Abstract
Islet-activating protein catalyzes the ADP-ribosylation of transducin, a guanine nucleotide-binding regulatory protein that mediates activation of a retinal cyclic GMP-selective phosphodiesterase. Radiolabel from [adenylate-32P]NAD+ was incorporated specifically into the alpha subunit of purified transducin. Maximal levels of incorporation approximated 0.8 mol of ADP-ribose/mol of transducin. A peptide containing the ADP-ribosyl moiety was purified from a tryptic digest of radiolabeled transducin. This peptide was characterized by chemical and enzymatic procedures and by fast atom bombardment mass spectrometry. The primary structure of this peptide was Glu-Asn-Leu-Lys-Asn(ADP-ribose)-Gly-Leu-Phe. It is probable that the peptide originated from the carboxyl terminus of the alpha subunit and that the ADP-ribosyl moiety is attached by an N-glycosidic linkage to the asparagine residue. Transducin associated with retinal disc membranes is also ADP-ribosylated by cholera toxin. Cholera toxin and islet-activating protein sequentially catalyze the incorporation of 1.9 mol of ADP-ribose/mol of transducin, indicating two distinct sites of ADP-ribosylation within transducin.
Highlights
Islet-activating protein catalyzes the ADP-ribosylation of transducin, a guanine nucleotide-binding regulatory protein that mediates activation of a retinal cyclicGMP-selective phosphodiesterase
Transducin is a guanine nucleotide-dependent regulatory protein of the retina that mediates activatoiof na cyclic GMPspecific phosphodiesterase by photolyzed rhodopsin.Transducin hasbeen purified to homogeneity from retinal rod outersegment disc membranesandhas been extensively characterized (2-6)
Abood et al ( 7 ) demonstrated that transducin associated with disc membranes, but not that purified by elution with GTP, could be ADP-ribosylated by cholera toxin
Summary
Purification of Transducin-Retinal rod outer segment discs were prepared from freshly dissected bovine retinas essentially by the procedures of Papermaster and Dreyer (8), with the exception that eyes werenot dark-adapted. Incorporation of radiolabel into transducin was quantitatively assessed by filtration of acid-precipitable material andscintillation spectrometry Incorporation determined by this procedure was comparable to that assessed directly following gel filtration. Tryptic Digestion-Transducin (1-2mg), ADP-ribosylated with IAP and [3’P]NAD’, was precipitated by addition of trichloroacetic acid to a concentration of 15%. Radiolabeled material was eluted with a linear gradient of NaCl(80 ml; 5-250 mM) (Fig. 1A): Fractions containing radioactivity werepooled, lyophilized, and dissoived in 1 mol1f00 mM NH4HC03.This sample was applied to a column of Bio-Gel P-6 Radiolabeledtryptic peptide was purified from transducin that had been ADP-ribosylated with IAP and [32P]NAD+P. urification steps are detailed under "Methods." Recovery of tryptic peptide is defined as the amount of radiolabel relative to thatinitially incorporated into transducin (22 nmol)
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