Cytochrome c oxidase inhibition by N-retinyl-N-retinylidene ethanolamine, a compound suspected to cause age-related macula degeneration.

The endogenous lipophilic and cationic compound N-retinyl-N-retinylidene ethanolamine (A2E) is suspected to cause age-related macula degeneration. It inhibits cytochrome c oxidase, detaches proapoptotic proteins from mitochondria, and induces apoptosis in mammalian retinal pigment epithelial cells (M. Suter, C. E. Remé, C. Grimm, A. Wenzel, M. Jäättela, P. Esser, N. Kociok, M. Leist, and C. Richter, 2000, J. Biol. Chem. 275, 39625-39630). The inhibition of cytochrome c oxidase is highly specific for A2E and is observed with the solubilized and reconstituted enzyme. In the dark, inhibition is overcome by cardiolipin or other acidic phospholipids. With illumination, inhibition is stronger, becomes complete with prolonged exposure, and is then no longer abrogated by cardiolipin. Cardiolipin effectively displaces A2E from cytochrome c oxidase, suggesting noncovalent binding of A2E to the enzyme. We conclude that A2E is a potent cytochrome c oxidase-specific inhibitor which interferes with the binding of cytochrome c to cytochrome c oxidase and, in the light, causes persistent modifications of the enzyme.

Recombinant human uncoupling protein-3 increases thermogenesis in yeast cells.

The long form of human uncoupling protein-3 (hUCP3L) is highly homologous to thermogenin (UCPI), the uncoupling protein of brown fat mitochondria, but is expressed predominantly in skeletal muscle. Its putative role is to regulate the coupling efficiency of oxidative phosphorylation and thus thermogenesis in skeletal muscle, a major thermogenic tissue in higher mammals. To study the functional relevance of hUCP3L, the protein was expressed in yeast cells under the control of the galactose promoter. Expression of hUCP3L induced a series of phenotype changes in the yeast cells. The cellular growth and the mitochondrial membrane potential were both diminished. The portion of cellular respiration coupled to oxidative phosphorylation decreased from 57% to 11% (P<0.001) and the cellular heat production, as measured by direct microcalorimetry, was increased by 33.3 +/- 3.2% (P<0.001) after induction of UCP3L. These observations demonstrate for the first time the intrinsic thermogenic properties of hUCP3L in intact cells.

Reconstitution of a voltage and calcium dependent potassium channel from rat cerebellum.

Membrane vesicles from rat cerebellum were reconstituted into lipid bilayers. The activity of two different potassium channels was recorded: (1) a small conducting voltage dependent potassium channel insensitive to [Ca2+]i, (2) a calcium and voltage dependent potassium channel (KCa). KCa channels had a conductance of (302+/-15) pS (n=5) and were activated by [Ca2+]i and membrane depolarizations. They were blocked by tetraethylamonium (TEA) and charybdotoxin (CTX) but insensitive to noxiustoxin (NTX). Finally, we showed the blocking effect of Androctonus australis Hector (AaH) scorpion venom on KCa channels from rat cerebellum.

Proteolysis of liver plectin by mu-calpain.

Rat liver plectin was found to be mainly associated with plasma membrane fractions enriched in junctional complexes. The membrane-associated plectin has been partially isolated. Plectin co-purifies with a 200 kDa polypeptide which, on the basis of sequence homology, has been identified as a myosin like-protein. The interaction of mu-calpain with liver plectin has been investigated. Plectin is very sensitive to mu-calpain and is digested to give a fragment of 240 kDa.

Phospholipids are necessary for calmodulin-stimulated activation of the Ca(2+)-ATPase of erythrocytes.

Treatment of red cell ghosts with increasing concentrations of the non-ionic detergent Triton X-100 caused a progressive loss of Ca(2+)-ATPase activity. Both the basal activity and the calmodulin-stimulated activity were affected and could be partially restored by acidic phospholipids. Lipid-free Ca(2+)-ATPase was prepared from solubilized ghosts by calmodulin affinity chromatography and extensive washing of the column with detergent to remove the endogenous phospholipids associated with the enzyme. The phospholipid-free, solubilized Ca(2+)-ATPase had very low activity and was not activated by calmodulin. The tryptic proteolytic pattern of the delipidated ATPase differed from the pattern of the phospholipid-associated enzyme, indicating that the delipidation had caused conformational changes. The activity was fully restored by phosphatidylserine, but was only partially restored by phosphatidylcholine. The phosphatidylcholine-activated enzyme was restored to maximal activity in the presence of calmodulin. The delipidated ATPase could be reconstituted in soybean lipid vesicles and was able to actively transport Ca2+.

The ionophore ETH 129 as Ca2+ translocator in artificial and natural membranes.

We have investigated the ability of the neutral ionophore ETH 129 to translocate Ca2+ across artificial and biological membranes. ETH 129 induces Ca2+ transport across planar lipid bilayer. The zero-current membrane potential in a gradient of Ca2+ concentration exhibits Nernst behavior. The dependence of the membrane conductance on ionophore and Ca2+ concentration indicates that three ionophore molecules are needed to transfer one Ca2+ across the hydrophobic region of the membrane. In mitochondria the neutral Ca2+ ionophore can move Ca2+ inside in response to a negative membrane potential under conditions in which the endogenous uniporter is blocked by ruthenium red. This electrophoretic transport of Ca2+ by ETH 129 occurs at a concentration much lower than the one previously reported with the neutral Ca2+ ionophore ETH 1001. Using sea urchin eggs, we have also shown that the efficiency of ETH 129 in inducing egg activation, as revealed by cortical granules exocytosis, is four orders of magnitude higher than that of the commonly used Ca2+ ionophore A21387. ETH 129 is a very efficient and useful tool for use in the investigation of Ca(2+)-dependent biological processes.

Partial purification and characterization of the Ca2(+)-pumping ATPase of the liver plasma membrane.

A Ca2(+)-pumping ATPase has been characterized in rat hepatocyte plasma membranes. The enzyme has high Ca2+ affinity, and properties typical of a P-type ion pump. At variance with the Ca2+ pumps of other eukaryotic plasma membranes, it is not stimulated by calmodulin. The steady state concentration of the phosphoenzyme formed in the presence of ATP is increased by La3+. The enzyme cross-reacts with a monoclonal antibody (mAb-5F10) raised against the human erythrocyte Ca2+ pump. The enzyme has been purified using a mAb-5F10 antibody affinity column. CNBr digestion of the isolated protein has yielded two peptides which have been sequenced. One of them matches perfectly a sequence contained in the erythrocyte Ca2+ pump, the other is very homologous to another domain in the erythrocyte pump. In spite of the absence of calmodulin stimulation, 125I-calmodulin overlay experiments on the purified liver ATPase under denaturing conditions have revealed that the enzyme binds calmodulin even more strongly than the erythrocyte pump. Immunocytochemical experiments on liver slices using the mAb-5F10 antibody have shown that the enzyme is located predominantly in the blood sinusoidal domain of the hepatocyte plasma membrane.

Isolation of rat liver spectrin and identification of functional domains.

Immunohistochemical studies carried out with liver sections show that spectrin is uniformly distributed along the whole plasma membrane of hepatocytes. The bilecanalicular spectrin is released during the purification of liver subplasma membrane fractions, whereas most of the basolateral spectrin remains tightly bound to the membrane. Spectrin associated with the basolateral membranes has been purified and its subunits isolated. The alpha-subunit retains the ability to bind both calmodulin and actin. Fragments have been obtained either by chemical or by proteolytical digestion of the 240 kDa alpha-subunit. Treatment with CNBr yields fragments of about 30 kDa which bind actin and calmodulin. Digestion with Staphylococcus aureus V-8 proteinase yields a calmodulin-binding fragment of 27 kDa and an actin-binding fragment of 31 kDa.

Stereospecific action of diltiazem on the mitochondrial Na-Ca exchange system and on sarcolemmal Ca-channels.

The benzothiazepine diltiazem is a potent Ca-channel blocker, which also inhibits the Na-dependent Ca-efflux from heart mitochondria. In this study, the action of the 4 stereoisomers of diltiazem has been investigated using guinea-pig heart and liver mitochondria. The rate of the Na-dependent Ca-efflux from liver mitochondria has been found to be 10 times smaller than in heart mitochondria. Otherwise, the exchange systems from the two tissues have been found to be pharmacologically indistinguishable. Both the (+)-optical isomers of the cis- and trans-forms of diltiazem inhibit Na-Ca exchange activity with comparable potency (IC50 of 10-20 microM), while the (-)-optical isomers are ineffective (IC50 greater than 200 microM). Radioligand binding experiments have revealed that only one stereoisomer of diltiazem, the (+)-cis form, interacts with high affinity with the Ca-channel receptors of guinea-pig heart sarcolemma preparations (KD = 120 nM). The results have shown that the Ca-channel of plasma membranes and the mitochondrial Na-Ca exchanger have different stereospecific requirements for the binding of diltiazem.

Identification of a fodrin-like protein in rat liver basolateral membranes.

A 240 KDa calmodulin- and actin-binding protein has been identified in the plasma membrane of rat liver. This protein is mainly associated with subplasmamembrane fractions enriched in the basolateral domain and very little of it is found in the canalicular membrane fraction. An 80 KDa actin-binding protein is found only in the canalicular fraction.

The interaction of calmodulin with rat liver plasma membrane.

Rat liver plasma membranes contain relatively high amounts of EGTA-insensitive calmodulin which seems to interact with cytoskeletal proteins. Calmodulin is particularly enriched in a subplasmamembrane fraction containing basolateral membranes. Two calmodulin-binding proteins with apparent Mr of 240 KDa and 145 KDa have been found associated with the purified plasmamembranes.

The association of calmodulin with subcellular fractions isolated from rat liver.

Calmodulin associated with rat liver mitochondria has been found to belong to a contaminant membranous fraction which contains different subcellular membranes. The concentration of calmodulin in this fraction is relatively high, about 1.6 micrograms/mg protein, and can not be decreased with EGTA. The calmodulin-rich membranous fraction seems to contain cytoskeletal proteins which could be responsible for the binding of calmodulin.

Calmodulin binding proteins in rat liver mitochondria.

Calmodulin binding proteins have been found in submitochondrial fractions obtained from highly purified rat liver mitochondria. The matrix fraction contains two major calmodulin binding proteins: one, having Mr of 145,000, apparently is carbamoyl-phosphate synthetase. Another has a Mr of 58,000 and has not been associated with enzyme activities. A major calmodulin binding protein of unknown function and having Mr of 32,000 has been found in the Triton X-100 solubilizate of the inner membrane. Minor amounts of two calmodulin binding proteins having Mr of about 37,000 and 56,000 have been found in the outer membrane.

Activity of phospholipase A2 in the inner membrane of rat-liver mitochondria.

The inner membrane of rat liver mitochondria contains a highly active phospholipase A2 which has alkaline pH optimum and requires Ca2+ in the micromolar range. The phospholipase is particularly active on the endogenous phosphatidylethanolamine and release relatively high amounts of docosahexanoic acid. The phospholipase A2 of mitochondria or mitoplasts is not dependent on calmodulin. Using fluorescamine-labelled mitoplasts there are indications that the enzyme is localized on both sides of the inner membrane.

Experiments on the mechanism of the inhibition of mitochondrial Ca2+ transport by La3+ and ruthenium red.

The effects of La3+ and ruthenium red on the energy-linked uptake of Ca2+ mediated by a synthetic neutral Ca2+ ionophore have been investigated in rat liver mitochondria. The results indicate that unspecific surface charge effects do not play a major role in the mechanism of inhibition of mitochondrial Ca2+ transport by La3+ and ruthenium red.

Characteristics of the active transport of Ca2+ by submitochondrial vesicles.

Inner membrane vesicles have been prepared by cholate treatment of rat liver mitoplasts. The vesicles can actively accumulate Ca2+ in the absence or presence of inorganic phosphate. The uptake is inhibited by ruthenium red and uncouplers of oxidative phosphorylation. Like in intact mitochondria the driving force for the uptake reaction seems to be the negative inside membrane potential generated during the oxidation of substrates. The level of antimycin-A-sensitive reduction of ferricyanide by succinate indicates that the cholate inner membrane vesicles are about 70% right side out. Using cytochrome-c-extracted inner membrane vesicles it can be shown that only those which have the same right-side-out polarity as intact mitochondria can actively accumulate Ca2+.

Ca2+ transport mediated by a synthetic neutral Ca2+ -ionophore in biological membranes.

The effect of a synthetic neutral ligand on the Ca2+ permeability of several biological membranes has been investigated. The ligand had been previously shown to possess Ca2+ -ionophoric activities in artificial phospholipid membranes. The neutral ionophore is able to transport Ca2+ across the membranes of erythrocytes and sarcoplasmic reticulum, when lipophilic anions such as tetraphenylborate and carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) are present, presumably to facilitate the diffusion of the charged Ca2+ -ionophore complex across the hydrophobic core of the membrane. In mitochondria, the neutral ionophore promotes the active transport of Ca2+ in response to the negative membrane potential generated by respiration, in the presence of the specific inhibitor of the natural carrier ruthenium red.

The translocation of Ca2+ across phospholipid bilayers induced by a synthetic neutral Ca2+ -ionophore.

The effect of a neutral synthetic Ca2+ -ligand, which induces selective Ca2+ transport in electrodialysis experiments in bulk membranes, on the Ca2+ permeability of phospholipid bilayers has been investigated. The ligand is able to promote the transport of Ca2+ across synthetic phospholipid bilayers and can therefore be classified as a Ca2+ -ionophore. Its activity is enhanced by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). The efficiency of the neutral carrier-mediated Ca2+ transport is rather low as compared with that of the charged Ca2+ -ionophore X537A. The Ca2+ selectivity of the nuetral ionophore is decreased by its incorporation in the low dielectric ambient of the phospholipid bilayer.