Identification of dinucleoside polyphosphates in adrenal glands

Abstract

Dinucleoside polyphosphates have been characterised as extracellular mediators controlling numerous physiological functions like vascular tone or cell proliferation. Here we describe the isolation and identification of dinucleoside polyphosphates Ap n A (with n=2–3), Ap n G (with n=2–6) as well as Gp n G (with n=2–6) from adrenal glands. These dinucleoside polyphosphates are localised in granules of the adrenal glands. The dinucleoside polyphosphates diadenosine diphosphate (Ap 2A), diadenosine triphosphate (Ap 3A), adenosine guanosine polyphosphates (Ap n G) and diguanosine polyphosphates (Gp n G), both with phosphate group (p) numbers ( n) ranging from 2 to 6, were identified by fractionating them to homogeneity by preparative size-exclusion- and affinity-chromatography as well as analytical anion-exchange and reversed-phase-chromatography from deproteinised adrenal glands and by analysis of the homogeneous dinucleoside polyphosphates containing fractions with post-source-decay (PSD) matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS). The identity of the dinucleoside polyphosphates was confirmed by retention time comparison with authentic dinucleoside polyphosphates. Enzymatic analysis demonstrated an interconnection of the phosphate groups with the adenosines in the 5 ′-positions of the riboses in all dinucleoside polyphosphates purified from adrenal glands. In conclusion, the identification of these dinucleoside polyphosphates in adrenal gland granules emphasises that these dinucleoside polyphosphates can be released from the adrenal glands upon stimulation into the circulation.