Ca2+-independent interaction of the γ subunit of phosphorylase kinase with dansyl-calmodulin

Chiun-Jye Yuan, Donald J. Graves*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

5 Scopus citations

Abstract

A strong Ca2+-independent interaction between the isolated, active γ subunit of phosphorylase kinase and dansyl-calmodulin (dansyl-CaM) was observed by monitoring changes in fluorescence intensity in the absence of calcium ion. The pure, active γ subunit of phosphorylase kinase was simply prepared by dialyzing the HPLC-purified, inactive γ subunit against 8 m urea, containing 0.1 mm DTT, 0.1 m Hepes at pH 6.8 or 0.1 m Tris at pH 8.2, followed by dilution of urea with pH 6.8 or 8.2 buffer. The dissociation constants determined by fluorescence spectroscopy for the γ subunit to dansyl-CaM are 25.7 ± 0.6 and 104 ± 12 nm at pH 6.8 in the presence and absence of CaCl2. At pH 8.2, these values are 4.9 ± 0.3 and 29 ± 8 nm in the presence and absence of CaCl2. As the free Ca2+ decreases to as low as 10-9m, the fluorescence intensity and the fluorescence polarization of the γ subunit and dansyl-CaM complex do not decrease in parallel, indicating that the complex does not come apart at low Ca2+ concentration. The presence of Mg2+ affects the interaction between dansyl-CaM and the γ subunit, as indicated by the increase in the polarization of fluorescence of dansyl-CaM. Mn2+ interferes with the interaction of the γ subunit and dansyl-CaM. Free ATP has little effect.

Original languageEnglish
Pages (from-to)317-326
Number of pages10
JournalArchives of Biochemistry and Biophysics
Volume274
Issue number2
DOIs
StatePublished - 1 Nov 1989

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