Immunological detection of degradation intermediates of skeletal-muscle glycogen phosphorylase in vitro and in vivo.
Over 95% of the pyridoxal phosphate (PLP) in skeletal is bound to one protein, glycogen phosphorylase. This, and the fact that phosphorylase constitutes approx. 5% of the soluble protein in skeletal muscle, introduce the possibility that PLP might be used as a specific label to identify degradation intermediates of the enzyme. In this investigation, we have developed immunological methods, using a monoclonal antibody to PLP and polyclonal antibodies to phosphorylase, to detect degradation intermediates in vitro and in vivo. We have identified a family of degradation intermediates of glycogen phosphorylase in the high-speed-supernatant fraction of mouse skeletal muscle. These peptides react with both types of antibodies and are in the size and concentration range expected for degradation intermediates in a model in which the committed step is followed by rapid clearance of the products. Changes in amounts of degradation intermediates are examined in physiological or pathological conditions in which the rate of degradation of phosphorylase is altered.
Cookson
E J
EJ
Department of Biochemistry, University of Liverpool, U.K.
Flannery
A V
AV
Cidlowski
J A
JA
Beynon
R J
RJ
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D001894:Borohydrides; D002645:Chickens; D007118:Immunoassay; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009124:Muscle Proteins / Q000378:metabolism*; D009132:Muscles / Q000201:enzymology*; D009137:Muscular Dystrophy, Animal / Q000201:enzymology; D010446:Peptide Fragments / Q000032:analysis*; D006005:Phosphorylases / Q000378:metabolism*; D011732:Pyridoxal Phosphate / Q000276:immunology / Q000378:metabolism; D011817:Rabbits; D014357:Trypsin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D001894:Borohydrides; D009124:Muscle Proteins; D010446:Peptide Fragments; D011732:Pyridoxal Phosphate; C009282:sodium cyanoborohydride; D006005:Phosphorylases; D014357:Trypsin