Existence of uncoupling protein‐2 antigen in isolated mitochondria from various tissues

Abstract

Antibodies against Escherichia coli-expressed uncoupling protein-2 (UCP2) and uncoupling protein-3 (UCP3) were raised by operating the blotted proteins into the spleen of minipigs. The antisera reacted more intensively with the recombinant UCP2 and UCP3 than with uncoupling protein-1 (UCP1) isolated from brown adipose tissue. Moreover, anti-UCP2 and cross-reacting anti-UCP3 antibodies identified the presence of the UCP2/3 antigen in isolated mitochondria from rat heart, rat kidney, rat brain, rabbit epididymal white adipose tissue, hamster brown adipose tissue, and rabbit skeletal muscle. It has been concluded that UCP2 is expressed in these tissues (UCP3 in skeletal muscle); however their existence in mitochondria had not previously been demonstrated.z 1999 Federation of European Biochemical Societies.

Activating ω-6 Polyunsaturated Fatty Acids and Inhibitory Purine Nucleotides Are High Affinity Ligands for Novel Mitochondrial Uncoupling Proteins UCP2 and UCP3

“…Protein extraction followed immediately. Changes in different fermentation yields were compensated by taking the same amount of yeast mitochondrial protein, usually 30 mg. 41). The cells were grown at 30°C to an OD 600 of 0.6 and then induced with 1 mM isopropyl-1-thio-␤-D-galactopyranoside at 30°C for 6 h. Cells from a 700-ml culture were lysed in a French Press in 20 ml of lysis buffer (10 mM Tris, pH 7, 1 mM EDTA, 1 mM dithiothreitol); the lysate was centrifuged at 27,000 ϫ g for 15 min, and the pellet was resuspended in 20 ml of the lysis buffer and centrifuged at 1000 ϫ g for 3 min.…”

“…Assay for 3 H-labeled Nucleotide Binding in Mitochondria-Isolation of mitochondria from rat lung, kidney, skeletal muscle, and liver was performed essentially as described elsewhere (41). Bovine serum albumin-washed mitochondria (0.2 mg of protein) were mixed with [ 3 H]GTP aliquots (8 -320 pmol and with 0.6 mM "cold" GTP in parallel series) and incubated for 30 min in 200 l of 100 mM sucrose, 20 mM Tris-HEPES, 1 mM EDTA, 2 M rotenone, 5 M CAT, pH 7.0.…”

Saccharomyces cerevisiae Lacking Btn1p Modulate Vacuolar ATPase Activity to Regulate pH Imbalance in the Vacuole

“…During later growth phases as oxidative phosphorylation takes over as the primary source of cellular ATP, less ATP is available to the V-ATPase, resulting in a decrease of the in vivo V-ATPase activity, and in btn1-⌬ releasing down-regulation V-ATPase activity. Therefore, Btn1p could be a protein involved in H ϩ -transport from the vacuole to the cytosol when the H ϩ concentration in the vacuolar lumen reaches high values in much the same way as uncoupling proteins do in mitochondria of mammalian cells (47)(48)(49)(50). These uncoupling proteins transport protons across the mitochondrial inner membrane and divert energy from ATP synthesis to thermogenesis in the mitochondria of brown adipose tissue (51,52) and are involved in decreasing the concentration of reactive oxygen species inside the mitochondria (53,54).…”

KATP Channel Openers Facilitate Glutamate Uptake by GluTs in Rat Primary Cultured Astrocytes

“…Activation of mitoK ATP channel induces K + influx, which partially restores the potential back toward basal level and enhances mitochondrial respiration by mitochondria matrix swelling, and subsequently yields a mild uncoupling (Starkov, 1997). Matrix swelling triggered by mitoK ATP channel activation changes the geometry between the inner and outer mitochondrial membranes, and thus decreases intermembrane space, inducing faster electron transport and decreases in ROS release (Starkov, 1997;Jezek et al, 2004). Thus, mitoK ATP channel acts as a potent governor to mitochondrial ROS release (Brookes et al, 2004).…”