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E Racker

Publications and source records attributed to E Racker.

At least 91 records · Page 5Linked to original sources
PubMed1981

Stimulation of Ca2+ uptake and protein phosphorylation in tumor cells by fibronectin.

Suspensions derived from attached HeLa cells transported 45Ca2+ considerably faster than those derived from spinner cultures grown in liquid medium. Incubation of spinner cells with fibronectin or cold-insoluble globulin in the presence of 5% calf serum at 37 degrees C for 1 to 2 h greatly increased the rate of Ca2+ flux into the cells. Suspensions of cells transformed by Rous sarcoma virus transported Ca2+ much more slowly than cell suspensions of the parent strain of normal rat kidney. Incubation of the transformed cells or Ehrlich ascites tumor cells with fibronectin increased the rate of Ca2+ uptake, while no effect was seen on Ca2+ transport by this treatment of normal kidney cells grown in tissue cultures. A 45,500-dalton protein was found to interact firmly with Ca2+ that entered into attached HeLa cells or fibronectin-treated spinner cells. This Ca2+-associated protein was detected by lithium dodecyl sulfate gel electrophoresis at 0 degrees C after 30 s of exposure to radioactive Ca2+. In tumor cells without fibronectin treatment, the radioactive band was not seen under the same conditions, even after 10 min incubation with 45Ca2+. In fibronectin-treated tumor cells, addition of Ca2+ to buffered solutions resulted in increased phosphorylation of a protein in the 45,000-dalton region. The phosphorylated protein band which appears to be associated with the cytoskeleton can be resolved by isoelectric focusing into four polypeptide chains. The relation of these observations to the cascade of protein kinases involved in the phosphorylation of the beta-subunit of the (Na+-K+)-ATPase is discussed.

Animals↗
PubMed1981

Isolation, characterization, and reconstitution of a solubilized fraction containing the hydrophobic sector of the mitochondrial proton pump.

The hydrophobic sector of the mitochondrial ATPase complex was purified by sequential extraction with cholate and octylglucoside, by further differential solubilization with guanidine and cholate in the presence of phosphatidylcholine, and by fractionation with ammonium sulfate. A polypeptide with a mass of 28,000 dalton was present in the purified hydrophobic section which was cleaved by trypsin, resulting in loss of reconstitution activity. In contrast, dicyclohexylcarbodiimide-binding proteolipid remained unimpaired after exposure to trypsin. The 32Pi-ATP exchange activity of the reconstituted ATPase complex was inhibited by p-hydroxymercuribenzoate, which reacted primarily with the 28,000-dalton protein, as monitored by acrylamide gel electrophoresis with 14C-labeled inhibitor. The function of a 22,000-dalton polypeptide and of some minor components in the region of the proteolipid remains unknown. An examination of the phospholipid requirements for reconstitution of an active complex revealed an unexpected discrepancy. With an excess of phosphatidylethanolamine, optimal reconstitution of 32Pi-ATP exchange and ATP synthesis in the presence of bacteriorhodopsin and light was achieved: at a high phosphatidylcholine:phosphatidylethanolamine ratio, the rate of ATP synthesis remained high, but the rate of 32Pi-ATP exchange dropped precipitously. A new procedure is described for the reconstitution of the ATPase complex with purified phospholipids which is stable for at least 15 days.

Adenosine Triphosphatases↗
PubMed1981

A mouse homolog to the avian sarcoma virus src protein is a member of a protein kinase cascade.

Recent work has identified a cascade of membrane bound protein kinases in Ehrlich ascites tumor cells. These enzymes, designated PKL, PKS and PKM, are present in both Ehrlich tumor and mouse brain, but the cascade is active only in the tumor tissue. We have now purified a fourth protein kinase, PKF, that is also associated with this cascade. Protein kinase F prosphorylates PKL and is phosphorylated by PKS. The position of this kinase in the cascade is as follows, where the arrows denote phosphorylation: [Formula: see text] The phosphorylation by PKF, like phosphorylation by the other kinases, is at a tyrosine residue and causes the substrate kinase (PKL) to become active. The role of the tyrosine phosphorylation in activating these kinases is described in detail elsewhere. One result of activation of the cascade is the phosphorylation of the beta subunit of the Na+K+-ATPase, which causes inefficient Na+ pumping and is at last in part responsible for the high aerobic glycolysis of Ehrlich ascites tumor cells. By several criteria protein kinase F from Ehrlich cells is homologous to the src gene product (pp60src) from avian sarcoma viruses. Antiserum raised against PKF and sera from rabbits bearing rous sarcoma virus (RSV)-induced tumors quantitatively precipitate the same 60 kd phosphoprotein from cell lysates of three different RSV-transformed cell lines. Both proteins phosphorylate PKL and a 130 kd cytoskeletal protein (vinculin). The tryptic maps of these proteins are closely similar. Both proteins bind specifically to PKL covalently coupled to Sepharose. We used this latter observation to facilitate the purification of pp60 src from RSV-transformed cells.

Amino Acids↗
PubMed1980

Calorimetric studies of ligand-induced modulation of calcium adenosine 5'-triphosphatase from sarcoplasmic reticulum.

The enthalpy change (delta H degrees ') associated with the binding of Mg2+ to the sarcoplasmic reticulum calcium adenosine 5'-triphosphatase [(Ca2+)ATPase] is -76 kcal/mol. The affinity constant for Mg2+ obtained from calorimetric measurements agrees with the Km value for Mg2+ in the phosphorylation of the enzyme by inorganic phosphate (Pi). The delta H degrees ' of binding of Pi to the enzyme is -23.5 kcal/mol, and the affinity constant for Pi obtained from the calorimetry also agrees with the Km value for Pi in the phosphorylation reaction. delta H degrees ' of Mg2+ binding is reduced to -35 kcal/mol in the presence of either 20 mM Pi or 1.2 mM Ca2+ without a significant change in the affinity of the enzyme for Mg2+. delta H degrees ' of Pi binding to the enzyme drops to -8.5 kcal/mol in the presence of 10 mM Mg2+ without a significant change in the affinity of the enzyme for Pi. On the other hand, the presence of Ca2+ does not affect the delta H degrees ' for the binding of the substrate analogue 5'-adenylyl beta,gamma-imidodiphosphate [App(NH)p], and the presence of this analogue does not affect the delta H degrees ' for Ca2+ binding. The results suggest a model in which a conformational change, largely controlled by Mg2+ binding to the enzyme, leads to the formation of the covalent phosphoprotein intermediate.

Animals↗
PubMed1980

Phosphorylation of the beta subunit of Na+K+-ATPase in Ehrlich ascites tumor by a membrane-bound protein kinase.

We have shown previously that proteoliposomes reconstituted with purified Na+K+-ATPase from Ehrlich ascites tumor cells, transport Na+ with low efficiency (Spector, M., O'Neal, S. and Racker, E. (1980) J. Biol. Chem., 255, 5504-5507). We now present evidence that this low efficiency (expressed in the ratio of Na+-transported/ATP-hydrolyzed) is caused by the phosphorylation of the beta subunit of the Na+K+-ATPase by an endogenous protein kinase. On addition of [gamma-32P]ATP, crude tumor plasma membrane preparations phosphorylated the beta subunit of the ATPase, whereas crude mouse brain plasma membranes did not. However, solubilized Na+K+-ATPase from either tumor or brain wre phosphorylated by purified protein kinase from the tumor plasma membrane and dephosphorylated by a phosphatase. In both cases, the phosphorylated enzyme was inefficient; the dephosphorylated enzyme was efficient after reconstitution into liposomes. During isolation of the Na+K+-ATPase from Ehrlich ascites tumor or mouse brain, an endogenous protease partially cleaved from the beta subunit a polypeptide of 29,000 daltons that contained the phosphorylation site. The proteolytic cleavage of the beta subunit was partially inhibited by phenylmethylsulfonyl fluoride and the major site of phosphorylation was then seen in the 53,000-dalton beta subunit of the enzyme. The isolated 29,000-dalton polypeptide from mouse brain ATPase was phosphorylated by tumor protein kinase with a stoichiometry of 1 mol of phosphate/mol of protein. When this 29,000-dalton polypeptide from mouse brain was incorporated into the tumor Na+K+-ATPase after mild proteolytic digestion, a marked increase in efficiency was observed after reconstitution of the Na+ pump.

Animals↗
PubMed1980

Inhibition of lactate transport in Ehrlich ascites tumor cells and human erythrocytes by a synthetic anhydride of lactic acid.

The synthesis and some of the physical and biological characteristics of a new inhibitor of lactate transport are described. The inhibitor is isobutylcarbonyl lactayl anhydride (iBCLA). It is formed by the condensation of lactic acid and isobutylchloroformate. It inhibits lactate transport 50% at 0.5 microgram/mg of protein in both Ehrlich ascites tumor cells and human erythrocytes. In contrast, 15 microgram of iBCLA/mg of protein is required for 50% inhibition of phosphate transport in erythrocytes, and phosphate transport in Ehrlich ascites tumor cells is unaffected at levels as high as 50 microgram of iBCLA/mg of protein. A time-dependent and concentration-dependent reversal of lactate transport inhibition took place on exposure of iBCLA-treated Ehrlich ascites cells to hydroxylamine or dithiothreitol. These data, along with the observed sensitivity of the lactate transporter to sulfhydryl reagents [Spencer, T. L., & Lehninger, A. L. (1976) Biochem. J. 154, 405-414], suggest that iBCLA acylates an essential sulfhydryl group on the transporter. When glycolyzing Ehrlich ascites tumor cells were treated with concentrations of iBCLA sufficient for complete inhibition of lactate transport, intracellular lactate levels increased, intracellular pH and extra-cellular lactate levels decreased, and overall lactate production was inhibited.

Anhydrides↗
PubMed1980

Reconstitution of the Na+K+ pump of Ehrlich ascites tumor and enhancement of efficiency by quercetin.

Plasma membranes from Ehrlich ascites tumor cells were solubilized by octylglucoside in the presence of phospholipids. The Na+K+-ATPase was purified from this extract by adsorption and elution from thio-Seph-arose 4B. The enzyme (specific activity, 7 mumoles of ATP hydrolyzed min-1 mg of protein -1) was reconstituted into liposomes by the octyglucoside dilution procedure. An ATP-dependent Na+ influx with low efficiency was observed. On addition of appropriate amounts of quercetin, the Na+ flux/ATP hydrolysis ratio was increased from 0.4 to 1.4.

Animals↗
PubMed1980

Solubilization and partial purification of the Ca2+/Na+ antiporter from the plasma membrane of bovine heart.

Vesicles derived from the plasma membrane of bovine heart muscle catalyze an uptake of 45Ca+ which is dependent on Na+ inside the vesicles and inhibited by external Na+ (Ki = 14 mM). The transporter was solubilized with 2% cholate at high ionic strength (0.5 M NaCl) in the presence of 2.4% soybean phospholipids. Appropriate dilution and sedimentation yielded a preparation of the transporter which could be reconstituted into liposomes. The proteoliposomes catalyzed Ca2+ uptake with a specific activity about 5 times higher than native vesicles. External Na+ inhibited Ca2+ uptake with a Ki of 50 mM. The uptake of Ca2+ was also inhibited when the Na+ gradient was collapsed with nigericin. Ca2+ uptake was stimulated when either valinomycin or dinitrophenol was added to the assay, in line with the known electrogenic nature of the transport system.

Animals↗
PubMed1980

Endogenous and exogenous proteolysis of the acetylcholine receptor from Torpedo californica.

Purified acetylcholine receptor reconstituted into liposomes catalyzes carbamylcholine-dependent ion flux [10]. An endogenous protease activated by Ca2+ gives rise to an acrylamide gel pattern of the receptor with the 40,000-dalton subunit apparently as the major component. Exogenous proteases nick the proteins so extensively that the acrylamide gel pattern reveals polypeptides of 20,000 daltons or less. In either case the receptor sediments at 9S, indicating that the polypeptide chains remain associated. Moreover, the nicked receptors bind alpha-bungarotoxin and catalyze carbamylcholine-dependent ion flux after reconstitution.

Animals↗
PubMed1980

A Light-dependent Protein Kinase Activity of Chloroplasts.

A protein kinase activity from spinach chloroplasts, tightly associated with the thylakoid membranes, has been solubilized and partially characterized. This membrane-bound protein kinase is stimulated by light and electron transport activity through photosystem II appears to be required for stimulation.Electron transport inhibitors like 3,4-dichlorophenyl-1,1-dimethylurea, Tris, and NH(2)OH treatments, inhibit the light activation process. Furthermore, after Tris inhibition, the protein kinase activity is restored by washing the Tris-treated chloroplasts with dichlorophenol indophenol plus ascorbate. The protein kinase remains active in the dark after short illumination periods, suggesting that a product of electron transport may be involved in light activation.Two endogenous substrates of the protein kinase in thylakoid membranes are the N,N'-dicyclohexylcarbodiimide-reactive proteolipid and the light-harvesting chlorophyll-protein complex. The membrane-bound protein kinase also phosphorylates externally added histone.

Journal Article↗