Proteoglycan synthesis by clonal skeletal muscle cells during in vitro myogenesis: differences detected in the types and patterns from primary cultures.
Proteoglycan synthesis by two clonal murine skeletal muscle cell lines, G8-1 and C2, was examined. Cultures of skeletal muscle cells at both the myoblast and myotube stages were radiolabeled using [35S]sulfate as a precursor. The proteoglycans of the cell layer and medium were separately extracted and isolated by ion exchange chromatography on DEAE-Sephacel followed by gel filtration chromatography on Sepharose CL-2B. The cell layer proteoglycans eluted from Sepharose CL-20 as a single peak with a Kav of 0.66 and contained glycosaminoglycan chains with an average molecular weight of 20,000. The glycosaminoglycan chains were composed of nearly equal mixtures of chondroitin sulfate and heparan sulfate with the exception that C2 myoblast cultures contained larger amounts of heparan sulfate. Of interest, this line differentiates more rapidly in our laboratory than G8-1. The medium proteoglycans also eluted from Sepharose CL-2B as a single peak with a Kav of 0.66 but contained glycosaminoglycan chains with an average molecular weight of 32,000. Based upon enzymatic and chemical analysis, the medium glycosaminoglycan chains were composed of a mixture of chondroitin sulfate (71-80%) and heparin sulfate (19-22%). Following chondroitinase ABC digestion, the predominant disaccharide released from all glycosaminoglycan fractions was chondroitin-4-sulfate. When the extracted cell layer proteoglycans were chromatographed on Sepharose CL-28 in the absence of detergent, a small but consistent proportion (14-18%) eluted in the void volume, suggesting the association of at least a portion of this proteoglycan with cellular lipid. These differences distinguish proteoglycan metabolism in fusing clonal lines from primary muscle cell cultures suggesting their utility in evaluating the contribution of these macromolecules in myogenesis.
Neurobiology Research Laboratory, Veterans Affairs Medical Center, Kansas City, MO 64128.
R R
RR
Miller
J S
JS
Rao
W V
WV
Burton
B W
BW
Festoff
D000818:Animals; D001485:Basement Membrane / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003902:Detergents / Q000494:pharmacology; D051379:Mice; D008970:Molecular Weight; D024510:Muscle Development; D009132:Muscles / Q000166:cytology / Q000378:metabolism*; D011509:Proteoglycans / Q000032:analysis / Q000096:biosynthesis*; D013462:Sulfur Radioisotopes
D003902:Detergents; D011509:Proteoglycans; D013462:Sulfur Radioisotopes