PubMed Health⌕ Search

Biomedical subjects

E Racker

Publications and source records attributed to E Racker.

At least 127 records · Page 7Linked to original sources
PubMed1979

A new method of preparing Ca2+-ATPase from sarcoplasmic reticulum: extraction with octylglucoside.

A fast method for preparing Ca2+-ATPase from rabbit muscle sarcoplasmic reticulum was devised. The method involves extracting extrinsic membrane proteins with the non-ionic detergent octylglucoside at high salt concentration. A Ca2+-ATPase of consistently high specific activity (about 25 mumoles/mg.min) is found in the insoluble residue. The method was optimized with respect to the concentrations of detergent and salt, pH, and other extraction conditions. By the criteria of the protein pattern in SDS-polyacrylamide gel electrophoresis, dependence of the hydrolytic activity on the presence of Ca2+, and the phosphoprotein formation, the preparation is identical with the Ca2+-ATPase isolated previously by MacLennan [10] and other authors. The main advantages of the new method are its rapidity, its reliability, and the high specific activity of the purified enzyme.

Animals↗
PubMed1979

Studies on the incorporation of the sodium channel of lobster nerve into soybean liposomes.

Na+ channels of lobster nerve membrane were incorporated into soybean liposomes by the freeze-thaw-sonication procedure. Electron microscopy showed that the vesicular pattern of membranes and liposomes was broken and reassembled during the process. The amount of membrane protein incorporated depended linearly on the amount of membrane protein added to the liposomes for reconstitution. The 22Na flux into the reconstituted vesicles was increased by veratridine (0.5 mM) or grayanotoxin I (150 microM) and the increment was abolished by tetrodotoxin (Ki = 4 to 5 nM). The drug-sensitive 22Na influx depended linearly on the amount of membrane protein incorporated. No response to the drugs was found in protein-free vesicles or in vesicles reconstituted with membrane heated at 50 degrees C for 15 min. A fraction of digitonin-treated membranes had a higher Na+ channel activity. The Na+ channel was able to discriminate between Na+ and K+ or Rb+.

Animals↗
PubMed1978

The mechanism of lactate transport in human erythrocytes.

Lactate accumulates in human erythrocytes stored at 4 degrees C in the presence of glucose. Efflux of lactate exhibits an activation energy of 22kcal/mole and is markedly stimulated with increasing medium pH. Lactate influx into erythrocytes that were depleted of intracellular lactate by incubation at 37 degrees at pH 8.0 was stimulated by decreasing medium pH. Under appropriate conditions the pH-dependent lactate flux was insensitive to 4-acet-amido-4'-isothiocyano-2,2'-disulfonic stilbene or 4,4'-diisothiocyano-2,2'-disulfonic stilbene, inhibitors of the inorganic anion channel, while, e.g., inorganic phosphate transport was fully sensitive. These experiments as well as measurements of H+ movements associated with lactate fluxes demonstrate that lactate transport takes place via a specific monocarboxylate transporter (distinct from the inorganic ion channel) by a H+-lactate symport mechanism.

Biological Transport, Active↗
PubMed1978

Reconstitution of carbamylcholine-dependent sodium ion flux and desensitization of the acetylcholine receptor from Torpedo californica.

Membranes rich in acetylcholine receptor were isolated from Torpedo californica by a modification of the procedure of Sobel et al. (Sobel, A., Weber, M., and Changeux, J.-P. (1977) Eur. J. Biochem. 80, 215-224). The receptor was extracted with 2% potassium cholate in the presence of 2.5% soybean phospholipids. After reconstitution by the cholate dialysis procedure, the vesicles exhibited a rapid, carbamylcholine-dependent uptake of 22Na+, which was inhibited by alpha-bungarotoxin and several other known inhibitors. At concentrations above 5 x 10(-5) M carbamylcholine, the fast phase of Na+ influx lasted less than 10 sec. At 5 x 10(-6)M, it lasted 30 sec but was only about 50% of the maximal total uptake observed at optimal agonist concentration. The phenomenon of desensitization was exhibited by the reconstituted vesicles. When 2 x 10(-4)M carbamylcholine was added, 15 sec before 22Na+, the rapid Na+ influx phase was no longer observed.

Acetylcholine↗
PubMed1977

Formation of ATP by the adenosine triphosphatase complex from spinach chloroplasts reconstituted together with bacteriorhodopsin.

The energy-linked ATPase complex has been isolated from spinach chloroplasts. This protein complex contained all the subunits of the chloroplast coupling factor (CF1) as well as several hydrophobic compoenents. When the activated complex was reconstituted with added soybean phospholipids, it catalyzed the exchange of radioactive inorganic phosphate with ATP. Sonication of the complex into proteoliposomes together with bacteriorhodopsin yield vesicles that catalyzed light-dependent ATP formation. Both the 32Pi-ATP exchange reactions and ATP formation were sensitive to uncouplers such as 3-tert-butyl-5,2'-dichloro-4'-nitrosalicylanilide, bis-(hexafluoroacetonyl)acetone and carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone, that act to dissipate a proton gradient. The energy transfer inhibitors dicyclohexylcarbodiimide, triphenyltin chloride and 2-beta-D-glucopyranosyl-4,6'-dihydroxydihydrochalcone were also effective inhibitors of both reactions.

Adenosine Triphosphatases↗
PubMed1977

Selective incorporation of membrane proteins into proteoliposomes of different compositions.

1. Cytochrome oxidase was incorporated into preformed liposomes containing phosphatidylserine. When confronted with a mixture of liposomes, some containing phosphatidylserine and some without it, the enzyme was incorporated only into the phosphatidylserine-containing liposomes. 2. The hydrophobic proteins of the oligomycin-sensitive ATPase incubated in the presence of a mixture of liposomes with and without cytochrome oxidase were preferentially incorporated into cytochrome oxidase-containing liposomes. This selectivity was abolished by either cytochrome c or ascorbate. 3. Cytochrome oxidase incubated in the presence of a mixture of liposomes with and without the hydrophobic proteins of the ATPase was preferentially incorporated into liposomes that did not contain the hydrophobic proteins. 4. Cytochrome oxidase and the oligomycin-sensitive ATPase were preferentially incorporated into pure liposomes over bacteriorhodopsin-containing vesicles. 5. Reduced coenzyme Q (QH2)-cytochrome c reductase was incorporated randomly when incubated in the presence of a mixture of pure liposomes and liposomes containing the hydrophobic proteins of the ATPase complex. 6. The significance of the incorporation procedure as a model for membrane biogenesis is discussed.

Adenosine Triphosphatases↗
PubMed1977

Requirement of the delta subunit of chloroplast coupling factor 1 for photophosphorylation.

1. Chloroplast coupling factor (CF1) can be prepared by a rapid and simple procedure after release of the protein from the membrane by brief exposure to chloroform. It is suitable for large scale preparation of the coupling factor. 2. The protein contains five subunits (alpha, beta, gamma, delta, and epsilon in order of decreasing molecular weight) and serves as a coupling factor for photophosphorylation. However, when this preparation of CF1 was passed through a DEAE-Sephadex A-50 column, a protein with four subunits, missing the delta subunit, was obtained. The four-subunit protein did not serve as a coupling factor. 3. Photophosphorylation was restored to CF4-depleted chloroplast by addition of the four-subunit protein together with a partially purified preparation of the delta subunit. The latter subunit, when added alone, not only did not stimulate photophosphorylation but consistently diminished the residual activity. 4. The delta subunit is required for the binding of CF1 and may represent the stalk seen in electron micrographs as a link between the protein and the membrane.

Chlorophyll↗
PubMed1977

Cholesterol Stimulation of Penetration of Unilamellar Liposomes by Hydrophobic Compounds.

The incorporation of cholesterol into unilamellar liposomes greatly increased the transmembranous movement of hydrophobic ionophores such as nigericin. In reconstituted liposomes containing rhodopsin as the only protein, the presence of cholesterol lowers by 10-fold or more the amount of negericin required to eliminate the light-driven proton gradient. These effects are seen both above and below the transition temperature of the phospholipid used for reconstitution. Cholesterol similarly increases the ability of A-23187, 1799, or NH4SCN to collapse the proton gradient of bacteriorhodopsin vesicles. Cholesterol also lowers the concentration of nigericin or valinomycin required for a rapid translocation of Rb+ into protein-free liposomes. It also lowers the concentration of A-23187 required for the release of Ca45 trapped in protein-free liposomes. In contrast to these observations and in confirmation of previous findings, we observed that cholesterol decreased the permeability of liposomes for glucose. Thus the effects of cholesterol on the permeability of the membrane vary with the chemical nature of the permeating compounds. We have also confirmed that in multilamellar liposomes cholesterol decreases the permeability of Rb+ in the presence of valinomycin. It therefore appears that the effect of cholesterol changes with the size and structural features of the model membranes.

Bacteriorhodopsins↗
PubMed1977

Reversible inhibition of (Na+, K+) ATPase by Mg2+, adenosine triphosphate, and K+.

Adenosine triphosphate (ATP) hydrolysis catalyzed by the plasma membrane (Na+,K+)ATPase isolated from several sources was inhibited by Mg+, provided that K+ and ATP were also present. Phosphorylation of the adenosine triphosphatase (ATPase) by ATP and by inorganic phosphate was also inhibited, as was p-nitrophenyl phosphatase activity. (Ethylenedinitrilo)tetraacetic acid (EDTA) and catecholamines protected from and reversed the inhibition of ATP hydrolysis by Mg2+, K+ and ATP. EDTA was protected by chelation of Mg2+ but catecholamines acted by some other mechanism. The specificities of various nucleotides as inhibitors (in conjunction with Mg2+ and K+) and as substrates for the (Na+, K+) ATPase were strikingly different. ATP, ADP, beta,gamma-CH2-ATP and alpha,beta-CH2-ADP were active as inhibitors, whereas inosine, cytidine, uridine, and guanosine triphosphates (ITP, CTP, UTP, and GTP) and adenosine monophosphate (AMP) were not. On the other hand, ATP and CTP were substrates and beta,gamma-NH-ATP was a competitive inhibitor of ATP hydrolysis, but not an inhibitor in conjunction with Mg2+ and K+. The Ca2+-ATPase from sarcoplasmic reticulum and F1, the Mg2+-ATPase from the inner mitochondrial membrane, were also inhibited by Mg2+. Catecholamines reversed inhibition of the Ca2+-ATPase, but not that of F1.

Adenosine Diphosphate↗
PubMed1977

Perspectives and limitations of resolutions-reconstitution experiments.

Reconstitutions of membranous activities can tell us how many components are required and what their functions are. The mitochondrial proton pump is used as an example. Moreover, the biological activity, such as Pi transport, can be used in reconstituted vesicles as an assay during the isolation of the transporter. Reconstitution experiments reveal the importance of membrane asymmetry and allow us to study conditions of vectorial assembly. The mechanism of action of ion pumps has been successfully analyzed in reconstituted liposomes. We can study the movement of ions and the electrogenicity of the system without interference by other unrelated processes. Based on studies with the resolved Ca2+-ATPase of sarcoplasmic reticulum, we propose a novel formulation of the mechanism of ATP-driven ion pumps in which cyclic binding of Mg2+ plays a key role.

Adenosine Triphosphatases↗