Isolation and characterization of an adenylyl-protein complex formed during the incubation of membranes from Dictyostelium discoideum with ATP

Edward F. Rossomando, Edmund V. Crean, Daniel Kestler

Research output: Contribution to journalArticle

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Abstract

When tritiated ATP is incubated with a membrane-enriched fraction prepared from the eukaryotic microorganism Dictyostelium discoideum significant levels of radioactivity can be precipitated with cold, 10% trichloroacetic acid. Reaction product was formed from ATP and dATP but not from GTP, CTP and UTP. Other studies showed that the maximum amount of the acid-insoluble product was formed about 1 min after the addition of the membranes and that, with further incubation, this reaction product was degraded. The rate of degradation of the reaction product was greatly reduced when the temperature was reduced to 4°C, and when either NaF, Na2SO4 or dithiothreitol was added to the reaction mixture. These additions or conditions had no effect on the product-formation reaction. The rate of degradation was also reduced following the addition of adenosine to the reaction and this result did not occur following the addition of ADP, AMP or cyclic AMP. The acid-insoluble reaction product could be solubilized with SDS and analysis by gel-filtration chromatography on Sephadex G-75 revealed that the radioactivity was associated with a macromolecule that was not sensitive to RNAase or DNAase but was degraded by pronase. The nucleotide-protein complex was stable at room temperature but radioactivity was released in hot acid, which, after analysis by thin-layer chromatography, was found to co-migrate with authentic AMP, suggesting the formation of an adenylyl-protein complex as the reaction intermediate. The complex bond was stable at neutral and alkaline pH, suggesting a phosphoamide linkage between the protein and the adenylyl moiety.

Original languageEnglish (US)
Pages (from-to)386-391
Number of pages6
JournalBBA - General Subjects
Volume675
Issue number3-4
DOIs
StatePublished - Jul 17 1981
Externally publishedYes

Fingerprint

Dictyostelium
Reaction products
Radioactivity
Adenosine Triphosphate
Adenosine Monophosphate
Membranes
Acids
Cytidine Triphosphate
Trichloroacetic Acid
Pronase
Uridine Triphosphate
Proteins
Temperature
Deoxyribonucleases
Dithiothreitol
Thin Layer Chromatography
Guanosine Triphosphate
Degradation
Thin layer chromatography
Cyclic AMP

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

Isolation and characterization of an adenylyl-protein complex formed during the incubation of membranes from Dictyostelium discoideum with ATP. / Rossomando, Edward F.; Crean, Edmund V.; Kestler, Daniel.

In: BBA - General Subjects, Vol. 675, No. 3-4, 17.07.1981, p. 386-391.

Research output: Contribution to journalArticle

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