Agarose/polyacrylamide minislab gel electrophoresis of intact cartilage proteoglycans and their proteolytic degradation products.
We have developed a procedure for the use of minislab gels to electrophoretically separate proteoglycans (PGs), large macromolecules with molecular masses greater than 2.5 million Da. Our procedure is a modification of the method of C.A. McDevitt and H. Muir (Anal. Biochem. 44, 612-622, 1971) for agarose/polyacrylamide, composite tube gels. These 1% agarose/1.2% acrylamide minigels are run at 35 mA for 75 min; bands are visualized by toluidine blue staining. The subtle size differences between the large aggregating PGs isolated from rat chondrosarcoma, bovine nasal septal cartilage, and adult bovine articular cartilage (which consists of two subpopulations) can be distinguished by their migration on these large pore gels. Chondroitin sulfate chains, added to all wells as a marker of constant mobility, ran immediately behind the dye front. The distance of migration into the gel of PGs incubated overnight with cathepsin B, carboxypeptidase A, papain, plasmin, elastase, or cathepsin G varied with the size of the cleavage products. We propose the use of this procedure for a convenient assessment of cartilage PGs and a rapid, reproducible assay for proteoglycanase activity.
Varelas
J B
JB
Department of Medicine, Evanston Hospital/Northwestern University, Illinois 60201.
Zenarosa
N R
NR
Froelich
C J
CJ
D000818:Animals; D002358:Cartilage, Articular / Q000737:chemistry; D002417:Cattle; D002813:Chondrosarcoma / Q000737:chemistry; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D009300:Nasal Septum / Q000737:chemistry; D010447:Peptide Hydrolases / Q000737:chemistry; D011509:Proteoglycans / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism
D011509:Proteoglycans; D010447:Peptide Hydrolases