Abstract
A new class of inositol phosphates containing energy-rich pyrophosphoryl residues has been characterized. D/L-1-Diphosphoinositol pentakisphosphate(s) and D/L-bis-(1,4)-diphosphoinositol tetrakisphosphate(s) are present as soluble ionic species in the cytosol of amoebae (Dictyostelium discoideum) at concentrations in the range of 0.05-0.25 mM. These compounds are rapidly metabolized in intact cells and can be synthesized in cell lysates from myo-inositol hexakisphosphate in the presence of ATP. Their phosphomonoester groups have predicted C-O-P bond energies of between 3.3 and 4 kcal mol-1; however, the bond energies of the P-O-P links in their diphosphate moieties are 6.6 kcal mol-1 and hence similar to the equivalent bonds in ADP, indicating a potential role for these compounds as phosphate donors in phosphotransferase reactions. Compounds with similar chromatographic properties are found in a variety of mammalian cell types.
Highlights
A new class of inositol phosphatescontaining energy- Ins(1,4,5)P3 isreleased, it is further metabolized to generate rich pyrophosphoryl residues has been characterized. a collection of metabolites largely associated with the synthe
Compounds with similar chromatographic properties are found in a variety of mammalian cell types
Recycling of these compounds back to inositol (6). This explains the fact that the levels of a large number of inositol phosphates rise upon agonist activationof many cells
Summary
Fractions of HPLC eluate containing inositol phosphates in phos- counted. phate bufferwere desalted with small columns of anion exchange Partial acid hydrolysis of compounds A and B was performed in resin (16, 25). Werecooled on ice, extracted four times with diethyl ether, and Analysis of PHlIns Contained in Inositol Polyphosphates-To es- aliquots were assayed for free inorganic phosphate or resolved and tablish that the 3H-labeledcarbon skeletons of compounds A and B quantified by MDD-HPLC (see Fig. 3) using a 25-cm MonoQ column Rther evidence that compounds A phosphate assay of the relevant HPLC-purified fractions; and (iii)by and B contained myo-inositolwas obtained by processing samples of labeling with 32Paind assuming InsP6, A and Bhad reached isotopic compounds A and B (derived from 50 ml of a 2 X lo cells ml" equilibrium; the concentration of A and B was calculated from their suspension of cells that had been prelabeled with 32Piand [3H]Insi)n 32P contentand the specific radioactivity of [32P]InsP6(see "Results parallel with those described above, except that the total phosphate content (25) of the HCl hydrolysates was determined prior to desaltand Discussion"). Of myo-inositol during the acid-catalyzed dephosphorylation reac- Molecular Modeling Techniques and Bond Energy Calculatiomtions
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