Miller R R, Waechter C J
Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore.
J Neurochem. 1988 Jul;51(1):87-94. doi: 10.1111/j.1471-4159.1988.tb04839.x.
Calf brain 3'-phosphoadenosine 5'-phosphosulfate (PAPS):proteoheparan sulfate (PHS) N-sulfotransferase activity is solubilized by extracting salt-washed microsomes with 1% Cutscum. A protocol is described for the partial purification of the sulfotransferase activity utilizing: (1) diethylaminoethyl (DEAE)-Sephacel, (2) heparin-Sepharose CL-6B, and (3) 3',5'-ADP-agarose as chromatographic supports. Sulfotransferase activity was followed by using 3'-phosphoadenosine 5'-phospho[35S]sulfate and endogenous acceptors in heat-inactivated microsomes as exogenous substrates. Two chromatographically distinct fractions (ST1 and ST2) of sulfotransferase activity are resolved on DEAE-Sephacel. Both sulfotransferase activities have been partially purified and characterized. An apparent purification of the two N-sulfotransferase fractions of 22- to 29-fold, relative to the microsomal activity, is achieved by this procedure. Since ST1 appears to represent approximately 24% of the total microsomal activity, a purification of 89-fold has been estimated for this fraction. Neither sulfotransferase activity was stimulated by MnCl2, MgCl2, or CaCl2 added at 10 mM, nor inhibited by the presence of 10 mM EDTA. ST1 and ST2 are optimally active at pH 7.5-8. Apparent Km values for PAPS of 2.3 microM and 0.9 microM have been determined for ST1 and ST2, respectively. ST1 exhibits N-sulfotransferase activity primarily and is inhibited by phosphatidylserine whereas the ST2 fraction contains a mixture of N- and O-sulfotransferase activity and is stimulated by phosphatidylserine, phosphatidylcholine, and lysophosphatidylcholine. The detection of two chromatographically distinct sulfotransferase activities raises the possibility that N-sulfation of proteoheparan sulfates could be catalyzed by more than one enzyme, and that N-sulfation and O-sulfation of proteoglycans are catalyzed by separate enzymes in nervous tissue.
小牛脑3'-磷酸腺苷5'-磷酸硫酸酯（PAPS）：蛋白聚糖硫酸酯（PHS）N-磺基转移酶活性可通过用1%的Cutscum提取盐洗微粒体来溶解。本文描述了一种利用以下方法对磺基转移酶活性进行部分纯化的方案：（1）二乙氨基乙基（DEAE）-Sephacel，（2）肝素-Sepharose CL-6B，以及（3）3',5'-ADP-琼脂糖作为色谱支持物。通过使用3'-磷酸腺苷5'-磷酸[35S]硫酸酯和热灭活微粒体中的内源性受体作为外源底物来跟踪磺基转移酶活性。在DEAE-Sephacel上分离出两种色谱上不同的磺基转移酶活性组分（ST1和ST2）。两种磺基转移酶活性均已得到部分纯化和表征。通过该方法，相对于微粒体活性，两种N-磺基转移酶组分的表观纯化倍数达到了22至29倍。由于ST1似乎约占微粒体总活性的24%，因此估计该组分的纯化倍数为89倍。添加10 mM的MnCl2、MgCl2或CaCl2均未刺激两种磺基转移酶活性，10 mM的EDTA也未抑制其活性。ST1和ST2在pH 7.5 - 8时活性最佳。已分别测定ST1和ST2对PAPS的表观Km值为2.3 microM和0.9 microM。ST1主要表现出N-磺基转移酶活性，并受到磷脂酰丝氨酸的抑制，而ST2组分则含有N-和O-磺基转移酶活性的混合物，并受到磷脂酰丝氨酸、磷脂酰胆碱和溶血磷脂酰胆碱的刺激。两种色谱上不同的磺基转移酶活性的检测增加了以下可能性：蛋白聚糖硫酸酯的N-硫酸化可能由不止一种酶催化，并且神经组织中蛋白聚糖的N-硫酸化和O-硫酸化由不同的酶催化。
