Flip-Flop of Phospholipids in Proteoliposomes Reconstituted from Detergent Extract of Chloroplast Membranes: Kinetics and Phospholipid Specificity

Eukaryotic cells are compartmentalized into distinct sub-cellular organelles by lipid bilayers, which are known to be involved in numerous cellular processes. The wide repertoire of lipids, synthesized in the biogenic membranes like the endoplasmic reticulum and bacterial cytoplasmic membranes are initially localized in the cytosolic leaflet and some of these lipids have to be translocated to the exoplasmic leaflet for membrane biogenesis and uniform growth. It is known that phospholipid (PL) translocation in biogenic membranes is mediated by specific membrane proteins which occur in a rapid, bi-directional fashion without metabolic energy requirement and with no specificity to PL head group. A recent study reported the existence of biogenic membrane flippases in plants and that the mechanism of plant membrane biogenesis was similar to that found in animals. In this study, we demonstrate for the first time ATP independent and ATP dependent flippase activity in chloroplast membranes of plants. For this, we generated proteoliposomes from Triton X-100 extract of intact chloroplast, envelope membrane and thylakoid isolated from spinach leaves and assayed for flippase activity using fluorescent labeled phospholipids. Half-life time of flipping was found to be 6±1 min. We also show that: (a) intact chloroplast and envelope membrane reconstituted proteoliposomes can flip fluorescent labeled analogs of phosphatidylcholine in ATP independent manner, (b) envelope membrane and thylakoid reconstituted proteoliposomes can flip phosphatidylglycerol in ATP dependent manner, (c) Biogenic membrane ATP independent PC flipping activity is protein mediated and (d) the kinetics of PC translocation gets affected differently upon treatment with protease and protein modifying reagents

Biochemical Evidence for Lead and Mercury Induced Transbilayer Movement of Phospholipids Mediated by Human Phospholipid Scramblase 1

Human phospholipid scramblase 1­(hPLSCR1) is a transmembrane protein involved in bidirectional scrambling of plasma membrane phospholipids during cell activation, blood coagulation, and apoptosis in response to elevated intracellular Ca²⁺ levels. Pb²⁺ and Hg²⁺ are known to cause procoagulant activation via phosphatidylserine exposure to the external surface in erythrocytes, resulting in blood coagulation. To explore its role in lead and mercury poisoning, hPLSCR1 was overexpressed in <i>Escherichia coli</i> BL21 (DE3) and purified using affinity chromatography. The biochemical assay showed rapid scrambling of phospholipids in the presence of Hg²⁺ and Pb²⁺. The binding constant (<i>K</i>_a) was calculated and found to be 250 nM^–1 and 170 nM^–1 for Hg²⁺ and Pb²⁺, respectively. The intrinsic tryptophan fluorescence and far ultraviolet circular dichroism studies revealed that Hg²⁺ and Pb²⁺ bind to hPLSCR1 and induce conformational changes. hPLSCR1 treated with protein modifying reagent N-ethylmaleimide before functional reconstitution showed 40% and 24% inhibition in the presence of Hg²⁺ and Pb²⁺, respectively. This is the first biochemical evidence to prove the above hypothesis that hPLSCR1 is activated in heavy metal poisoning, which leads to bidirectional transbilayer movement of phospholipids

Investigation of a robust pretreatment technique based on ultrasound-assisted, cost-effective ionic liquid for enhancing saccharification and bioethanol production from wheat straw

Abstract Application of cost-effective pretreatment of wheat straw is an important stage for massive bioethanol production. A new approach is aimed to enhance the pretreatment of wheat straw by using low-cost ionic liquid [TEA][HSO4] coupled with ultrasound irradiation. The pretreatment was conducted both at room temperature and at 130 °C with a high biomass loading rate of 20% and 20% wt water assisted by ultrasound at 100 W-24 kHz for 15 and 30 min. Wheat straw pretreated at 130 °C for 15 and 30 min had high delignification rates of 67.8% and 74.9%, respectively, and hemicellulose removal rates of 47.0% and 52.2%. Moreover, this pretreatment resulted in producing total reducing sugars of 24.5 and 32.1 mg/mL in enzymatic saccharification, respectively, which corresponds to saccharification yields of 67.7% and 79.8% with commercial cellulase enzyme CelluMax for 72 h. The ethanol generation rates of 38.9 and 42.0 g/L were attained for pretreated samples for 15 and 30 min, equivalent to the yields of 76.1% and 82.2% of the maximum theoretical yield following 48 h of fermentation. This demonstration provided a cheap and promising pretreatment technology in terms of efficiency and shortening the pretreatment time based on applying low-cost ionic liquid and efficient ultrasound pretreatment techniques, which facilitated the feasibility of this approach and could further develop the future of biorefinery