Dual function of calmodulin (delta) in phosphorylase kinase.
The Ca2+-independent activity of fast skeletal muscle phosphorylase kinase, A0, can be reversibly stimulated by heparin more than 20-fold; concomitantly the Ca2+-dependent A2 activity is abolished completely. Heparin also drastically changes the aggregation state of the enzyme; aggregated species contain significantly less delta and show an about fivefold higher A0 activity than the tetrameric form containing delta stoichiometrically. We interpret this to mean that delta has two functions in the phosphorylase kinase: an inhibitory one with respect to A0 and an activating one with respect to A2. The inhibition of A0 by Ca2+-free delta is released, i.e. A0 increases when this subunit dissociates from the holoenzyme. The maximally heparin-stimulated A0 activity, A0,hep, is enriched from a crude extract to the same degree and approximately with the same yield as the major activity, A2. The phosphorylase kinase is not eluted from DEAE-cellulose as a symmetrical bell-shaped protein peak. The peak fraction contains the activities A2 and A0,hep superimposed and yields a nearly homogeneous sedimentation boundary with an S20,w value of 25.5 S. The A0 yields a much broader eluation profile showing a distinct maximum from the A2 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 A2/A0 decreases about sevenfold whereas the ratio A2/A0,hep is constant on average. This shows that A0 is an intrinsic activity of phosphorylase kinase. The heparin-activated A0 activity or A0 itself in the presence of the phosphorylase phosphatase inhibitor, fluoride, can trigger a Ca2+-independent flash activation of phosphorylase in a protein-glycogen complex. Thus, A0 could be responsible for the conversion of phosphorylase b to a at 20 nM free Ca2+ in resting, hormone-stimulated, muscle.
D000818:Animals; D002118:Calcium / Q000502:physiology; D002147:Calmodulin / Q000502:physiology*; D002384:Catalysis; D011232:Chemical Precipitation; D002918:Chymotrypsin; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D006493:Heparin / Q000494:pharmacology; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology; D010764:Phosphorylase Kinase / Q000378:metabolism*; D010766:Phosphorylation; D011817:Rabbits; D014461:Ultracentrifugation
D002147:Calmodulin; D006493:Heparin; D010764:Phosphorylase Kinase; D002918:Chymotrypsin; D002118:Calcium