TY - JOUR
T1 - Cholera-toxin-dependent ADP-ribosylation of the adenylate cyclase regulatory protein in turkey erythrocyte membranes
AU - Downs, Robert W.
AU - Reen, Sharon A.
AU - Levine, Michael A.
AU - Aurbach, Gerald D.
AU - Spiegel, Allen M.
PY - 1981
Y1 - 1981
N2 - Incubation of turkey erythrocyte membranes with cholera toxin and [32P]NAD caused toxin-dependent incorporation of 32P into a 42,000 Mr peptide which could be distinguished from toxin-independent 32P incorporation into other membrane proteins. The radiolabeled 42,000 Mr peptide could be extracted from the membranes using Lubrol PX. When toxin-treated membranes were incubated with isoproterenol and GMP before detergent solubilization, the 42,000 Mr labeled peptide was adsorbed by GTP-γ-agarose which, with the same conditions, adsorbed the adenylate cyclase guanine nucleotide regulatory protein. The labeled peptide and guanine nucleotide regulatory protein activity were coeluted from the affinity matrix by guanylyl-β,γ-imidodiphosphate, GDP, and GMP. Guanosine 5′-O-(2-thiodiphosphate), an analog of GDP which blocks guanine nucleotide- and fluoride-stimulated adenylate cyclase activity, caused elution of labeled peptide which exhibited no regulatory protein activity. Our data support the view that the 42,000 Mr peptide is part of the adenylate cyclase guanine nucleotide regulatory protein. The labeled peptide allows identification of both active and inactive regulatory protein and should be useful in monitoring the purification of the regulatory protein from turkey erythrocytes.
AB - Incubation of turkey erythrocyte membranes with cholera toxin and [32P]NAD caused toxin-dependent incorporation of 32P into a 42,000 Mr peptide which could be distinguished from toxin-independent 32P incorporation into other membrane proteins. The radiolabeled 42,000 Mr peptide could be extracted from the membranes using Lubrol PX. When toxin-treated membranes were incubated with isoproterenol and GMP before detergent solubilization, the 42,000 Mr labeled peptide was adsorbed by GTP-γ-agarose which, with the same conditions, adsorbed the adenylate cyclase guanine nucleotide regulatory protein. The labeled peptide and guanine nucleotide regulatory protein activity were coeluted from the affinity matrix by guanylyl-β,γ-imidodiphosphate, GDP, and GMP. Guanosine 5′-O-(2-thiodiphosphate), an analog of GDP which blocks guanine nucleotide- and fluoride-stimulated adenylate cyclase activity, caused elution of labeled peptide which exhibited no regulatory protein activity. Our data support the view that the 42,000 Mr peptide is part of the adenylate cyclase guanine nucleotide regulatory protein. The labeled peptide allows identification of both active and inactive regulatory protein and should be useful in monitoring the purification of the regulatory protein from turkey erythrocytes.
UR - http://www.scopus.com/inward/record.url?scp=0019854796&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0019854796&partnerID=8YFLogxK
U2 - 10.1016/0003-9861(81)90282-4
DO - 10.1016/0003-9861(81)90282-4
M3 - Article
C2 - 6269497
AN - SCOPUS:0019854796
SN - 0003-9861
VL - 209
SP - 284
EP - 290
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
IS - 1
ER -