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An automated method to evaluate the enzyme kinetics of ?-glucosidases.

ABSTRACT: Enzyme kinetic measurements are important for the characterization and engineering of biocatalysts, with applications in a wide range of research fields. The measurement of initial reaction velocity is usually slow and laborious, which motivated us to explore the possibilities for automating this process. Our model enzyme is the maize ?-glucosidase Zm-p60.1. Zm-p60.1 plays a significant role in plant growth and development by regulating levels of the active plant hormone cytokinin. Zm-p60.1 belongs to a wide group of hydrolytic enzymes. Members of this group hydrolyze several different types of glucosides, releasing glucose as a secondary product. Enzyme kinetic measurements using artificial substrates are well established, but burdensome and time-consuming. Thus, they are a suitable target for process automation. Simple optical methods for enzyme kinetic measurements using natural substrates are often impossible given the optical properties of the enzymatic reaction products. However, we have developed an automated method based on glucose detection, as glucose is released from all substrates of glucosidase reactions. The presented method can obtain 24 data points from up to 15 substrate concentrations to precisely describe the enzyme kinetics. The combination of an automated liquid handling process with assays that have been optimized for measuring the initial hydrolysis velocity of ?-glucosidases yields two distinct methods that are faster, cheaper, and more accurate than the established protocols.

SUBMITTER: Klimes P

PROVIDER: S-EPMC5275727 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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An automated method to evaluate the enzyme kinetics of β-glucosidases.

Klimeš Pavel P Mazura Pavel P Turek Dušan D Brzobohatý Břetislav B

Protein science : a publication of the Protein Society 20161124 2

Enzyme kinetic measurements are important for the characterization and engineering of biocatalysts, with applications in a wide range of research fields. The measurement of initial reaction velocity is usually slow and laborious, which motivated us to explore the possibilities for automating this process. Our model enzyme is the maize β-glucosidase Zm-p60.1. Zm-p60.1 plays a significant role in plant growth and development by regulating levels of the active plant hormone cytokinin. Zm-p60.1 belo ...[more]

PMID: 27862518

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Project description:BACKGROUND:Mesenchymal stromal cells (MSC) demonstrate innate and regulatory immunologic functions and have been widely explored for cell therapy applications. Mechanisms by which MSCs achieve therapeutic effects are theorized, though appropriate dosing and duration of these mechanisms in vivo warrant deeper investigation. One, rapid immunosuppressive function of MSCs is through ectoenzyme expression of CD73 and CD39 which cooperatively hydrolyze inflammatory, extracellular adenosine triphosphate (ATP) to anti-inflammatory adenosine. Extracellular ATP has a key role in autoimmune and inflammatory diseases, which administered MSCs have the potential to modulate in a timescale that is befitting of shorter acting therapeutic function. METHODS:In vitro experiments were performed to determine the hydrolysis rates of ATP by MSCs. Through kinetic modeling from experimental results, the rate at which a single cell can metabolize ATP was determined. Based on these rates, the ability of MSCs to downregulate inflammatory signaling pathways was prospectively validated using model system parameters with respect to two different mechanisms: extracellular ATP stimulates lymphocytes to suppress proliferation and induce apoptosis and with co-stimulation, it stimulates monocytes to release pro-inflammatory IL-1?. MSCs were co-cultured with immune cells using transwell inserts and compared to immune cell only groups. RESULTS:Hydrolysis of ATP was efficiently modeled by first-order enzyme kinetics. For in vitro culture, the rate at which a single cell can hydrolyize ATP is 8.9 nmol/min. In the presence of extracellular ATP, cocultures of MSCs reduced cytotoxicity and allows for proliferation of lymphocytes while limiting IL-1? secretion from monocytes. CONCLUSIONS:Such use of these models may allow for better dosing predictions for MSCs to be used therapeutically for chronic inflammatory diseases such as rheumatoid arthritis, diabetes, pancreatitis, and other systemic inflammatory response syndromes. For the first time, the effect of MSCs on ATP hydrolysis in immune cell response is quantitatively analyzed on a cell-molecule basis by modeling the hydrolysis as an enzyme-substrate reaction. The results also give insight into MSCs' dynamic response mechanisms to ameliorate effects of extracellular ATP whether it be through positive or negative regulation.

Dataset Information

An automated method to evaluate the enzyme kinetics of ?-glucosidases.

Publications

An automated method to evaluate the enzyme kinetics of β-glucosidases.

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