Tips on the analysis of phosphatidic acid by the fluorometric coupled enzyme assay

Azam Hassaninasab; Gil Soo Han; George M. Carman

doi:10.1016/j.ab.2017.03.020

Tips on the analysis of phosphatidic acid by the fluorometric coupled enzyme assay

Azam Hassaninasab, Gil Soo Han, George M. Carman

School of Environmental and Biological Sciences, Food Science

Research output: Contribution to journal › Article › peer-review

9 Scopus citations

Abstract

The fluorometric coupled enzyme assay to measure phosphatidic acid (PA) involves the solubilization of extracted lipids in Triton X-100, deacylation, and the oxidation of PA-derived glycerol-3-phosphate to produce hydrogen peroxide for conversion of Amplex Red to resorufin. The enzyme assay is sensitive, but plagued by high background fluorescence from the peroxide-containing detergent and incomplete heat inactivation of lipoprotein lipase. These problems affecting the assay reproducibility were obviated by the use of highly pure Triton X-100 and by sufficient heat inactivation of the lipase enzyme. The enzyme assay could accurately measure the PA content from the subcellular fractions of yeast cells.

Original language	English (US)
Pages (from-to)	69-70
Number of pages	2
Journal	Analytical Biochemistry
Volume	526
DOIs	https://doi.org/10.1016/j.ab.2017.03.020
State	Published - Jun 1 2017

All Science Journal Classification (ASJC) codes

Biophysics
Biochemistry
Molecular Biology
Cell Biology

Keywords

Coupled enzyme assay
Fluorometric assay
Phosphatidic acid
Phosphatidic acid phosphatase
Yeast

Access to Document

10.1016/j.ab.2017.03.020

Cite this

@article{7e83afc649e347e08b680ac50656f706,

title = "Tips on the analysis of phosphatidic acid by the fluorometric coupled enzyme assay",

abstract = "The fluorometric coupled enzyme assay to measure phosphatidic acid (PA) involves the solubilization of extracted lipids in Triton X-100, deacylation, and the oxidation of PA-derived glycerol-3-phosphate to produce hydrogen peroxide for conversion of Amplex Red to resorufin. The enzyme assay is sensitive, but plagued by high background fluorescence from the peroxide-containing detergent and incomplete heat inactivation of lipoprotein lipase. These problems affecting the assay reproducibility were obviated by the use of highly pure Triton X-100 and by sufficient heat inactivation of the lipase enzyme. The enzyme assay could accurately measure the PA content from the subcellular fractions of yeast cells.",

keywords = "Coupled enzyme assay, Fluorometric assay, Phosphatidic acid, Phosphatidic acid phosphatase, Yeast",

author = "Azam Hassaninasab and Han, {Gil Soo} and Carman, {George M.}",

note = "Funding Information: This work was supported, in whole or in part, by National Institutes of Health Grant GM028140. Publisher Copyright: {\textcopyright} 2017 Elsevier Inc.",

year = "2017",

month = jun,

day = "1",

doi = "10.1016/j.ab.2017.03.020",

language = "English (US)",

volume = "526",

pages = "69--70",

journal = "Analytical Biochemistry",

issn = "0003-2697",

publisher = "Academic Press Inc.",

}

TY - JOUR

T1 - Tips on the analysis of phosphatidic acid by the fluorometric coupled enzyme assay

AU - Hassaninasab, Azam

AU - Han, Gil Soo

AU - Carman, George M.

PY - 2017/6/1

Y1 - 2017/6/1

N2 - The fluorometric coupled enzyme assay to measure phosphatidic acid (PA) involves the solubilization of extracted lipids in Triton X-100, deacylation, and the oxidation of PA-derived glycerol-3-phosphate to produce hydrogen peroxide for conversion of Amplex Red to resorufin. The enzyme assay is sensitive, but plagued by high background fluorescence from the peroxide-containing detergent and incomplete heat inactivation of lipoprotein lipase. These problems affecting the assay reproducibility were obviated by the use of highly pure Triton X-100 and by sufficient heat inactivation of the lipase enzyme. The enzyme assay could accurately measure the PA content from the subcellular fractions of yeast cells.

AB - The fluorometric coupled enzyme assay to measure phosphatidic acid (PA) involves the solubilization of extracted lipids in Triton X-100, deacylation, and the oxidation of PA-derived glycerol-3-phosphate to produce hydrogen peroxide for conversion of Amplex Red to resorufin. The enzyme assay is sensitive, but plagued by high background fluorescence from the peroxide-containing detergent and incomplete heat inactivation of lipoprotein lipase. These problems affecting the assay reproducibility were obviated by the use of highly pure Triton X-100 and by sufficient heat inactivation of the lipase enzyme. The enzyme assay could accurately measure the PA content from the subcellular fractions of yeast cells.

KW - Coupled enzyme assay

KW - Fluorometric assay

KW - Phosphatidic acid

KW - Phosphatidic acid phosphatase

KW - Yeast

UR - http://www.scopus.com/inward/record.url?scp=85016285668&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85016285668&partnerID=8YFLogxK

U2 - 10.1016/j.ab.2017.03.020

DO - 10.1016/j.ab.2017.03.020

M3 - Article

C2 - 28359787

AN - SCOPUS:85016285668

SN - 0003-2697

VL - 526

SP - 69

EP - 70

JO - Analytical Biochemistry

JF - Analytical Biochemistry

ER -

Tips on the analysis of phosphatidic acid by the fluorometric coupled enzyme assay

Abstract

All Science Journal Classification (ASJC) codes

Keywords

Access to Document

Other files and links

Fingerprint

Cite this