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Studies on the calcium- and phospholipid- dependent protein kinase activity from heart Allen, Bruce Gordon


Protein kinase C (PKC) exists as a family of at least 10 (β1, β2 ,y,σ, ɛ,ɛ’,ξ, ƞ, L) phospholipid-dependent protein kinase activities. The PKC family may be subdivided into two groups: group A ( α, β1, β2, y) which require free calcium ions (Ca2+) in order to become fully active; and group B (σ, ɛ,ɛ’,ξ, ƞ, L) which may not. Expression, and possibly subcellular localization, of each isoform varies with cell type. The aim of the present study was to isolate, identify, and characterize the Ca2-and phospholipid-dependent protein kinase activities from heart. In addition, the presence of specific protein substrates in cardiac junctional and free sarcoplasmic reticulum would be determined for the PKC subtype(s). Cardiac PKC was isolated by chromatography on DEAE-Sephacel, phenyl-Sepharose and poly(L lysine) Agarose columns. Estimates based upon enzyme recovery indicate the presence of 10-20 mnollmin of protein kinase C activity per gram of bovine ventricular myocardium. Hydroxylapatite column chromatography resolved the preparation into two peaks of calcium- and phospholipid-dependent protein kinase activity. By Western blot analysis, peaks 1 and 2 from the hydroxylapatite column contained PKCβ (type II) and PKCα (type III), respectively. No cross-reactivity was observed, indicating that separation was complete. PKCα, the major subtype detected, was subsequently purified to apparent homogeneity by chromatography on phosphatidylserine acrylamide. PKCβ activity could not be recovered following phosphatidylserine affinity chromatography. Phosphoamino acid analysis showed that PKCα autophosphorylated at a serine residue, whereas PKCβ autophosphorylated at both serine and threonine residues. Among the various phospholipids tested for activity, phosphatidylserine was the most effective. Both subtypes were activated further by 1-stearoyl-2-arachidonylglycerol in the presence of phosphatidylserine and calcium. Activation of both subtypes occured at free calcium concentrations of less than 1 .µM. In addition to several similarities, these two subtypes showed differences in activation and kinetic properties: PKCβ was activated by cardiolipin, 1,2- and 1,3-dioleoylglycerol, and both cis- and trans unsaturated fatty acids. PKCα was activated to a lesser degree by cardiolipin and showed no response to 1,3-dioleoylglycerol. PKCα was activated to a greater extent by 1,2-diacyiglycerols and by cis-unsaturated fatty acids. In the presence of phosphatidylserine and 1-stearoyl-2-arachidonylglycerol, PKCβ exhibited substantial activity in the presence of 1 mM ethyleneglycol-bis-(β-aminoethyl ether)-N,N,N’,N’-tetraacetic acid (EGTA) without added calcium. Activation of PKCβ and PKCα by unsaturated fatty acids was independent of phospholipid and showed a lower apparent calcium affinity than observed for activation by phosphatidylserine. These results show that cardiac protein kinase C isoforms β and ( α are functionally distinguishable and may play unique roles in the regulation of cardiac function. Protein phosphorylation is an important mechanism in the regulation of cardiac function. Phosphoproteins are associated with the sarcolemma, contractile apparatus, and sarcoplasmic reticulum (SR). The major phosphoprotein in the SR is phospholamban: a 27 kDa homopentameric proteolipid, made up of 6,080-Da subunits, which is believed to associate with and regulate the activity of the SR calcium-transport ATPase. In the present study, canine cardiac SR vesicles were separated into junctional (JSR) and free (FSR) fractions by phosphate-facilitated calcium loading followed by centrifrigation into discontinuous sucrose density gradients. Several potentially novel SR-associated polypeptides were observed in this preparation. The substrate specificities of PKCα and PKCEβ, for phosphorylation in junctional and free sarcoplasmic reticulum, were similar. In addition, similar rates for the phosphorylation of phospholamban were observed for these two isoforms. Two other proteins, enriched in the FSR, appeared to be selectively phosphorylated by PKA. The identity of these two proteins and the functional significance of this phosphorylation event are unknown.

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