Book chapter
Detecting Purinosome Metabolon Formation with Fluorescence Microscopy: Methods and Protocols
pp.279-289
Methods in Molecular Biology, Humana Press Inc
01/01/2018
DOI: 10.1007/978-1-4939-7759-8_17
PMCID: PMC6396681
PMID: 29605921
Abstract
A long-standing hypothesis in the de novo purine biosynthetic pathway is that there must be highly coordinated processes to allow for enhanced metabolic flux when a cell demands purines. One mechanism by which the pathway meets its cellular demand is through the spatial organization of pathway enzymes into multienzyme complexes called purinosomes. Cellular conditions known to impact the activity of enzymes in the pathway or overall pathway flux have been reflected in a change in the number of purinosome-positive cells or the density of purinosomes in a given cell. The following general protocols outline the steps needed for purinosome detection through transient expression of fluorescent protein chimeras or through immunofluorescence in purine-depleted HeLa cells using confocal laser scanning microscopy. These protocols define a purinosome as a colocalization of FGAMS with one additional pathway enzyme, such as PPAT or GART, and provide insights into the proper identification of a purinosome from other reported cellular bodies.
Details
- Title: Subtitle
- Detecting Purinosome Metabolon Formation with Fluorescence Microscopy: Methods and Protocols
- Creators
- Anthony M. Pedley - Pennsylvania State UniversityStephen J. Benkovic - Pennsylvania State University
- Contributors
- J A Marsh (Editor)
- Resource Type
- Book chapter
- Publication Details
- pp.279-289
- Series
- Methods in Molecular Biology
- DOI
- 10.1007/978-1-4939-7759-8_17
- PMID
- 29605921
- PMCID
- PMC6396681
- NLM abbreviation
- Methods Mol Biol
- eISSN
- 1940-6029
- ISSN
- 1064-3745
- Publisher
- Humana Press Inc; TOTOWA
- Number of pages
- 11
- Grant note
- NIH GM024129 / National Institutes of Health; United States Department of Health & Human Services; National Institutes of Health (NIH) - USA
- Language
- English
- Date published
- 01/01/2018
- Academic Unit
- Biochemistry and Molecular Biology
- Record Identifier
- 9984771650602771
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