Enzymatic fluorometric assay for myo-inositol trisphosphate

James A. Shayman; Aubrey R. Morrison; Oliver H. Lowry

doi:10.1016/0003-2697(87)90434-9

Enzymatic fluorometric assay for myo-inositol trisphosphate

James A. Shayman, Aubrey R. Morrison, Oliver H. Lowry

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21 Scopus citations

Abstract

The determination of myo-inositol trisphosphate by an enzymatic fluorometric assay is presented. The method involves the acid extraction of water-soluble inositol polyphosphates followed by separation by anion-exchange chromatography. Samples are subsequently neutralized by passage over a Dowex Cl^- resin and elution with lithium chloride. Samples are then desalted with ethanol. Following dephosphorylation with alkaline phosphatase, free myo-inositol is measured enzymatically via the NAD-dependent oxidation to scyllo-inosose with myo-inositol dehydrogenase. The efficiency of recovery, assay specificity, and an application to the measurement of inositol polyphosphates in hormone-stimulated tissue are discussed.

Original language	English
Pages (from-to)	562-568
Number of pages	7
Journal	Analytical Biochemistry
Volume	162
Issue number	2
DOIs	https://doi.org/10.1016/0003-2697(87)90434-9
State	Published - May 1 1987

Keywords

enzymatic assay
fluorometry
inositol polyphosphate

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10.1016/0003-2697(87)90434-9

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title = "Enzymatic fluorometric assay for myo-inositol trisphosphate",

abstract = "The determination of myo-inositol trisphosphate by an enzymatic fluorometric assay is presented. The method involves the acid extraction of water-soluble inositol polyphosphates followed by separation by anion-exchange chromatography. Samples are subsequently neutralized by passage over a Dowex Cl- resin and elution with lithium chloride. Samples are then desalted with ethanol. Following dephosphorylation with alkaline phosphatase, free myo-inositol is measured enzymatically via the NAD-dependent oxidation to scyllo-inosose with myo-inositol dehydrogenase. The efficiency of recovery, assay specificity, and an application to the measurement of inositol polyphosphates in hormone-stimulated tissue are discussed.",

keywords = "enzymatic assay, fluorometry, inositol polyphosphate",

author = "Shayman, {James A.} and Morrison, {Aubrey R.} and Lowry, {Oliver H.}",

note = "Funding Information: {\textquoteright} This work was supported by U.S.P.H.S. Grants 1 KO8 AM 0 1479-O 1 (JAS), AM 030542 (ARM), and NS 08862 and American Cancer Society BC-4-28(OHL). {\textquoteleft}To whom reprint requests should be addressed at current address: Nephrology Division, Department of Internal Medicine, The University of Michigan Medical Center, 39 14 Taubman Center, Ann Arbor, MI 48 109-0364.",

year = "1987",

month = may,

day = "1",

doi = "10.1016/0003-2697(87)90434-9",

language = "English",

volume = "162",

pages = "562--568",

journal = "Analytical Biochemistry",

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TY - JOUR

T1 - Enzymatic fluorometric assay for myo-inositol trisphosphate

AU - Shayman, James A.

AU - Morrison, Aubrey R.

AU - Lowry, Oliver H.

N1 - Funding Information: ’ This work was supported by U.S.P.H.S. Grants 1 KO8 AM 0 1479-O 1 (JAS), AM 030542 (ARM), and NS 08862 and American Cancer Society BC-4-28(OHL). ‘To whom reprint requests should be addressed at current address: Nephrology Division, Department of Internal Medicine, The University of Michigan Medical Center, 39 14 Taubman Center, Ann Arbor, MI 48 109-0364.

PY - 1987/5/1

Y1 - 1987/5/1

N2 - The determination of myo-inositol trisphosphate by an enzymatic fluorometric assay is presented. The method involves the acid extraction of water-soluble inositol polyphosphates followed by separation by anion-exchange chromatography. Samples are subsequently neutralized by passage over a Dowex Cl- resin and elution with lithium chloride. Samples are then desalted with ethanol. Following dephosphorylation with alkaline phosphatase, free myo-inositol is measured enzymatically via the NAD-dependent oxidation to scyllo-inosose with myo-inositol dehydrogenase. The efficiency of recovery, assay specificity, and an application to the measurement of inositol polyphosphates in hormone-stimulated tissue are discussed.

AB - The determination of myo-inositol trisphosphate by an enzymatic fluorometric assay is presented. The method involves the acid extraction of water-soluble inositol polyphosphates followed by separation by anion-exchange chromatography. Samples are subsequently neutralized by passage over a Dowex Cl- resin and elution with lithium chloride. Samples are then desalted with ethanol. Following dephosphorylation with alkaline phosphatase, free myo-inositol is measured enzymatically via the NAD-dependent oxidation to scyllo-inosose with myo-inositol dehydrogenase. The efficiency of recovery, assay specificity, and an application to the measurement of inositol polyphosphates in hormone-stimulated tissue are discussed.

KW - enzymatic assay

KW - fluorometry

KW - inositol polyphosphate

UR - http://www.scopus.com/inward/record.url?scp=0023191309&partnerID=8YFLogxK

U2 - 10.1016/0003-2697(87)90434-9

DO - 10.1016/0003-2697(87)90434-9

M3 - Article

C2 - 3496810

AN - SCOPUS:0023191309

SN - 0003-2697

VL - 162

SP - 562

EP - 568

JO - Analytical Biochemistry

JF - Analytical Biochemistry

IS - 2

ER -

Enzymatic fluorometric assay for myo-inositol trisphosphate

Abstract

Keywords

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