15-kDa Polypeptide Research Articles

Secretion of novel and homologous neutrophil-activating peptides by LPS-stimulated human endothelial cells.

Human umbilical vein endothelial cells in culture produce two chemotactic polypeptides when stimulated with LPS. The chemotactic factors could be purified to apparent homogeneity by HPLC techniques and were identified as 7.5-kDa and 15-kDa polypeptides by SDS-PAGE under nonreducing conditions. Both factors are potent chemotaxins for human neutrophils demonstrating half-maximal chemotaxis at 2 ng/ml and g ng/ml, respectively. In addition both peptides elicited release of azurophilic granule constituents when neutrophils were pretreated with cytochalasin B. Cross-desensitization experiments by using human neutrophils revealed cross-reactivities between both chemotaxins, not, however, with C5a or FMLP, indicating that both endothelial cell-derived neutrophil activating peptides (ENAP) are homologous. In addition, the 7.5-kDa factor (beta-ENAP) proved to be the quantitatively dominating and more potent chemotaxin as compared to the 15 kDa factor (alpha-ENAP). beta-ENAP shows biochemical and biologic similarities to monocyte- and lymphocyte-derived neutrophil-activating peptides MONAP and LYNAP, which recently were purified and sequenced.

The 110-kDa reaction center protein of photosystem I, P700-chlorophyll a-protein 1, is an iron-sulfur protein.

Germination and growth of barley (Hordeum vulgare L.) in the presence of 59Fe2+ or 35SO4(2-) allows heavy incorporation of both isotopes into the thylakoid membranes and into isolated photosystem I particles. Analysis of 59Fe-labeled preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under mild conditions demonstrates that a minimum of four iron atoms/P700 is carried on P700-chlorophyll a-protein 1. When isolated from 35S-labeled preparations, P700-chlorophyll a-protein 1 binds zero valence 35S, which is converted into acid-labile [35S]sulfide by dithiothreitol reduction. Isolated photosystem I particles contain 14 acid-labile sulfide atoms and 10 iron atoms for each molecule of P700 and are composed of polypeptides of 110, 18, 15, 10, and 8 kDa of which the 10-kDa component is loosely bound. Under the electrophoretic conditions used, none of the low molecular weight polypeptides could be shown to be specifically associated with iron or acid-labile sulfide. Carboxymethylation of cysteine residues shows a high cysteine content in the 8-kDa polypeptide and an intermediate content in the 110- and 18-kDa polypeptides, whereas the 15-kDa polypeptide is devoid of sulfur amino acids. The experiments with the 59Fe-labeled thylakoids reveal other labeled polypeptides not associated with photosystem I, namely cytochrome f and possibly cytochromes b6 and b559.

NusB: a protein factor necessary for transcription antitermination in vitro by phage lambda N gene product.

We demonstrate that the protein product of the Escherichia coli nusB gene is essential for transcription antitermination in vitro by phage lambda N gene product. We recently have described a convenient biochemical assay for N protein activity in the S30-coupled transcription translation system and demonstrated that N action requires the 69-kDa L factor (nusA), the product of E. coli nusA gene. Using a complementation assay for the restoration of N activity specifically in the nusB mutant extract, we have purified the nusB complementing activity. This activity is due to a 15-kDa polypeptide that is overproduced in E. coli containing multiple copies of the nusB gene. We find that nusA and nusB are required for N activity to suppress a rho-dependent as well as a rho-independent terminator. The requirement for nusB protein in antitermination could not be overcome by an excess of nusA or N protein, nor could an excess of nusB overcome the requirements for nusA in antitermination. Our results suggest that the formation of an antitermination apparatus by N requires nusA and nusB proteins in equimolar amounts.