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E Racker

Publications and source records attributed to E Racker.

At least 73 records · Page 4Linked to original sources
PubMed1983

An ATP-driven proton pump in clathrin-coated vesicles.

Clathrin containing coated vesicles prepared from bovine brain catalyzed ATP-driven proton translocation and a 32Pi-ATP exchange reaction. Both activities were measured in the presence of 5 micrograms of oligomycin/mg of protein which completely inhibited these reactions catalyzed by submitochondrial particles. Analyses performed during the purification procedure demonstrated that the oligomycin-resistant pump was concentrated and highly purified in the fractions containing coated vesicles. Moreover, vesicles precipitated by either monoclonal or polyclonal antibodies against clathrin contained the H+ pump activity. Dicyclohexylcarbodiimide (0.5 mM) and N-ethylmaleimide (1 mM) added to the assay mixture inhibited the pump completely, whereas neither vanadate, sodium azide, efrapeptin, or mitochondrial ATPase inhibitor had an effect.

Adenosine Triphosphatases↗
PubMed1983

The role of ATPase in glycolysis of Ehrlich ascites tumor cells.

Glycolysis in Ehrlich ascites tumor cells suspended in buffer containing 5 mM Pi was 50% inhibited by ouabain. In the absence of Pi the inhibition was less striking. Permeabilization of the cells with filipin abolished glycolysis, but glycolysis was restored by addition of Pi and AMP. Neither ouabain nor quercetin inhibited glycolysis in these permeabilized cells. We conclude that quercetin did not inhibit hexokinase sufficiently to affect glycolysis. An extract of Ehrlich ascites tumor cells glycolyzed weakly unless either Pi or an ATPase (e.g. (Na+K+)-ATPase) was added. The low rate of glycolysis of the extract was even further reduced when an endogenous ATPase was removed by precipitation with CaATP. The glycolytic activity of this ATPase-deficient extract was restored by addition of purified (Na+K+)-ATPase or of CaATP-precipitable ATPase. Addition of hexokinase without Pi did not restore glycolytic activity to the extract. An explanation for the contradictory conclusions by Bustamante, E., Morris, H.P., and Pedersen, P.L. (J. Biol. Chem. (1981) 265, 8699-8704) is presented.

Adenosine Triphosphatases↗
PubMed1983

A membrane-bound human placental protein kinase activated by endogenous polypeptides.

A protein kinase (PPdPK) was purified from plasma membranes of human placenta. Phosphorylation of casein, but not of phosvitin or lactalbumin, by [gamma-32 P]ATP in the presence of PPdPK was stimulated about 10-fold by naturally occurring polypeptides prepared from a variety of sources similar to the procedure of Roberts et al. (Proc. Natl. Acad. Sci. U.S.A. 77, 3494-3498, 1980). The amino acid phosphorylated on casein was serine. According to gel exclusion chromatography the mol.wt. of PPdPK was 95 000. In autoradiograms, following polyacrylamide-gel electrophoresis, the autophosphorylation of PPdPK was greatly enhanced by the polypeptide activators.

Cell Compartmentation↗
PubMed1983

A rapid assay for protein kinases phosphorylating small polypeptides and other substrates.

A new and rapid method of protein kinase assay is presented which is suitable for low-molecular-weight substrates, irrespective of their electrophoretic or chromatographic mobility. It depends on the phosphorylation of the substrates with [gamma-32P]ATP, hydrolysis of the pyrophosphate bonds by boiling in 1 N HCl, extraction of 32P with isobutanol-benzene, and measurement of the radioactivity of 32P-labeled phosphoesters in the water phase. The method is shown to be suitable for both tyrosine- and serine-phosphorylating protein kinases.

Amino Acid Sequence↗
PubMed1983

Resolution and reconstitution of biological pathways from 1919 to 1984.

The central theme of this paper is the reconstitution of the Warburg effect, the high aerobic glycolysis of malignant tumors. The history of resolution-reconstitution started with the isolation of glycolytic enzymes. In 1945 Meyerhof prepared an extract from yeast that did not ferment unless an ATPase was added. An extract of Ehrlich ascites tumor cells that does not glycolyze in the presence of catalytic amounts of Pi and nucleotides without addition of an ATPase is presented as a model for future reconstitutions of the Warburg effect. Natural polypeptide preparations from placenta and hypothalamus 1) stimulate a protein kinase from tumor plasma membranes, 2) serve as substrates for another protein kinase from tumor plasma membranes, and 3) stimulate glycolysis of normal rat or chick embryo fibroblasts and may be related to the transforming tumor growth factors. We can hope that an exploration of the mechanism by which these polypeptides stimulate glycolysis could lead to the successful reconstitution of the Warburg effect in an in vitro system. It may also help us understand how tumor RNA or malignant DNA induces the various other biochemical changes that take place when normal cells are transformed to tumor cells.

Animals↗
PubMed1982

Inhibition of tyrosine protein kinases by halomethyl ketones.

A chloromethyl ketone derivative of lactic acid was shown to inhibit protein phosphorylation in plasma membranes of Ehrlich ascites tumor cells [Johnson, H. J., Zimniak, A., & Racker, E. (1982) Biochemistry 21, 2984-2989]. We now show that this inhibitor as well as three halomethyl ketone derivatives of amino acids and peptides specifically inhibits tyrosine protein kinase activity in intact plasma membranes and Triton extracts of plasma membrane of A-431 tumor cells. The most effective inhibitor is a bromomethyl ketone derivative of leucine that inhibits the phosphorylation of a protein that migrates to the same position as the EGF receptor in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Inhibition of phosphorylation took place in the presence or absence of added EGF, and the inhibitor did not interfere with the binding of EGF to the receptor nor with the dephosphorylation of the EGF-stimulated phosphoprotein. EGF-dependent phosphorylation in a Triton extract of plasma membranes from normal placenta was considerably less sensitive to the bromomethyl ketone derivative of leucine. The tyrosine protein kinase activity of the transformation gene product of Fujinami virus was particularly sensitive to the bromomethyl ketone derivative of leucine, while the src gene product of Rous sarcoma virus was comparatively less sensitive. The bromomethyl ketone inhibitor interfered with the phosphorylation of the EGF receptor by [gamma-32P]-8-azido-ATP but much less with the light-sensitive binding. This observation and the lack of interference with EGF binding suggest that the inhibitor interacts with the protein kinase portion of the receptor complex.

Avian Sarcoma Viruses↗
PubMed1982

Protein kinases from spinach chloroplasts. I. Purification and identification of two distinct protein kinases.

Two protein kinases (chloroplast protein kinases 1 and 2 (ChlPK1 and ChlPK2)) were isolated from spinach chloroplasts. After solubilization of the chloroplasts with octylglucoside and cholate, these kinases were purified by ammonium sulfate precipitation, sucrose gradient centrifugation, and hydroxylapatite chromatography. ChlPK1 traveled as a single band in polyacrylamide gel electrophoresis corresponding to 25,000 daltons; ChlPK2 traveled as a single band corresponding to 38,000 daltons. After exposure to 8-azido-[gamma-32P]ATP, the radioactive bands appeared in the same positions revealed by Coomassie blue staining. However, a trace of ChlPK2 was detected in the ChlPK1 preparation and a faint second lower molecular weight radioactive band was seen in the ChlPK2 preparations. Both enzymes acted on casein or histone IIIS as substrate and phosphorylated a serine residue. The proteolytic peptide maps, however, were clearly distinguishable in autoradiograms, suggesting that different serine residues were phosphorylated by ChlPK1 and ChlPK2.

Centrifugation, Density Gradient↗
PubMed1982

Inhibition of hexokinase and protein kinase activities of tumor cells by a chloromethyl ketone derivative of lactic acid.

A chloromethyl ketone derivative of lactic acid is a potent inhibitor of glycolysis of Ehrlich ascites tumor cells. It inhibited glycolysis of intact cells by about 50% at 200 microM (100 nmol/mg of protein) while cell-free extracts were inhibited 50% at 50 microM (50 nmol/mg of protein). N alpha-(p-Tosyl)-L-lysine chloromethyl ketone and N alpha-(p-tosyl)-L-phenylalanine chloromethyl ketone inhibited only slightly or not at all at this concentration. The inhibition was localized at the hexokinase and phosphofructokinase steps since these two enzymes added to an inactivated extract restored the glycolytic activity, whereas none of the other glycolytic enzymes did. In fact, addition of pyruvate kinase or lactate dehydrogenase, which stimulated glycolysis, resulted in a more pronounced inhibition. Glycolysis and hexokinase activities in extracts of Rous sarcoma virus transformed cells were considerably more sensitive to the inhibitor than the activities from normal chick embryo fibroblasts. Hexokinase from mouse brain required 50 times higher concentrations for inhibition than the enzyme from mouse Ehrlich ascites tumor cells. Yeast hexokinase was unaffected at all concentrations tested. Since 5,5'-dithiobis(2-nitrobenzoate) protected against the inhibition, the chloromethyl ketone appeared to inhibit by interaction with an essential SH group. A pronounced inhibition of protein kinase activity of plasma membranes of Ehrlich ascites tumor cells was observed in the presence of the chloromethyl ketone. As in the case of glycolysis, the chloromethyl ketone of lactic acid was a more potent inhibitor of protein kinase activity than several other chloromethyl ketones that were tested.

Animals↗
PubMed1982

ATP-dependent phosphate transport in sarcoplasmic reticulum and reconstituted proteoliposomes.

During uptake of Ca2+ by rabbit sarcoplasmic reticulum, about 1 mumol of 32Pi was taken up per mumol 45Ca2+ transported. The uptake of Pi was dependent on external Ca2+, Mg2+ and ATP. Intravesicular Ca2+ did not substitute for external Ca2+. In contrast to the accumulation of Ca2+ which was abolished by the ionophore A23187, the uptake of Pi continued to take place provided sufficient Ca2+ was present in the medium. Thus, a Ca2+ gradient did not seem to be required. Similar observations were made with proteoliposomes reconstituted with membrane preparations of sarcoplasmic reticulum and soybean phospholipids. However, when purified Ca2+ -ATPase was used for reconstitution, there was ATP-dependent Ca2+ uptake but no ATP-dependent Pi transport was observed. These data show that the mechanism of Pi transport cannot be a passive movement in response to a Ca2+ gradient but appears to be catalyzed by a specific protein, which is inactivated during purification of the Ca2+ -ATPase. A protein that catalyzes Pi transport in reconstituted vesicles has been solubilized by extraction of sarcoplasmic reticulum with sodium cholate.

Adenosine Triphosphate↗
PubMed1982

Purification and characterization of two protein kinases from bovine heart mitochondrial membrane.

Two protein kinases (MPK1 and MPK2) were isolated from bovine heart mitochondria. After the solubilization of submitochondrial particles with cholate, these protein kinases were purified by ammonium sulfate precipitation, Sepharose 6B gel filtration, and affinity chromatography on phosvitin-Sepharose. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the final preparation of MPK1 contained two major polypeptide bands (40,000 and 36,000 daltons). MPK2 contained one major polypeptide of 34,000 daltons. Under nondenaturing conditions, the molecular weights of MPK1 and MPK2 were estimated to be approximately 250,000 and 70,000-90,000, respectively. MPK1 had a pH optimum at 9.0 and MPK2 at 7.5. Both enzymes required Mg2+ for activity and responded poorly to Mn2+ or Ca2+. Both had similar apparent Km values for ATP and were not affected by either cyclic AMP or Ca2+-calmodulin. MKP1 phosphorylated threonine residues and MPK2 serine residues of casein. With beta casein as substrate, MPK1 was more active than MPK2, whereas with alpha casein, MPK2 was more active than MPK1.

Animals↗
PubMed1982

Mechanism of calcium release from skeletal sarcoplasmic reticulum.

Ca2+ -induced Ca2+ release at the terminal cisternae of skeletal sarcoplasmic reticulum was demonstrated using heavy sarcoplasmic reticulum vesicles. Ca2+ release was observed at 10 mum Ca2+ in the presence of 1.25 mm free Mg2+ and was sensitive to low concentrations of ruthenium red and was partially inhibited by valinomycin. These results suggest that the Ca2+ -induced Ca2+ release is electrogenic and that an inside negative membrane potential created by the Ca2+ flux opens a second channel that releases Ca2+. Results in support of this formulation were obtained by applying a Cl- gradient or K+ gradient to sarcoplasmic reticulum vesicles to initiate Ca2+ release. Based on experiments the following hypothesis for the excitation-contraction coupling of skeletal muscle was formulated. On excitation, small amounts of Ca2+ enter from the transverse tubule and interact with a Ca2+ enter from the transverse tubule and interact with a Ca2+ receptor at the terminal cisternae and cause Ca2+ release (Ca2+ -induced Ca2+ release). This Ca2+ flux generates an inside negative membrane potential which opens voltage-gated Ca2+ channels (membrane potential-dependent Ca2+ release) in amounts sufficient for contraction.

Adenosine Triphosphate↗