Biochemistry

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    Protocols in Current Issue
    Measurement of ATPase Activity of Valosin-containing Protein/p97
    [Abstract] Valosin-containing protein (VCP; also known as p97) is a type II ATPase regulating several cellular processes. Using proteomic techniques, we identified a chemical compound that binds to the D1 ATPase domain of VCP. The protocol described here was to study the effect of the compound on ATPase activity in vitro of purified VCP protein. ...
    Sulfatase Assay to Determine Influence of Plants on Microbial Activity in Soil
    Authors:  Anna Koprivova, Achim Schmalenberger and Stanislav Kopriva, date: 01/20/2020, view: 526, Q&A: 0
    [Abstract] Sulfatase activity is often used as a measure of the activity of soil microorganisms. It is thus a suitable tool to investigate the response of microbes to plants. Here we present a method to determine the influence of various Arabidopsis genotypes on the function of soil microbiota using the sulfatase as a quantitative measure. We grew ...
    A Radioactive-free Kinase Inhibitor Discovery Assay Against the Trypanosoma brucei Glycogen Synthase Kinase-3 short (TbGSK-3s)
    Authors:  Antonia Efstathiou and Despina Smirlis, date: 01/20/2020, view: 192, Q&A: 0
    [Abstract] The identification of small molecules possessing inhibitory activity in vitro, against a given target kinase, is the first step in the drug discovery process. Herein, we describe a non radioactive protocol using luciferase-based ATP assay for the identification of inhibitors for the short isoform of the Trypanosoma brucei’s ...
    Measurement of Acid Ecto-phosphatase Activity in Live Leishmania donovani Parasites
    Authors:  Amalia Papadaki and Haralabia Boleti, date: 10/05/2019, view: 547, 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
    [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: 1805, 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: 998, 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: 1504, 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
    [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: 2033, 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 ...



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