Modulation of phosphorylase kinase activity by sphingolipids.

Psychosine (galactosyl sphingosine) potently inhibits the activity of both nonactivated and activated by covalent modification (autophosphorylation and limited proteolysis) rabbit skeletal muscle phosphorylase kinase. Half-maximal inhibition was observed at 44 microM or 66 microM when the kinase activity was assayed at pH 6.8 or 8.2 respectively. Sphingosine was also inhibitory, but only at pH 6.8 (half-maximal inhibition was observed at 130 microM). In this respect, sphingomyelin, cerebroside and cerebroside sulfate were ineffective. On the other hand, a number of gangliosides stimulated the activity of nonactivated phosphorylase kinase at neutral pH. Among the individual gangliosides tested the activation potency was GD1a greater than GT1b greater than GM1, while GM3 was without effect. Most important, GD1a dramatically increases the activity of the kinase at low Ca2+ concentrations. Both psychosine and GD1a increased the rate of kinase autophosphorylation on alpha- subunit only, but although ganglioside-induced stimulation of autophosphorylation was accompanied with an enhancement of the rate of autoactivation at pH 6.8, psychosine completely blocked autoactivation.

Ca2+- and Mg2+-dependent association of phosphorylase kinase with human erythrocyte membranes.

The interaction of rabbit muscle phosphorylase kinase (EC 2.7.1.38) with human erythrocyte membranes was investigated. It was found that at pH 7.0 the kinase binds to the inner face of the erythrocyte membrane (inside-out vesicles) and that this binding is Ca2+- and Mg2+-dependent. The sharpest increase in the binding reaction occurs at concentrations between 70 and 550 nM free Ca2+. Erythrocyte ghost or right-side out erythrocyte vesicles showed a significantly lower capacity to interact with phosphorylase kinase. Autophosphorylated phosphorylase kinase shows a similar Ca2+-dependent binding profile, while trypsin activation of the kinase and calmodulin decrease the original binding capacity by about 50%. Heparin (200 micrograms/ml) and high ionic strength (50 mM NaCl) almost completely blocks enzyme-membrane interaction; glycogen does not affect the interaction.

Stimulation of glycogen phosphorylase kinase by phospholipids.

The acidic phospholipids phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-biphosphate (PIP2) and the neutral phospholipid lysophosphatidylcholine (LPC) were found to stimulate (3 to 8-fold) the activity of nonactivated rabbit skeletal muscle phosphorylase kinase at pH 6.8, without significantly affecting the activity at pH 8.2. In this respect, phosphatidylcholine and phosphatidylethanolamine were ineffective, while the anionic detergent sodium dodecyl sulfate (SDS) and the anionic steroid dehydroisoandrosterone sulfate (DIAS) were able to mimic the action of phospholipids. SDS was also found to be a very efficient activator of the autophosphorylation of phosphorylase kinase (20-fold activation at 200 microM). The activating effect of phospholipids largely depends on the size of lipid vesicles, which is connected with the procedure of their preparation. These results suggest that phosphorylase kinase belongs to the class of Ca2+-dependent enzymes, which are sensitive to stimulation by calmodulin, limited proteolysis and anionic amphiphiles.

Interaction of flavonoids with rabbit muscle phosphorylase kinase.

We have examined the effect of several flavonoids on the activity of phosphorylase kinase from rabbit skeletal muscle. From 14 flavonoids tested, the flavones quercetin and fisetin were found to be efficient inhibitors of nonactivated phosphorylase kinase when assayed at pH 8.2, causing 50% inhibition at a concentration of about 50 microM, while the flavanone hesperetin stimulated phosphorylase kinase activity about 2-fold when tested at 250 microM. The efficiency of quercetin in inhibiting the kinase is higher when the enzyme is stimulated either by ethanol or by alkaline pH. Both casein and troponin phosphorylation by phosphorylase kinase and the autophosphorylation of the kinase were inhibited by quercetin. In addition, quercetin was found to be a competitive inhibitor of ATP for the phosphorylation of phosphorylase b at pH 8.2. These observations suggest that the inhibitory effect of the flavone is directly on the phosphorylase kinase molecule. Trypsin-activated phosphorylase kinase was inhibited by quercetin and stimulated by hesperetin, as for the native enzyme.