Dual function of calmodulin (δ) in phosphorylase kinase

The Ca 2+ ‐independent activity of fast skeletal muscle phosphorylase kinase, A 0 , can be reversibly stimulated by heparin more than 20‐fold; concomitantly the Ca 2+ ‐dependent A 2 activity is abolished completely. Heparin also drastically changes the aggregation state of the enzyme; aggregated species contain significantly less δ and show an about fivefold higher A 0 activity than the tetrameric form containing δ stoichiometrically. We interpret this to mean that δ has two functions in the phosphorylase kinase: an inhibitory one with respect to A 0 and an activating one with respect to A 2 . The inhibition of A 0 by Ca 2+ ‐free δ is released, i.e. A 0 increases when this subunit dissociates from the holoenzyme. The maximally heparin‐stimulated A 0 activity, A 0,hep , is enriched from a crude extract to the same degree and approximately with the same yield as the major activity, A 2 . The phosphorylase kinase is not eluted from DEAE‐cellulose as a symmetrical bell‐shaped protein peak. The peak fraction contains the activities A 2 and A 0,hep , superimposed and yields a nearly homogeneous sedimentation boundary with an s 20,w value of 25.5 S. The A 0 yields a much broader eluation profile showing a distinct maximum from the A 2 activity which contains slower sedimenting species of 12.1 S, some tetrameric enzyme of 22.7 S and higher aggregated material. Over the whole profile the activity ratio A 2 /A 0 decreases about sevenfold whereas the ratio A 2 /A 0,hep is constant on average. This shows that A 0 is an intrinsic activity of phosphorylase kinase. The heparin‐activated A 0 activity or A 0 itself in the presence of the phosphorylase phosphatase inhibitor, fluoride, can trigger a Ca 2+ ‐independent flash activation of phosphorylase in a protein‐glycogen complex. Thus, A 0 could be responsible for the conversion of phosphorylase b to a at 20 nM free Ca 2+ in resting, hormone‐stimulated, muscle.