Characterization of proteoglycans synthesized by a rat parathyroid cell line.
The structure, biosynthesis, and distribution of cell-associated proteoglycans in a clonal line of parathyroid cells, which exhibit differentiated characteristics such as calcium-regulated hormone secretion and cell growth, were studied by metabolic labeling with [3H] glucosamine and [35S]sulfate as precursors. Proteoglycans were isolated by two consecutive ion exchange chromatography steps and then analyzed by gel filtration, polyacrylamide gel electrophoresis, and specific enzyme and chemical reactions. The cells synthesize almost exclusively (greater than 95%) heparan sulfate (HS) proteoglycans with a glycosaminoglycan synthesis rate of approximately 0.5 micrograms/10(6) cells/24 h. Two major HS proteoglycan species were identified. HS proteoglycan-I has a mass of approximately kDa with a single HS chain (approximately 12 kDa) and a core protein of approximately 150 kDa including oligosaccharides. HS proteoglycan-II has a mass of approximately 170 kDa with 3-4 HS chains (approximately 30 kDa) and a core protein of 70-80 kDa including oligosaccharides. In the medium with low ionized calcium (0.05 mM), HS proteoglycan-I is synthesized at approximately 1.6 times the rate and HS proteoglycan-II at a similar rate as for cells cultured in the medium with high ionized calcium (2.1 mM). The distribution of proteoglycans, examined by the accessibility of the molecules to trypsin, was dramatically influenced by environmental calcium concentration; at low calcium levels 70-80% of the HS proteoglycans are trypsin-accessible while only 20-30% are accessible at high calcium levels. This suggests that the proteoglycans are primarily on the cell surface in low calcium and in trypsin-inaccessible compartments in high calcium conditions.
Yanagishita
M
M
Bone Research Branch, National Institute of Dental Research, Bethesda, Maryland 20892.
Brandi
M L
ML
Sakaguchi
K
K
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D002999:Clone Cells; D005944:Glucosamine / Q000378:metabolism; D006025:Glycosaminoglycans / Q000302:isolation & purification; D007700:Kinetics; D009844:Oligosaccharides / Q000302:isolation & purification; D010280:Parathyroid Glands / Q000378:metabolism*; D011509:Proteoglycans / Q000096:biosynthesis* / Q000302:isolation & purification; D051381:Rats; D013431:Sulfates / Q000378:metabolism; D013462:Sulfur Radioisotopes; D014316:Tritium
D006025:Glycosaminoglycans; D009844:Oligosaccharides; D011509:Proteoglycans; D013431:Sulfates; D013462:Sulfur Radioisotopes; D014316:Tritium; D005944:Glucosamine; D002118:Calcium