Immunoprecipitation of adenylate cyclase with an antibody to a carboxyl-terminal peptide from Gsα

Diane Morris, Elizabeth McHugh-Sutkowski, Malcolm Moos, William F. Simonds, Allen M. Spiegel, Allen M. Spiegel

Research output: Contribution to journalArticle

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Abstract

An antibody (RM) raised against the carboxyl-terminal decapeptide of the α subunit of the stimulatory guanine nucleotide regulatory protein (Gsα) has been used to study the interaction of Gsα with bovine brain adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. RM antibody immunoprecipitated about 60% of the solubilized adenylate cyclase preactivated with either GTP-γ-S or AlF4-. In contrast, RM antibody immunoprecipitated about 5% of the adenylate cyclase not preactivated (control) and 15% of the adenylate cyclase pretreated with forskolin. Adenylate cyclase solubilized from control membranes or GTP-γ-S preactivated membranes was partially purified by using forskolin-agarose affinity chromatography. The amount of Gsα protein in the partially purified preparations was determined by immunoblotting with RM antibody. There was 3-fold more Gsα detected in partially purified adenylate cyclase from preactivated membranes than in the partially purified adenylate cyclase from control membranes. Partially purified adenylate cyclase from preactivated membranes was immunoprecipitated with RM antibody and the amount of adenylate cyclase activity immunoprecipitated (65% of total) corresponded to the amount of Gsα protein immunoprecipitated. Only 15% of the partially purified adenylate cyclase from control membranes was immunoprecipitated. The presence of other G proteins in the partially purified preparations of adenylate cyclase was investigated by using specific antisera that detect Goα, Giα, and Gβ. The Gβ protein was the only subunit detected in the partially purified preparations of adenylate cyclase and the amount of Gβ was about the same in adenylate cyclase from preactivated membranes and from control membranes. Examination of the RM antibody immunoprecipitates from control and GTP-γ-S preactivated solubilized membranes failed to detect any of these G proteins. These results indicate that the complex between the adenylate cyclase catalytic subunit and the activated Gsα protein can be isolated by immunoprecipitation with an anti-Gsα antibody. There does not appear to be a specific association of Goα, Giα, or Gβ with the preactivated complex of the catalytic subunit and the activated Gsα subunit.

Original languageEnglish (US)
Pages (from-to)9079-9084
Number of pages6
JournalBiochemistry®
Volume29
Issue number38
StatePublished - 1990
Externally publishedYes

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Immunoprecipitation
Adenylyl Cyclases
Peptides
Antibodies
Membranes
GTP-Binding Proteins
Guanosine Triphosphate
Catalytic Domain
Affinity chromatography
Agarose Chromatography
Proteins
Colforsin
Affinity Chromatography
Immunoblotting
Immune Sera
Anti-Idiotypic Antibodies
Brain

ASJC Scopus subject areas

  • Biochemistry

Cite this

Morris, D., McHugh-Sutkowski, E., Moos, M., Simonds, W. F., Spiegel, A. M., & Spiegel, A. M. (1990). Immunoprecipitation of adenylate cyclase with an antibody to a carboxyl-terminal peptide from Gsα. Biochemistry®, 29(38), 9079-9084.

Immunoprecipitation of adenylate cyclase with an antibody to a carboxyl-terminal peptide from Gsα. / Morris, Diane; McHugh-Sutkowski, Elizabeth; Moos, Malcolm; Simonds, William F.; Spiegel, Allen M.; Spiegel, Allen M.

In: Biochemistry®, Vol. 29, No. 38, 1990, p. 9079-9084.

Research output: Contribution to journalArticle

Morris, D, McHugh-Sutkowski, E, Moos, M, Simonds, WF, Spiegel, AM & Spiegel, AM 1990, 'Immunoprecipitation of adenylate cyclase with an antibody to a carboxyl-terminal peptide from Gsα', Biochemistry®, vol. 29, no. 38, pp. 9079-9084.
Morris D, McHugh-Sutkowski E, Moos M, Simonds WF, Spiegel AM, Spiegel AM. Immunoprecipitation of adenylate cyclase with an antibody to a carboxyl-terminal peptide from Gsα. Biochemistry®. 1990;29(38):9079-9084.
Morris, Diane ; McHugh-Sutkowski, Elizabeth ; Moos, Malcolm ; Simonds, William F. ; Spiegel, Allen M. ; Spiegel, Allen M. / Immunoprecipitation of adenylate cyclase with an antibody to a carboxyl-terminal peptide from Gsα. In: Biochemistry®. 1990 ; Vol. 29, No. 38. pp. 9079-9084.
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abstract = "An antibody (RM) raised against the carboxyl-terminal decapeptide of the α subunit of the stimulatory guanine nucleotide regulatory protein (Gsα) has been used to study the interaction of Gsα with bovine brain adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. RM antibody immunoprecipitated about 60{\%} of the solubilized adenylate cyclase preactivated with either GTP-γ-S or AlF4-. In contrast, RM antibody immunoprecipitated about 5{\%} of the adenylate cyclase not preactivated (control) and 15{\%} of the adenylate cyclase pretreated with forskolin. Adenylate cyclase solubilized from control membranes or GTP-γ-S preactivated membranes was partially purified by using forskolin-agarose affinity chromatography. The amount of Gsα protein in the partially purified preparations was determined by immunoblotting with RM antibody. There was 3-fold more Gsα detected in partially purified adenylate cyclase from preactivated membranes than in the partially purified adenylate cyclase from control membranes. Partially purified adenylate cyclase from preactivated membranes was immunoprecipitated with RM antibody and the amount of adenylate cyclase activity immunoprecipitated (65{\%} of total) corresponded to the amount of Gsα protein immunoprecipitated. Only 15{\%} of the partially purified adenylate cyclase from control membranes was immunoprecipitated. The presence of other G proteins in the partially purified preparations of adenylate cyclase was investigated by using specific antisera that detect Goα, Giα, and Gβ. The Gβ protein was the only subunit detected in the partially purified preparations of adenylate cyclase and the amount of Gβ was about the same in adenylate cyclase from preactivated membranes and from control membranes. Examination of the RM antibody immunoprecipitates from control and GTP-γ-S preactivated solubilized membranes failed to detect any of these G proteins. These results indicate that the complex between the adenylate cyclase catalytic subunit and the activated Gsα protein can be isolated by immunoprecipitation with an anti-Gsα antibody. There does not appear to be a specific association of Goα, Giα, or Gβ with the preactivated complex of the catalytic subunit and the activated Gsα subunit.",
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AU - Spiegel, Allen M.

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N2 - An antibody (RM) raised against the carboxyl-terminal decapeptide of the α subunit of the stimulatory guanine nucleotide regulatory protein (Gsα) has been used to study the interaction of Gsα with bovine brain adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. RM antibody immunoprecipitated about 60% of the solubilized adenylate cyclase preactivated with either GTP-γ-S or AlF4-. In contrast, RM antibody immunoprecipitated about 5% of the adenylate cyclase not preactivated (control) and 15% of the adenylate cyclase pretreated with forskolin. Adenylate cyclase solubilized from control membranes or GTP-γ-S preactivated membranes was partially purified by using forskolin-agarose affinity chromatography. The amount of Gsα protein in the partially purified preparations was determined by immunoblotting with RM antibody. There was 3-fold more Gsα detected in partially purified adenylate cyclase from preactivated membranes than in the partially purified adenylate cyclase from control membranes. Partially purified adenylate cyclase from preactivated membranes was immunoprecipitated with RM antibody and the amount of adenylate cyclase activity immunoprecipitated (65% of total) corresponded to the amount of Gsα protein immunoprecipitated. Only 15% of the partially purified adenylate cyclase from control membranes was immunoprecipitated. The presence of other G proteins in the partially purified preparations of adenylate cyclase was investigated by using specific antisera that detect Goα, Giα, and Gβ. The Gβ protein was the only subunit detected in the partially purified preparations of adenylate cyclase and the amount of Gβ was about the same in adenylate cyclase from preactivated membranes and from control membranes. Examination of the RM antibody immunoprecipitates from control and GTP-γ-S preactivated solubilized membranes failed to detect any of these G proteins. These results indicate that the complex between the adenylate cyclase catalytic subunit and the activated Gsα protein can be isolated by immunoprecipitation with an anti-Gsα antibody. There does not appear to be a specific association of Goα, Giα, or Gβ with the preactivated complex of the catalytic subunit and the activated Gsα subunit.

AB - An antibody (RM) raised against the carboxyl-terminal decapeptide of the α subunit of the stimulatory guanine nucleotide regulatory protein (Gsα) has been used to study the interaction of Gsα with bovine brain adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. RM antibody immunoprecipitated about 60% of the solubilized adenylate cyclase preactivated with either GTP-γ-S or AlF4-. In contrast, RM antibody immunoprecipitated about 5% of the adenylate cyclase not preactivated (control) and 15% of the adenylate cyclase pretreated with forskolin. Adenylate cyclase solubilized from control membranes or GTP-γ-S preactivated membranes was partially purified by using forskolin-agarose affinity chromatography. The amount of Gsα protein in the partially purified preparations was determined by immunoblotting with RM antibody. There was 3-fold more Gsα detected in partially purified adenylate cyclase from preactivated membranes than in the partially purified adenylate cyclase from control membranes. Partially purified adenylate cyclase from preactivated membranes was immunoprecipitated with RM antibody and the amount of adenylate cyclase activity immunoprecipitated (65% of total) corresponded to the amount of Gsα protein immunoprecipitated. Only 15% of the partially purified adenylate cyclase from control membranes was immunoprecipitated. The presence of other G proteins in the partially purified preparations of adenylate cyclase was investigated by using specific antisera that detect Goα, Giα, and Gβ. The Gβ protein was the only subunit detected in the partially purified preparations of adenylate cyclase and the amount of Gβ was about the same in adenylate cyclase from preactivated membranes and from control membranes. Examination of the RM antibody immunoprecipitates from control and GTP-γ-S preactivated solubilized membranes failed to detect any of these G proteins. These results indicate that the complex between the adenylate cyclase catalytic subunit and the activated Gsα protein can be isolated by immunoprecipitation with an anti-Gsα antibody. There does not appear to be a specific association of Goα, Giα, or Gβ with the preactivated complex of the catalytic subunit and the activated Gsα subunit.

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