Detecting purinosome metabolon formation with fluorescence microscopy

Anthony M. Pedley, Stephen Benkovic

Research output: Chapter in Book/Report/Conference proceedingChapter

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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages279-289
Number of pages11
DOIs
StatePublished - Jan 1 2018

Publication series

NameMethods in Molecular Biology
Volume1764
ISSN (Print)1064-3745

Fingerprint

Fluorescence Microscopy
Enzymes
Multienzyme Complexes
Purines
Biosynthetic Pathways
HeLa Cells
Confocal Microscopy
Fluorescent Antibody Technique
Cell Count
Proteins
purine

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Genetics

Cite this

Pedley, A. M., & Benkovic, S. (2018). Detecting purinosome metabolon formation with fluorescence microscopy. In Methods in Molecular Biology (pp. 279-289). (Methods in Molecular Biology; Vol. 1764). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-7759-8_17
Pedley, Anthony M. ; Benkovic, Stephen. / Detecting purinosome metabolon formation with fluorescence microscopy. Methods in Molecular Biology. Humana Press Inc., 2018. pp. 279-289 (Methods in Molecular Biology).
@inbook{56aeb8a5004f46d49152ac22c197d83a,
title = "Detecting purinosome metabolon formation with fluorescence microscopy",
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.",
author = "Pedley, {Anthony M.} and Stephen Benkovic",
year = "2018",
month = "1",
day = "1",
doi = "10.1007/978-1-4939-7759-8_17",
language = "English (US)",
series = "Methods in Molecular Biology",
publisher = "Humana Press Inc.",
pages = "279--289",
booktitle = "Methods in Molecular Biology",

}

Pedley, AM & Benkovic, S 2018, Detecting purinosome metabolon formation with fluorescence microscopy. in Methods in Molecular Biology. Methods in Molecular Biology, vol. 1764, Humana Press Inc., pp. 279-289. https://doi.org/10.1007/978-1-4939-7759-8_17

Detecting purinosome metabolon formation with fluorescence microscopy. / Pedley, Anthony M.; Benkovic, Stephen.

Methods in Molecular Biology. Humana Press Inc., 2018. p. 279-289 (Methods in Molecular Biology; Vol. 1764).

Research output: Chapter in Book/Report/Conference proceedingChapter

TY - CHAP

T1 - Detecting purinosome metabolon formation with fluorescence microscopy

AU - Pedley, Anthony M.

AU - Benkovic, Stephen

PY - 2018/1/1

Y1 - 2018/1/1

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=85044831677&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85044831677&partnerID=8YFLogxK

U2 - 10.1007/978-1-4939-7759-8_17

DO - 10.1007/978-1-4939-7759-8_17

M3 - Chapter

T3 - Methods in Molecular Biology

SP - 279

EP - 289

BT - Methods in Molecular Biology

PB - Humana Press Inc.

ER -

Pedley AM, Benkovic S. Detecting purinosome metabolon formation with fluorescence microscopy. In Methods in Molecular Biology. Humana Press Inc. 2018. p. 279-289. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-4939-7759-8_17