One of the neuronal growth-associated proteins, GAP-43 (molecular mass, -43 kDa; pI 4.3), is abundant in growth-cone membranes and corresponds to a major protein kinase C substrate, the 46-kDa phosphoproteiw (pp46), of a growth-cone-enriched subcellular fraction. This protein has the following additional designations (depending on context): B-50 (phospholipid metabolism), F1 (synaptic plasticity), and p57 (calmodulin binding). We show that a protein with the same molecular mass and isoelectric point as GAP-43, which interacts with anti-GAP-43 antibodies on immunoblots, is present in the plasma membranes of cultured neonatal rat cortical astrocytes. Double-immunofluorescence labeling of cells with a serum against glial fibrillary acidic protein and anti-GAP-43 antibody was observed. Furthermore, astrocytic protein was phosphorylated in vitro by protein kinase C and comigrated in two-dimensional PAGE with GAP-43. The data indicate that GAP-43, heretofore believed to be neuronspecific, is present in at least one class of glial cells.Astrocytes constitute about one-half of all glial cells in the central nervous system. Although their precise functions are unknown, data indicate that they may be involved in a symbiotic relationship with neurons and thereby play important roles in neuronal development and function. This suggests that a significant aspect of astrocytic activity involves its plasma membrane. However, astrocytic plasma membranes remain to a large extent biochemically uncharacterized and, as such, their capacity for interacting with their environment is unknown. In this report, the proteins of the astrocytic plasma membrane are characterized by twodimensional PAGE, immunoblotting, and comparison with membranes from other cell types.We have described two brain-specific plasma membrane proteins that are very abundant in rat cortex (1). The smaller polypeptide was identified as growth-associated protein 43 (GAP-43) because of its relative molecular mass (=43 kDa), pI value (pI 4.3), in vitro phosphorylation by protein kinase C (PKC), and reactivity with anti-GAP-43 antibody. A polypeptide with these characteristics has been identified by several groups and designated F1, B-50, and a 46-kDa phosphoprotein (pp46) (2). Since this protein is developmentally regulated and its synthesis increases in regenerating axons, it was named growth-associated protein 43 (3). We show here that a protein with a molecular mass and a pI value similar to those of neuronal GAP-43 is present in the plasma membranes of cultured rat astrocytes. Two anti-GAP-43 antibodies interacted with this protein on immunoblots and mediated fluorescent labeling of cultured astrocytes. It is currently believed that GAP-43 is exclusively a neuronal protein. The presence of GAP-43 in astrocytic membranes may have important implications for the role of this protein in nervous system development.
Glutathione S-transferase (GSH-transferase) was purified from human placenta and kidney by affinity chromatography on S-glutathione-carbamidomethyl-e-aminolysyl- Sepharose CL 4B and gel filtration chromatography on Sephadex G-75. Electrophoretically pure enzyme with the specific activities of 50.7 and 55.9 U/mg, respectively, were obtained. In addition to the known acidic isoenzyme from human placenta (isoelectric point, pi, 4.5), we describe here for the first time the presence of 6 basic forms with pi values between 8.0 and 9.0. The kidney GSH-transferase contained 2 acidic forms with isoelectric points at 4.6 and 4.65, and 6 basic forms with pi values between 8.7 and 9.4. The basic and acidic isoenzymes from placenta were separated by ion exchange chromatography on Sephadex DEAE A-25. The acidic form accounted for 36% of the total GSH-transferase activity from placenta. Antibodies against the kidney enzyme were raised in rabbit. Total cross-reactivity of placental GSH-transferase with antikidney-GSH-transferase antibodies was obtained, suggesting that the kidney and placental enzymes are immunologically closely related.
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