To study PLB (phospholamban) inhibition of the cardiac Ca(2+) pump [SERCA2a (sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase 2a)], a fusion protein (SER-20G-PLB) was engineered by tethering SERCA2a with PLB through a 20-glycine residue chain, allowing the PLB tether to either bind to or dissociate from the inhibition site on SERCA2a. When expressed in insect cells, SER-20G-PLB produced active ...

Carbon and nitrogen are the two elements that most affect plant organogenesis. In vitro, usually as part of the macronutrients. Some anecdotal evidence from the literature suggests that the ammonium (NH4+) to nitrate (NO3–) ratio may affect orchid organogenesis. In this study, to test this hypothesis, different NH4+: NO3– ratios were tested on the development of protocorm-like bodies (PLBs) of ...

We have used functional co-reconstitution of purified sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA) with phospholamban (PLB), its inhibitor in the heart, to test the hypothesis that loss-of-function (LOF) PLB mutants (PLB(M)) can compete with wild-type PLB (PLB(W)) to relieve SERCA inhibition. Co-reconstitution at varying PLB-to-SERCA ratios was conducted using synthetic PLB(W), gain-of-fun...

To investigate the effect of phosphorylation on the interactions of phospholamban (PLB) with itself and its regulatory target, SERCA, we measured FRET from CFP-SERCA or CFP-PLB to YFP-PLB in live AAV-293 cells. Phosphorylation of PLB was mimicked by mutations S16E (PKA site) or S16E/T17E (PKA+CaMKII sites). FRET increased with protein concentration up to a maximum (FRET(max)) that was taken to ...

We have studied the differential effects of phospholamban (PLB) phosphorylation states on the activity of the sarcoplasmic reticulum Ca-ATPase (SERCA). It has been shown that unphosphorylated PLB (U-PLB) inhibits SERCA and that phosphorylation of PLB at Ser-16 or Thr-17 relieves this inhibition in cardiac sarcoplasmic reticulum. However, the levels of the four phosphorylation states of PLB (U-P...

Phospholamban (PLB), a 52-amino acid integral membrane protein, regulates the Ca-ATPase (calcium pump) in cardiac sarcoplasmic reticulum through PLB phosphorylation mediated by beta-adrenergic stimulation. Based on site-directed mutagenesis and coexpression with Ca-ATPase (SERCA2a) in Sf21 insect cells or in HEK 293 cells, and on spin label detection of PLB oligomeric state in lipid bilayers, i...

Using a chemically defined reconstitution system, we performed a systematic study of key factors in the regulation of the Ca-ATPase by phospholamban (PLB). We varied both the lipid/protein and PLB/Ca-ATPase ratios, determined the effects of PLB phosphorylation, and compared the regulatory effects of several PLB mutants, as a function of Ca concentration. The reconstitution system allowed us to ...

We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to characterize the interaction between phospholamban (PLB) and the sarcoplasmic reticulum (SR) Ca-ATPase (SERCA) under conditions that relieve SERCA inhibition. Unphosphorylated PLB inhibits SERCA in cardiac SR, but inhibition is relieved by either micromolar Ca(2+) or PLB phosphorylation. In both cases, it has been pr...

Phosphorylation of phospholamban (PLB) at Ser16 (protein kinase A site) and at Thr17 [Ca2+/calmodulin kinase II (CaMKII) site] increases sarcoplasmic reticulum Ca2+ uptake and myocardial contractility and relaxation. In perfused rat hearts submitted to ischemia-reperfusion, we previously showed an ischemia-induced Ser16 phosphorylation that was dependent on beta-adrenergic stimulation and an is...

Phospholamban (PLB) is a membrane protein that regulates heart muscle relaxation rates via interactions with the sarcoplasmic reticulum Ca(2+) ATPase (SERCA). When PLB is phosphorylated or Arg9Cys (R9C) is mutated, inhibition of SERCA is relieved. (13)C and (15)N solid-state NMR spectroscopy is utilized to investigate conformational changes of PLB upon phosphorylation and R9C mutation. (13)C═O ...