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Measurement of picomole amounts of any inositol phosphate isomer separable by h.p.l.c. by means of a bioluminescence assay.

ABSTRACT: An assay is described which allows the determination of the mass of any individual inositol phosphate (InsP) isomer by combining a popular h.p.l.c. separation method with simple desalting, dephosphorylation and final measurement of Ins liberated using an inositol dehydrogenase-NADH-linked bioluminescence reaction. The limit of sensitivity of this assay is about 1 pmol of Ins. routinely 5 pmol. About 40 mg wet wt. of guinea pig small intestine longitudinal smooth muscle contains 5 pmol of Ins(1,4,5)P3. For Inst(1,3,4)P3 or Ins(1,3,4,5)P4 slightly more smooth muscle is needed, and for major isomers of InsP1 or InsP2 10 mg wet wt. or less of tissue can be used. A 35 mm tissue culture plate with a confluent layer of rat fibroblasts contains about 30 pmol of Ins(1,4,5)P3. The method was applied to the measurement of the masses of Ins1P1. Ins4P1, Ins(1,4)P2, Ins(1,3,4)P3, Ins(1,4,5)P3, Ins(1,3,4,5)P4 and InsP5. The h.p.l.c. elution profiles of radiolabelled InsPS generated from [32P]Pi-labelled human Erythrocytes, [3H]Ins-labelled cultured rat fibroblasts and [3H]Ins-labelled smooth muscle fragments from guinea pig small intestine were compared with the h.p.l.c. elution profiles of their masses.

SUBMITTER: Prestwich SA

PROVIDER: S-EPMC1149963 | biostudies-other | 1991 Mar

REPOSITORIES: biostudies-other

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Project description:Trehalose is a non-reducing disaccharide that is used as an osmolyte, transport sugar, carbon reserve and stress protectant in a wide range of organisms. In plants, trehalose 6-phosphate (Tre6P), the intermediate of trehalose biosynthesis, is thought to be a signal of sucrose status. Trehalose itself may play a role in pathogenic and symbiotic plant-microbe interactions, in responses to abiotic stress and in developmental signalling, but its precise functions are unknown. A major obstacle to investigating its function is the technical difficulty of measuring the very low levels of trehalose usually found in plant tissues, as most of the established trehalose assays lack sufficient specificity and/or sensitivity.A kinetic assay for trehalose was established using recombinant Escherichia coli cytoplasmic trehalase (treF), which was shown to be highly specific for trehalose. Hydrolysis of trehalose to glucose is monitored fluorometrically and the trehalose content of the tissue extract is determined from an internal calibration curve. The assay is linear for 0.2-40 pmol trehalose, and recoveries of trehalose were ?88%. A. thaliana Col-0 rosettes contain about 20-30 nmol g-1FW of trehalose, increasing to about 50-60 nmol g-1FW in plants grown at 8°C. Trehalose is not correlated with sucrose content, whereas a strong correlation between Tre6P and sucrose was confirmed. The trehalose contents of ear inflorescence primordia from the maize ramosa3 mutant and wild type plants were 6.6±2.6 nmol g-1FW and 19.0±12.7 nmol g-1FW, respectively. The trehalose:Tre6P ratios in the ramosa3 and wild-type primordia were 2.43±0.85 and 6.16±3.45, respectively.The fluorometric assay is highly specific for trehalose and sensitive enough to measure the trehalose content of very small amounts of plant tissue. Chilling induced a 2-fold accumulation of trehalose in A. thaliana rosettes, but the levels were too low to make a substantial quantitative contribution to osmoregulation. Trehalose is unlikely to function as a signal of sucrose status. The abnormal inflorescence branching phenotype of the maize ramosa3 mutant might be linked to a decrease in trehalose levels in the inflorescence primordia or a downward shift in the trehalose:Tre6P ratio.

Dataset Information

Measurement of picomole amounts of any inositol phosphate isomer separable by h.p.l.c. by means of a bioluminescence assay.

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