Conformations of the alpha 39, alpha 41, and beta.gamma components of brain guanine nucleotide-binding proteins. Analysis by limited proteolysis.
We have recently purified two proteins, alpha 39 and alpha 41, from bovine cerebral cortex which are substrates for ADP-ribosylation by pertussis toxin (Neer, E. J., Lok, J. M., and Wolf, L. G. (1984) J. Biol. Chem. 259, 14222-14229). Both proteins bind guanine nucleotides and interact with beta.gamma units. We have used limited proteolysis by trypsin to probe the structure and the conformational states of these proteins. The guanosine 5'-O-(thiotriphosphate) (GTP gamma S)-liganded alpha 41 protein is cleaved into stable 39- and 24/25-kDa products which appear at the same rate. In addition, an 18-kDa peptide is seen. These products are also formed from GDP- or GTP-liganded alpha 41 but are less stable. Cleavage of alpha 39 is different. With GTP gamma S stable 37-kDa product predominates while with GTP or GDP the 37-kDa fragment appears transiently, followed by 24/25-kDa fragments which are stable in the presence of guanine nucleotides but rapidly cleaved without ligand. A 17-kDa peptide is also formed with GTP or GDP. The beta.gamma unit is cleaved by trypsin to stable peptides, a 26/27-kDa doublet and a 14-kDa peptide. Addition of beta.gamma slows tryptic cleavage of alpha 41 but not alpha 39. ADP-ribosylation of alpha 39 and alpha 41 by pertussis toxin affects their conformation in distinct ways which are clearly brought out by the GTP-liganded state. In contrast to unmodified alpha 41, ADP-ribosylated and GTP-liganded alpha 41 is proteolyzed very slowly and without formation of a 39-kDa intermediate. GTP gamma S seems to override the effect of ADP-ribosylation so that cleavage is more rapid and goes via the 39-kDa product. ADP-ribosylation affects alpha 39 more subtly. The GTP-liganded protein is first cleaved to the 37-kDa product and then degraded without forming the 24/25-kDa fragment. These results suggest that ADP-ribosylation might affect the conformation and function of these related proteins differently. The site of [32P]ADP-ribosylation is on the 18-kDa product of alpha 41 and on the 17-kDa product of alpha 39. We have raised polyclonal antibodies against alpha 39 and beta in rabbits and used the antibodies to examine antigenic sites on alpha 39 and beta. The antigenic determinants of alpha 39 are located over most of the native tryptic peptides. Tryptic cleavage of alpha 41 leads to rapid loss of cross-reactivity with anti-alpha 39 antibody.(ABSTRACT TRUNCATED AT 400 WORDS)
J W
JW
Winslow
J R
JR
Van Amsterdam
E J
EJ
Neer
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002540:Cerebral Cortex / Q000032:analysis*; D000939:Epitopes / Q000032:analysis; D019204:GTP-Binding Proteins / Q000032:analysis / Q000276:immunology; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006153:Guanosine Diphosphate / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D037342:Pertussis Toxin; D010761:Phosphorus Radioisotopes; D011487:Protein Conformation; D011817:Rabbits; D013873:Thionucleotides / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D000939:Epitopes; D010761:Phosphorus Radioisotopes; D013873:Thionucleotides; D010566:Virulence Factors, Bordetella; D006153:Guanosine Diphosphate; D000246:Adenosine Diphosphate Ribose; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D037342:Pertussis Toxin; D014357:Trypsin; D019204:GTP-Binding Proteins