Purification and characterization of GP-55, a protein associated with actin-based cytoplasmic gels derived from brain tissue.
Incubation of rat brain cytoplasmic extracts results in the formation of three-dimensional gels that can be collapsed by low-speed centrifugation. Electrophoresis in polyacrylamide gels indicate that these cytoplasmic gels are composed of actin and several associated proteins. Among the latter we have identified a component with an apparent molecular mass of 55,000 daltons. The results of two-dimensional electrophoresis, peptide mapping, and in vivo labeling experiments with [35S]methionine, indicate that the 55-kDa protein is composed of two different gene products we have named alpha and beta; a third polypeptide, named beta', probably derives from the beta polypeptide as a result of some posttranslational modification(s). These three polypeptides, which have the same molecular weight but different isoelectric points, are present in a rather pure preparation of the 55-kDa protein, obtained from cytoplasmic gels by ion-exchange chromatography. In vitro, purified 55-kDa protein interacts in a specific manner with F-actin, as shown by viscosimetry and electron microscopy, leading to the formation of a complex network of cross-linked microfilament bundles. The results of immunofluorescence experiments indicate that the 55-kDa protein is located intracellularly in structures belonging to the Golgi apparatus, and that it is not only present in every type of rat cell tested but also in primary cultures of chick embryo neurons. Based on its intracellular location, we have named the 55-kDa protein as Golgi Protein-55 (GP-55).
D000199:Actins / Q000032:analysis*; D000818:Animals; D001221:Aspartate Carbamoyltransferase; D001921:Brain / Q000648:ultrastructure; D001923:Brain Chemistry; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); D002642:Chick Embryo; D003078:Colchicine / Q000494:pharmacology; D003571:Cytochalasin B / Q000378:metabolism; D003593:Cytoplasm / Q000032:analysis; D004080:Dihydroorotase; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D006056:Golgi Apparatus / Q000032:analysis; D008715:Methionine / Q000378:metabolism; D008854:Microscopy, Electron; D008970:Molecular Weight; D008985:Monensin / Q000494:pharmacology; D009097:Multienzyme Complexes; D009419:Nerve Tissue Proteins / Q000302:isolation & purification*; D009474:Neurons / Q000032:analysis; D011506:Proteins / Q000032:analysis; D051381:Rats; D014783:Viscosity
D000199:Actins; C033626:CAD trifunctional enzyme; D009097:Multienzyme Complexes; D009419:Nerve Tissue Proteins; D011506:Proteins; D003571:Cytochalasin B; D008985:Monensin; D008715:Methionine; D001221:Aspartate Carbamoyltransferase; D004080:Dihydroorotase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); D003078:Colchicine