The Granulin/Epithelin Precursor Abrogates the Requirement for the Insulin-like Growth Factor 1 Receptor for Growth in Vitro

3T3 cells null for the type 1 insulin-like growth factor receptor are refractory to stimulation by a variety of purified growth factors that are known to be required for the stimulation of other 3T3 cells. However, these cells, known as R- cells, grow in serum-supplemented medium and also in media conditioned by certain cell lines. We report here the purification of a growth factor that stimulates DNA synthesis (and growth) of R- cells. The growth factor, purified to homogeneity by SDS-polyacrylamide gel electrophoresis, was identified as the granulin/epithelin precursor by an accurate determination of the masses of endoproteinase Lys-C peptides using matrix-assisted laser desorption ionization mass spectrometry, followed by a data base search. The granulin/epithelin precursor is a little known growth factor, secreted by a variety of epithelial and hemopoietic cells. It is at present the only purified growth factor that can stimulate the growth of mouse embryo fibroblasts null for the type 1 insulin-like growth factor receptor

Isolation and characterization of the Bacillus subtilis sigma 28 factor.

RNA polymerase preparations isolated from vegetatively growing Bacillus subtilis cells contain the core subunits beta, beta', and alpha, together with multiple sigma factors and other core-associated polypeptides such as delta, omega 1, and omega 2. We have developed an improved, large-scale purification procedure that yields RNA polymerase fractions enriched in both the sigma 28 and delta proteins. These fractions have been used to isolate sigma 28 protein for biochemical characterization and for preparation of highly specific anti-sigma 28 antisera. The amino acid composition of purified sigma 28 protein and the amino acid sequences of tryptic peptide fragments have been determined. Anti-sigma 28 antisera specifically inhibit transcription by the purified sigma 28 -dependent RNA polymerase, yet do not affect transcription by sigma 43 -dependent RNA polymerase. Immunochemical analysis confirms that the sigma 28 protein copurifies with total RNA polymerase activity through the majority of the purification procedure and allows the steps when sigma 28 protein is lost to be identified and optimized. Immunochemical techniques have also been used to monitor the structure and abundance of the sigma 28 protein in vivo. A single form of antibody-reactive protein was detected by two-dimensional gel electrophoresis-isoelectric focusing. Its abundance corresponds to a maximal content of 220 molecules of sigma 28 per B. subtilis cell during late-logarithmic-phase growth