Biomedical subjects

E Racker

Publications and source records attributed to E Racker.

At least 55 records · Page 3Linked to original sources

PubMed1986

Induction of anchorage-independent growth by epidermal growth factor and altered sensitivity to type beta transforming growth factor in partially transformed rat kidney cells.

A partially transformed cell line (NRK-PT14) was isolated from normal rat kidney (NRK) cells. Like NRK cells, NRK-PT14 cells required epidermal growth factor for anchorage-independent growth, but lost the additional requirement for exogenous type beta transforming growth factor (TGF-beta). Compared to NRK cells, NRK-PT14 cells did not secrete elevated levels of TGF-beta, but exhibited an altered response to this growth factor. Monolayer growth of NRK cells in a serum-free medium was inhibited by TGF-beta, whereas growth of NRK-PT14 cells was stimulated by TGF-beta. In addition, TGF-beta stimulated epidermal growth factor binding to high affinity sites in NRK cells, but decreased epidermal growth factor binding to NRK-PT14 cells during growth of the cells in serum-free medium. These qualitative changes in the response to TGF-beta may be representative of an intermediate stage in the spontaneous transformation of NRK cells.

Animals↗

PubMed1986

Effect of growth factors and methionine on glycolysis and methionine transport in rat fibroblasts and fibroblasts transfected with myc and ras genes.

The inhibition of glycolysis in tumor cells by methionine requires that the cells be incubated with methionine for several hours in the presence of serum. We now show that in the case of confluent rat-1 fibroblasts transfected with the ras gene the serum can be substituted by insulin and insulin-like growth factor I or II. No other growth factor tested was effective. In subconfluent ras cells additional growth factors (transferrin and high density lipoproteins) were required for maximal inhibition of glycolysis by methionine. Exploration of the mechanism of action of methionine revealed that the accumulation of [35S]methionine into rat-1 fibroblasts was only marginally increased by insulin. We propose that methionine inhibits an adenosine triphosphatase activity because addition of low concentrations of Nonidet P-40 greatly enhanced glycolysis even in the presence of methionine, suggesting that it did not affect the glycolytic enzymes directly. Methionine also affected growth both in monolayer and soft agar. Rat-1 fibroblasts transfected with the ras gene were markedly more sensitive to methionine than cells transfected with the myc gene.

Animals↗

PubMed1985

Interaction of duramycin with artificial and natural membranes.

Duramycin is a polypeptide antibiotic (molecular weight 2012) obtained from culture filtrates of Streptomyces cinnamomeus forma azacoluta. In this work, we show that low concentrations of duramycin induced aggregation of lipid vesicles containing unsaturated phosphatidylethanolamine and unsaturated monogalactosyl diglyceride, and of sarcoplasmic reticulum vesicles from rabbit skeletal muscle. Furthermore, duramycin inhibited the ATP-dependent Ca2+ uptake in sarcoplasmic reticulum vesicles without affecting the hydrolysis of ATP or the permeability of Ca2+. Also, duramycin only inhibited the bacteriorhodopsin proton pump reconstituted into phospholipid vesicles containing phosphatidylethanolamine. We have isolated a duramycin-resistant strain of Bacillus subtilis and have mapped the location of duramycin resistance. In this strain, the secretion of protons and influx of calcium were resistant to duramycin, and its lipid composition was profoundly different from that of the parent strain. No phosphatidylethanolamine was detected in the resistant strain. Our findings are consistent with the idea that duramycin recognizes a particular membrane conformation determined by the presence of phosphatidylethanolamine or monogalactosyl diglyceride.

Adenosine Triphosphate↗

PubMed1985

On the mechanism of glycolysis stimulation by neutral detergents in 3T3 and Ehrlich ascites tumor cells.

Glycolysis of 3T3 and Ehrlich ascites tumor cells was greatly enhanced by Nonidet P-40 or Triton X-100 at about 100 micrograms/mg cell protein. This enhanced glycolysis was partly sensitive to rutamycin and partly sensitive to ouabain, suggesting that the detergent released the control of the ATPase of the mitochondria and of the plasma membrane Na+K+-ATPase. Nonidet P-40 had no effect on glycolysis in cell-free extracts from Ehrlich ascites tumor cells to which soluble mitochondrial ATPase was added. Measuring ouabain-sensitive 22Na efflux and using ouabain-sensitive lactate production as a measure of ATP hydrolysis by the Na+K+ pump, it was shown that Nonidet P-40 greatly decreased the efficiency of the Na+K+ pump. Quercetin increased the efficiency of pumping in EAT cells both in the absence and presence of the detergent.

Adenosine Triphosphate↗

PubMed1985

Glycolysis and methylaminoisobutyrate uptake in rat-1 cells transfected with ras or myc oncogenes.

A high rate of aerobic glycolysis was catalyzed by rat-1 cells transfected with a ras oncogene (ras cells); rat-1 cells and rat-1 cells transfected with myc oncogene (myc cells) showed a low rate of glycolysis that was increased after exposure of the cells to type B transforming growth factor (TGF-beta). The uptake of radioactive methylaminoisobutyric acid or L-methionine via system A of amino acid transport also was accelerated after exposure of these cells to TGF-beta, with the myc cells being most sensitive and the ras cells least sensitive. Methionine was found to be a potent inhibitor of glycolysis in ras cells as well as in rat-1 or myc cells that were exposed to TGF-beta. We propose a relationship between the product of the ras oncogene (p21) and the protein(s) induced by exposure to TGF-beta.

Aminoisobutyric Acids↗

PubMed1985

Methionine-sensitive glycolysis in transformed cells.

Glycolysis in several tumor cell lines grown in tissue culture was inhibited by methionine. Kirsten murine sarcoma virus-transformed rat kidney cells (K-NRK) were inhibited 60-75% by 10 mM methionine, whereas normal rat kidney (NRK-49F) cells showed little or no inhibition. Inhibition of glycolysis in K-NRK cells was manifest 2-4 hr after exposure to the amino acid. Glycolysis in a chemically transformed cell line of Madin-Darby canine kidney cells was also sensitive to methionine, but maximal inhibition (75%) required 18-24 hr of incubation with the amino acid. Under the same conditions glycolysis in the nontransformed canine cells was less than 20% inhibited by methionine. In Ehrlich ascites tumor cells grown in tissue culture, 10 mM methionine inhibited glycolysis by about 50%. Inhibition of glycolysis, even by 50 mM methionine, was rapidly reversible. Within 2 hr after removal of methionine the rate of glycolytic activity was restored to that observed in control cells. Furthermore, inhibition by methionine required a minimum level (7%) of serum in the growth medium and inhibition was not sensitive to cycloheximide. Only amino acids that are transported by system A (including the nonmetabolized analogue methylaminoisobutyric acid) specifically inhibited glycolysis in tumor cells. The only exception was phenylalanine, which was toxic to both transformed and normal cell lines.

Amino Acids↗

PubMed1985

Stimulation of glycolysis and amino acid uptake in NRK-49F cells by transforming growth factor beta and epidermal growth factor.

Glycolysis in normal resting rat kidney cells (NRK-49F) was stimulated by a 2-hr exposure to transforming growth factors prior to assay. Transforming growth factor beta (TGF-beta) was effective when added alone, and further addition of epidermal growth factor (EGF) had little effect. The stimulation by TGF-beta was abolished when cycloheximide was present during the incubation, suggesting that protein synthesis is required for the effect. Incubation of the cells with 25 mM methionine abolished the stimulation of glycolysis by TGF-beta. The uptake of methylaminoisobutyrate via system A was stimulated by either TGF-beta or EGF. The greater than 3-fold stimulation of uptake by 1 ng of pure TGF-beta per ml was usually somewhat enhanced on addition of 0.5 ng of EGF per ml. Moreover, an antiserum against EGF receptor partially depressed the response to TGF-beta, suggesting some overlapping interactions of EGF and TGF-beta.

Amino Acids↗

PubMed1984

Activation and partial purification of the ATPase of clathrin-coated vesicles and reconstitution of the proton pump.

A N-ethylmaleimide-sensitive ATPase was extracted and partially purified from clathrin-coated vesicles of bovine brain. During purification the enzyme lost activity which was restored by a purified phospholipid fraction from brain. Phosphatidylserine, but no other commercial phospholipids tested, replaced the brain lipid fraction as activator. Particles depleted of the ATPase exhibited no H+ pump activity when reconstituted with brain phospholipids by the cholate dilution procedure. H+ pump activity was restored by incubating the reconstituted vesicles with the partially purified ATPase.

Animals↗

PubMed1984

Inhibition of clathrin-coated vesicle acidification by duramycin.

Clathrin-coated vesicles contain a proton translocating ATPase which operates in parallel with a chloride transporter (Xie, X.-S., Stone, D.K., and Racker, E. (1983) J. Biol. Chem. 258, 14834-14838). The polypeptide antibiotic, duramycin, has a dual inhibitory effect on clathrin-coated vesicle acidification. Low amounts of duramycin (5 micrograms/100 micrograms of protein) inhibit by 50% the proton translocation facilitated by chloride translocation. Under these conditions duramycin inhibits also 36Cl uptake when driven by either the electrogenic proton pump or by inward directed K+ movement in the presence of valinomycin. Higher amounts of duramycin (20 micrograms/100 micrograms of protein) are needed to inhibit by 50% the proton pump itself, as evidenced by reduced proton translocation facilitated by an outward potassium movement in the presence of valinomycin. In addition, the amount of duramycin needed to inhibit the proton pump corresponded well with the amount needed to inhibit the ouabain-insensitive, N-ethylmaleimide-sensitive ATPase activity of clathrin-coated vesicles.

Animals↗

PubMed1984

Synthetic tyrosine polymers as substrates and inhibitors of tyrosine-specific protein kinases.

Several synthetic random polymers of tyrosine containing glutamic acid, alanine, and lysine in various proportion served as substrates for tyrosine-specific protein kinases. The Km values for these substrates were much lower than for small polypeptides such as angiotensin. For the protein kinase coded by Fujinami virus, the best substrates (with the lowest Km) were polymers containing glutamic acid, alanine, and 8 to 10% tyrosine; for the insulin receptor protein kinase, the best substrate was a polymer containing 80% glutamic acid and 20% tyrosine. These polymers serve as inexpensive and tyrosine-specific substrates that can be used even with crude extracts and analyzed by the convenient filter paper assay. Several synthetic polymers with ordered sequences were found to be potent inhibitors of these tyrosine-specific protein kinases.

Animals↗

PubMed1984

Inhibitory effect of duramycin on partial reactions catalyzed by (Na+,K+)-adenosinetriphosphatase from dog kidney.

The polypeptide antibiotic duramycin inhibited the (Na+,K+)-adenosinetriphosphatase purified from dog kidney. An analysis of its mode of action revealed that the formation of phosphoenzyme from Pi but not from ATP was inhibited. The rate of dephosphorylation of the phosphoenzyme formed from ATP was markedly reduced. In contrast to quercetin, duramycin did not inhibit K+-dependent p-nitrophenylphosphatase activity. The effect of duramycin was completely reversed by phospholipids.

Adenosine Diphosphate↗

PubMed1984

Effect of lipid composition on the calcium/adenosine 5'-triphosphate coupling ratio of the Ca2+-ATPase of sarcoplasmic reticulum.

The Ca2+-ATPase of sarcoplasmic reticulum was purified and depleted of proteolipids by solubilization in Triton X-100 and by fractionation on a DE-52 column. The protein reconstituted by deoxycholate-cholate dialysis at low lipid to protein ratios (2-5 mg of lipid/mg of protein), with either dioleoylphosphatidylethanolamine or monogalactosyldiglyceride, exhibited high initial rates of ATP-dependent Ca2+ uptake [300-900 nmol min-1 (mg of protein)-1] and coupling ratios (Ca2+ transported/ATP hydrolyzed) up to 1.2. Ca2+-ATPase reconstituted with lipids of increasing degrees of methylation (dioleoylphosphatidylethanolamine, dioleoylmonomethylphosphatidylethanolamine, dioleoyldimethylphosphatidylethanolamine and dioleoylphosphatidylcholine) or increasing degrees of glycosylation (monogalactosyldiglyceride and digalactosyldiglyceride) revealed a progressive decrease in both ATP-dependent Ca2+-uptake and coupling ratios. The rate and extent of Ca2+ uptake decreased as the dioleoylphosphatidylethanolamine/dioleoylphosphatidylcholine or monogalactosyldiglyceride/dioleoylphosphatidylcholine molar ratios in the reconstituted vesicles were reduced. Vesicles reconstituted with high molar ratios of dioleoylphosphatidylethanolamine/dioleoylphosphatidylcholine or monogalactosyldiglyceride/dioleoylphosphatidylcholine and at a high lipid to protein ratio became leaky and released the Ca2+ accumulated inside the vesicles when the temperature of the incubation mixture was increased (e.g., from 20 to 37 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗

PubMed1984

Three proton pumps, morphology and movements.

The diameter of F1 coupling factor and the distance it protrudes from the membrane of bovine heart submitochondrial particles were measured quantitatively using horse spleen ferritin as a standard. Employing the freeze-etch technique, particles of similar size were found on membranes of submitochondrial particles and on membranes of particles first depleted by F1, then reconstituted by addition of F1. The extramembranous size of F1 is 9.7 nm and F1 protrudes from the membrane surface by about 13.6 nm. Bacteriorhodopsin and cytochrome oxidase were incorporated into lipids derived from membranes of extremely thermoacidophilic microorganisms by the octylglucoside dilution method. The bacteriorhodopsin pump was fully functional provided high concentrations of valinomycin were added. With decanoyl-N-methylglucamide as detergent the pump was very active in the absence of valinomycin. Concentrations of gramicidin that collapsed the delta pH in bacteriorhodopsin liposomes prepared with soybean phospholipid had little or no effect on these rigid proteoliposomes. Very high concentrations (30 micrograms per ml) were partially effective, suggesting a mechanism other than formation of a gramicidin dimer channel. Cytochrome oxidase lost virtually all activity when incorporated into these rigid liposomes but was fully reactivated on addition of suitable detergents.

Animals↗

PubMed1984

New protein kinase from plasma membrane of Ehrlich ascites tumor cells activated by natural polypeptides.

A polypeptide-dependent protein kinase was purified about 80-fold from an extract of plasma membranes of Ehrlich ascites tumor cells. The membranes were extracted with Nonidet P-40, and the extract was purified by ammonium sulfate fractionation and hydroxylapatite and affinity chromatography. The activity was stimulated 10-fold or more by polypeptide preparations from a variety of tissues, including placenta and hypothalamus. Polypeptide-dependent protein kinase had a pH optimum of about 7.5 and required Mg2+ for activity. Mn2+ at low concentrations (200 microM) stimulated enzyme activity somewhat but inhibited activity strongly at higher concentrations. The best available substrate for polypeptide-dependent protein kinase was beta-casein, and little or no phosphorylation was observed with alpha-casein, kappa-casein, phosvitin, alpha-lactalbumin, alpha-lactoglobulin, and histone. However, several endogenous substrates from plasma membranes of Ehrlich ascites tumor cells were phosphorylated. Polypeptide-dependent protein kinase activity was not inhibited by 10 mM N-ethylmaleimide, and this resistance was useful in differentiating this protein kinase from other protein kinases that were present in crude fractions and sensitive to the inhibitor.

Animals↗

PubMed1984

Amino acid sequence of bovine heart coupling factor 6.

The amino acid sequence of bovine heart mitochondrial coupling factor 6 (F6) has been determined by automated Edman degradation of the whole protein and derived peptides. Preparations based on heat precipitation and ethanol extraction showed allotypic variation at three positions while material further purified by HPLC yielded only one sequence that also differed by a Phe-Thr replacement at residue 62. The mature protein contains 76 amino acids with a calculated molecular weight of 9006 and a pI of approximately equal to 5, in good agreement with experimentally measured values. The charged amino acids are mainly clustered at the termini and in one section in the middle; these three polar segments are separated by two segments relatively rich in nonpolar residues. Chou-Fasman analysis suggests three stretches of alpha-helix coinciding (or within) the high-charge-density sequences with a single beta-turn at the first polar-nonpolar junction. Comparison of the F6 sequence with those of other proteins did not reveal any homologous structures.

Adenosine Triphosphatases↗

PubMed1984

A placental polypeptide activator of a membranous protein kinase and its relation to histone 1.

Crude transforming growth factor preparations of placenta contain a polypeptide that is required for the activity of a protein kinase that has been purified from plasma membrane preparations of Ehrlich ascites tumor cells. The kinase activator has been separated from transforming growth factor beta by reversed-phase HPLC and affinity chromatography. Like the transforming growth factor, it is heat stable and trypsin labile, but it is not inactivated by dithiothreitol. In sodium dodecyl sulfate/polyacrylamide gel electrophoresis the purified preparation shows a major double band at about 31,000 daltons. Comparisons of electrophoretic mobility, protein kinase stimulatory activity, and cross-reactivity with an antibody against histone 1 suggest that the placental activator is identical with histone 1.

Cell Membrane↗

PubMed1984

Stimulation of glycolysis by placental polypeptides and inhibition by duramycin.

Placental polypeptides present in crude preparations of transforming growth factors stimulate glycolysis when added to quiescent 3T3 cells, normal rat kidney, and chick embryo fibroblasts. The stimulation was apparent over a time period of at least 90 min and was seen at glucose concentrations ranging from 1 to 30 mM. Duramycin, an antibiotic isolated from Streptomyces cinnamomeus, inhibits the polypeptide-stimulated and nonstimulated glycolysis of intact cells, since it permeabilizes cells to Pi and nucleotides. However, duramycin also inhibits the Na+-K+-ATPase as well as the ouabain-insensitive Mg2+-ATPase of plasma membranes. Duramycin has no effect on glycolysis catalyzed by cell-free extracts of Ehrlich ascites tumor cells in the presence of mitochondrial ATPase but partially inhibits glycolysis when ADP and Pi are generated by ATPases of plasma membrane preparations.

Adenosine Triphosphatases↗

PubMed1983

Determinants of clathrin-coated vesicle acidification.

The proton-translocating ATPase of clathrin-coated vesicles of bovine brain is characterized by ATP specificity. Chloride or bromide serve as co-ions balancing electrogenic proton pumping. ATP-driven proton translocation can be observed in the absence of chloride, provided the membrane potential is collapsed by K+ moving out in the presence of valinomycin. Chloride transport can be observed independent of proton movements in the absence of ATP, provided an inside positive membrane potential is generated by K+ moving from the outside to the inside in the presence of valinomycin.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗