Effects of brefeldin A on the localization of chondroitin sulfate-synthesizing enzymes. Activities in subfractions of the Golgi from chick embryo epiphyseal cartilage.
Membranes from brefeldin A-treated and untreated chick embryo epiphyseal cartilage were fractionated separately by equilibrium sucrose density gradient centrifugation. Fractions were assayed for Gal I transferase, Gal II transferase, Gal ovalbumin transferase, chondroitin polymerization on endogenous acceptors, GalNAc transfer to exogenous chondroitin hexasaccharide, and sulfate transfer to exogenous chondroitin. Gal I transferase and Gal II transferase activities were found in heavier cis- and medial-Golgi fractions, but with distributions different from each other. Brefeldin A had no effect on either their distribution or their total activity. Gal ovalbumin transferase activity in fractions from untreated cartilage was found as a dual peak in medial- and trans-Golgi areas. The latter peak was diminished in the fractions from the brefeldin A-treated cartilage, whereas the former peak was correspondingly increased. A similar dual medial- and trans-Golgi distribution for chondroitin polymerization on endogenous acceptors was seen with fractions from untreated cartilage. This was modified in fractions from brefeldin A-treated cartilage with a complete loss of synthesis in the trans-Golgi peak and a slight increase in synthesis in the medial-Golgi peak. However, the distribution of GalNAc transferase activity using exogenous chondroitin hexasaccharide indicated that considerable chondroitin-synthesizing activity still remained in these trans-Golgi fractions. This demonstrated that brefeldin A had caused a block in movement of endogenous proteochondroitin acceptors to the trans-Golgi site of synthesis. Sulfotransferase activity was also found in a dual distribution similar to that of the chondroitin polymerization and GalNAc transferase, with a small reduction in activity in the trans-Golgi fractions of brefeldin A-treated cartilage. Thus, treatment of cartilage with brefeldin A resulted in the loss of considerable trans-Golgi chondroitin sulfate-synthesizing enzyme activity and a block in the transport of one form of proteochondroitin precursor to the trans-Golgi membranes.
Sugumaran
G
G
Connective Tissue Research Laboratory, Department of Veterans Affairs Outpatient Clinic, Bedford, MA 01730.
Katsman
M
M
Silbert
J E
JE
D000818:Animals; D020126:Brefeldin A; D002240:Carbohydrate Sequence; D002642:Chick Embryo; D002809:Chondroitin Sulfates / Q000096:biosynthesis*; D003517:Cyclopentanes / Q000494:pharmacology*; D005700:Galactosyltransferases / Q000378:metabolism*; D006056:Golgi Apparatus / Q000187:drug effects* / Q000201:enzymology; D006132:Growth Plate / Q000187:drug effects* / Q000201:enzymology; D007425:Intracellular Membranes / Q000187:drug effects / Q000201:enzymology; D008861:Microsomes / Q000187:drug effects / Q000201:enzymology; D008969:Molecular Sequence Data
D003517:Cyclopentanes; D020126:Brefeldin A; D002809:Chondroitin Sulfates; D005700:Galactosyltransferases; C063053:xylosylprotein 4-beta-galactosyltransferase; C063054:galactosylxylosylprotein 3-beta-galactosyltransferase