Characterization of 3′‐Phosphoadenosine 5′‐Phospho[35S]Sulfate Transport Carrier from Rat Brain Microsomes

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Abstract
3′‐Phosphoadenosine 5′‐phospho[35S]sulfate ([35S]PAPS) specific binding properties of rat brain tissue were studied. [35S]PAPS specific binding was optimal at pH 5.8 in either Tris‐maleate or potassium phosphate buffers. Association was maximal at low temperature, reaching equilibrium in 20 min. Dissociation was rapid, with a dissociation time of 80 s. Scatchard analysis of [35S]PAPS specific binding was consistent with a single site having a KD of 0.46 ± 0.06 μM and a Bmax of 20.8 ± 2.0 pmol/mg of protein. Low concentrations of Triton X‐100 (0.025%) were effective in increasing the number of binding sites to a Bmax of 44.5 ± 4.6 pmol/mg of protein without affecting the affinity. [35S]PAPS specific binding was enriched in crude synaptic membranes (P2) and microsomes (P3). Regional distribution of [35S]PAPS specific binding was quite homogeneous in all brain structures studied. The pharmacological profile of [35S]PAPS specific binding in rat brain microsomes was consistent with a membrane protein having a high selectivity for the 3′‐O‐phosphoryl group substitution on the ribose moiety. Thus, 3′‐phosphoadenosine 5′‐phosphate was more potent than 2′‐phosphoadenosine 5′‐phosphate in competing for [35S]PAPS specific binding. Adenosine 5′‐phosphosulfate was a good inhibitor of [35S]PAPS specific binding. ATP and ADP were also good displacers. Dipyridamole, a highly selective marker for adenosine uptake sites, was ineffective. 4,4‐Diisothiocyanostilbene‐2,2‐disulfonic acid, the chloride transporter inhibitor, showed an IC50 of 36 ± 5.1 μM for inhibition of [35S]PAPS specific binding. 2,6‐Dichloro‐4‐nitrophenol had a low selectivity in competing for the [35S]PAPS binding site. The properties of the [35S]PAPS binding in rat brain tissue observed in this work correspond closely to the membrane transport carrier for the PAPS molecule, which is different from adenosine uptake sites or adenosine receptors.