J. Biol. Chem., Vol. 263, Issue 10, 4673-4678, 04, 1988

Sulfation of chondroitin. Specificity, degree of sulfation, and detergent effects with 4-sulfating and 6-sulfating microsomal systems

G Sugumaran and JE Silbert
Connective Tissue Research Laboratory, Veterans Administration Outpatient Clinic, Boston, Massachusetts 02108.

Microsomal preparations from chondroitin 6-sulfate-producing chick embryo epiphyseal cartilage, and from chondroitin 4-sulfate-producing mouse mastocytoma cells, were incubated with UDP-[14C]glucuronic acid and UDP-N-acetylgalactosamine to form non-sulfated proteo[14C]chondroitin. Aliquots of the incubations were then incubated with 3'-phosphoadenylylphosphosulfate (PAPS) in the presence or absence of various detergents. In the absence of detergents, there was good sulfation of this endogenous proteo[14C]chondroitin by the original microsomes from both sources. Detergents, with the exception of Triton X-100, markedly inhibited sulfation in the mast cell system but not in the chick cartilage system. These results indicate that sulfation and polymerization are closely linked on cell membranes and that in some cases this organization can be disrupted by detergents. When aliquots of the original incubation were heat inactivated, and then reincubated with new microsomes from chick cartilage and/or mouse mastocytoma cells plus PAPS, there was no significant sulfation of this exogenous proteo[14C] chondroitin with either system unless Triton X-100 was added. Sulfation of exogenous chondroitin and chondroitin hexasaccharide was compared with sulfation of endogenous and exogenous proteo[14C]chondroitin. Sulfate incorporation into hexasaccharide and chondroitin decreased as their concentrations (based on uronic acid) approached that of the proteo[14C]chondroitin. At the same time, the degree of sulfation in percent of substituted hexosamine increased. However, the degree of sulfation did not reach that of the endogenous proteo[14C]chondroitin. Hexasaccharide and chondroitin sulfation were stimulated by the presence of Triton X-100. However, in contrast to the exogenous proteo[14C]chondroitin, there was some sulfation of hexasaccharide and chondroitin in the absence of this detergent. These results indicate that the intact microsomal system was not accessible to the larger substrates, and that even with detergents exogenous substrates were not sulfated as effectively as newly formed proteo[14C]chondroitin in an intact microsomal system. When the proteo[14C]chondroitin formed by the chick cartilage microsomal system was incubated together with the mast cell microsomal system and PAPS, sulfation only occurred at the 4-position. When the proteo[14C]chondroitin formed by the mouse mast cell microsomal system was incubated together with the chick cartilage microsomal system and PAPS, sulfation only occurred at the 6-position.(ABSTRACT TRUNCATED AT 400 WORDS)
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