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Production and Bioassay of a Diffusible Factor That Induces Gametophyte-to-Sporophyte Developmental Reprogramming in the Brown Alga Ectocarpus

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The ATPase Activity of Escherichia coli Expressed AAA⁺-ATPase Protein

Authors: Amita Kaundal, Vemanna S. Ramu and Kirankumar S. Mysore, date: 08/05/2020, view: 573, Q&A: 0

[Abstract] ATPases are the enzymes that breakdown ATP to ADP and release inorganic phosphate (Pi). Here we provide a detailed protocol to determine the ATPase activity of a recombinant AAA⁺-ATPase protein (GENERAL CONTROL NON-REPRESSIBLE-4 [GCN4]) by spectrophotometric absorption at 360 nm to measure the accumulated inorganic phosphate. In ...

Quantification of Protein Kinase A (PKA) Activity by An in vitro Radioactive Assay Using the Mouse Sperm Derived Enzyme

Authors: Cintia Stival, Carolina Baro Graf, Pablo E. Visconti and Dario Krapf, date: 06/20/2020, view: 923, Q&A: 0

[Abstract] In order to acquire fertilizing potential, mammalian sperm must undergo a process known as capacitation, which relies on the early activation of Protein Kinase A (PKA). Frequently, PKA activity is assessed in whole-cell experiments by analyzing the phosphorylation status of its substrates in a western-blot. This technique ...

Preparation of HeLa Total Membranes and Assay of Lipid-inhibition of Serine Palmitoyltransferase Activity

Authors: Muthukumar Kannan, Deanna L. Davis, John Suemitsu, Christopher D. Oltorik and Binks Wattenberg, date: 06/20/2020, view: 809, Q&A: 0

[Abstract] Serine palmitoyltranferase (SPT) is a pyridoxal 5′ phosphate (PLP)-dependent enzyme that catalyzes the first and rate-limiting step of de novo synthesis of sphingolipids. SPT activity is homeostatically regulated in response to increased levels of sphingolipids. This homeostatic regulation of SPT is mediated through small ER membrane ...

A Sensitive Coupled Enzyme Assay for Measuring Kinase and ATPase Kinetics Using ADP-Specific Hexokinase

Authors: Ciaran R. McFarlane and James W. Murray, date: 05/05/2020, view: 967, Q&A: 0

[Abstract] Kinases and ATPases perform essential biological functions in metabolism and regulation. Activity of these enzymes is commonly measured by coupling ATP consumption to the synthesis of a detectable product. For most assay systems the ATP concentration during the reaction is unknown, compromising the precision of the assay.

Using the ...

Colorimetric RhoB GTPase Activity Assay

Authors: Kossay Zaoui and Stephanie Duhamel, date: 05/05/2020, view: 1467, Q&A: 0

[Abstract] The Ras homologous protein (Rho) GTPase subfamily, including RhoA, RhoB, and RhoC are small molecules (~21 kDa) that act as molecular switches in a wide range of signaling pathways to orchestrate biological processes associated with both physiological and tumorigenic cellular states. The Rho GTPases are crucial regulators of actin cytoskeleton ...

In vivo Quantification of Alkanes in Escherichia coli

Authors: Tabinda Shakeel, Zia Fatma and Syed Shams Yazdani, date: 04/20/2020, view: 1198, Q&A: 0

[Abstract] Microbial production of alkanes employing synthetic biology tools has gained tremendous attention owing to the high energy density and similarity of alkanes to existing petroleum fuels. One of the most commonly studied pathways includes the production of alkanes by AAR (acyl-ACP (acyl carrier protein) reductase)-ADO (aldehyde deformylating ...

An in vitro Assay to Screen for Substrates of PKA Using Phospho-motif-specific Antibodies

Authors: David M. Gau and Partha Roy, date: 04/20/2020, view: 917, Q&A: 0

[Abstract] Kinases function as regulators of many cellular processes such as cell migration. These enzymes typically phosphorylate target motif sequences. Mass spec or phospho-specific antibody detection can be used to determine whether a kinase can phosphorylate proteins of interest, however, mass spec can be expensive and phospho-antibodies for the protein ...

Spectrophotometric Assessment of Heme Oxygenase-1 Activity in Leishmania-infected Macrophages

Authors: Moumita Basu, Shriya Saha and Anindita Ukil, date: 04/05/2020, view: 914, Q&A: 0

[Abstract] Heme oxygenase-1 (HO-1) is a stress responsive enzyme that metabolizes heme and releases free iron, carbon monoxide (CO), and biliverdin (BV), which rapidly undergoes conversion to bilirubin (BL). Estimation of bilirubin is the basis of HO-1 assay. HO-1 activity is widely employed to determine antioxidant response of cells under different ...

Manganese Superoxide Dismutase Activity Assay in the Yeast Saccharomyces cerevisiae

Authors: Louise Thines and Pierre Morsomme, date: 03/05/2020, view: 1128, Q&A: 0

[Abstract] Superoxide dismutases (SODs) act as a primary defence against reactive oxygen species (ROS) by converting superoxide anion radicals (O₂^-) into molecular oxygen (O₂) and hydrogen peroxide (H₂O₂). Members of this enzyme family include CuZnSODs, MnSODs, FeSODs, and NiSODs, depending on the nature ...

Measurement of ATPase Activity of Valosin-containing Protein/p97

Authors: Kruthi Suvarna, Kaori Honda, Makoto Muroi, Yasumitsu Kondoh, Hiroyuki Osada and Nobumoto Watanabe, date: 02/05/2020, view: 1513, Q&A: 0

[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. ...

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