Ca 2+-independent interaction of the γ subunit of phosphorylase kinase with dansyl-calmodulin

• Published in: Archives of biochemistry and biophysics Vol. 274; no. 2; pp. 317 - 326
• Main Authors: Yuan, Chiun-Jye, Graves, Donald J.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 01.11.1989
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/0003-9861(89)90445-1

A strong Ca 2+-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 m m 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 n m at pH 6.8 in the presence and absence of CaCl 2. At pH 8.2, these values are 4.9 ± 0.3 and 29 ± 8 n m in the presence and absence of CaCl 2. As the free Ca 2+ decreases to as low as 10 −9 m, 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 Ca 2+ concentration. The presence of Mg 2+ affects the interaction between dansyl-CaM and the γ subunit, as indicated by the increase in the polarization of fluorescence of dansyl-CaM. Mn 2+ interferes with the interaction of the γ subunit and dansyl-CaM. Free ATP has little effect.