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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.

Monoclonal antibodies to rabbit skeletal muscle phosphorylase kinase were produced by the conventional hybridoma cell technique. 90 out of 600 hybridomas were found to produce phosphorylase kinase binding antibodies from which only five secreted also phosphorylase kinase activity affecting antibodies. Three of them were cloned; two hybridomas resisted all cloning efforts. Employing immunoblot technique all monoclonal antibodies show cross-reactivity with the alpha, beta, and gamma subunits of phosphorylase kinase indicating that similar, if not identical, epitopes are present on these three subunits. No cross-reactivity with delta is observed. Monoclonal antibodies secreted by two clones which bind to the alpha subunit stimulate the Ca2+-independent A0 activity of phosphorylase kinase more than 30-fold, whereas all other monoclonal antibodies obtained are ineffective in this respect. Monoclonal antibodies binding to the beta subunit inhibit the Ca2+-dependent activities significantly. Antibody produced by one hybridoma binds to the alpha, beta, and gamma subunits with approximately the same affinity. Based on the dual function of calmodulin in phosphorylase kinase (Hessová, Z., Varsányi, M., and Heilmeyer, L.M.G., Jr. (1985) Eur. J. Biochem. 146, 107-115) we conclude that binding of anti-alpha monoclonal antibodies to a regulatory domain in the alpha subunit results in an uncoupling of the inhibitory function of the Ca2+-free delta from the holoenzyme which leads to a concomitant increase in A0 activity. Furthermore, binding of anti-beta monoclonal antibodies to the beta subunit prevents a signal transfer from the Ca2+-saturated delta to the catalytic site of the holoenzyme which inhibits the Ca2+-dependent activities.