Abstract
Glial fibrillary acidic (GFA) protein and desmin were purified from bovine brain and large intestine, respectively, and used in a comparison of the major protein components of two classes of intermediate filaments, the glial and smooth muscle filaments. The proteins are similar in size, charge, and amino acid composition, but clearly distinct. By sodium dodecyl sulfate-gel electrophoresis, GFA protein is about 5,000 daltons smaller than desmin. GFA protein is composed of three isoelectric variants which are all slightly more basic than the two variants observed for desmin. One-dimensional peptide mapping following limited proteolysis under denaturing conditions or following cyanogen bromide cleavage demonstrates that the proteins are not closely related in primary structure. Assembly-disassembly experiments reveal that the proteins share solubility properties and that negatively stained preparations of in vitro polymerized filaments are very similar. Limited proteolysis under native conditions demonstrates substructural similarities; comparative peptide mapping following digestion with chymotrypsin and trypsin suggests related core polypeptides of about 37,000 and 21,000 daltons. We conclude that GFA protein and desmin are distinct with respect to primary structure, but probably represent two of the more closely related classes of intermediate filament proteins.
Highlights
Than desmin.Glial fibrillary acidic (GFA) protein is composed of three isoelectric variants which are asllightly more basic than the two variantsobservedfordesmin.One-dimensional peptide mapping following limited proteolysis under denaturing conditions or following cyanogen bromide cleavage demonstrates that the proteins are cnloostely relatedinprimarystructure.Assembly-disassembly experiments reveal that the proteins share solubility
GFA Protein Preparation-GFA protein was purified from bovine brain extracts by the immunoaffinity chromatographyprocedure were performedwithbothpreparationsto controlfor any adverse effects of the presence of SDS during purification
The purified preparations of both GFA protein and desmin showed evidenceof degradation productsmigrating just ahead described in detail previously [10, 11]
Summary
Tris-HCI buffer (pH 8.5 a t 4 "C) containing0.5 r n d~ithiothreitol and cleared by centrifugation a t 150,000 X g for 90 min. Gels are shown of desmin before and tion step, the desmin pellet was dissolved in 1 M acetic acid with 20 after SDS chromatography since in some cases experiments mM NaCI, dialyzed for 3 days a t 4 "C against 2 mM Tris-HCI buffer (pH 8.5 at 4 "C) containing 0.5 mM dithiothreitol, and centrifuged a t 150,000 X g for 90 min. The purified preparations of both GFA protein and desmin showed evidenceof degradation productsmigrating just ahead described in detail previously [10, 11]. GFA protein preparations protein were dialyzed for days a t 4 "Cagainst 2 mM Tris-HCI buffer contained three such componentsw, hile desmin preparations (pH 8.5) containing 0.5 mM dithiothreitol and cleared by centrifugation a t 150,000 X g for 90 min prior to use. Enzymatic digestions of assembly-competent proteins were per- rations of GFA protein (Gel 1 ) and desmin before (Gel 2) and after formed with either chymotrypsionr trypsin (Worthington, tosylphen- (Gel 3) SDS-gel chromatography
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