ISSN:
1573-4943
Keywords:
Rod GTP-binding protein
;
G t , transducin
;
proteolysis
;
chymotrypsin
Source:
Springer Online Journal Archives 1860-2000
Topics:
Chemistry and Pharmacology
Notes:
Abstract The limited proteolytic pattern of transducin,G t , and its purified subunits with chymotrypsin were analyzed and the cleavage sites on the α t subunit were identified. The α t subunit in the GTPγS bound form was cleaved into a major 38 kD fragment, whereas α t -GDP was progressively digested into 38, 23, 21, and 15 kD fragments. The βγ t subunit was not very sensitive to proteolytic digestion with chymotrypsin. The γ t subunit was not cleaved and only a small portion of β t was digested into several fragments. In order to determine which proteolytic fragment of α t still contained the carboxyl terminal region, chymotrypsinization was carried out usingG t previously32P-labeled at Cys347 by petrussis toxin-catalyzed ADP-ribosylation. The32P-label was mainly associated with the α t subunit and a 15 kD fragment. The 23 and 21 kD fragments were not32P-labeled. Analysis of amino terminal sequences of 38, 21, and 15 kD proteolytic bands allowed the identification of the major cleavage sites. Chymotrypsin had two cleavage sites in the amino terminal region of α t , at Leu15 and Leu19. Chymotrypsin removed 15–19 amino acid residues from the amino terminus of α t , generating two peptides (38 kD) which comigrates in gel electrophoresis. Chymotrypsin also cleaved at Trp207 in a conformation-dependent manner. Trp207 of α t -GTPγS was resistant to proteolysis but α t -GDP and the 38 kD fragments of α t -GDP produced the 23 and 21 kD fragments, respectively, and a 15 kD fragment containing the carboxyl terminus. This proves that the environment of Trp207 changes when GTP or GTPγS is bound, leading to its inaccessibility to chymotrypsin.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF01026043
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