Activation of cardiac muscle sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) by 
beta1-agonists involves cAMP- and PKA-dependent phosphorylation of phospholamban 
(PLB), which relieves the inhibitory effects of PLB on SERCA2a. To investigate 
the mechanism of SERCA2a activation, we compared the kinetic properties of 
SERCA2a expressed with (+) and without (-) PLB in High Five insect cell 
microsomes to those of SERCA1 and SERCA2a in native skeletal and cardiac muscle 
SR. Both native SERCA1 and expressed SERCA2a without PLB exhibited high-affinity 
(10-50 microM) activation of pre-steady-state catalytic site dephosphorylation 
by ATP, steady-state accumulation of the ADP-sensitive phosphoenzyme (E1P), and 
a rapid phase of EGTA-induced phosphoenzyme (E2P) hydrolysis. In contrast, 
SERCA2a in native cardiac SR vesicles and expressed SERCA2a with PLB lacked the 
high-affinity activation by ATP and the rapid phase of E2P hydrolysis, and 
exhibited low steady-state levels of E1P. The results indicate that the kinetic 
differences in Ca2+ transport between skeletal and cardiac SR are due to the 
presence of phospholamban in cardiac SR, and not due to isoform-dependent 
differences between SERCA1 and SERCA2a. Therefore, the results are discussed in 
terms of a model in which PLB interferes with SERCA2a oligomeric interactions, 
which are important for the mechanism of Ca2+ transport in skeletal muscle 
SERCA1 [Mahaney, J. E., Thomas, D. D., and Froehlich, J. P. (2004) Biochemistry 
43, 4400-4416]. We propose that intermolecular coupling of SERCA2a molecules 
during catalytic cycling is obligatory for the changes in Ca2+ transport 
activity that accompany the relief of PLB inhibition of the cardiac SR 
Ca2+-ATPase.