The limited proteolytic pattern of transducin,
G
t
, and its purified subunits with chymotrypsin were analyzed and the cleavage sites on the
t
subunit were identified. The
t
subunit in the GTPS bound form was cleaved into a major 38 kD fragment, whereas
t
-GDP was progressively digested into 38, 23, 21, and 15 kD fragments. The
t
subunit was not very sensitive to proteolytic digestion with chymotrypsin. The
t
subunit was not cleaved and only a small portion of
t
was digested into several fragments. In order to determine which proteolytic fragment of
t
still contained the carboxyl terminal region, chymotrypsinization was carried out using
G
t
previously
32P-labeled at Cys
347 by petrussis toxin-catalyzed ADP-ribosylation. The
32P-label was mainly associated with the
t
subunit and a 15 kD fragment. The 23 and 21 kD fragments were not
32P-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
t
, at Leu
15 and Leu
19. Chymotrypsin removed 15–19 amino acid residues from the amino terminus of
t
, generating two peptides (38 kD) which comigrates in gel electrophoresis. Chymotrypsin also cleaved at Trp
207 in a conformation-dependent manner. Trp
207 of
t
-GTPS was resistant to proteolysis but
t
-GDP and the 38 kD fragments of
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 Trp
207 changes when GTP or GTPS is bound, leading to its inaccessibility to chymotrypsin.
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