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Biomedical subjects

E Racker

Publications and source records attributed to E Racker.

At least 145 records · Page 8Linked to original sources
PubMed1976

Inhibition of (Na+, K+)adenosine triphosphatase and its partial reactions by quercetin.

The bioflavonoid, quercetin, inhibited the (Na+, K+)adenosine triphosphatase purified from the electric organ of electric eel (Electrophorus electricus) or from lamb kidney. An analysis of its mode of action revealed that the formation of phosphoenzyme from Pi but not from ATP was inhibited. Quercetin increased the amount of ADP-sensitive phosphoenzyme (E1--P), indicating an inhibition of the conversion of E1--P to the ADP-insensitive form (E2--P). The rate of dephosphorylation of the phosphoenzyme formed from ATP was slowed by quercetin. These results suggest that quercetin inhibits the formation of E2--P from either Pi or E1-P as well as the hydrolysis of the phosphoenzyme. Its mode of action is therefore different from that of ouabain and other inhibitors of the Na+, K+)adenosine triphosphatase.

Adenosine Triphosphatases↗
PubMed1976

Calorimetric studies of the interaction of magnesium and phosphate with Na+, K+) ATPase: evidence for a ligand-induced conformational change in the enzyme.

The phosphorylation of (Na+, K+)ATPase from the electric organ of the electric eel is dependent on Mg2+. The amount of phosphoenzyme formed was increased by K+ and decreased by Na+. Kinetic analyses indicate that a ternary complex of ATPase, Pi and Mg2+ is formed prior to phosphorylation of the protein. Calorimetric studies revealed extraordinarily large enthalpy changes associated with the binding of Mg2+ (-49 kcal/mol) and of Pi (-42 kcal/mol), indicating a thermodynamically significant conformational change in the enzyme. The dissociation constant for the binding of Mg2+ and Pi derived from calorimetric measurements is in good agreement with the value obtained from the kinetic studies. These results indicate that ion binding induces a conformational change in the enzyme which is a prerequisite for phosphorylation by Pi.

Adenosine Triphosphatases↗
PubMed1976

Incorporation of mitochondrial membrane proteins into liposomes containing acidic phospholipids.

Cytochrome oxidase, QH2-cytochrome c reductase, and the oligomycin-sensitive adenosine triphosphatase were incorporated into liposomes by a new procedure which yielded unidirectional orientation of the proteins. Cytochrome oxidase was reconstituted in the mitochrondrial orientation and the adenosine triphosphatase in the submitochondrial orientation. Reconstitutions were achieved by incubating the proteins at room temperature with liposomes which contained phosphatidylcholine, phosphatidylethanolamine, and an acidic phospholipid (cardiolipin, phosphatidylinositol, or phosphatidylserine). The incorporation occurred without added detergent or sonication. This incorporation procedure may serve as a model for the insertion of proteins in vivo.

Adenosine Triphosphatases↗
PubMed1976

Phospholipid-protein interactions in the Ca2+-adenosine triphosphatase of sarcoplasmic reticulum.

Ca2+-adenosine triphosphatase from sarcoplasmic reticulum has been delipidated by gel filtration through a Sephadex G-200 column equilibrated with buffer containing cholate. The delipidated Ca2+-adenosine triphosphatase had negligible adenosine triphosphatase activity, but up to 50% of the ATPase activity was restored when the delipidated enzyme was recombined with phosphilipids. It was shown with the delipidated preparation that the phosphorylation of the enzyme by either ATP or Pi was entirely dependent on phospholipids. Among the purified phospholipids, phosphatidylcholine reactivated the adenosine triphosphatase activity better than phosphatidylethanolamine. Vesicles capable of translocating Ca2+ were reconstituted from delipidated Ca2+-adenosine triphosphatase and phosphatidylethanolamine, but not with phosphatidylcholine alone. We conclude that the firmly bound phospholipids which are purified together with the adenosine triphosphatase protein are not essential for the pump since they can be substituted by phosphatidylethanolamine isolated from soybeans.

Adenosine Triphosphatases↗
PubMed1976

Purification and properties of the proton-translocating adenosine triphosphatase complex of bovine heart mitochondria.

1. The proton-translocating adenosine triphosphatase (ATPase) of bovine heart mitochondria was highly purified by extraction of submitochondrial particles with cholate, fractionation with ammonium sulfate, and sucrose gradient centrifugation in the presence of methanol, deoxycholate, and lysolecithin. 2. The preparation had a very low content of phospholipids, respiratory components, and adenine nucleotide transporter. The ATPase activity (14 o 16 micromoles/min/mg at 30 degrees) was dependent on addition of phospholipids. The purified enzyme was reconstituted with phospholipids, coupling factor 1 (F1), and the oligomycin sensitivity-conferring protein (OSCP) yielding vesicles with highly active 32Pi-ATP exchange (up to 260 nanomoles/min/mg at 30 degrees), and a proton pump driven by ATP. Site III oxidative phosphorylation was reconstituted when purified cytochrome oxidase was included. 3. The 32Pi-ATP exchange of the reconstituted vesicles was sensitive to both rutamycin and dichylohexylcarbodiimide but the ATPase activity was sensitive to rutamycin and not to dicyclohexylcarbodiimide. 4. In sodium dodecyl sulfate-acrylamide gel scans of the complex, the subunits of F1, OSCP, and three other major bands with apparent molecular weights of 32,000, 23,000, and about 11,000 were noted. Three other minor bands with estimated molecular weights of 80,000, 70,000, and 52,000 were also detected. These bands apparently represent residual trace amounts of respiratory components. Quantitative assays of individual respiratory components revealed between 0 and 3% contamination. 5. We conclude that the rutamycin-sensitive ATPase complex functions as a reversible ATP-driven proton pump.

Adenosine Triphosphatases↗
PubMed1976

Reconstitution and characterization of the adenine nucleotide transporter derived from bovine heart mitochondria.

1. Adenine nucleotide exchange-transport was reconstituted in vesicles prepared from phospholipids and protein fractions derived from bovine heart submitochondrial particles. The transport, which was specific for ATP and ADP was measured either as ADP/ADP, ATP/ATP, or ADP/ATP exchange. The highest specific activity (370 nanomoles of ADP/ADP exchange/min/mg of protein at room temperature) was obtained with a protein fraction prepared by cholate extraction of partly resolved submitochondrial particles followed by ammonium sulfate fractionation. 2. At 200 muM external nucleotide, the exchange reactions were inhibited by low concentrations of bongkrekate, atractyloside, and palmitoyl-CoA, with Ki values of 1.8, 3.0, and 7.5 muM, respectively. The ADP/ADP nucleotide exchange was stimulated about 5-fold by 500 muM MgCl2 or MnCl2(km of 40 muM) and about 3-fold by 500 muM CaCl2(Km of 90 muM). It was optimal between pH 6.0 and 7.0 and decreased rapidly above pH 7.5. Arrhenius plots between 0 degrees and 40 degrees showed a break point at 15 degrees with soybean phospholipids and an activation energy of 29.5 kcal/mole from 0 degrees-15 degrees and 9.0 kcal/mole from 15 degrees-40 degrees. With mitochondrial phospholipids the break point was at 9 degrees and activation energies were 42.4 kcal/mole from 0 degrees-9 degrees and 7.6 kcal/mole from 9 degrees-40 degrees. 3. The phospholipid requirements for adenine nucleotide exchange were similar to those of oxidative phosphorylation. Optimal rates were observed with a phosphatidylethanolamine to phosphatidylcholine ratio of 4:1. Cardiolipin had a slight stimulatory effect. 4. The uptake of ADP into vesicles containing ATP was stimulated by KCl or by KPi as well as by hexafluoracetonylacetone, and uncoupler of oxidative phosphorylation. The uptake of ATP into vesicles containing ADP was inhibited by KCl or by KPi, but was also stimulated by hexafluoracetonylacetone. In both cases valinomycin reversed the effects of KCl, while mersalyl or N-ethylmaleimide prevented the effects of KPi. In contrast, none of these salts nor hexafluoracetonylactone affected the ADP/ADP or ATP/ATP exchange. These findings suggest that in the reconstituted system the ADP/ATP exchange is electrogenic.

Animals↗
PubMed1976

Why do tumor cells have a high aerobic glycolysis?

In Ehrlich ascites cells and several other tumors, the high aerobic glycolysis is maintained by generation of ADP and Pi by the plasma membrane Na+K+ ATPase. The high ATP activity is caused by a defective pump that operates at a low efficiency. Studies of the mechanism of action of the Na+K+ ATPase and other pump ATPases suggest several alternative mechanisms that might account for the decreased efficiency. The possibility of involvement of a proteolipid is under investigation.

Adenosine Triphosphatases↗
PubMed1976

Fusion of phospholipid vesicles reconstituted with cytochrome c oxidase and mitochondrial hydrophobic protein.

Reconstituted cytochrome oxidase liposomes were fused with liposomes reconstituted with mitochondrial hydrophobic protein, which acts as a membrane-bound uncoupler of cytochrome oxidase. Fusion was assayed by the loss of respiratory control of cytochrome oxidase as measured by the increased rate of ascorbate oxidation induced by hydrophobic protein when both proteins shared the same vesicles. Fusion was dependent on the presence of phosphatidylserine in the liposomes Ca++ in the aqueous medium. Phosphatidylcholine-phosphatidylserine liposomes required higher concentrations of phosphatidylserine and Ca++ than did phosphatidylethanolamine-phosphatidylserine liposomes. Cytochrome oxidase vesicles containing high concentrations of phosphatidylserine showed little or no respiratory control, while those with lower concentrations showed high respiratory control; respiratory control could be induced by fusing cytochrome oxidase vesicles containing high phosphatidylserine with protein-free liposomes containing low phosphatidylserine concentration. If cytochrome oxidase vesicles and hydrophobic protein vesicles were prefused separately for 15 min, they lost the ability to fuse upon being subsequently mixed together. The reconstituted vesicles had diameters of about 200 A; fusion yielded vesicles with diameters in excess of 1000 A.

Animals↗
PubMed1976

Ca++-induced fusion of proteoliposomes: dependence on transmembrane osmotic gradient.

The fusion of cytochrome oxidase liposomes with liposomes reconstituted with mitochondrial hydrophobic protein is dependent on the presence of an acidic phospholipid in the liposomes and on the addition of Ca++ions. Liposomes which have grown, by fusion, to diameters in excess of 1000 A lose the ability to fuse further, unless an osmotic gradient across the liposome membrane is established, with the internal osmotic pressure higher than the external. At a given Ca++ concentration, the extent to which this second fusion step takes place is determined by the ratio of internal to external osmolarity. Single-walled liposomes with diameters exceeding 1 mumM have been produced by this technique. The data suggest that the thermodynamic driving force for the Ca++-induced fusion is an excess surface free energy which can be supplied by membrane curvature or transmembrane osmotic gradients.

Calcium↗
PubMed1976

Ca++-induced fusion of fragmented sarcoplasmic reticulum with artificial planar bilayers.

Addition of fragmented sarcoplasmic reticulum (SR) vesicles to the aqueous phase of a black lipid membrane (BLM) causes a large increase in BLM conductance within 10 min. The conductance increase is absolutely dependent on three conditions: The presence of at least 0.5 mM Ca++, an acidic phospholipid such as phosphatidylserine or diphosphatidylglycerol in the BLM phospholipid mixture, and an osmotic gradient across the SR vesicle membrane, with the internal osmolarity greater than the external. These requirements are identical to conditions under which the fusion of phospholipid vesicles occurs. When the early part of the time course of conductance rise is examined at high sensitivity, the conductance is seen to increase in discrete steps. The probability of a step increases with the concentration of Ca++ in the medium, with the fraction of acidic phospholipid in the BLM, and with the size of the osmotic gradient across the SR vesicle membrane. On the other hand, the average conductance change per step is independent of the above parameters, but varies with the type and concentration of ions present in the aqueous phase. For a given ion, the mean specific conductance per step is independent of the ion's concentration between 10 and 100 mM. The probability distribution of the step-conductances agrees well with the distribution of SR vesicle surface areas, both before and after sonication of the vesicles. The evidence indicates that SR vesicles fuse with the BLM, thereby inserting SR membrane conductance pathways into it. Each discrete conductance jump appears to be the result of the fusion of a single SR vesicle with the BLM. This technique may serve as a general method for inserting membrane vesicles into an electrically accessible system.

Animals↗
PubMed1976

Protein synthesis in dextran sulfate-treated ascites tumor cells.

Treatment of ascites tumor cells with dextran sulfate resulted in a marked inhibition of the incorporation of [14C]valine into protein in the presence of a high Na+ medium. Amino acid incorporation was restored after i.p. injection of these cells into mice or by exposure of the cells to ascites fluid in vitro. In a medium high in K+ and low in Na+, [14C]valine incorporation into protein took place in dextran treated cells. Rotenone inhibited the reaction, which could be restored by addition of both inorganic phosphate and either glucose or glucose 6-phosphate. Quercetin, an inhibitor of the Na+-K+-ATPase, markedly depressed the incorporation of [14C]valine into protein in intact sdviyrd tumor cells in a high Na+ medium. There was little or no inhibition of protein synthesis in dextran sulfate treated cells when tested in a high K+-low Na+ medium. These experiments suggest a relationship between protein synthesis and the operation of the membranous Na+-K+-ATPase.

Adenosine Triphosphatases↗
PubMed1976

Characterization of dextran sulfate-treated ascites tumor cells and their repair by ascites fluid.

Exposure of Ehrlich ascites tumor cells to the polyanion dextran sulfate altered the permeability of the plasma membrane resulting in decreased glycolysis and respiration, enhanced permeability to Rb+ and adenosine 5'-triphosphate, and increased staining by erythrosin B. The inhibition of respiration was reversed by the addition of inorganic phosphate, whereas restoration of glycolysis required both inorganic phosphate and adenosine 5'-monophosphate. The membrane lesion was repaired by injection of dextran sulfate-treated cells into mice or by in vitro exposure to a heat-stable extract from ascites fluid. Reparied cells were no longer stained by erythrosin B and glycolyzed without added adenosine 5'-monophosphate. The time course of repaire was separable into a rapid initial repair of the membrane lesion followed by a slower recovery of the normal intracellular ionic composition. Both phases were insensitive to inhibitors of protein synthesis. The repair activity of ascites fluid was heat stable and was composed of both dialyzable and nondialyzable factors. Incubation of dextran sulfate-treated cells with ascites fluid restored Rb+ uptake and abolished the adenosine 5'-triphosphate-stimulated Ca2+ uptake.

Adenosine Monophosphate↗
PubMed1975

Resolution and reconstitution of ion-transport systems.

1. Oxidative phosphorylation was reconstituted with a mitochondrial proton pump (oligomycin-sensitive ATPase) and segments of the oxidation chain (cytochrome oxidase or DPNH-Q1 reductase). A proton pump of bacteriorhodopsin substituted for the respiratory chain components, giving rise to light-induced ATP formation. 2. Since oxidative phosphorylation has thus become a special case of the problem of ion translocation in general, we have investigated and reconsituted other pumps. The reconstituted Ca++ pump of sarcoplasmic reticulum consists of two factors, the Ca++-dependent ATPase and a heat-stable coupling factor. 3. Other information obtained from reconstitution experiments include the role of asymmetry in organized membranes and the specificity of protein-phospholipid interaction. 4. Purified preparations of Ca++-ATPase catalyze the formation of ATP from Pi and ADP in a stepwise reaction stoichiometric with the enzyme and dependent on Ca++.

Adenosine Diphosphate↗
PubMed1975

Arrangement of the subunits in solubilized and membrane-bound cytochrome c oxidase from bovine heart.

The arrangement of the six cytochrome c oxidase subunits in the inner membrane of bovine heart mitochondria was investigated. The experiments were carried out in three steps. In the first step, exposed subunits were coupled to the membrane-impermeant reagent p-diazonium benzene [32S]sulfonate. In the second step, the membranes were lysed with cholate anc cytochrome c oxidase was isolated by immunoprecipitation. In the third step, the six cytochrome c oxidase subunits were separated from each other by dodecyl sulfate-acrylamide gel electrophoresis and scanned for radioactivity. Exposed subunits on the outer side of the mitochondrial inner membrane were identified by labeling intact mitochondria. Exposed subunits on the matrix side of the inner membrane were identified by labeling sonically prepared submitochondrial particles in which the matrix side of the inner membrane is exposed to the suspending medium. Since sonic irradiation leads to a rearrangement of cytochrome c oxidase in a large fraction of the resulting submitochondrial particles, an immunochemical procedure was developed for isolating particles with a low content of displaced cytochrome c oxidase. With mitochondria, subunits II, V, and VI were labeled, whereas in purified submitochondrial particles most of the label was in subunit III. The arrangement of cytochrome c oxidase in the mitochondrial inner membrane is thus transmembraneous and asymmetric; subunits II, V, and VI are situated on the outer side, subunit III is situated on the matrix side, and subunits I and IV are buried in the interior of the membrane. In a study of purified cytochrome c oxidase labeled with p-diazonium benzene [32S]sulfonate, the results were similar to those obtained with the membrane-bound enzyme. Subunits I and IV were inaccessible to the reagent, whereas the other four subunits were accessible. In contrast, all six subunits became labeled if the enzyme was dissociated with dodecyl sulfate before being exposed to the labeling reagent.

Animals↗