Isolation of phospholamban and a second proteolipid component from canine cardiac sarcoplasmic reticulum

doi:10.1016/0006-291X(81)91235-3

Biochemical and Biophysical Research Communications

Volume 99, Issue 3, 15 April 1981, Pages 796-803

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Abstract

We have isolated two proteolipid fractions from canine cardiac sarcoplasmic reticulum by chromatography on columns of Sepharose CL-6B, and Sephadex LH-60. One, “fraction B”, is phosphorylated by cyclic AMP-dependent protein kinase and was identified as phospholamban, the activator of cardiac sarcoplasmic reticulum. The other, “fraction A”, is not phosphorylated and has an amino acid composition very similar to those of proteolipids we previously isolated from (Na,K)-ATPase.

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Na⁺/K⁺-ATPase from animal cell membranes is known to consist of an α-subunit and a β-subunit. Amino acids within the α-subunit have been shown to participate in the catalytic functions of the enzyme and in the binding of cardioactive steroids. Although the function of the β-subunit is not known, expression of both α- and β-subunits is required for the functional enzyme. A putative third subunit, the γ-subunit, has been suggested to be a part of the functional Na⁺/K⁺-ATPase complex, based on experiments showing that both the catalytic α-subunit and a small peptide of $M_{r} = 11 000$ can be labeled by a photoreactive ouabain analog. Although the primary structure for the putative γ-subunit from rat and sheep was recently deduced from cDNA clones, participation of this small protein in the catalytic activity of the Na⁺/K⁺-ATPase has not been demonstrated. In experiments described here, the heterologous expression of Na⁺/K⁺-ATPase in yeast cells was used to investigate whether the γ-subunit is an essential component of the Na⁺/K⁺-ATPase. Yeast cells do not contain an endogenous Na⁺/K⁺-ATPase. The α- and β-subunits or the α-, β- and the putative γ-subunits of Na⁺/K⁺-ATPase were expressed in the yeast. Saccharomyces cerevisiae and ouabain-sensitive ATPase, $p- nitrophenylphosphatase$ , and ⁸⁶Rb uptake activities were measured either in membranes prepared from transformed yeast cells, or in intact yeast cells. Nontransformed yeast cells or yeast cells transformed with the γ-subunit alone served as controls. Northern analysis and Western blots demonstrated that yeast cells do not contain an endogenous peptide with significant sequence homology to the putative γ-subunit. Yeast samples containing only Na⁺/K⁺-ATPase α and β subunits were capable of ouabain-inhibitable enzymatic activity and ⁸⁶Rb transport. No γ-subunit-dependent differences in the measured enzymatic activities or transport properties were detected in the different samples. These observations establish that the αβ-subunit complex is the minimum structural unit required for all the ouabain-sensitive reactions of Na⁺/K⁺-ATPase.
Purification of Phospholamban from Canine Cardiac Sarcoplasmic Reticulum Vesicles by Use of Sulfhydryl Group Affinity Chromatography
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This chapter focuses on the purification of phospholamban from canine cardiac sarcoplasmic reticulum vesicles by use of sulfhydryl group affinity chromatography. Phospholamban is an integral membrane protein localized to cardiac sarcoplasmic reticulum, which regulates the activity of the sarcoplasmic reticulum Ca^2÷-pump in response to phosphorylation. Phospholamban in isolated sarcoplasmic reticulum vesicles is a substrate for cAMP-dependent protein kinase, Ca²⁺/calmodulin-dependent protein kinase, and protein kinase C. Phospholamban is also readily phosphorylated in sarcoplasmic reticulum in intact hearts in response to β-adrenergic stimulation. A prerequisite for studying the protein in an isolated, reconstituted system is a method for preparing phospholamban from myocardium in reasonable yield and purity. Central to the isolation scheme is the use of sulfhydryl group affinity chromatography at the final stage of purification. The purpose of this chapter is to describe isolation method in detail. Hopefully, the availability of a highly purified phospholamban preparation will be useful in future studies directed to probe more deeply into the molecular structure and function of the protein.
Regulation of Ca<sup>2+</sup>-Pump from Cardiac Sarcoplasmic Reticulum
1988, Methods in Enzymology
This chapter extensively surveys, and summarizes the technical and procedural aspects, as well as the enzymatic properties of cardiac sarcoplasmic reticulum (SR) Ca²⁺-pump and its relationship to the putative regulator, phospholamban. Structural and functional aspects of the latter protein are thoroughly reviewed, with emphasis on its regulatory role of the Ca²⁺-pumping function by SR membranes. Cardiac SR membranes are obtained by differential centrifugation of the homogenate from canine ventricular muscle. SR membranes, largely condensed in the microsomal fraction, are found to form resealed right-side-out vesicles with a diameter of 0.1–0.2 μm. Ca²⁺-pump ATPase, representing the major protein component within cardiac SR, forms a tight complex with bilayer lipids. Like skeletal SR, cardiac ATPase protein is asymmetrically distributed across the membrane. The ATPase of the cardiac SR is an amphipathic single polypeptide whose hydrophobic region is embedded in the lipid bilayer of the SR membrane and whose hydrophilic region is exposed to the cytoplasmic surface of the SR.
Regulation of Calcium Transport in Cardiac Sarcoplasmic Reticulum
1985, Current Topics in Membranes and Transport
Cardiac sarcoplasmic reticulum (SR) is one of the major sites in which calcium transport can be regulated and thus influence the levels of intracellular calcium. This chapter describes the effect of phospholamban on kinetics of Ca²⁺ transport and Ca²⁺, Mg²⁺-ATPase; characterization and purification of Ca²⁺, Mg²⁺-ATPase; characterization of the functional association among Ca²⁺, Mg²⁺-ATPase, phospholamban, and calmodulin in cardiac sarcoplasmic reticulum (SR); and reconstitution of the Ca²⁺, Mg²⁺-ATPase with the 22,000-da protein in the absence and presence of phosphate. Phospholamban contains two sites of phosphorylations, P₁ and P₂, where P₁ is Ca²⁺-calmodulin dependent and P₂ is cyclic adenosine monophosphate (cAMP)-protein kinase dependent. When, both P₁, and P₂ of phospholamban are not occupied by the phosphate, the Ca²⁺, Mg²⁺-ATPase is uncoupled. After P₁ is occupied, the enzyme is coupled, and the normal basal pumping levels of calcium are observed. However, when P₂ is occupied, the extra stimulation of calcium transport takes place. This kind of regulation, therefore, is unique in terms of down-regulation and up-regulation through the regulatory protein. Once a membrane-bound protein is solubilized or inactivated, it is not feasible to reconstitute the protein back to the same site, even though the substance that caused the inactivation or solubilization has been removed.
Regulation of calcium transport by the ATPase-phospholamban system
1983, Journal of Molecular and Cellular Cardiology

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Isolation of phospholamban and a second proteolipid component from canine cardiac sarcoplasmic reticulum

Abstract

Biochim. Biophys. Acta

Biochim. Biophys. Acta

J. Biol. Chem

J. Biol. Chem

Biochim. Biophys. Acta

Biochem. Biophys. Res. Commun

J. Biol. Chem

J. Biol. Chem

Arch. Biochem. Biophys

Circulation