scholarly journals Purification of phospholamban, a 22,000-dalton protein from cardiac sarcoplasmic reticulum that is specifically phosphorylated by cyclic AMP-dependent protein kinase.

1982 ◽  
Vol 257 (8) ◽  
pp. 4501-4506
Author(s):  
J M Bidlack ◽  
I S Ambudkar ◽  
A E Shamoo
Keyword(s):  
Protein Kinase ◽  
Sarcoplasmic Reticulum ◽  
Cyclic Amp ◽  
Dependent Protein Kinase ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Dependent Protein

Enhanced phosphorylation of myocardial sarcoplasmic reticulum in experimental hyperthyroidism

1978 ◽  
Vol 234 (4) ◽  
pp. H426-H431
Author(s):  
C. J. Limas
Keyword(s):  
Protein Kinase ◽  
Sarcoplasmic Reticulum ◽  
Cyclic Amp ◽  
Gel Electrophoresis ◽  
Calcium Transport ◽  
Polyacrylamide Gel Electrophoresis ◽  
Dependent Protein Kinase ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Phosphoprotein Phosphatase ◽  
Dependent Protein

Calcium transport by cardiac sarcoplasmic reticulum (SR) was compared in hyperthyroid (HT) and euthyroid (ET) rats. Both Ca2+ uptake (97 +/- 3.1 nmol/mg per min in HT vs. 63 +/- 2.9 nmol/mg per min in ET, P less than 0.01) and CA2+ -stimulated ATPase activity (61 +/- 4.1 vs. 37 +/- 1.6 nmol Pi/mg per min, P less than 0.01) were higher in the thyroxine-treated animals. These changes were accompanied by enhanced cyclic AMP-dependent phosphorylation of cardiac SR in hyperthyroid rats (180 +/- 4.3 pmol Pi/mg per min vs. 117 +/- 4.2 pmol Pi/mg per min, P less than 0.01). SDS-polyacrylamide gel electrophoresis of cardiac SR showed that phosphorylation of a 22,000-dalton protein (phospholamban) primarily accounted for the differences between the two groups. There was no difference in the rate of SR dephosphorylation by endogenous phosphoprotein phosphatase between HT and ET rats. Differences in cyclic AMP-dependent phosphorylation between the two groups were blunted in the presence of excess exogenous cyclic AMP-dependent protein kinase. These results suggest that increased levels or activity of endogenous cyclic AMP-dependent protein kinases may partially explain enhanced calcium transport by the cardiac SR of hyperthyroid animals.

scholarly journals Effect of cyclic AMP-dependent phosphorylation on the reactivity of sulphydryl groups in cardiac sarcoplasmic reticulum

1980 ◽  
Vol 192 (3) ◽  
pp. 867-872 ◽  
Author(s):  
C J Limas
Keyword(s):  
Protein Kinase ◽  
Sarcoplasmic Reticulum ◽  
Cyclic Amp ◽  
Thiol Groups ◽  
Dependent Protein Kinase ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Dependent Atpase ◽  
Dependent Protein ◽  
Sulphydryl Groups ◽  
Atpase Inhibition

Phosphorylation of cardiac sarcoplasmic reticulum by cyclic AMP-dependent protein kinase results in enhanced Ca2+ transport even though Ca2+-dependent ATPase is not a substrate for the kinase. The mechanisms involved in this enhancement are not clear. In the present study, we used the reactivity of sulphydryl groups in the Ca2+-dependent ATPase as an index of conformational change during the Ca2+ transport cycle and examined the effects of protein kinase-catalysed phosphorylation. N-Ethylmaleimide alkylation allowed the distinction of several thiol groups with variable functional significance for the ATPase. A sulphydryl group involved in the formation of the phosphorylated intermediate (EP) of the Ca2+-dependent ATPase was protected by adenosine 5′-[beta, gamma-imido]triphosphate. Reactivity of an additional thiol group was related to EP dephosphorylation and was dependent on Ca2+. The Ca2+ concentration for change in the reactivity of this sulphydryl group and ATPase inhibition occurred within the range for Ca2+ binding to the high-affinity sites. Phosphorylation of cardiac sarcoplasmic reticulum by cyclic AMP-dependent protein kinase resulted in decreased N-ethyl[1-14C]-maleimide binding and the ATPase inhibition; the thiol groups involved in EP dephosphorylation were selectively protected. The results indicate that protein kinase-catalysed phosphorylation results in conformational changes of the ATPase, which renders certain thiol groups inaccessible to N-ethylmaleimide. This conformational change may facilitate functional movements involved in Ca2+ transport.

scholarly journals Anti-phospholamban and protein kinase A alter the Ca2+ sensitivity and maximum velocity of Ca2+ uptake by the cardiac sarcoplasmic reticulum

1998 ◽  
Vol 331 (1) ◽  
pp. 245-249 ◽  
Author(s):  
Margaret E. KARGACIN ◽  
Zenobia ALI ◽  
Gary J. KARGACIN
Keyword(s):  
Protein Kinase ◽  
Sarcoplasmic Reticulum ◽  
Maximum Velocity ◽  
Functional Effect ◽  
Dependent Protein Kinase ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Dependent Protein ◽  
Uptake Velocity ◽  
Phospholamban Phosphorylation ◽  
Kinase A

The activity of the SERCA2a Ca2+ pump in the sarcoplasmic reticulum (SR) of cardiac muscle is inhibited by phospholamban. When phospholamban is phosphorylated by cyclic-AMP-dependent protein kinase (PKA) this inhibition is relieved. It is generally agreed that this results in an increase in the Ca2+ sensitivity of the SR Ca2+ pump; however, some investigators have also reported an increase in the maximum velocity of the pump. We have used a sensitive fluorescence method to measure net Ca2+ uptake by native cardiac SR vesicles and compared the effects of a constitutively active subunit of PKA (cPKA) with those of a monoclonal antibody (A1) that binds to phospholamban and is thought to mimic the effect of phosphorylation. Both the Ca2+ sensitivity and the maximum velocity of uptake were increased by cPKA and by A1. The effects of cPKA and A1 on uptake velocity were only slightly additive. No changes in uptake were detected with denatured cPKA or denatured A1. These results indicate that the functional effect of phospholamban phosphorylation is to increase both the Ca2+ sensitivity and the maximum velocity of net Ca2+ uptake into the SR.

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