31P NMR studies of nucleotide storage in the dense granules of pig platelets.

Abstract

Detailed 31P NMR measurements have been conducted on pig platelet dense granules and aqueous mixtures of ATP, ADP, MgCl2, and 5HT. The resonance line widths of the dense granule nucleotides were temperature independent above approximately 30 degrees C; below this temperature they exhibit a strong temperature dependence, becoming undetectably broad at approximately 5 degrees C. The temperature of transition is determined by the effective solute concentrations within the granules. Spin-spin relaxation time (T2) and line-width measurements indicate that the 31P resonances of dense granule nucleotides are not homogeneously broadened at 35 and 21 degrees C. However, the temperature-dependent changes in the intrinsic widths calculated from T2 values parallel the changes in the measured line widths. From the T1 and the T2 data, a rotational correlation time of 13 ns is calculated for the dense granule nucleotides at 35 degrees C. Removal of 5HT and HA from the dense granules induce significant but relatively small changes in the temperature dependence of the resonance line widths. Analogous effects are seen with a gel phase separated from aqueous mixtures of ATP, ADP, and MgCl2 in the presence or absence of 5HT. These results demonstrate that interactions involving the nucleotides and the divalent cations are predominant in determining the physicochemical state of the granule contents; in pig platelet dense granules the nucleotides and Mg2+ form a relatively fluid aggregate which serves as a matrix for 5HT and possibly HA binding. Incorporation of the amines into this matrix tends to increase its fluidity.