Purification and reconstitution of a collagen-binding heat-shock glycoprotein from L6 myoblasts.
A major collagen-binding heat-shock glycoprotein from L6 myoblasts, designated gp46, was purified by gelatin-agarose chromatography and ion-exchange chromatography. Purified gp46 was functionally active, as shown by its ability to rebind to gelatin-agarose, and was homogeneous as determined by SDS/polyacrylamide-gel electrophoresis. This is the first reported purification of myoblast gp46 in an active state. Triton X-100-soluble gp46 was found to bind preferentially to immobilized pepsin-treated type IV collagen compared with native type I collagen. gp46, reconstituted into phospholipid vesicles, retained collagen-binding activity. This activity could be destroyed by chemical modification with chloramine-T, but was decreased by only 20-30% following treatment with iodoacetamide or N-ethylmaleimide. Since gp46 is a heat-shock protein, we examined the possibility that it may confer protection on type I collagen from thermal denaturation at temperatures above its normal melting temperature of 41 degrees C. In the presence of gp46 liposomes the apparent melting temperature of type I collagen was marginally increased to 42 degrees C. This change was considered to be too small to be of physiological significance. We have therefore concluded that the role of gp46 in collagen metabolism is unlikely to be related to any thermal-stabilizing function.
Vaillancourt
J P
JP
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
Cates
G A
GA
D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification*; D002460:Cell Line; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003094:Collagen / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000302:isolation & purification*; D006360:Heat-Shock Proteins / Q000302:isolation & purification*; D006358:Hot Temperature; D008081:Liposomes; D009132:Muscles / Q000378:metabolism*; D051381:Rats
D002352:Carrier Proteins; D006023:Glycoproteins; D006360:Heat-Shock Proteins; D008081:Liposomes; C046289:colligin; D003094:Collagen