Dual function of calmodulin (delta) in phosphorylase kinase

Area of Science:

• Biochemistry
• Muscle Physiology
• Enzymology

Background:

• Fast skeletal muscle phosphorylase kinase (PhK) regulates glycogen metabolism.
• The enzyme exhibits both Ca2+-dependent (A2) and Ca2+-independent (A0) activities.
• The role of the delta subunit and the A0 activity in muscle regulation remains incompletely understood.

Purpose of the Study:

• To investigate the regulation and function of the Ca2+-independent activity (A0) of fast skeletal muscle phosphorylase kinase.
• To elucidate the role of the delta subunit in modulating PhK activity.
• To determine if A0 activity is intrinsically linked to PhK and its physiological relevance.

Main Methods:

• Enzyme purification using DEAE-cellulose chromatography.
• Heparin stimulation assay to assess A0 activity.
• Ultracentrifugation to analyze enzyme aggregation states.
• Enzyme activity assays to measure A2 and A0 activities.

Main Results:

• Heparin stimulates Ca2+-independent PhK activity (A0) over 20-fold while abolishing Ca2+-dependent activity (A2).
• Heparin alters enzyme aggregation, with aggregated forms showing higher A0 activity.
• The delta subunit inhibits A0 activity and activates A2 activity; its dissociation releases A0 inhibition.
• A0 activity is an intrinsic property of PhK and can trigger Ca2+-independent activation of phosphorylase.

Conclusions:

• The delta subunit plays a dual role, inhibiting A0 and activating A2 activity.
• Heparin acts as a potent activator of the intrinsic A0 activity of PhK.
• The A0 activity of PhK may be crucial for phosphorylase activation in resting, hormone-stimulated muscle.