Dataset Information

Further characterization of the acid-soluble phosphoprotein (SDS/PAGE apparent molecular mass of 22 kDa) in rat fat-cells by peptide sequencing and immuno-analysis: effects of insulin and isoprenaline.

ABSTRACT: 1. Earlier studies have shown that exposure of fat-cells to insulin results in the rapid increased phosphorylation of an acid-soluble protein which migrates as a doublet on SDS/PAGE with an apparent molecular mass of close to 22 kDa; agents such as isoprenaline, which increase cell concentrations of cyclic AMP, also increase phosphorylation, but to a lesser extent [Belsham, Brownsey, Hughes and Denton (1980) Diabetologia 18, 307-312; Diggle and Denton (1992) Biochem. J. 282, 729-736]. 2. The protein has been purified from rat epididymal adipose tissue, and the sequences of six tryptic peptides were determined. All six peptides are present in the deduced sequence of a protein of similar properties, designated PHAS-I by Hu, Pang, Kong, Velleca and Lawrence [(1994) Proc. Natl. Acad. Sci. U.S.A. 91, 3730-3734]. Hence the proteins are the same or extremely similar. 3. A rabbit anti-peptide antibody has been raised against one of the peptides (AGGDESQFEMD). The antibody was found to be highly specific for the phosphorylated and non-phosphorylated forms of the acid-soluble 22 kDa protein in Western blots and by immunoprecipitation. Studies with the antibody preparation have shown that both phosphorylated and non-phosphorylated forms of the protein appear to be exclusively located in the cytoplasm, and that exposure of cells to isoprenaline causes increased phosphorylation of the same acid-soluble 22 kDa protein as does insulin treatment. 4. Western blots carried out with the antibody preparation indicate that the protein is also present in other insulin-sensitive tissues, including liver, skeletal muscle, heart and brown adipose tissue. The protein was also detected in lung and spleen, but not brain and kidney. It is concluded that the protein may play an important role in some of the actions of insulin.

SUBMITTER: Diggle TA

PROVIDER: S-EPMC1136492 | biostudies-other | 1995 Feb

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:Recombinant protein technology provides an invaluable source of proteins for use in structure-function studies, as immunogens, and in the development of therapeutics. Recombinant proteins are typically engineered with "tags" that allow the protein to be purified from crude host cell extracts using affinity based chromatography techniques. Amelogenin is the principal component of the developing enamel matrix and a frequent focus for biomineralization researchers. Several groups have reported the successful production of recombinant amelogenins but the production of recombinant amelogenin free of any tags, and at single band purity on silver stained SDS PAGE is technically challenging. This is important, as rigorous structure-function research frequently demands a high degree of protein purity and fidelity of protein sequence. Our aim was to generate His-tagged recombinant amelogenin at single band purity on silver stained SDS PAGE for use in functionality studies after His-tag cleavage. An acetic acid extraction technique (previously reported to produce recombinant amelogenin at 95% purity directly from E. coli) followed by repeated rounds of nickel column affinity chromatography, failed to generate recombinant amelogenin at single band purity. This was because following an initial round of nickel column affinity chromatography, subsequent cleavage of the His-tag was not 100% efficient. A second round of nickel column affinity chromatography, used in attempts to separate the cleaved His-tag free recombinant from uncleaved His-tagged contaminants, was still unsatisfactory as cleaved recombinant amelogenin exhibited significant affinity for the nickel column. To solve this problem, we used preparative SDS PAGE to successfully purify cleaved recombinant amelogenins to single band purity on silver stained SDS PAGE. The resolving power of preparative SDS PAGE was such that His-tag based purification of recombinant amelogenin becomes redundant. We suggest that acetic acid extraction of recombinant amelogenin and subsequent purification using preparative SDS PAGE provides a simple route to highly purified His-tag free amelogenin for use in structure-function experiments and beyond.

Dataset Information

Further characterization of the acid-soluble phosphoprotein (SDS/PAGE apparent molecular mass of 22 kDa) in rat fat-cells by peptide sequencing and immuno-analysis: effects of insulin and isoprenaline.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets