Biosynthesis of heparin. Purification of a 110-kDa mouse mastocytoma protein required for both glucosaminyl N-deacetylation and N-sulfation.
The polymer modification process in the biosynthesis of heparin/heparan sulfate is initiated by N-deacetylation, followed by N-sulfation, of N-acetylglucosamine units. Chromatography of a detergent extract from mouse mastocytoma on wheat germ agglutinin-Sepharose yielded a protein fraction, eluted with 0.3 M N-acetylglucosamine, that expressed N-deacetylase activity, but only after recombination with proteins that did not bind to the lectin column. In subsequent purification of the active lectin-bound component, all assays were performed following addition of the unbound protein fraction. After two additional chromatography steps, on blue Sepharose and 3',5'-ADP-agarose, the lectin-binding N-deacetylase component had been purified about 4300-fold with an 11% yield and showed essentially a single band, corresponding to an apparent molecular weight of approximately 110,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analysis of the purified 110-kDa protein showed that it contained, in addition to the N-deacetylase, N-sulfotransferase activity; however, the expression of N-sulfotransferase activity was independent of additional proteins. Backtracking the N-sulfotransferase through the purification scheme previously applied to the N-deacetylase showed the two enzyme activities to the N-deacetylase showed the two enzyme activities to be cofractionated in each separation step. It is proposed that the expression of glucosaminyl N-deacetylase activity depends on the concerted action of (at least) two protein components, one of which also possesses glucosaminyl N-sulfotransferase activity.
Pettersson
I
I
Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, Uppsala.
Kusche
M
M
Unger
E
E
Wlad
H
H
Nylund
L
L
Lindahl
U
U
Kjellén
L
L
D000581:Amidohydrolases / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006493:Heparin / Q000096:biosynthesis*; D008415:Mastocytosis / Q000378:metabolism*; D051379:Mice; D009363:Neoplasm Proteins / Q000302:isolation & purification / Q000378:metabolism; D015238:Sulfotransferases / Q000302:isolation & purification / Q000378:metabolism; D013552:Swine
D009363:Neoplasm Proteins; D006493:Heparin; D015238:Sulfotransferases; D000581:Amidohydrolases; C023079:N-acetylglucosamine deacetylase