ISSN:
1471-4159
Source:
Blackwell Publishing Journal Backfiles 1879-2005
Topics:
Medicine
Notes:
Calf brain 3′-phosphoadenosine 5′-phosphosul-fate (PAPS):proteoheparan sulfate (PHS) N-sulfotransfer-ase 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 chromato-graphic 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 μM and 0.9 μM 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 ly-sophosphatidylcholine. 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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1111/j.1471-4159.1988.tb04839.x
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