Sulfation of p-nitrophenyl-N-acetyl-beta-D-galactosaminide with a microsomal fraction from cultured chondrocytes.
Chick embryo chondrocyte microsomes containing intact Golgi vesicles took up 3'-phosphoadenosine-5'-phospho[35S]sulfate ([35S]PAPS) in a time- and temperature-dependent, substrate-saturable manner. When [35S]PAPS and p-nitrophenyl-N-acetyl-beta-D-galactosaminide (pNP-GalNAc) were added to the incubation in the absence of detergent, the microsomes catalyzed the transfer of sulfate from [35S]PAPS to pNP-GalNAc to form pNP-GalNAc-6-35SO4. The apparent Km values for PAPS in the uptake and the pNP-GalNAc sulfation reactions were 2 X 10(-7) and 2 X 10(-6) M, respectively. The sulfation of pNP-GalNAc by the microsomal preparation was inhibited by detergent. The microsomal fraction also catalyzed the transfer of sulfate from [35S]PAPS to oligosaccharides prepared from chondroitin. However, in contrast to the sulfation of pNP-GalNAc, the rate of sulfation of these oligosaccharides was low in the absence of detergent and was markedly stimulated when detergent was added. Sulfation of pNP-GalNAc by the freeze-thawed microsomes was inhibited when the octasaccharide prepared from chondroitin was present in the reaction mixture. As the PAPS that had been internalized in the microsomal vesicles was consumed in the sulfation of pNP-GalNAc, more [35S]PAPS was taken up and the sulfated pNP-GalNAc was released from the vesicles. These observations suggest that pNP-GalNAc may serve as a model membrane-permeable substrate for study of the 6-sulfo-transferase reaction involved in sulfation of chondroitin sulfate in intact Golgi vesicles.
D000116:Acetylgalactosamine / Q000031:analogs & derivatives* / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002356:Cartilage / Q000648:ultrastructure*; D002592:Cetomacrogol / Q000494:pharmacology; D002642:Chick Embryo; D005615:Freezing; D005688:Galactosamine / Q000031:analogs & derivatives*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008861:Microsomes / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism; D010724:Phosphoadenosine Phosphosulfate / Q000378:metabolism; D013431:Sulfates / Q000378:metabolism*; D013462:Sulfur Radioisotopes; D013696:Temperature
D009844:Oligosaccharides; D013431:Sulfates; D013462:Sulfur Radioisotopes; C014123:4-nitrophenyl-2-acetamido-2-deoxygalactopyranoside; D010724:Phosphoadenosine Phosphosulfate; D000244:Adenosine Diphosphate; D005688:Galactosamine; D000255:Adenosine Triphosphate; D002592:Cetomacrogol; D000116:Acetylgalactosamine