Immunologic identification of a glycogen synthase 93,000-dalton subunit from rat heart and liver.
A polyclonal sheep antibody to rat heart glycogen synthase has been used for immunoblot analysis and immunoprecipitation of both rat heart and liver synthase. The purified antibody completely inhibits glycogen synthase activity in rat heart preparations and specifically blots to a 93-kDa band in the 10,000 X g supernatants of both heart and liver homogenates. Immunoprecipitation of in vitro translation products from rat heart or liver poly(A+) RNA yields a unique band with a molecular mass of 93 kDa. Thus the subunit molecular mass of active glycogen synthase in rat heart is 93 kDa. In rat liver at least one form of glycogen synthase also appears to have a molecular mass of 93 kDa. Protocols used to purify rat liver synthase yield a subunit of 80-87 kDa, which retains activity, but which is no longer recognized by the antibody. This suggests that 1) a specific antigenic sequence has been proteolytically removed from the NH2 or COOH terminus of the protein, or 2) that limited proteolysis has led to a conformational change in the enzyme such that the antibody binding site is no longer recognized. Either or both of these possibilities represent a significant alteration in the enzyme due to proteolysis. In vitro studies using synthase preparations having molecular masses less than 93 kDa must be interpreted with caution due to possible structural changes which occur during purification which may alter the regulation or covalent modification of synthase.
Rulfs
J
J
Wolleben
C D
CD
Miller
T B
TB
Johnson
G L
GL
D000818:Animals; D006006:Glycogen Synthase / Q000032:analysis* / Q000276:immunology; D007163:Immunosorbent Techniques; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008970:Molecular Weight; D009206:Myocardium / Q000201:enzymology*; D011061:Poly A / Q000378:metabolism; D014176:Protein Biosynthesis; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger; D051381:Rats; D012756:Sheep
D046911:Macromolecular Substances; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D006006:Glycogen Synthase