1,721,091 research outputs found
Interaction of transducin with light-activated rhodopsin protects it from proteolytic digestion by trypsin
No full textThe tryptic cleavage pattern of transducin (G(t)) in solution was compared with that in the presence of phospholipid vesicles, rod outer segment (ROS) membranes kept in the dark, or ROS membranes containing light-activated rhodopsin, metarhodopsin II (Rh*). When G(t) was in the high affinity complex with Rh*, the alpha(t) subunit was almost completely protected from proteolysis. The protection of at at Arg(310) was complete, while Arg(204) was substantially protected. The cleavage of alpha(t) at Lys's was protected in the presence of phospholipid vesicles, ROS membranes kept in the dark, or ROS membranes containing Rh*. The cleavage of beta(t) was slower in the presence of ROS membranes or phospholipid vesicles. When the Rh*. G(t) complex was incubated with guanyl-5'-yl thiophosphate, a guanine nucleotide analog known to release the high affinity interaction between G(t) and Rh*, the protection at Arg(310) and Arg(304) was diminished. From our results, we propose that Rh* either physically blocks access of trypsin to Arg(204) and Arg(310) or maintains the heterotrimer in such a conformation that these cleavage sites are not available. Since Arg(204) is involved in the switch interface with beta gamma(t) (Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 311-319), it may be that beta gamma(t) is implicated in protecting this cleavage site in the receptor-bound, stabilized heterotrimer. Arg(310) is not near the beta gamma(t) subunit, thus we believe that the high affinity binding of G(t) to Rh* physically or sterically blocks access of trypsin to this site. Thus, Arg(310), only a few angstroms away from the carboxyl terminus of a,, which is known to directly bind to Rh*, is likely to also be a part of the Rh* binding site. This is in agreement with other studies and has implications for the mechanism by which receptors catalyze GDP release from G proteins. The protection of Lys(18) in the presence of phospholipid vesicles suggests that the amino-terminal region is in contact with the membrane, consistent with the crystal structure of the heterotrimer (Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., and Sigler, P. B.(1996) Nature 379, 311-319)
STRUCTURAL-ANALYSIS OF ROD GTP-BINDING PROTEIN, GT - LIMITED PROTEOLYTIC DIGESTION PATTERN OF GT WITH 4 PROTEASES DEFINES MONOCLONAL-ANTIBODY EPITOPE
No full textThe epitope of monoclonal antibody (mAb 4A), which recognizes the alpha-subunit of the rod G protein, G(t), has been suggested to be both at the carboxyl terminus (Deretic, D., and Hamm, H. E. (1987) J. Biol. Chem. 262, 10839-10847) and the amino terminus (Navon, S. E., and Fung, B. K.-K. (1988) J. Biol. Chem. 263, 489-496) of the molecule. To characterize further the mAb 4A binding site on alpha-t and to resolve the discrepancy between these results limited proteolytic digestion of G(t) or alpha-t using four proteases with different substrate specificities has been performed. Endoproteinase Arg-C, which cleaves the peptide bond at the carboxylic side of arginine residues, cleaved the majority of alpha-t into two fragments of 34 and 5 kDa. The alpha-t 34-kDa fragment in the holoprotein, but not alpha-t-guanosine 5'-O-(3-thiotriphosphate), was converted further to a 23-kDa fragment. A small fraction of alpha-t-GDP was cleaved into 23- and 15-kDa fragments. Endoproteinase Lys-C, which selectively cleaves at lysine residues, progressively removed 17 and then 8 residues from the amino terminus, forming 38- and 36-kDa fragments. Staphylococcus aureus V8 protease is known to remove 21 amino acid residues from the amino-terminal region of alpha-t, with the formation of a 38-kDa fragment. L-1-Tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin cleaved alpha-t progressively into fragments of known amino acid sequences (38, then 32 and 5, then 21 and 12 kDa) and a transient 34 kDa fragment. The binding of mAb 4A to proteolytic fragments was analyzed by Western blot and immunoprecipitation. The major fragments recognized by mAb 4A on Western blots were the 34- and 23-kDa fragments obtained by endoproteinase Arg-C and tryptic digestion. Under conditions that allowed sequencing of the 15- and 5-kDa fragments neither the 34- nor the 23-kDa fragments could be sequenced by Edman degradation, indicating that they contained a blocked amino terminus. The smallest fragment that retained mAb 4A binding was the 23-kDa fragment containing Met1 to Arg204. Thus the main portion of the mAb 4A antigenic site was located within this fragment, indicating that the carboxyl-terminal residues from Lys205 to Phe350 were not required for recognition by the antibody. Additionally, the antibody did not bind the 38- and 36-kDa or other fragments containing the carboxyl terminus, showing that the amino-terminal residues from Met1 to Lys17 were essential for antibody binding to alpha-t
EFFECT OF MONOCLONAL-ANTIBODY BINDING ON ALPHA-BETA-GAMMA-SUBUNIT INTERACTIONS IN THE ROD OUTER SEGMENT G-PROTEIN, GT
No full text
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
- …
