Generation of continuous ligand–macromolecule binding isotherms. Use of exponential concentration gradients

Mountcastle, Donald B.; Freire, Ernesto; Biltonen, Rodney L.

doi:10.1002/bip.1976.360150212

Cited by 29 publications

(6 citation statements)

References 18 publications

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“…Our microcalorimetric assays were typically performed in a point by point manner with about five substrate concentrations. In a few cases (as noted in Table I), a continuous concentration gradient of substrate was used (Mountcastle et al, 1976;Eftink et al, 1981b). In the latter experiments, a very large number (~200) of [P]T vs. [S]0 data points were collected, thus allowing an accurate analysis for Fmax and Km to be made.…”

mentioning

confidence: 99%

Energetics of ribonuclease A catalysis. 1. pH, ionic strength, and solvent isotope dependence of the hydrolysis of cytidine cyclic 2',3'-phosphate

Eftink¹,

Biltonen²

1983

Biochemistry

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The pH, ionic strength, and solvent deuterium isotope dependence of the steady-state kinetics of the ribonuclease A catalyzed hydrolysis of cytidine cyclic 2',3'-phosphate has been investigated by using, primarily, the technique of flow microcalorimetry to monitor the kinetics. The pH dependence of the Michaelis-Menten parameters has been analyzed by assuming the participation of His-12 and -119 of the enzyme and a third ionizing group, postulated to be on the pyrimidine ring of the substrate, to determine the pH-independent rate constant kc, and Michaelis constant Km. The reported pH analysis, together with existing NMR data and chemical modification studies, allows an assignment of the functional roles of His-12 and -119 as being those of general acid and general base catalytic residues, respectively. At high pH, the apparent Km value is found to increase to unity. This drop in affinity between the enzyme and the substrate at high pH indicates that the substrate binds to the enzyme primarily through an electrostatic interaction with the active-site histidine residues, particularly His-12. The apparent absence of an interaction with the riboside portion of the substrate is suggested to be due to the fact that the substrate exists in a syn conformation about its glycosidic bond and thus cannot interact optimally with the enzyme's binding pocket. This will result in a relative destabilization of the enzyme-substrate complex, which can then be relieved upon the formation of the transition state. The ionic strength dependence of ribonuclease activity is shown to be primarily a result of its effect on the pKa of the histidine residues and a concomitant change in the value of Km.

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confidence: 99%

Energetics of ribonuclease A catalysis. 1. pH, ionic strength, and solvent isotope dependence of the hydrolysis of cytidine cyclic 2',3'-phosphate

Eftink¹,

Biltonen²

1983

Biochemistry

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“…Heat of dilution data were generated continuously through measurement of a sample having an exponential gradient in DiC6 concentration established, as described by Mountcastle et al (1976), by use of an open vessel with an effective volume of about 6 mL. The gradient had a half-time of about 18 min.…”

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confidence: 99%

“…Approximately 1500 data points were collected during an experiment. The data were corrected for the response time of the calorimeter (Mountcastle et al, 1976) and then converted, using the initial DiC6 concentration and the time constant for the exponential gradient determined from the HC1 dilution experiment, to an array of DiC6 concentrations and corresponding heats of dilution. For analysis of the data, approximately 100 eight-point averages were extracted from the full data set; these were selected to be as closely as possible evenly spaced in concentration.…”

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confidence: 99%

Thermodynamics of dihexanoylphosphatidylcholine aggregation

Johnson¹,

Wells²,

Rupley³

1981

Biochemistry

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Heats of dilution of aqueous solutions of dihexanoylphosphatidylcholine were determined by use of a flow microcalorimeter to monitor an exponential dilution gradient. Three different models of micelle formation were tested: monomer in equilibrium with micelles of fixed size, with micelles of varied size, or with small aggregates and micelles. The heat of dilution data for low solute concentration could be fit only by assuming the existence of premicellar aggregates. The critical micelle concentration determined calorimetrically is 0.016 +/- 0.002 M and is independent of the model. The enthalpy change for transfer of monomer into the micelle is 1.6 +/- 0.2 kcal/mol; about one-third of this heat effect is produced in formation of the premicellar aggregation. Comparison of the calorimetric measurements with results obtained by using other methods indicates the complexity of the micellization process.

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“…Both GG types have impacted a wide range of biological studies, including cancer metastasis, immune response, angiogenesis, and others. [34][35][36] In addition, microfluidic GGs have been used to investigate stem cell behavior, 24 e.g., the influence of a mixture of growth factors on human neural stem cell proliferation and differentiation 12 and the effect of a bovine serum albumin concentration gradient on human embryonic stem cell viability and morphology. 37 Despite the variety of microfluidic devices for stem cell applications, 17 there is a dearth of simple-to-use, yet potentially powerful experimental platforms with the capability to test cellular response to a chemical concentration gradient.…”

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confidence: 99%

Polyester μ-assay chip for stem cell studies

et al. 2012

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The application of microfluidic technologies to stem cell research is of great interest to biologists and bioengineers. This is chiefly due to the intricate ability to control the cellular environment, the reduction of reagent volume, experimentation time and cost, and the high-throughput screening capabilities of microscale devices. Despite this importance, a simple-to-use microfluidic platform for studying the effects of growth factors on stem cell differentiation has not yet emerged. With this consideration, we have designed and characterized a microfluidic device that is easy to fabricate and operate, yet contains several functional elements. Our device is a simple polyester-based microfluidic chip capable of simultaneously screening multiple independent stem cell culture conditions. Generated by laser ablation and stacking of multiple layers of polyester film, this device integrates a 10 Â 10 microwell array for cell culture with a continuous perfusion system and a non-linear concentration gradient generator. We performed numerical calculations to predict the gradient formation and calculate the shear stress acting on the cells inside the device. The device operation was validated by culturing murine embryonic stem cells inside the microwells for 5 days. Furthermore, we showed the ability to maintain the pluripotency of stem cell aggregates in response to concentrations of leukemia inhibitory factor ranging from 0 to $1000 U/ml. Given its simplicity, fast manufacturing method, scalability, and the cell-compatible nature of the device, it may be a useful platform for long-term stem cell culture and studies.

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Generation of continuous ligand–macromolecule binding isotherms. Use of exponential concentration gradients

Cited by 29 publications

References 18 publications

Energetics of ribonuclease A catalysis. 1. pH, ionic strength, and solvent isotope dependence of the hydrolysis of cytidine cyclic 2',3'-phosphate

Energetics of ribonuclease A catalysis. 1. pH, ionic strength, and solvent isotope dependence of the hydrolysis of cytidine cyclic 2',3'-phosphate

Thermodynamics of dihexanoylphosphatidylcholine aggregation

Polyester μ-assay chip for stem cell studies

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