Photoaffinity labeling of atrial natriuretic factor analog atriopeptin III with [gamma-32P]8N3GTP.
We have determined that atrial natriuretic factor (ANF) analog atriopeptin III (AP III) is a guanosine 5'-triphosphate (GTP) binding protein as shown by photoaffinity labeling with the GTP-analog [gamma-32P]8N3GTP (azido-GTP). The photo-insertion was saturable at 40 microM azido-GTP. Half-maximal protection from photo-insertion was observed using 40-microM concentrations of the native nucleotide GTP. The photolabeling was inhibited approximately 70% by addition of micromolar concentrations of the divalent cations MgCl2, MnCl2 or CaCl2. The related purine-containing nucleotide adenosine 5'-triphosphate (ATP) was able to elicit half-maximal protection of AP III from photo-insertion with 20 microM azido-GTP at a concentration of 120 microM. No significant protection from photoinsertion was observed with other compounds such as GDP, GMP, cGMP, UTP and pyrophosphate. Additionally, a carboxy-terminal F-R-Y deleted analog of ANF, AP I; reduced and carboxymethylated AP III; and resact, a peptide released from sea urchin eggs, were not photolabeled by azido-GTP. These results demonstrate that the AP III analog of ANF contains a functional GTP binding site that would be saturated at physiological concentrations of GTP. At present, the role of this interaction in mediating the biological effects of ANF is unknown, but we speculate that direct GTP interaction with AP III may be involved in the mechanism of action of this peptide.
Mann
D M
DM
Veterans Administration Hospital Research Office, Lexington, KY 40511.
Haley
B E
BE
Greenberg
R N
RN
D000345:Affinity Labels; D009320:Atrial Natriuretic Factor / Q000737:chemistry*; D001386:Azides / Q000737:chemistry*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry; D010446:Peptide Fragments; D013329:Structure-Activity Relationship
C021961:8-azidoguanosine triphosphate; D000345:Affinity Labels; D001386:Azides; D010446:Peptide Fragments; C062525:atrial natriuretic factor prohormone (103-126); D009320:Atrial Natriuretic Factor; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins