Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

WD repeat proteins are a family of proteins that contain a series of highly conserved internal repeat motifs, usually ending with WD (Trp-Asp). The G beta subunit of heterotrimeric guanine nucleotide binding protein is a member of this family, and its crystal structure has been recently solved at high resolution (Wall et al. (1995) Cell 83, 1047-1058; Sondek et al. (1996) Nature 379, 369-374). Based on the coordinates of G beta, we have constructed a model for the structure of Sec13, a 33 kDa WD repeat protein from Saccharomyces cerevesiae essential for vesicular traffic. The model has been tested using a combination of biophysical and biochemical methods. Sec13 was expressed in Escherichia coli as a hexa-His-tagged protein (H6Sec13) and purified to homogeneity. In contrast to some other WD repeat proteins that are unable to fold into monomeric structures when expressed in E. coli, H6Sec13 was soluble and monomeric in the absence of detergent. The far-UV circular dichroism (CD) spectra of H6Sec13 indicated less than 10% alpha-helix consistent with the model which predicts primarily beta-sheets. H6Sec13 shows a cooperative and irreversible thermal denaturation curve consistent with a tightly packed structure. The CD spectrum shows an unusual positive ellipticity at 229 nm that was attributed to interactions of surface tryptophans since the 229 nm maximum could be abolished by modification of 6.3 +/- 0.3 (n = 3) tryptophans (out of 15 total in the molecule) with N-bromosuccinimide. Our model predicts that three sets of tryptophans are clustered near the surface. As predicted by the model, purified H6Sec13 was completely resistant to trypsin digestion. The concordance of the model of Sec13 presented in this paper with the biochemical and biophysical studies suggests that this model can be useful as a guide to further experiments designed to elucidate the function of Sec13 in vesicular traffic.
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PMID:Analysis of the physical properties and molecular modeling of Sec13: A WD repeat protein involved in vesicular traffic. 895 69

Thrombin is an agonist inducing platelet activation. We combined two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry (MALDI-TOF MS) to analyse differentially expressed proteins secreted from thrombin-stimulated platelets. Human washed platelets, from healthy volunteers, were stimulated with thrombin 0.5 U/ml at 37 degrees C without stirring and the secreted proteins were resolved by 2D-DIGE. By image analysis, 1094 spots were detected in the 2D gel. The spots whose mean intensity showed at least a five-fold change intensity increase or decrease in the thrombin-activated platelet gel in comparison with unstimulated control were digested by trypsin and subjected to MALDI-TOF MS analysis. Peptides from mass spectra of in-gel digest samples were matched against available databases, using the Mascot search engine (Matrix Science) for peptide mass fingerprint. In the activated platelet secretome, transferrin, glutathione-transferase, WD repeat protein, ER-60, thrombospondin-1 precursor and thrombospondin were the most abundant. Also lamin A, a nuclear protein, not previously identified in platelets, appeared to be released. The novel strategy to combine 2D-DIGE with MALDI-TOF MS is a useful approach for a quantitative analysis of the effect of thrombin on the secretome profile of human platelets.
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PMID:Application of 2-dimensional difference gel electrophoresis (2D-DIGE) to the study of thrombin-activated human platelet secretome. 1823 37