Sulphation of proteochondroitin and 4-methylumbelliferyl beta-D-xyloside-chondroitin formed by mouse mastocytoma cells cultured in sulphate-deficient medium.
Mouse mastocytoma cells were cultured in medium containing [3H]GlcN and concentrations of [35S]sulphate varying from 0.01 to 0.5 mM. Intracellular [35S]sulphate incorporation increased severalfold from the lowest concentrations, reaching a maximum at 0.1-0.2 mM, whereas incorporation of [3H]hexosamine remained constant at all sulphate concentrations. Proteo[3H]-chondroitin [35S]sulphate was isolated and incubated with chondroitin ABC lyase, yielding 35S-labelled and/or 3H-labelled delta Di-0S and delta Di-4S disaccharide products. The increasing percentage of delta Di-4S was consistent with the increasing sulphate incorporation at each higher [35S]sulphate concentration. Examination of proteochondroitin [35S]sulphate size by Sepharose CL-6B chromatography indicated a range consistent with various numbers of glycosaminoglycan chains on the protease-resistant serglycin core protein. Alkali-cleaved chondroitin [35S]sulphate products indicated similar size distributions at all sulphate concentrations with no indication of preferential sulphation being related to smaller or larger size. DEAE-cellulose chromatography of [3H]chondroitin [35S]sulphate glycosaminoglycans indicated a random undersulphation as [35S]sulphate concentration was lowered. Addition of 4-methylumbelliferyl beta-D-xyloside to the cultures resulted in a 2-2.5-fold stimulation of [3H]chondroitin [35S]sulphate synthesis with formation of beta-xyloside-[3H]chondroitin [35S]sulphate which was much smaller, as estimated by Sepharose CL-6B chromatography, than the decreased amount of [3H]chondroitin [35S]sulphate derived from proteo[3H]chondroitin [35S]sulphate. Much higher concentrations of sulphate were necessary to produce sulphation of the beta-xyloside-[3H]chondroitin comparable with that of proteo[3H]-chondroitin, as indicated by chondroitin ABC lyase products and DEAE-cellulose chromatography. The specific radioactivities of the [3H]GalN in the proteo[3H]chondroitin [35S]sulphate and beta-xyloside-[3H]chondroitin [35S]sulphate were calculated from the 3H and 35S c.p.m. of isolated dual-labelled delta Di-4S from each, and indicated that the presence of the beta-xyloside resulted in a dilution of the [3H]GlcN by endogenous GlcN that was 4 times higher than that of cultures lacking the beta-xyloside. The higher sulphate concentrations needed for sulphation of beta-xyloside-chondroitin suggests that the membrane-bound nature of the proteochondroitin acceptor in juxtaposition to a chondroitin sulphate-synthesizing enzyme complex effectively reduces the apparent Km for adenosine 3'-phosphate 5'-phosphosulphate.
Silbert
J E
JE
Connective Tissue Research Laboratory, Department of Veterans Affairs Medical Center, Bedford, MA.
Sugumaran
G
G
Cogburn
J N
JN
D000818:Animals; D002460:Cell Line; D002807:Chondroitin / Q000302:isolation & purification / Q000378:metabolism*; D002808:Chondroitin Lyases / Q000378:metabolism; D011508:Chondroitin Sulfate Proteoglycans / Q000302:isolation & purification / Q000378:metabolism*; D002848:Chromatography, DEAE-Cellulose; D002854:Chromatography, Paper; D003470:Culture Media; D005944:Glucosamine / Q000378:metabolism; D006923:Hymecromone / Q000031:analogs & derivatives* / Q000378:metabolism; D007700:Kinetics; D012515:Mast-Cell Sarcoma / Q000378:metabolism*; D051379:Mice; D013431:Sulfates / Q000378:metabolism*; D013462:Sulfur Radioisotopes; D014316:Tritium; D014407:Tumor Cells, Cultured
D011508:Chondroitin Sulfate Proteoglycans; D003470:Culture Media; D013431:Sulfates; D013462:Sulfur Radioisotopes; D014316:Tritium; D006923:Hymecromone; C014111:methylumbelliferyl-beta-D-xyloside; D002807:Chondroitin; D002808:Chondroitin Lyases; D005944:Glucosamine