Purification of glycogen phosphorylase from bovine brain and immunocytochemical examination of rat glial primary cultures using monoclonal antibodies raised against this enzyme.
The physiological function in brain of glycogen and the enzyme catalyzing the rate-limiting step in glycogenolysis, glycogen phosphorylase (EC 2.4.1.1), is unknown. As a first step toward elucidating such a function, we have purified bovine brain glycogen phosphorylase isozyme BB 1,700-fold to a specific activity of 24 units/mg protein. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent silver staining, a single major protein band corresponding to an apparent molecular mass of 97 kDa was observed. Mouse monoclonal antibodies raised against the enzyme were purified and shown to be monospecific as indicated by immunoblotting. Immunocytochemical examination of astroglia-rich primary cultures of rat brain cells revealed a colocalization of glycogen phosphorylase with the astroglial marker glial fibrillary acidic protein in many cells. The staining for the enzyme appeared at two levels of intensity. There were other cells in the culture showing no specific staining under the experimental conditions employed. Neurons in neuron-rich primary cultures did not show positive staining. The data suggest that glycogen phosphorylase may be predominantly an astroglial enzyme and that astroglia cells play an important role in the energy metabolism of the brain.
Reinhart
P H
PH
Physiologisch-chemisches Institut, Universität Tübingen, F.R.G.
Pfeiffer
B
B
Spengler
S
S
Hamprecht
B
B
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001253:Astrocytes / Q000201:enzymology*; D001921:Brain / Q000166:cytology / Q000201:enzymology*; D002417:Cattle; D002478:Cells, Cultured; D007150:Immunohistochemistry / Q000379:methods; D009474:Neurons / Q000201:enzymology; D006005:Phosphorylases / Q000276:immunology / Q000302:isolation & purification*; D051381:Rats; D013194:Staining and Labeling
D000911:Antibodies, Monoclonal; D006005:Phosphorylases