Kinetics studies of the cardiac Ca-ATPase expressed in Sf21 cells: New insights on Ca-ATPase regulation by phospholamban

James E. Mahaney, Joseph M. Autry, Larry Jones

Research output: Contribution to journalArticle

19 Citations (Scopus)

Abstract

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 (max)mol·nmol Ca- ATPase-1·min-1 at 15 μM Ca2+), but that wild-type phospholamban and L37A-phospholamban increased the Ca-ATPase K(Ca) 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 K(Ca) 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 (P(i)) release concomitant with the phosphoenzyme formation studies showed that L37A-phospholamban decreased the steady-state rate of P(i) 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.

Original languageEnglish
Pages (from-to)1306-1323
Number of pages18
JournalBiophysical Journal
Volume78
Issue number3
StatePublished - Mar 2000

Fingerprint

Sf9 Cells
Adenosine Triphosphatases
Adenosine Triphosphate
phospholamban
Hydrolysis

ASJC Scopus subject areas

  • Biophysics

Cite this

Kinetics studies of the cardiac Ca-ATPase expressed in Sf21 cells : New insights on Ca-ATPase regulation by phospholamban. / Mahaney, James E.; Autry, Joseph M.; Jones, Larry.

In: Biophysical Journal, Vol. 78, No. 3, 03.2000, p. 1306-1323.

Research output: Contribution to journalArticle

@article{09e91a703dde441abe3af4d0e6ec94ca,
title = "Kinetics studies of the cardiac Ca-ATPase expressed in Sf21 cells: New insights on Ca-ATPase regulation by phospholamban",
abstract = "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 (max)mol·nmol Ca- ATPase-1·min-1 at 15 μM Ca2+), but that wild-type phospholamban and L37A-phospholamban increased the Ca-ATPase K(Ca) 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 K(Ca) 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 (P(i)) release concomitant with the phosphoenzyme formation studies showed that L37A-phospholamban decreased the steady-state rate of P(i) 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.",
author = "Mahaney, {James E.} and Autry, {Joseph M.} and Larry Jones",
year = "2000",
month = "3",
language = "English",
volume = "78",
pages = "1306--1323",
journal = "Biophysical Journal",
issn = "0006-3495",
publisher = "Biophysical Society",
number = "3",

}

TY - JOUR

T1 - Kinetics studies of the cardiac Ca-ATPase expressed in Sf21 cells

T2 - New insights on Ca-ATPase regulation by phospholamban

AU - Mahaney, James E.

AU - Autry, Joseph M.

AU - Jones, Larry

PY - 2000/3

Y1 - 2000/3

N2 - 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 (max)mol·nmol Ca- ATPase-1·min-1 at 15 μM Ca2+), but that wild-type phospholamban and L37A-phospholamban increased the Ca-ATPase K(Ca) 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 K(Ca) 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 (P(i)) release concomitant with the phosphoenzyme formation studies showed that L37A-phospholamban decreased the steady-state rate of P(i) 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.

AB - 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 (max)mol·nmol Ca- ATPase-1·min-1 at 15 μM Ca2+), but that wild-type phospholamban and L37A-phospholamban increased the Ca-ATPase K(Ca) 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 K(Ca) 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 (P(i)) release concomitant with the phosphoenzyme formation studies showed that L37A-phospholamban decreased the steady-state rate of P(i) 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.

UR - http://www.scopus.com/inward/record.url?scp=0034090664&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0034090664&partnerID=8YFLogxK

M3 - Article

C2 - 10692318

AN - SCOPUS:0034090664

VL - 78

SP - 1306

EP - 1323

JO - Biophysical Journal

JF - Biophysical Journal

SN - 0006-3495

IS - 3

ER -