Abstract
A heterobifunctional cross-linking reagent, 125I-N-(3-iodo-4-azidophenylpropionamido-S-(2-thiopyridyl) cysteine (125-ACTP), has been synthesized. 125I-ACTP has been used to derivative reduced sulfhydryls of the retinal G protein, transducin (Gt), to form a mixed disulfide bond under mild, nondenaturing conditions (pH 7.4, 4 degrees C). The resulting disulfide was easily cleaved using reducing reagents. A 200-fold molar excess of 125I-ACTP relative to Gt resulted in the incorporation of 1-1.3 mol of the 125I-N-(3-iodo-4-azidophenylpropionamido)cysteine moiety of ACTP into Gt alpha. In contrast to 125I-ACTP, dithionitrobenzoate and dithiopyridone derivatized six sulfhydryls in native Gt. Incubation of a 10-fold molar excess of 125I-ACTP relative to Gt resulted in the derivatization of 0.75-0.9 and 0.1 mol of reduced sulfhydryls/mol Gt alpha and beta, respectively. Gt gamma was not derivatized by 125I-ACTP. Thus, Gt alpha was preferentially derivatized by 125I-ACTP. Tryptic digestion and amino acid sequencing of Gt alpha indicated that both Cys-347 near the carboxyl terminus and Cys-210 between the second and third consensus sequences forming the GTP-binding site were derivatized by 125I-ACTP in a ratio of approximately 70 and 30%, respectively. Thus, both Cys-210 and Cys-347 are labeled, even though derivatization by 125I-ACTP does not exceed 1 mol of SH/mol Gt alpha. It appears that derivatization of one sulfhydryl, either Cys-210 or Cys-347, excludes labeling of the second cysteine either by steric hindrance or induced conformational change making the second cysteine inaccessible to 125I-ACTP. Consistent with this finding was the observation that pertussis toxin-catalyzed ADP-ribosylation of Cys-347 inhibited 125I-ACTP derivatization of Cys-210. Derivatization of Gt alpha at either Cys-210 or Cys-347 by 125I-ACTP inhibited rhodopsin-catalyzed guanosine 5'-3-O-(thio)triphosphate binding to Gt, mimicking the effect of ADP-ribosylation of Cys-347 by pertussis toxin. ACTP contains a radioiodinated phenylazide moiety which, upon activation, can cross-link the derivatized cysteine to an adjacent polypeptide domain. Following reduction of the disulfide, the [125I] iodophenyl moiety will be transferred to the azide-inserted polypeptide. When photoactivation of the phenylazide moiety of 125I-ACTP after sulfhydryl derivatization was performed, insertion of the Cys-347 which contains Cys-210, was found.(ABSTRACT TRUNCATED AT 400 WORDS)
Highlights
The finding that Cys-347 reacts with lZ5I-ACTPis signifiRadioactivity eluted as a single major peak (A1),which was cant since this residue is ADP-ribosylated by pertussis toxin reapplied to the C3 column and isolated using a shallower in thenative protein [28].To explore the relationship between gradient of acetonitrile
The radioactive these two events, Gt was treated withpertussistoxin to peptide was once again injected into theC3column and eluted generate nearmole per mol ADP-ribosylation of the a subunit with ammonium acetate as thaequeous phase for acetonitrile, and subsequently derivatized with '251-ACTP.Fig. 6 shows of Transducin a Subunit
Cys-347 of the carboxyl-terminal 3-kDa domain and Cys-210 of the 12-kDa domain of Gta are rapidly and preferentially derivatized by lZ51-ACTPM. odification of either cysteine by ACTP or ADP-ribosylation of Cys-347 disrupts Gt function, measured by the ability of rhodopsin to catalyze guanine nucleotide exchange. Even though both Cys-210 and Cys-347 appear to be labeled, derivatization is no greater than 1 mol of-SH/mol of Gta even in the presence of 200-fold molar excess lZ5I-ACTPrelative to Gt.Two related but distinct mechanisms may account for this property of 'T-ACTP derivatization of Gta
Summary
The synthesis of the reagent is described in detail under “Materials and Methods.”. This compound contains a number of special features: it is radioactive (can be prepared, theoretically, to carrier-free specific activity), it is photoactivatable, and it derivatizes sulfhydryl groups in an asymmetric fashion which, under the reaction conditions used in these experiments, resulted in quantitative release of thipyridone (see below).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
