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E Racker

Publications and source records attributed to E Racker.

At least 109 records · Page 6Linked to original sources
PubMed1980

From Pasteur to Mitchell: a hundred years of bioenergetics.

The discovery in 1861 by Louis Pasteur that more yeast is formed aerobically than anaerobically per gram of glucose was the first clue to the difference in efficiency of glycolysis and oxidative phosphorylation. During the first half of the 20th century the pathway of glycolysis was untraveled. Individual enzymes and cofactors were isolated and characterized. A reconstituted system of all enzymes and cofactors catalyzed steady-state glycolysis, provided an appropriate ATPase was added. The need for an ATPase, clearly demonstrated in 1945 by Otto Meyerhof, remains an important aspect of glycolysis that has been sorely neglected by textbooks. The coupling of oxidation and phosphorylation and the formation of the high-energy intermediate 1,3-diphosphoglycerate, discovered by Otto Warburg, are the key reactions of glycolysis. A high-energy intermediate formed during this process was identified as a thiolester. Early concepts of the mechanism of oxidative phosphorylation based on this model led to some frustrating and confusing years of search for high-energy intermediates. Important contributions from the laboratories of Boyer, Cohn, Chance, Green, Lardy, and Lehninger elucidated the properties of the mitochondrial process. Then Peter Mitchell proposed in 1961, 100 years after the publication by Pasteur, that the "high-energy intermediate" is an electrochemical proton gradient generated by the electron transport chain and utilized by a proton turbine (the mitochondrial ATPase complex) to generate ATP. This concept is now widely accepted. Several problems remain to be solved. How are the protons translocated during electron transport? How many protons per site? What is the mechanism of ATP generation during proton flux via the mitochondrial ATPase?

Adenosine Triphosphatases↗
PubMed1979

Ca2+ translocation in Ehrlich ascites tumor cells.

Ca2+ uptake into Ehrlich ascites tumor cells was studied at 0 degrees C in the presence of mitochondrial inhibitors, conditions that minimized complications caused by sequestration of Ca2+ into organelles or by excretion. Under these conditions Ruthenium Red inhibited Ca2+ uptake, but other previously implicated ions, such as Pi or Mg2+, had no effect. Valinomycin either inhibited or slightly stimulated Ca2+ uptake depending on the presence of excess K+ on the outside or inside of the cell, respectively. Nigericin inhibited Ca2+ transport. Based on these data we propose an electrogenic uptake of Ca2+, possibly via a Ca2+/H+ antiport mechanism. The observation that glucose inhibited Ca2+ uptake suggested that in Ehrlich ascites tumor cells an energy-driven Ca2+ expulsion mechanism is operative, similar to that in erythrocytes. Plasma membrane preparations of ascites tumor cells were found to contain a Ca2+-dependent ATPase. These preparations, when incorporated into liposomes in an inside-out orientation, catalyzed an ATP-dependent uptake of Ca2+.

Animals↗
PubMed1979

Inhibition of lactate transport and glycolysis in Ehrlich ascites tumor cells by bioflavonoids.

Bioflavonoids are potent inhibitors of lactate transport in Ehrlich ascites tumor cells. The most effective bioflavonoids have four to five hydroxyl groups. Sugar substitution at carbon three, or reduction of the double bond between carbons two and three, decreases their inhibitory activity. Quercetin, the most extensively studied of these compounds, inhibits lactate efflux by 50% at 0.1 micrograms/mg of protein. On addition of quercetin to glycolyzing Ehrlich ascites tumor cells, lactate accumulates inside the cell and the intracellular pH drops. Total lactate production is also inhibited. Nigericin prevents the internal acidification that occurs in the presence of quercetin and also reduces the inhibition of glycolysis. Thus, it appears that inhibition of lactate efflux can affect glycolysis through a lowering of the intracellular pH. The inhibitory effect of quercetin on glycolysis can be explained by its effect on lactate efflux and its previously reported effect on the Na+--K+ ATPase [Suolinna, E.--M., et al. (1974) J. Natl. Cancer Inst. 53, 1515].

Animals↗
PubMed1979

Purification and reconstitution of the Ca2+-ATPase from plasma membrane of pig erythrocytes.

The Ca2+-ATPase from plasma membranes of pig erythrocytes was purified by mixed micelle gel chromatography (Wolf, H.U., Diekvoss, G., and Lichtner, R. (1977) Acta Biol. Med. Germ. 36, 847-858). The enzyme was activated at high concentrations of Tween 20 (10 mg/ml) or by appropriate mixtures of Triton X-100 and phospholipids. It was highly unstable in the absence of Ca2+ and activator protein. The Ca2+-ATPase was incorporated into liposomes by freeze-thaw sonication. After removal of non-ionic detergent by passage through a phenyl Sepharose 4B column, the reconstituted vesicles catalyzed a rapid ATP-dependent uptake of Ca2+. Modulator protein from brain substituted for the natural activator protein and stimulated Ca2+ uptake in reconstituted vesicles.

Animals↗
PubMed1979

Purification and reconstitution of the N,N'-dicyclohexylcarbodiimide-sensitive ATPase complex from spinach chloroplasts.

The dicyclohexylcarbodiimide-sensitive ATPase from spinach chloroplast has been isolated. On sodium dodecyl sulfate gels, seven different polypeptides were seen. Five of these polypeptides coincided with the CF1 subunits, a 7,500-dalton peptide was identified as the proteolipid which interacts with [14C]dicyclohexylcarbodiimide, and there was a 15,500-dalton hydrophobic polypeptide with unknown function. In two-dimentional gels, two additional peptides were resolved, one 17,500 daltons (co-migrating in sodium dodecyl sulfate gels with subunit delta) and one 13,500 daltons (co-migrating with subunit epsilon). Reconstitution was obtained by freezing and thawing the complex with a crude mixture of phospholipids. After reconstitution the complex catalyzed 32P1-ATP exchange (rates of 200 to 400 nmoles x mg-1 x min-1) and ATP formation during acid-to-base transition. These reactions were inhibited by dicyclohexylcarbodiimide and uncouplers. Uncouplers at low concentrations stimulated and at high concentrations inhibited the Mg2+-ATPase activity. ATP hydrolysis and 32P1-ATP exchange were catalyzed by the complex in the presence of either Mg2+ or Mn2+ but not with Ca2+ or Co2+. ATP and GTP were substrates for the exchange reaction but not ADP or CTP.

Adenosine Triphosphatases↗
PubMed1979

Inhibition of the (Ca2+)ATPase from sarcoplasmic reticulum by dicyclohexylcarbodiimide: evidence for location of the Ca2+ binding site in a hydrophobic region.

Dicyclohexylcarbodiimide (DCCD) inhibits the (Ca2+)ATPase, Ca2+ uptake by sarcoplasmic reticulum vesicles and Ca2+ binding to the (Ca2+)ATPase from sarcoplasmic reticulum. Ca2+ (at micron concentrations) specifically protects against DCCD inhibition. The inhibition can, therefore, be readily demonstrated only in the presence of Ca2+ chelating agents such as EGTA. In the presence of EGTA, the ionophore A-23187 increased the sensitivity to DCCD. The ionophore also increased the phosphorylation of the enzyme by inorganic phosphate in the presence of Mg2+. These results indicate that tightly bound Ca2+ is located in a hydrophobic region of the enzyme which is not accessible to EGTA. Complete inhibition of the (Ca2+)ATPase is accompanied by binding of 4--5 nmol of [14C]DCCD per mg of ATPase protein in the absence of Ca2+ compared with 2 nmol bound per mg in the presence of Ca2+ with no ATPase inhibition. Assuming a molecular weight of 100 000 for the ATPase monomer, about 1 nmol of DCCD inhibits 4 nmol of ATPase. This result suggests that the minimal functional unit of the enzyme is a tetramer. Following trypsin digestion of the [14C]DCCD-labeled ATPase most of the radioactivity appears in the 20 000-dalton fragment. We propose that DCCD reacts with the Ca2+-binding site of the ATPase.

Animals↗
PubMed1979

Components and mechanism of action of ATP-driven proton pumps.

We have studied the composition of ATP-driven proton pumps from bovine heart mitochondria and have reconstituted the oligomycin-sensitive ATPase complex from its individual components. The complex contains 9 to 10 subunits of which 5 are assembled in the soluble F1 protein, 2 are required for the attachment of F1 to the membrane and 2 form the proton channel within the membrane. With the help of information obtained from studies of the chloroplast and the bacterial proton pumps, we can tentatively assign a function to each of the subunits of the pump. The position of F1 outside of the membrane seen in electron micrographs of negatively stained preparations, does not appear to be an artifact. Evidence from immunological studies, chemical derivatizations as well as further electron microscopy (positive staining and freeze-etching), support this statement. We describe in this paper a 28 000-dalton polypeptide which has been isolated from the mitochondria membrane and is required for the reconstitution of oligomycin-sensitive ATPase and 32Pi-ATP exchange activity. We propose a mechanism of action of the proton pump in which the key energy-yielding reaction is the binding of Mg2+ to the protein. The function of the proton gradient is to displace Mg2+ from this site to permit cyclic repetition of the binding process. Essential for this scheme is the cyclic opening and closing of the proton channel. We have outlined our present approaches to test this hypothesis.

Adenosine Triphosphatases↗
PubMed1979

Partial purification and characterization of the phosphate transporter from bovine heart mitochondria.

A highly active phosphate transporter was extracted with octylglucoside from bovine heart submitochondrial particles that were first partially depleted of other membrane components. It was then partially purified by ammonium sulfate fractionation. After reconstitution of the transporter into liposomes prepared with a crude mixture of soybean phospholipids, the Pi/OH exchange, but not the Pi/Pi exchange, was stimulated three- to fourfold by valinomycin and nigericin in the presence of K+. Both Pi/OH and Pi/Pi exchange activities were sensitive to mercurials and other SH reagents. The rutamycin-sensitive ATPase complex from mitochondria was reconstituted together with the phosphate transporter and adenine nucleotide transporter into liposomes. After inhibition of externally located ATPase, the hydrolysis of ATP was sensitive to atractyloside and mersalyl.

Adenosine Triphosphatases↗