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(TVS)sulfate ((TVS)PAPS) in a time- and temperature-dependent, substrate-saturable manner. When (TVS)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 (TVS)PAPS to pNP-GalNAc to form pNP-GalNAc-6-TVSO4. 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 (TVS)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 (TVS)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.
- Research Organization:
- Univ. of Illinois, Urbana
- OSTI ID:
- 6182046
- Journal Information:
- J. Biol. Chem.; (United States), Vol. 24
- Country of Publication:
- United States
- Language:
- English
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CELL MEMBRANES
PERMEABILITY
SULFATES
BIOCHEMICAL REACTION KINETICS
MEMBRANE TRANSPORT
AMINES
ATP
CARTILAGE
CELL CONSTITUENTS
CHICKENS
DETERGENTS
EMBRYOS
FREEZING
MICROSOMES
OLIGOSACCHARIDES
SUBSTRATES
SULFUR 35
TEMPERATURE DEPENDENCE
THAWING
TIME DEPENDENCE
TRACER TECHNIQUES
ADDITIVES
ANIMAL TISSUES
ANIMALS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIRDS
BODY
CARBOHYDRATES
CONNECTIVE TISSUE
DAYS LIVING RADIOISOTOPES
EMULSIFIERS
EVEN-ODD NUCLEI
FOWL
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LIGHT NUCLEI
MEMBRANES
NUCLEI
NUCLEOTIDES
ORGANIC COMPOUNDS
ORGANOIDS
OXYGEN COMPOUNDS
RADIOISOTOPES
REACTION KINETICS
SACCHARIDES
SULFUR COMPOUNDS
SULFUR ISOTOPES
SURFACTANTS
TISSUES
VERTEBRATES
WETTING AGENTS
550201* - Biochemistry- Tracer Techniques