Amorphin is phosphorylase; phosphorylase is an alpha-actinin-binding protein.
In a study of myofibrillar proteins, Chowrashi and Pepe [1982: J. Cell Biol. 94:565-573] reported the isolation of a new, 85-kD Z-band protein that they named amorphin. We report that partial sequences of purified amorphin protein indicate that amorphin is identical to phosphorylase, an enzyme important in the metabolism of glycogen. Anti-amorphin antibodies also reacted with purified chicken and rabbit phosphorylase. To explore the basis for phosphorylase's (amorphin's) localization in the Z-bands of skeletal muscles, we reacted biotinylated alpha-actinin with purified amorphin and with purified phosphorylase and found that alpha-actinin bound to each. Radioimmune assays also indicated that phosphorylase (amorphin) bound to alpha-actinin, and, with lower affinity, to F-actin. Negative staining of actin filaments demonstrated that alpha-actinin mediates the binding of phosphorylase to actin filaments. There are several glycolytic enzymes that bind actin (e.g., aldolase, phosphofructokinase, and pyruvate kinase), but phosphorylase is the first one demonstrated to bind alpha-actinin. Localization of phosphorylase in live cells was assessed by transfecting cultures of quail embryonic myotubes with plasmids expressing phosphorylase fused to Green Fluorescent Protein (GFP). This resulted in targeting of the fusion protein to Z-bands accompanied by a diffuse pattern in the cytoplasm.
Chowrashi
Prokash
P
Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, 19104-6058, USA.
Mittal
Balraj
B
Sanger
Jean M
JM
Sanger
Joseph W
JW
D000185:Actinin / Q000378:metabolism*; D000199:Actins / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000378:metabolism; D002645:Chickens; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism / Q000648:ultrastructure; D010763:Phosphorylase b / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011784:Quail; D011863:Radioimmunoassay; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D000199:Actins; D000906:Antibodies; D008164:Luminescent Proteins; D009124:Muscle Proteins; C035770:Phosphorylase b protein, Gallus gallus; D011993:Recombinant Fusion Proteins; D000185:Actinin; D049452:Green Fluorescent Proteins; D010763:Phosphorylase b