Biosynthesis of Sulfated Glycoprotein in the Endometrium of Rabbit Uterus1

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Abstract
The endometrial scrapings obtained from the uteri of estrogen-treated rabbits were incubated with N-acetyl-D-[1-3H]glucosamine and [35S]sulfate, and then the incubation medium (M-Fr) was separated from the tissue. The tissue was subsequently homogenized exhaustively in 0.25 M sucrose, and the insoluble residue (R-Fr) was separated. The supernatant at 8, 500× for 10 min of the homogenate was subjected to subcel-lular fractionation by discontinuous sucrose gradient ultracentrifugation, and a thiamine pyrophosphatase-rich fraction (G-Fr) was obtained. Complex carbohydrates were then separated from M-Fr, R-Fr, and G-Fr. The radioactivities incorporated into these complex carbohydrates suggested that sulfated glycoprotein synthesized in G-Fr was secreted into M-Fr. In order to confirm the above observation, labelled sulfated glycoprotein was isolated from the incubation medium. Subsequently, N-acetylglucosamine sulfate doubly labelled with 3H and 35S was separated from an acid hydrolyzate of the sulfated glycoprotein. The results indicated that N-acetyl-D-[l-3H]glucosamine was incorporated into N-acetylglucosamine residues and [35S]sulfate into sulfates located most probably at the 6-position of N-acetylglucosamine residues of sulfated glycoprotein.