Purification and Characterization of Lymphokines: An Approach to the Study of Molecular Mechanisms of Cell-Mediated Immunity1

2015 ◽  
pp. 106-162
Author(s):  
Maurice K. Gately ◽  
Manfred M. Mayer
Keyword(s):  
Molecular Mechanisms ◽  
Purification And Characterization

scholarly journals Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics

Science ◽  
2021 ◽  
Vol 373 (6553) ◽  
pp. eabf8761
Author(s):  
C. J. Markin ◽  
D. A. Mokhtari ◽  
F. Sunden ◽  
M. J. Appel ◽  
E. Akiva ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Enzyme Kinetics ◽  
High Throughput ◽  
Active Site ◽  
Molecular Mechanisms ◽  
Extensive Investigation ◽  
Purification And Characterization ◽  
Multiple Substrates ◽  
Catalytic Effects

Systematic and extensive investigation of enzymes is needed to understand their extraordinary efficiency and meet current challenges in medicine and engineering. We present HT-MEK (High-Throughput Microfluidic Enzyme Kinetics), a microfluidic platform for high-throughput expression, purification, and characterization of more than 1500 enzyme variants per experiment. For 1036 mutants of the alkaline phosphatase PafA (phosphate-irrepressible alkaline phosphatase of Flavobacterium), we performed more than 670,000 reactions and determined more than 5000 kinetic and physical constants for multiple substrates and inhibitors. We uncovered extensive kinetic partitioning to a misfolded state and isolated catalytic effects, revealing spatially contiguous regions of residues linked to particular aspects of function. Regions included active-site proximal residues but extended to the enzyme surface, providing a map of underlying architecture not possible to derive from existing approaches. HT-MEK has applications that range from understanding molecular mechanisms to medicine, engineering, and design.

scholarly journals Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics

2020 ◽  
Author(s):  
C.J. Markin ◽  
D.A. Mokhtari ◽  
F. Sunden ◽  
M.J. Appel ◽  
E. Akiva ◽  
...  
Keyword(s):  
High Throughput ◽  
Active Site ◽  
Molecular Mechanisms ◽  
Extensive Investigation ◽  
Purification And Characterization ◽  
Functional Architecture ◽  
Microfluidic Platform ◽  
Multiple Substrates ◽  
Catalytic Effects

AbstractSystematic and extensive investigation of enzymes is needed to understand their extraordinary efficiency and meet current challenges in medicine and engineering. We present HT-MEK, a microfluidic platform for high-throughput expression, purification, and characterization of >1500 enzyme variants per experiment. For 1036 mutants of the alkaline phosphatase PafA, we performed >670,000 reactions to determine >5000 kinetic and physical constants for multiple substrates and inhibitors. These constants allowed us to uncover extensive kinetic partitioning to a misfolded state and isolate catalytic effects, revealing spatially contiguous “regions” of residues linked to particular aspects of function. These regions included active-site proximal residues but also extended to the enzyme surface, providing a map of underlying architecture that could not be derived from existing approaches. HT-MEK, using direct and coupled fluorescent assays, has future applications to a wide variety of problems ranging from understanding molecular mechanisms to medicine to engineering and design.One Sentence SummaryHT-MEK, a microfluidic platform for high-throughput, quantitative biochemistry, reveals enzyme architectures shaping function.

scholarly journals Purification and characterization of GAD65-specific monoclonal autoantibodies

F1000Research ◽  
2015 ◽  
Vol 4 ◽  
pp. 135 ◽  
Author(s):  
Wei Jiang ◽  
Henriette Macmillan ◽  
Anne-Marie Madec ◽  
Elizabeth D. Mellins
Keyword(s):  
Molecular Mechanisms ◽  
Critical Role ◽  
Antigen Binding ◽  
Healthy Individuals ◽  
Β Cells ◽  
Purification And Characterization ◽  
Antigen Binding Site ◽  
Patients At Risk

Autoantibodies against antigens expressed by insulin-producing β cells are circulating in both healthy individuals and patients at risk of developing Type 1 diabetes. Recent studies suggest that another set of antibodies (anti-idiotypic antibodies) exists in this antibody/antigen interacting network to regulate auto-reactive responses. Anti-idiotypic antibodies may block the antigen-binding site of autoantibodies or inhibit autoantibody expression and secretion. The equilibrium between autoantibodies and anti-idiotypic antibodies plays a critical role in mediating or preventing autoimmunity. Herein, using GAD65/anti-GAD65 autoantibodies as a model system, we aimed at establishing reliable approaches for purification of highly pure autoantibodies for the downstream investigation of molecular mechanisms underlying such a network.

Purification and characterization of cholesterol sulfotransferase from rat skin

2001 ◽  
Vol 79 (4) ◽  
pp. 499-506 ◽  
Author(s):  
James I Rearick ◽  
Eric S Calhoun
Keyword(s):  
Gel Electrophoresis ◽  
Molecular Mechanisms ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Partial Purification ◽  
Epidermal Keratinocytes ◽  
Rat Skin ◽  
Purification And Characterization ◽  
Coomassie Blue

Previous work has demonstrated that the activity of the enzyme cholesterol sulfotransferase is rapidly and dramatically increased upon squamous differentiation of a variety of epithelial cells in culture, including epidermal keratinocytes. As a step toward understanding the molecular mechanisms underlying this differentiation-related change, we now report the partial purification and characterization of this enzyme activity from rat skin. Supernatant solutions from rat skin homogenates were subjected to a series of column chromatography steps including anion exchange, gel filtration, chromatofocusing and hydrophobic interaction chromatography. The purification procedure resulted in cholesterol sulfotransferase activity purified 2700-fold with a 11% recovery. The most purified preparation yielded a major Coomassie blue-stained band on denaturing polyacrylamide gel electrophoresis of an apparent molecular weight (MW) of 40 000 Da. Photoaffinity labeling with the donor substrate, 3'-phosphoadenosine-5'-phospho-[35S]-sulfate resulted in a single radiolabeled protein band on denaturing polyacrylamide gel electrophoresis, again of apparent MW 40 000 Da, strongly suggesting that the major Coomassie blue-stained band in the most purified preparation is the cholesterol sulfotransferase protein. Among 3β-hydroxysteroids with a Δ5 double bond that were tested, each served as a substrate, while androgens, estrogens, corticosteroids, p-nitrophenol and DOPA did not serve as substrates. Apparent Michaelis constants for the 3β-hydroxysteroid substrates ranged from 0.6 to 8 µM.Key words: sulfotransferase, ichthyosis, cholesterol, skin, enzymology.

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