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Measurement of Acid Ecto-phosphatase Activity in Live Leishmania donovani Parasites

Authors: Amalia Papadaki and Haralabia Boleti, date: 10/05/2019, view: 314, Q&A: 0

[Abstract] Acid ecto-phosphatases are enzymes that hydrolyze phosphomonoesters in the acidic pH range with their active sites facing the extacellular medium. Their activities can be measured in living cells. In bacteria and protozoan pathogens, acid ecto-phosphatases have been associated with the survival of intracellular pathogens within phagocytes through ...

Subcellular Fractionation of Hela Cells for Lysosome Enrichment Using a Continuous Percoll-density Gradient

Authors: Julian M. Carosi, Kathryn J. Hattersley, Yi Cui, Zhe Yang, Rohan D. Teasdale and Timothy J. Sargeant, date: 09/20/2019, view: 700, Q&A: 0

[Abstract] The enrichment of lysosomes is a useful way to study their structure and function. These dynamic vesicles can be enriched from cell cultures in a variety of ways including immunoprecipitation and fluorescence-activated organelle sorting. These methods are extremely precise but often require the transfection and expression of an affinity or ...

Fluorescence HPLC Analysis of the in-vivo Activity of Glucosylceramide Synthase

Authors: Kartik R. Roy, Sachin K. Khiste, Zhijun Liu and Yong-Yu Liu, date: 06/20/2019, view: 1596, Q&A: 0

[Abstract] Almost all functions of cells or organs rely on the activities of cellular enzymes. Indeed, the in-vivo activities that directly represent the cellular effects of enzymes in live organs are critical importance to appreciate the roles enzymes play in modulating physiological or pathological processes, although assessments of such in-vivo ...

Probing Conformational States of a Target Protein in Escherichia coli Cells by in vivo Cysteine Cross-linking Coupled with Proteolytic Gel Analysis

Authors: Sujeet Kumar and Natividad Ruiz , date: 06/20/2019, view: 781, Q&A: 0

[Abstract] Transporters are dynamic membrane proteins that are essential to the physiology of cells. To function, transporters must cycle between various conformational states, so to understand their mechanistic details, it is critical to characterize how their structure changes during the transport cycle. One approach to studying the dynamics of ...

Non-radiometric Cell-free Assay to Measure the Effect of Molecular Chaperones on AMP-activated Kinase Activity

Authors: Elizabeth B. Wilson, Carrie E. Rubel and Jonathan C. Schisler, date: 04/20/2019, view: 1311, Q&A: 0

[Abstract] AMP-activated kinase (AMPK) is a trimeric protein holoenzyme with kinase activity. AMPK plays an important role in cellular metabolism and is thought to function as a fuel sensor within the cell, exerting kinase activity to activate energy-conserving pathways and simultaneously inhibit energy-consuming pathways. Traditional in vitro ...

Insulin Tolerance Test under Anaesthesia to Measure Tissue-specific Insulin-stimulated Glucose Disposal

Authors: Daniel J. Fazakerley, Andreas M. Fritzen, Marin E. Nelson, Ida H. Thorius, Kristen C. Cooke, Sean J. Humphrey, Gregory J. cooney and David E. James, date: 01/20/2019, view: 2059, Q&A: 0

[Abstract] Insulin resistance is a pathophysiological state defined by impaired responses to insulin and is a risk factor for several metabolic diseases, most notably type 2 diabetes. Insulin resistance occurs in insulin target tissues including liver, adipose and skeletal muscle. Methods such as insulin tolerance tests and hyperinsulinaemic-euglycaemic ...

Detection of D-glutamate Production from the Dual Function Enzyme, 4-amino-4-deoxychorismate Lyase/D-amino Acid Transaminase, in Mycobacterium smegmatis

Authors: Helen K. Opel-Reading, Roman Mortuza and Kurt L. Krause, date: 01/05/2019, view: 1713, Q&A: 0

[Abstract] D-amino acid transaminase (D-AAT) is able to synthesize both D-glutamate and D-alanine, according to the following reaction: D-alanine + α-ketoglutarate ⇌ D-glutamate + pyruvate. These two D-amino acids are essential components of the peptidoglycan layer of bacteria. In our recently published work, MSMEG_5795 from Mycobacterium smegmatis ...

Quantitation of Regulatory Activity for the Complement Alternative Pathway Using an Adaptation of the AP₅₀ in vitro Assay

Authors: Sheila Cabezas Falcon, David L Gordon and Jillian M Carr, date: 12/20/2018, view: 1408, Q&A: 2

[Abstract] Complement pathways function to identify and remove pathogens and infected cells. There are three complement pathways: the classical, lectin and alternative pathway (AP). While all pathways are activated following pathogen stimuli, the AP is constitutively active and tightly controlled by activators (e.g., Factor B, Factor D) and negative ...

Integrin Dependent RhoB Activation Assay Using Leukocytes

Authors: Janne Lehtonen, Katarzyna Potrzebowska, Malin Samuelsson and Lena Svensson, date: 12/05/2018, view: 928, Q&A: 0

[Abstract] To be able to migrate, leukocyte needs to re-use its adhesion molecules to move forward. These adhesion molecules are called integrins and are intracellularly transported via endocytosis and exocytosis in order to translocate to a new site on the cell membrane. The intracellular transportation is regulated by different small GTPases including ...

Kinetic Characterization of the Shigella Type Three Secretion System ATPase Spa47 Using α-³²P ATP

Authors: Heather B. Case and Nicholas E. Dickenson, date: 11/05/2018, view: 1617, Q&A: 0

[Abstract] ATPases represent a diverse class of enzymes that utilize ATP hydrolysis to support critical biological functions such as driving ion pumps, providing mechanical work, unfolding/folding proteins, and supporting otherwise thermodynamically unfavorable chemical reactions. We have recently shown that the Shigella protein Spa47 is an ATPase ...

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