Premium
Characterization of 3′‐Phosphoadenosine 5′‐Phospho[ 35 S]Sulfate Transport Carrier from Rat Brain Microsomes
Author(s) -
Vargas Froylan
Publication year - 1990
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1990.tb02323.x
Subject(s) - chemistry , microsome , binding site , biochemistry , dissociation constant , adenosine , stereochemistry , enzyme , receptor
3′‐Phosphoadenosine 5′‐phospho[ 35 S]sulfate ([ 35 S]PAPS) specific binding properties of rat brain tissue were studied. [ 35 S]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 [ 35 S]PAPS specific binding was consistent with a single site having a K D of 0.46 ± 0.06 μ M and a B max 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 B max of 44.5 ± 4.6 pmol/mg of protein without affecting the affinity. [ 35 S]PAPS specific binding was enriched in crude synaptic membranes (P 2 ) and microsomes (P 3 ). Regional distribution of [ 35 S]PAPS specific binding was quite homogeneous in all brain structures studied. The pharmacological profile of [ 35 S]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 [ 35 S]PAPS specific binding. Adenosine 5′‐phosphosulfate was a good inhibitor of [ 35 S]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 IC 50 of 36 ± 5.1 μ M for inhibition of [ 35 S]PAPS specific binding. 2,6‐Dichloro‐4‐nitrophenol had a low selectivity in competing for the [ 35 S]PAPS binding site. The properties of the [ 35 S]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.