Ca2+-independent interaction of the gamma subunit of phosphorylase kinase with dansyl-calmodulin.
A strong Ca2+-independent interaction between the isolated, active gamma 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 gamma subunit of phosphorylase kinase was simply prepared by dialyzing the HPLC-purified, inactive gamma 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 gamma 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(-9) M, the fluorescence intensity and the fluorescence polarization of the gamma 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 gamma subunit, as indicated by the increase in the polarization of fluorescence of dansyl-CaM. Mn2+ interferes with the interaction of the gamma subunit and dansyl-CaM. Free ATP has little effect.
Yuan
C J
CJ
Department of Biochemistry and Biophysics, Iowa State University, Ames 50011.
Graves
D J
DJ
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D002118:Calcium / Q000502:physiology*; D002147:Calmodulin / Q000302:isolation & purification / Q000378:metabolism; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004533:Egtazic Acid; D004789:Enzyme Activation / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010764:Phosphorylase Kinase / Q000302:isolation & purification / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
C035684:5-dimethylaminonaphthalene-1-sulfonyl-calmodulin; D002147:Calmodulin; D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D010764:Phosphorylase Kinase; D008274:Magnesium; D002118:Calcium