Purification and characterization of phospholamban from canine cardiac sarcoplasmic reticulum.

Jones, Larry R.; Simmerman, Heather K.B.; Wilson, WH; Gurd, Frank R. N.; Wegener, A. D.

doi:10.1016/s0021-9258(17)39667-9

Cited by 135 publications

(26 citation statements)

References 39 publications

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“…Thus it has been proposed that betaadrenergic stimulation of phosphorylation of the putative sarcolemmal-associated phospholamban might enhance the inotropic state of myocardial cells (7,8,26,27). However, this interpretation has been disputed on the basis of biochemical studies suggesting that the sarcolemmal-associated phospholamban is not an integral component of the sarcolemma but rather a contaminant originating from the sarcoplasmic reticuhm (9,10,22). This interpretation is further supported by the recent finding that the phospholamban content of highly purified sarcolemmal vesicles is only 8 % of that of purified sarcoplasmic reticulum vesicles when com- The entire length of network sarcoplasmic reticulum or sarcolemma was measured in each micrograph.…”

Section: Discussionmentioning

confidence: 99%

Section: Preparation and Characterization Of Affinity-purified Antibodies To Canine Cardiac Phospholambanmentioning

confidence: 99%

“…Canine cardiac phospholamban was purified according to the procedure of Jones et al (10). Rabbit antiserum to canine cardiac phospholamban was prepared and characterized as previously described by Jones et al (10). Affinity-purified rabbit antibodies to cardiac phospholamban were prepared according to the procedure of Fowler and Bennett (5) by adsorption of the rabbit antiserum to phospholamban electrophoretically transferred to nitrocellulose sheets (12).…”

Section: Preparation and Characterization Of Affinity-purified Antibodies To Canine Cardiac Phospholambanmentioning

confidence: 99%

“…The gamma globulin fraction of goat anti-rabbit serum conjugated to fluorescein (diluted 1:20; Miles-Yeder, Rehevat, Israel) was used as the secondary reagent. For preadsorption, 10 ~tg of affinity purified antibodies to canine cardiac phospholamban were incubated with either 0 or 7.5 I~g highly purified phospholamban (10) as previously described (13). The supernatants obtained by centrifugation were used in the indirect immunofluorescence-labeling assay.…”

Section: Indirect Immunofluorescence Labelingmentioning

confidence: 99%

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Immunoelectron microscopical localization of phospholamban in adult canine ventricular muscle.

Jorgensen

Jones²

1987

The Journal of Cell Biology

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Abstract. The subcellular distribution of phospholamban in adult canine ventricular myocardial cells was determined by the indirect immunogold-labeling technique. The results presented suggest that phospholamban, like the Ca2+-ATPase, is uniformly distributed in the network sarcoplasmic reticulum but absent from the junctional portion of the junctional sarcoplasmic reticulum. Unlike the Ca2÷-ATPase, but like cardiac calsequestrin, phospholamban also appears to be present in the corbular sarcoplasmic reticulum. Comparison of the relative distribution of phospholamban immunolabeling in the sarcoplasmic reticulum with that of the sarcolemma showed that the density of phospholamban in the network sarcoplasmic reticulum was •35-fold higher than that of the cytoplasmic side of the sarcolemma, which in turn was found to be threeto fourfold higher than the density of the background labeling. However, a majority of the specific phospholamban labeling within 30 nm of the cytoplasmic side of the sarcolemma was clustered and present over the sarcoplasmic reticulum in the subsarcolemmal region of the myocardial cells, suggesting that phospholamban is confined to the junctional regions between the sarcolemma and the sarcoplasmic reticulum, but absent from the nonjunctional portion of the sarcolemma. Although the resolution of the immunogoldlabeling technique used (60 nm) does not permit one to determine whether the specific labeling within 30 nm of the cytoplasmic side of the sarcolemma is associated with the sarcolemma and/or the junctional sarcoplasmic reticulum, it is likely that the low amount of labeling in this region represents phospholamban associated with sarcoplasmic reticulum. These results suggest that phospholamban is absent from the sarcolemma and confined to the sarcoplasmic reticulum in cardiac muscle.

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Section: Discussionmentioning

confidence: 99%

Section: Preparation and Characterization Of Affinity-purified Antibodies To Canine Cardiac Phospholambanmentioning

confidence: 99%

Section: Preparation and Characterization Of Affinity-purified Antibodies To Canine Cardiac Phospholambanmentioning

confidence: 99%

Section: Indirect Immunofluorescence Labelingmentioning

confidence: 99%

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Immunoelectron microscopical localization of phospholamban in adult canine ventricular muscle.

Jorgensen

Jones²

1987

The Journal of Cell Biology

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“…p HOSPHOLAMBAN (PL) ~ is a low molecular mass, integral membrane protein that has been purified from cardiac sarcoplasmic reticulum (SR) and well characterized both biochemically and functionally (5,7). In vitro phosphorylation of PL by three different kinases, at distinct sites, enhances Ca 2+ uptake by cardiac microsomes (2,9,10,14,15,17,19,20).…”

mentioning

confidence: 99%

Localization of phospholamban in smooth muscle using immunogold electron microscopy.

Ferguson

Young

Raeymaekers

et al. 1988

The Journal of Cell Biology

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Abstract. Phospholamban, the putative regulator of the Ca2+-ATPase in cardiac sarcoplasmic reticulum, was immunolocalized in canine visceral and vascular smooth muscle. Gently disrupted tissues were labeled with an afffinity-purified phospholamban polyclonal antibody and indirect immunogold, using preembedding techniques. The sarcoplasmic reticulum of smooth muscle cells was specifically labeled with patches of immunogold distributed in a nonuniform fashion, while the sarcolemma did not appear to contain any phospholamban. The outer nuclear envelopes were also observed to be heavily labeled with the affinitypurified phospholamban polyclonal antibody. These findings suggest that phospholamban may play a role in the regulation of cytoplasmic and intranuclear calcium levels in smooth muscle .ceils.

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A Structural Model of the Complex Formed by Phospholamban and the Calcium Pump of Sarcoplasmic Reticulum Obtained by Molecular Mechanics

Hutter

Krebs²,

Meiler

et al. 2002

ChemBioChem

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Phospholamban (PLN) is an intrinsic membrane protein of 52 amino acids that modulates the activity of the reticular Ca 2 ion pump. We recently solved the three-dimensional structure of chemically synthesized, unphosphorylated, monomeric PLN (C41F) by high-resolution nuclear magnetic resonance spectroscopy in chloroform/methanol. The structure is composed of two a-helical regions connected by a b turn (Type III). We used this structure and the crystallographic structure of the sarcoplasmic reticulum calcium pump (SERCA) recently determined by Toyoshima and co-workers and modeled into its E 2 form by Stokes (1KJU) or by Toyoshima (1FQU). We applied restrained and unrestrained energy optimizations and used the AMBER molecular mechanics force field to model the complex formed between PLN and the pump. The results indicate that transmembrane helix 6 (M6) of the SERCA pump is energetically favored, with respect to the other transmembrane helices, as the PLN binding partner within the membrane and is the only one of these helices that also permits contact between the N-terminal residues of PLN and the critical cytosolic binding loop region of the pump. This result is in agreement with published biochemical data and with the predictions of previous mutagenesis work on the membrane sector of the pump. The model reveals that PLN does not span the entire width of the membrane, that is, its hydrophobic C-terminal end is located near the center of the transmembrane region of the SERCA pump. The model also shows that interaction with M6 is stabilized by additional contacts made by PLN to M4. The contact between the N-terminal portion of PLN and the pump is stabilized by a number of salt and hydrogen-bond bridges, which may be abolished by phosphorylation of PLN. The contacts between the cytosolic portions of PLN and the pump are only observed in the E 2 conformation of the pump. Our model of the complex also offers a plausible structural explanation for the preference of protein kinase A for phosphorylation of Ser16 of PLN.

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Purification and characterization of phospholamban from canine cardiac sarcoplasmic reticulum.

Cited by 135 publications

References 39 publications

Immunoelectron microscopical localization of phospholamban in adult canine ventricular muscle.

Immunoelectron microscopical localization of phospholamban in adult canine ventricular muscle.

Localization of phospholamban in smooth muscle using immunogold electron microscopy.

A Structural Model of the Complex Formed by Phospholamban and the Calcium Pump of Sarcoplasmic Reticulum Obtained by Molecular Mechanics

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