Biosynthesis of proteokeratan sulfate in the bovine cornea. 2) Isolation of subcellular membrane fragments from bovine cornea cells with keratan sulfate synthesizing activity.
Cornea cells were isolated from bovine corneae after collagenase treatment. Subcellular fragments were fractionated by density gradient centrifugation. The density gradient run was monitored by determination of the marker enzyme activities for mitochondria, plasma membranes, lysosomes and endoplasmatic reticulum, of the enzyme activities involved in keratan sulfate synthesis and of the protein content. The fractions were further investigated by electron microscopy. Two membrane fractions with keratan sulfate-synthesizing activity (UDP-N-acetylglucosamine:keratan-N-acetylglucosaminyl-transferase, UDPgalactose:keratan galactosyltransferase and keratan sulfotransferase) were detected: a heavy fraction separated from the other organells investigated and a light fraction exhibiting the same density as plasma membranes. The activities of the three enzymes were found in the same density gradient fractions with a similar distribution pattern between the fractions, which suggests a joint localization of these 3 enzymes at the same intracellular sites.
D000818:Animals; D002417:Cattle; D011508:Chondroitin Sulfate Proteoglycans / Q000096:biosynthesis*; D003315:Cornea / Q000378:metabolism* / Q000648:ultrastructure; D005347:Fibroblasts / Q000378:metabolism; D005700:Galactosyltransferases / Q000302:isolation & purification; D005964:Glucosyltransferases / Q000302:isolation & purification; D006025:Glycosaminoglycans / Q000096:biosynthesis*; D066298:In Vitro Techniques; D007632:Keratan Sulfate / Q000096:biosynthesis*; D000071234:Lumican; D008566:Membranes / Q000378:metabolism; D017351:N-Acetylglucosaminyltransferases; D011509:Proteoglycans / Q000096:biosynthesis*; D013347:Subcellular Fractions / Q000378:metabolism*; D015238:Sulfotransferases; D013466:Sulfurtransferases / Q000302:isolation & purification; D000097811:Carbohydrate Sulfotransferases
D011508:Chondroitin Sulfate Proteoglycans; D005700:Galactosyltransferases; D005964:Glucosyltransferases; D006025:Glycosaminoglycans; D007632:Keratan Sulfate; D000071234:Lumican; D017351:N-Acetylglucosaminyltransferases; D011509:Proteoglycans; D015238:Sulfotransferases; D013466:Sulfurtransferases; D000097811:Carbohydrate Sulfotransferases; C038188:UDP-N-acetylglucosamine-keratan-N-acetylglucosaminyltransferase; C038189:UDPgalactose-keratan galactosyltransferase