Harnessing¹³C-labeledmyo-inositol to interrogate inositol phosphate messengers by NMR

Abstract

The analysis of inositol poly- and pyrophosphates, an important group of eukaryotic messengers, is enabled by applying 13C-labeled inositol.

An ATP-responsive metabolic cassette comprised of inositol tris/tetrakisphosphate kinase 1 (ITPK1) and inositol pentakisphosphate 2-kinase (IPK1) buffers diphosphosphoinositol phosphate levels

“…With 100-fold more enzyme, we confirmed the production of 5-InsP 7 from InsP 6 ( Figure 5C), as reported [12,13]. Because a central tenet of the energy status signaling role of diphosphoinositol phosphates (5-InsP 7 , specifically) is the high (1-1.2 mM) K m for ATP of mammalian IP6K [40] and kcs1 [41,42], a more recent estimate for IP6K1 is 0.35 mM [43], we measured kinetic parameters for ATP with these two substrates ( Figure 5D,E). Ins(3,4,5,6)P 4 was, by more than two-orders of magnitude (V max 8640 ± 025 nmol min −1 mg −1 vs 40 ± 3 nmol min −1 mg −1 ), the stronger substrate.…”

Inositol Pyrophosphates: Signaling Molecules with Pleiotropic Actions in Mammals

“…Establishing a tight link between PP-IPs and clinical findings will be important to translate scientific knowledge into clinical diagnostic and therapeutic applications. Mass spectrometric measurement of PP-IPs was recently attempted [95,96], applying a highly sensitive platform to the detection of PP-IPs. We strongly believe that advanced detection techniques and a comprehensive understanding of the roles of PP-IPs in cellular signaling networks will lead to the development of valuable tools to manage leading human diseases, like metabolic syndromes and cancer.…”

Two bifunctional inositol pyrophosphate kinases/phosphatases control plant phosphate homeostasis

“…The reaction was incubated at 37°C overnight, quenched with 50 µl 0.7 M EDTA, lyophilized and resuspended in 600 µl of 100% (v/v) D 2 O. The samples were measured as previously described (Harmel et al, 2019). In brief, Bruker AV-III spectrometers (Bruker Biospin, Rheinstetten, Germany) using cryogenically cooled 5 mm TCI-triple resonance probe equipped with one-axis self-shielded gradients and operating at 600 MHz for proton nuclei, 151 MHz for carbon nuclei, and 244 MHz for phosphorous nuclei were used.…”