Purification and characterization of protease-resistant secretory granule proteoglycans containing chondroitin sulfate di-B and heparin-like glycosaminoglycans from rat basophilic leukemia cells.
Proteoglycans were extracted from nuclease-digested sonicates of 10(9) rat basophilic leukemia (RBL-1) cells by the addition of 0.1% Zwittergent 3-12 and 4 M guanidine hydrochloride and were purified by sequential CsCl density gradient ultracentrifugation, DE52 ion exchange chromatography, and Sepharose CL-6B gel filtration chromatography under dissociative conditions. Between 0.3 and 0.8 mg of purified proteoglycan was obtained from approximately 1 g initial dry weight of cells with a purification of 200-800-fold. The purified proteoglycans had a hydrodynamic size range of Mr 100,000-150,000 and were resistant to degradation by a molar excess of trypsin, alpha-chymotrypsin, Pronase, papain, chymopapain, collagenase, and elastase. Amino acid analysis of the peptide core revealed a preponderance of Gly (35.4%), Ser (22.5%), and Ala (9.5%). Approximately 70% of the glycosaminoglycan side chains of RBL-1 proteoglycans were digested by chondroitinase ABC and 27% were hydrolyzed by treatment with nitrous acid. Sephadex G-200 chromatography of glycosaminoglycans liberated from the intact molecule by beta-elimination demonstrated that both the nitrous acid-resistant (chondroitin sulfate) and the chondroitinase ABC-resistant (heparin/heparan sulfate) glycosaminoglycans were of approximately Mr 12,000. Analysis of the chondroitin sulfate disaccharides in different preparations by amino-cyano high performance liquid chromatography revealed that 9-29% were the unusual disulfated disaccharide chondroitin sulfate di-B (IdUA-2-SO4----GalNAc-4-SO4); the remainder were the monosulfated disaccharide GlcUA----GalNAc-4-SO4. Subpopulations of proteoglycans in one preparation were separated by anion exchange high performance liquid chromatography and were found to contain chondroitin sulfate glycosaminoglycans whose disulfated disaccharides ranged from 9-49%. However, no segregation of subpopulations without both chondroitin sulfate di-B and heparin/heparan sulfate glycosaminoglycans was achieved, suggesting that RBL-1 proteoglycans might be hybrids containing both classes of glycosaminoglycans. Sepharose CL-6B chromatography of RBL-1 proteoglycans digested with chondroitinase ABC revealed that less than 7% of the molecules in the digest chromatographed with the hydrodynamic size of undigested proteoglycans, suggesting that at most 7% of the proteoglycans lack chondroitin sulfate glycosaminoglycans.(ABSTRACT TRUNCATED AT 400 WORDS)
D C
DC
Seldin
K F
KF
Austen
R L
RL
Stevens
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001491:Basophils / Q000276:immunology / Q000378:metabolism; D000001:Calcimycin / Q000494:pharmacology; D002460:Cell Line; D002807:Chondroitin / Q000031:analogs & derivatives*; D002809:Chondroitin Sulfates / Q000032:analysis*; D003594:Cytoplasmic Granules / Q000032:analysis*; D010450:Endopeptidases; D006025:Glycosaminoglycans / Q000032:analysis*; D006493:Heparin; D006636:Histamine Release / Q000187:drug effects; D007942:Leukemia, Experimental / Q000378:metabolism*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D011509:Proteoglycans / Q000302:isolation & purification*; D051381:Rats; D013431:Sulfates / Q000378:metabolism; D013462:Sulfur Radioisotopes
D000596:Amino Acids; D006025:Glycosaminoglycans; D010446:Peptide Fragments; D011509:Proteoglycans; D013431:Sulfates; D013462:Sulfur Radioisotopes; D000001:Calcimycin; D006493:Heparin; D002807:Chondroitin; D002809:Chondroitin Sulfates; D010450:Endopeptidases