary L.

doi:10.1096/fasebj.2.13.2844615

Cited by 47 publications

(32 citation statements)

References 29 publications

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“…Ϫ1 s Ϫ1 (24)). As described in detail elsewhere (25)(26)(27), diffusion-limited kinetics of multisite particles are complex. Precise mathematical modeling requires knowledge of particle size, the number of sites per particle (n), the distribution of sites over all particles (even or biased), as well as their distribution on the surface of each particle (clustered or dispersed).…”

Section: Figmentioning

confidence: 99%

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Elevated Function of Blood Clotting Factor VIIa Mutants That Have Enhanced Affinity for Membranes

Nelsestuen¹,

Stone²,

Martinez³

et al. 2001

Journal of Biological Chemistry

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Blood clotting factor VIIa is involved in the first step of the blood coagulation cascade, as a membrane-associated enzyme in complex with tissue factor (TF). Factor VIIa is also an important therapeutic agent for hemophilia where its function may include TF-independent as well as TF-dependent mechanisms. This study compared the activity of wild type factor VIIa (WT-VIIa) with that of a mutant with elevated affinity for membrane (P10Q/Q32E, QE-VIIa). Phospholipid and cellbased assays showed the mutant to have up to 40-fold higher function than WT-VIIa in both TF-dependent and TF-independent reactions. Tissue factor-dependent reactions displayed the maximum enhancement when binding had reached equilibrium in competition with another TF-binding protein. In liposome-based assays, the association rate of WT-VIIa with TF occurred at a physical maximum and could not be improved by sitedirected mutagenesis. A practical consequence was identical function of WT-VIIa and QE-VIIa in assays that depended entirely on assembly kinetics. Thus, factor VIIa mutants provided unique reagents for probing the mechanism of factor VIIa action. They may also offer superior agents for therapy.

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Section: Figmentioning

confidence: 99%

“…28). One feature is slow ligand dissociation under conditions of excess receptor but rapid dissociation under conditions of excess ligand (26,27). Indeed, work that is in progress shows that this behavior is very pronounced for the factor VIIa⅐TF interaction with Innovin and other reconstituted liposomes.…”

Section: Figmentioning

confidence: 99%

Elevated Function of Blood Clotting Factor VIIa Mutants That Have Enhanced Affinity for Membranes

Nelsestuen¹,

Stone²,

Martinez³

et al. 2001

Journal of Biological Chemistry

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“…Interestingly our kinetics for the dissociation of FPR-GFP or wild type FPR from Gα i in the detergent solubilized system are much faster than was reported in cells from experiments involving FRET between β 2 AR and Gα s [19]. We do not know whether this reflects differences between the intact cells and detergent, the difference between specific receptors and G proteins, the possibility that their measurement has contributions from a higher order topographical redistribution, or the possibility that ligand dissociation and rebinding [44][45][46] is rate-limiting in their measurement of RG dissociation.…”

Section: Whether Gα Gβγ and Gpcrs Remain Associated After Activationmentioning

confidence: 62%

Rapid-mix flow cytometry measurements of subsecond regulation of G protein-coupled receptor ternary complex dynamics by guanine nucleotides

Buranda

Simons

et al. 2007

Analytical Biochemistry

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We have used rapid mix flow cytometry to analyze the early subsecond dynamics of the disassembly of ternary complexes of G-protein coupled receptors (GPCRs) immobilized on beads to examine individual steps associated with guanine nucleotide activation. Our earlier studies suggested that the slow dissociation of Gα and Gβγ subunits was unlikely to be an essential component of cell activation. However, these studies did not have adequate time resolution to define precisely the disassembly kinetics. Ternary complexes were assembled using three formyl peptide receptor constructs (wild type, FPR-Gα i2 fusion, and FPR-GFP fusion) and two isotypes of the α subunit (α i2 and α i3 ) and βγ dimer (β 1 γ 2 and β 4 γ 2 ). At saturating nucleotide levels, the disassembly of a significant fraction of ternary complexes occurred on a subsecond time frame for α i2 complexes and τ 1/2 ≤ 4 sec for α i3 complexes, time scales which are compatible with cell activation. β 1 γ 2 isotype complexes were generally more stable than β 4 γ 2 associated complexes. The comparison of the three constructs however proved that the fast step was associated with the separation of receptor and G protein and that the dissociation of the ligand or of the α and βγ subunits was slower. These results are compatible with a cell activation model involving G protein conformational changes rather than disassembly of Gαβγ heterotrimer.

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“…Upon collision between an enzyme and a polymer, we assume a catalytic reaction. The outcomes of the above-mentioned collision types are based on the theory of collisional limit (39), which predicts that the close proximity of many different receptors and enzymes on a cell surface makes the cell surface uniformly reactive, resulting in a binding or catalytic event with every encounter. However, in the case of enzymes not present on the cell surface (i.e., a free-enzyme strategy), the polymer is ultimately reflected.…”

Section: Methodsmentioning

confidence: 99%

A Model of Extracellular Enzymes in Free-Living Microbes: Which Strategy Pays Off?

Traving

Thygesen

Riemann

et al. 2015

Appl Environ Microbiol

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An initial modeling approach was applied to analyze how a single, nonmotile, free-living, heterotrophic bacterial cell may optimize the deployment of its extracellular enzymes. Free-living cells live in a dilute and complex substrate field, and to gain enough substrate, their extracellular enzymes must be utilized efficiently. The model revealed that surface-attached and free enzymes generate unique enzyme and substrate fields, and each deployment strategy has distinctive advantages. For a solitary cell, surface-attached enzymes are suggested to be the most cost-efficient strategy. This strategy entails potential substrates being reduced to very low concentrations. Free enzymes, on the other hand, generate a radically different substrate field, which suggests significant benefits for the strategy if free cells engage in social foraging or experience high substrate concentrations. Swimming has a slight positive effect for the attached-enzyme strategy, while the effect is negative for the free-enzyme strategy. The results of this study suggest that specific dissolved organic compounds in the ocean likely persist below a threshold concentration impervious to biological utilization. This could help explain the persistence and apparent refractory state of oceanic dissolved organic matter (DOM). Microbial extracellular enzyme strategies, therefore, have important implications for larger-scale processes, such as shaping the role of DOM in ocean carbon sequestration. Dissolved organic matter (DOM) in the oceans is one of the largest active reservoirs of organic carbon in the biosphere (1). An intricate web of biotic (e.g., photosynthesis, viral lysis, and grazing) and abiotic (e.g., photodegradation and aggregation) processes contribute to the physical and chemical complexity of DOM (2-4). However, DOM is almost exclusively exploited by the bacterioplankton (5, 6). Microbes face several challenges when consuming DOM. While small molecules approximately 600 to 800 Da in size can be taken up directly (7,8), larger compounds need to be enzymatically hydrolyzed outside the cell before uptake. This two-phase system of hydrolysis and subsequent uptake is a rate-limiting step of microbial enzymatic degradation (9-11), and a mechanistic understanding of these processes, therefore, is of global biogeochemical relevance. Furthermore, the bacterioplankton are faced with the challenges of spatial heterogeneity (12, 13) and individual compounds at extremely dilute concentrations (14-16). For a microbe, there is presumably a delicate balance between the substrate encounter rate and the energy cost associated with carrying and maintaining an enzymatic apparatus for substrate uptake.It has been proposed that bacteria have adapted to the "landscape" of marine DOM by two very different trophic strategies. Copiotrophs use a "feast and famine" strategy where they proliferate from low abundances upon exposure to high substrate levels (17), such as in association with detrital aggregates or phytoplankton blooms. Copiotrophs are distinguished...

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The collisional limit: an important consideration for membrane‐associated enzymes and receptors

Cited by 47 publications

References 29 publications

Elevated Function of Blood Clotting Factor VIIa Mutants That Have Enhanced Affinity for Membranes

Elevated Function of Blood Clotting Factor VIIa Mutants That Have Enhanced Affinity for Membranes

Rapid-mix flow cytometry measurements of subsecond regulation of G protein-coupled receptor ternary complex dynamics by guanine nucleotides

A Model of Extracellular Enzymes in Free-Living Microbes: Which Strategy Pays Off?

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