A simple enzymic method to separate [3H]inositol 1,4,5- and 1,3,4-trisphosphate isomers in tissue extracts.
A novel method to separate [3H]Ins(1,4,5)P3 and [3H]Ins(1,3,4)P3 in tissue extracts is described. It is based on the selective metabolism of Ins(1,3,4)P3 by a crude cerebral supernatant in a Mg2+-free buffer followed by separation of [3H]inositol trisphosphates using conventional anion-exchange chromatography. Evaluation of the assay was performed using [3H]Ins(1,3,4)P3 standards and tissue extracts containing different proportions of [3H]Ins(1,4,5)P3 and [3H]Ins(1,3,4)P3. Parallel h.p.l.c. separations of extracts established the selective and complete metabolism of [3H]Ins(1,3,4)P3 under the above conditions and demonstrated that the enzymic method provides an accurate estimate of the trisphosphate isomers in rat cerebral cortex, parotid gland and bovine tracheal smooth muscle.
Kennedy
E D
ED
Department of Pharmacology and Therapeutics, University of Leicester, U.K.
Batty
I H
IH
Chilvers
E R
ER
Nahorski
S R
SR
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002417:Cattle; D002540:Cerebral Cortex / Q000032:analysis / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid; D006868:Hydrolysis; D015544:Inositol 1,4,5-Trisphosphate; D007295:Inositol Phosphates / Q000302:isolation & purification*; D007536:Isomerism; D051379:Mice; D009130:Muscle, Smooth / Q000032:analysis; D010306:Parotid Gland / Q000032:analysis; D051381:Rats; D013403:Sugar Phosphates / Q000302:isolation & purification*; D014132:Trachea / Q000032:analysis
D007295:Inositol Phosphates; D013403:Sugar Phosphates; D015544:Inositol 1,4,5-Trisphosphate