| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Biophys J, March 2000, p. 1306-1323, Vol. 78, No. 3
and
*Department of Biochemistry, West Virginia University School of
Medicine, Morgantown, West Virginia 26506-9142, and
Department of Medicine and the Krannert Institute of
Cardiology, Indiana University School of Medicine, Indianapolis,
Indiana 46202 USA
Kinetics studies of the cardiac Ca-ATPase expressed in
Sf21 cells (Spodoptera frugiperda insect cells) have
been carried out to test the hypotheses that phospholamban inhibits
Ca-ATPase cycling by decreasing the rate of the E1·Ca to E1'·Ca
transition and/or the rate of phosphoenzyme hydrolysis. Three sample
types were studied: Ca-ATPase expressed alone, Ca-ATPase coexpressed
with wild-type phospholamban (the natural pentameric inhibitor), and Ca-ATPase coexpressed with the L37A-phospholamban mutant (a more potent
monomeric inhibitor, in which Leu37 is replaced by Ala).
Phospholamban coupling to the Ca-ATPase was controlled using a
monoclonal antibody against phospholamban. Gel electrophoresis and
immunoblotting confirmed an equivalent ratio of Ca-ATPase and
phospholamban in each sample (1 mol Ca-ATPase to 1.5 mol
phospholamban). Steady-state ATPase activity assays at 37°C, using 5 mM MgATP, showed that the phospholamban-containing samples had nearly
equivalent maximum activity (~0.75 µmol·nmol Ca-ATPase
1·min1 at 15 µM
Ca2+), but that wild-type phospholamban and
L37A-phospholamban increased the Ca-ATPase
KCa values by 200 nM and 400 nM,
respectively. When steady-state Ca-ATPase phosphoenzyme levels were
measured at 0°C, using 1 µM MgATP, the
KCa values also shifted by 200 nM and 400 nM, respectively, similar to the results obtained by measuring ATP
hydrolysis at 37°C. Measurements of the time course of phosphoenzyme formation at 0°C, using 1 µM MgATP and 268 nM ionized
[Ca2+], indicated that L37A-phospholamban decreased the
steady-state phosphoenzyme level to a greater extent (45%) than did
wild-type phospholamban (33%), but neither wild-type nor
L37A-phospholamban had any effect on the apparent rate of phosphoenzyme
formation relative to that of Ca-ATPase expressed alone. Measurements
of inorganic phosphate (Pi) release concomitant with the
phosphoenzyme formation studies showed that L37A-phospholamban
decreased the steady-state rate of Pi release to a greater
extent (45%) than did wild-type phospholamban (33%). However,
independent measurements of Ca-ATPase dephosphorylation after the
addition of 5 mM EGTA to the phosphorylated enzyme showed that neither
wild-type phospholamban nor L37A-phospholamban had any effect on the
rate of phosphoenzyme decay relative to Ca-ATPase expressed alone.
Computer simulation of the kinetics data indicated that phospholamban
and L37A-phospholamban decreased twofold and fourfold, respectively,
the equilibrium binding of the first Ca2+ ion to the
Ca-ATPase E1 intermediate, rather than inhibiting rate of the E·Ca to
E'·Ca transition or the rate of phosphoenzyme decay. Therefore, we
conclude that phospholamban inhibits Ca-ATPase cycling by decreasing
Ca-ATPase Ca2+ binding to the E1 intermediate.
Biophys J, March 2000, p. 1306-1323, Vol. 78, No. 3
© 2000 by the Biophysical Society 0006-3495/00/03/1306/18 $2.00
This article has been cited by other articles:
 |
|
 |
|
  E. Hughes and D. A. Middleton Solid-state NMR Reveals Structural Changes in Phospholamban Accompanying the Functional Regulation of Ca2+-ATPase J. Biol. Chem., May 30, 2003; 278(23): 20835 - 20842. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. R. Jones, R. L. Cornea, and Z. Chen Close Proximity between Residue 30 of Phospholamban and Cysteine 318 of the Cardiac Ca2+ Pump Revealed by Intermolecular Thiol Cross-linking J. Biol. Chem., July 26, 2002; 277(31): 28319 - 28329. [Abstract] [Full Text] [PDF]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
