Brion F, Schwartz J C, Vargas F
J Neurochem. 1987 Apr;48(4):1171-7. doi: 10.1111/j.1471-4159.1987.tb05643.x.
The formation of the sulfate donor [35S]3'-phosphoadenosine 5'-phosphosulfate (PAPS) from inorganic [35S]sulfate was studied using a novel assay. The assay was based on the quantitative transfer of radioactivity from [35S]PAPS to beta-naphthol under the action of phenolsulfotransferase activity from rat brain cytosol, with the [35S]beta-naphthyl sulfate formed being isolated by polystyrene bead chromatography. This simple assay was validated by comparison of results with those derived from direct assay of [35S]PAPS isolated by either TLC or ion exchange chromatography. [35S]PAPS formation by a high-speed supernatant of rat cerebral cortex occurred with an optimal pH of approximately 7.6, varied linearly with time and protein concentration, and depended on the presence of Mg2+-ATP. The latter could not be replaced by other nucleotides such as GTP, UTP, or CTP, which at 1-5 mM concentrations inhibited the reaction. Mg2+ could not be replaced by Mn2+, which at micromolar concentrations inhibited the reaction. The apparent Km values of Mg2+-ATP (at 0.1 mM [35S]sulfate) and inorganic sulfate (at 5 mM Mg2+-ATP) were 2.7 and 0.2 mM, respectively. These kinetics parameters corresponded to those reported for purified ATP sulfurylase (EC 2.7.7.4), the enzyme responsible for the first step of PAPS synthesis in liver. The product of its reaction, [35S]adenosine 5'-phosphosulfate (APS), could not be detected after incubations, an observation implying that the action of APS kinase was not rate limiting in cerebral extracts tested under the selected experimental conditions. [35S]PAPS formation was detectable in cytosolic fractions from various brain regions, which displayed only limited differences in synthesizing activity.(ABSTRACT TRUNCATED AT 250 WORDS)
利用一种新型检测方法研究了由无机[35S]硫酸盐形成硫酸盐供体[35S]3'-磷酸腺苷5'-磷酸硫酸酯（PAPS）的过程。该检测方法基于在大鼠脑细胞质中酚磺基转移酶活性作用下，放射性从[35S]PAPS定量转移至β-萘酚，所形成的[35S]硫酸β-萘酯通过聚苯乙烯珠色谱法分离。通过将结果与通过薄层层析（TLC）或离子交换色谱法分离的[35S]PAPS直接检测结果进行比较，验证了这种简单的检测方法。大鼠大脑皮质高速上清液形成[35S]PAPS的最佳pH约为7.6，随时间和蛋白质浓度呈线性变化，且依赖于Mg2+-ATP的存在。后者不能被其他核苷酸如GTP、UTP或CTP替代，这些核苷酸在1 - 5 mM浓度下会抑制反应。Mg2+不能被Mn2+替代，Mn2+在微摩尔浓度下会抑制反应。Mg2+-ATP（在0.1 mM [35S]硫酸盐时）和无机硫酸盐（在5 mM Mg2+-ATP时）的表观Km值分别为2.7和0.2 mM。这些动力学参数与报道的纯化ATP硫酸化酶（EC 2.7.7.4）的参数相对应，该酶是肝脏中PAPS合成第一步的负责酶。孵育后未检测到其反应产物[35S]腺苷5'-磷酸硫酸酯（APS），这一观察结果表明在所选实验条件下测试的脑提取物中，APS激酶的作用不是限速因素。在来自不同脑区的细胞溶质组分中可检测到[35S]PAPS的形成，这些组分在合成活性上仅表现出有限差异。（摘要截短于250字）
