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Biomedical subjects

E Racker

Publications and source records attributed to E Racker.

At least 37 records · Page 2Linked to original sources
PubMed1988

Induction of system A amino acid transport through long-term treatment with ouabain: correlation with increased (Na+/K+)-ATPase activity.

Mouse embryo fibroblast cells (C3H-10T1/2) and the methylcholanthrene-transformed derivative (MCA-10T1/2) were treated with basal modified Eagle's medium (BME) containing 10% fetal bovine serum and varying concentrations of ouabain ranging from 0.05 mM to 0.7 mM for 16 h in culture. After replacing the ouabain-containing medium with Earl's balanced salts solution, System A amino acid transport activity increased from approximately 40 to 500 pmol AIB accumulated.mg protein-1.min-1 in the C3H-10T1/2 cells and from approximately 300 to 700 pmol AIB accumulated.mg protein-1.min-1 in the MCA-10T1/2 cells. The (Na+/K+)-ATPase pump activity also increased from approximately 12 to 46 nmol Rb+ accumulated.mg protein-1.min-1 in the normal cells and from approximately 20 to 42 nmol Rb+ accumulated.mg protein-1.min-1 in the transformed cells. System A and the (Na+/K+)ATPase activity were maximally increased at approximately 0.4-0.6 mM ouabain in the normal cells in contrast to the transformed cells which were maximally stimulated at a concentration of approximately 0.2 mM ouabain. This treatment with ouabain increased the [Na+]i/[K+]i as measured by atomic absorption spectroscopy, and thereby decreased the Na+ and K+ electrochemical gradients. Our data show that the internal ion gradients inverted at a lower concentration of ouabain in the transformed cells compared to the normal cells. The ouabain-induced increase in pump and System A activity shown here was used as a tool to further investigate the coordinated ion transport regulation in the control of cell growth.

Amino Acids↗
PubMed1988

Energetic mechanism of system A amino acid transport in normal and transformed mouse fibroblasts.

Ouabain treatment (0.4 mM) of normal and transformed C3H-10T1/2 cells caused a progressive increase in 2-aminoisobutyrate (AIB) transport reaching a maximum after 16 to 18 h exposure. There was a virtually complete blockage of this stimulated rate when 3 microM cycloheximide (CHX) was added together with ouabain at T = 0. In the transformed cell, addition of CHX after 14 h had no effect; in the normal cell, it inhibited (ca. 50%) the final AIB transport rate achieved after 24 h. The t1/2 for reaching maximal activity (insensitive to CHX exposure) was thus shifted from 8 h in the transformed cell to 15 h in the normal cell. Since the rate of achieving maximal activity in the absence of CHX was about the same in the two cells, the shift in t1/2 in the presence of CHX suggests that the rate of degradation is more rapid in the normal cell. Following ouabain treatment, the apparent Km for Na+ was decreased in both cells. The Km returned to the basal level 1 h after ouabain removal in the normal cell, but remained low in the transformed cell during this time period. The stimulation of AIB transport following ouabain removal was largely abolished by a proton ionophore (1799), a lipophilic cation (tetraphenyl-phosphonium), or ouabain. These results suggest that, under the conditions of ouabain stress, there is a switch in the bioenergetic mechanism. The Na+/K+ pump and System A transporter appear to be linked and the membrane potential generated by the Na+/K+ pump activity becomes a major driving force for AIB uptake.

Amino Acids↗
PubMed1988

Motility, heat, and lactate production in ejaculated bovine sperm.

Effects of various inhibitors on motility, heat, and lactate production of ejaculated bovine sperm were determined in the presence of antimycin A and rotenone. erythro-9-[3-(2-Hydroxynonyl)]adenine (EHNA) and polyvinylpyrrolidone (PVP-360) stopped motility and reduced heat or lactate production by 30-50%. Carbodiimides resulted in loss of motility and a reduction of metabolism by 60-75%. Quercetin treatment, which enhanced rather than inhibited motility, depressed heat and lactate production by 50-60%. Since mechanical immobilization reduced heat production by only 30%, the question arises as to what other cellular processes are major contributors to the energy budget. Inhibitors of ion flux had little-to-no effect on heat or lactate production, suggesting that neither mitochondrial nor Na+/K+ ATPases were major ATP-requiring processes. Calcium flux at the plasma membrane also was minimal and previous reports eliminated glycolytic substrate cycling as major consuming processes for ATP. Although quercetin inhibited lactate production in intact cells, no effect of quercetin on cell-free glycolysis and the ATPase activities of isolated dynein was detected. Quercetin did, however, inhibit ATPase activity of plasma membrane, suggesting that this unidentified ATPase may contribute to the formation of ADP and Pi required for lactate production by the intact cell. We propose (a) that the bioenergetic costs of motility are divided between regulatory events and dynein-microtubule interaction (dynein ATPase), (b) that some of the membrane-related processes may be "inefficient," and (c) that quercetin may render these steps more "efficient," in a manner analogous to its action on the Na+/K+ pump of Ehrlich ascites tumor cells.

Adenosine Triphosphatases↗
PubMed1988

Functional reconstitution of skeletal muscle Ca2+ channels: separation of regulatory and channel components.

Regulatory properties of a partially purified Ca2+ -channel preparation from isolated rabbit skeletal muscle triads were examined in proteoliposomes. These properties included (i) inhibition by phenylalkylamine antagonists, such as verapamil, (ii) inhibition by the GTP-binding protein Go in the presence of guanosine 5'-[gamma-thio]triphosphate, and (iii) regulation of phenylalkylamine inhibition as a result of phosphorylation by a polypeptide-dependent protein kinase (PK-P). By selective reconstitution of protein fractions obtained by wheat germ lectin and ion-exchange chromatography, a separation of Ca2+-channel activity (fraction C) from regulatory component(s) (fraction R) responsible for verapamil sensitivity was achieved. Reconstitution of fraction C alone yielded vesicles that exhibited channel-mediated 45Ca2+ uptake that could be directly inhibited by coreconstitution of Go in the presence of guanosine 5'-[gamma-thio]triphosphate. However, the 45Ca2+ uptake obtained with fraction C was not inhibited by verapamil. Coreconstitution of fractions C and R yielded vesicles in which the sensitivity of 45Ca2+ uptake to verapamil was restored. The verapamil sensitivity of this preparation could be inhibited by PK-P. Fraction C, obtained by wheat germ agglutinin-Sepharose chromatography followed by DEAE-Sephacel chromatography, included a 180-kDa protein that was phosphorylated by cAMP-dependent protein kinase (PK-A) but not by PK-P and a 145-kDa protein (180 kDa under nonreducing conditions) that was not phosphorylated by either kinase. Fraction R contained proteins that did not adsorb to wheat germ lectin and included 165-kDa and 55-kDa proteins that were phosphorylated by PK-P but not by PK-A. These results suggest a complex model for Ca2+-channel regulation in skeletal muscle involving a number of distinct, separable protein components.

Animals↗
PubMed1988

Specific dephosphorylation of phosphoproteins by protein-serine and -tyrosine kinases.

Five protein kinases are shown to serve as specific phosphatases in the absence of ADP. Although the rates of hydrolysis are very slow compared to the forward phosphorylation rates under optimal conditions, they are of the same order as the reverse reaction in the presence of ADP. Because cells contain approximately equal to 3 mM ATP, neither the reverse reaction nor the phosphatase is likely to play a physiological role. beta-casein B phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (protein kinase A) is specifically dephosphorylated by protein kinase A but not by polypeptide-dependent protein kinase (protein kinase P). beta-casein B phosphorylated by protein kinase P is specifically dephosphorylated by protein kinase P but not by protein kinase A. Histone H1 phosphorylated by protein kinase C is dephosphorylated by the same enzyme in the absence of ADP. In all cases tested addition of ADP and F1-ATPase accelerates moderately the rate of dephosphorylation. Native H+-ATPase from yeast plasma membranes is isolated mainly in the phosphorylated form. It is dephosphorylated and rephosphorylated by protein kinase P but not by protein kinase A. Protein-tyrosine kinase of the epidermal growth factor receptor phosphorylates the random synthetic polypeptide poly(Glu80Tyr20). The phosphorylated polymer is specifically dephosphorylated in the absence of ADP by epidermal growth factor receptor preparations but not by insulin receptor preparations. The same polymer phosphorylated by insulin receptor is dephosphorylated by insulin receptor but not by epidermal growth factor receptor preparations. By using a cycle of dephosphorylation-rephosphorylation, it is possible to identify proteins that are phosphorylated by these protein kinases in vivo. Should this method be applicable to additional protein kinases, it should be possible to estimate the quantitative contribution of each protein kinase to a single phosphoprotein.

ErbB Receptors↗
PubMed1988

Phosphorylation of synthetic random polypeptides by protein kinase P and other protein-serine (threonine) kinases and stimulation or inhibition of kinase activities by microbial toxins.

A synthetic random polymer of threonine and glutamate (1:4.4) is readily phosphorylated by protein kinase P but not by five other protein-serine (threonine) kinases. A synthetic random polymer of serine and arginine (1:3) is readily phosphorylated by protein kinase A and protein kinase C but not by protein kinase P. Although the amino acid sequences surrounding the phosphorylated serine (threonine) residue have been demonstrated in studies with small synthetic polypeptides to be decisive factors in the rate at which they are phosphorylated, the findings with the large synthetic polypeptides suggest that in the case of proteins the size, the tertiary structure, and particularly the electrostatic interactions are equally or more important contributing factors. Syringomycin, a toxin from Pseudomonas syringae, and polymyxin B, from Bacillus polymyxa, stimulate protein kinase P, strongly inhibit protein kinase C, and have no effect on protein kinase A. Basic polypeptides with high lysine content are phosphorylated by ATP nonenzymatically.

Adenosine Triphosphate↗
PubMed1988

Phosphorylation of the RAS2 gene product by protein kinase A inhibits the activation of yeast adenylyl cyclase.

The RAS2 gene product of Saccharomyces cerevisiae expressed in Escherichia coli was phosphorylated by protein kinase A in vitro to approximately 0.5-0.7 mol of phosphate per mol of protein. Neither protein kinase C nor protein kinase P phosphorylated the RAS2 protein significantly. The RAS2 protein is known to activate, in the presence of either Mg2+ and GTP or Mn2+, a yeast membrane preparation with an overexpressed adenylyl cyclase and a deficiency in endogenous RAS1 and RAS2 proteins. When the RAS2 protein was phosphorylated by protein kinase A prior to exposure to the yeast membranes, its capacity to activate the adenylyl cyclase was diminished by 40-60%, while activation by Mn2+ remained unaffected. The phosphorylated protein retained, however, its ability to bind GTP. Incubation of protein kinase A with a specific protein kinase A inhibitor prior to phosphorylation prevented the inhibition. Furthermore, the hydrolysis of GTP was not required for the observed inhibition. These data suggest that phosphorylation of the RAS2 gene product by protein kinase A may function as one mechanism by which the intracellular level of cAMP in yeast is regulated.

Adenylyl Cyclases↗
PubMed1988

Stimulation of inositol incorporation into lipids of PC12 cells by nerve growth factor and bradykinin.

The effects of bradykinin (BK) and lithium on the phosphatidylinositol cycle were examined in PC12 cells cultured for 20 h in the presence [PC12(+)] or in the absence [PC12(-)] of nerve growth factor (NGF). BK (1 microM) induced a small stimulation of the incorporation of myo-[2-3H]inositol into the lipids of PC12(-) cells and a three- to fourfold stimulation of such incorporation into the lipids of PC12 (+) cells. About 15 h of incubation with NGF and greater than 10 min of incubation with BK were needed for maximal stimulation of inositol incorporation by BK. In the presence of 25 mM LiCl, BK stimulated the inositol monophosphate levels nine-fold in PC12 (-) and 30-fold in PC12 (+) cells. After incubation for 20 h with NGF, an increased binding of [3H]BK to the PC12 (+) cells was observed at 4 degrees C. Exposure of the cells for 30 min to 25 mM LiCl enhanced the effect of BK on the inositol incorporation into total inositol lipids, especially in PC12(+) cells. In these cells, LiCl in the presence of BK also increased several-fold the intracellular levels of inositol bisphosphate and inositol trisphosphate.

Adrenal Gland Neoplasms↗
PubMed1988

Lithium stimulation of membrane-bound phospholipase C from PC12 cells exposed to nerve growth factor.

LiCl stimulated the formation of inositol monophosphate in PC12 cells that had been exposed to nerve growth factor (NGF) for 4-5 days. Half-maximal accumulation was observed at approximately 8 mM LiCl. Stimulation of formation of inositol bisphosphate plus inositol trisphosphate was half-maximal at approximately 1 mM LiCl. With membranes isolated from PC12 cells differentiated with NGF, the hydrolysis of added phosphatidylinositol 4,5-bisphosphate (PIP2) was stimulated by LiCl in a biphasic manner, with the first stimulation half-maximal at approximately 0.7 mM and the second half-maximal at approximately 15 mM LiCl. The apparent Km for PIP2 was lowered in the presence of 1.1 mM LiCl from approximately 200 to approximately 70 microM. Membranes from cells grown in the absence of NGF did not respond to LiCl. Although observations with intact cells are difficult to interpret without ambiguity, the results obtained with isolated membranes support our interpretation of the stimulatory action of lithium in the intact PC12 cells.

Adrenal Gland Neoplasms↗
PubMed1987

A tyrosine-specific protein kinase from Ehrlich ascites tumor cells.

A protein tyrosine kinase that phosphorylates both alpha and beta subunits of inactivated (Na+,K+)-ATPase from dog kidney was purified about 500-fold from Ehrlich ascites tumor cell membranes. The enzyme required divalent cations Mn2+, Mg2+, or Fe2+ but was inhibited by Cu2+ or Zn2+. The purified enzyme phosphorylated the beta subunit about five times faster than the alpha subunit of the (Na+,K+)-ATPase. The random polymer poly(Glu80Tyr20) was an excellent substrate while casein was only marginally phosphorylated. In contrast, the purified transforming gene product of Rous sarcoma virus phosphorylated all three substrates and the (Na+,K+)-ATPase was preferentially phosphorylated on the alpha subunit. The transforming gene product of Fujinami sarcoma visue and EGF receptor kinase from A431 cells phosphorylated (Na+,K+)-ATPase poorly whereas casein was an excellent substrate. The molecular weight of the partially purified protein tyrosine kinase from Ehrlich ascites tumor cells determined by gel filtration was about 60,000. One of two major phosphorylated phosphopeptides resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis had an Mr of 60 kDa, thus suggesting that it might be the autophosphorylated protein tyrosine kinase. A phosphatase that hydrolyzes phosphorylated histones or poly(Glu80Tyr20) was partially purified from the same membrane.

Animals↗
PubMed1987

Effect of protein kinase P on phosphorylations catalyzed by the epidermal growth factor.

Protein kinase P (PK-P) activated by histones or certain other basic compounds has been purified previously from yeast [Yanagita, Y., Abdel-Ghany, M., Raden, D., Nelson, N. & Racker, E. (1987) Proc. Natl. Acad. Sci. USA 84, 925-929]. It is shown here that PK-P is present in solubilized membranes of A-431 carcinoma cells where it changes the epidermal growth factor (EGF) receptor kinase activity. Polylysine, a weak PK-P activator, inhibited the autophosphorylation of the EGF receptor both in the absence and presence of EGF. Increased PK-P activity induced by histone 1, a potent activator, gave rise to increased autophosphorylation of the EGF receptor as well as phosphorylation at tyrosine residues of numerous other endogenous membrane components. The stimulation by histone was particularly striking in the presence of EGF. A similar stimulation was achieved with polylysine and EGF on addition of yeast PK-P. However, addition of yeast PK-P in the presence of histone 1 markedly inhibited the EGF-stimulated phosphorylation of endogenous membrane proteins. We conclude from these results that the effect of PK-P on the EGF receptor takes place in three phases: at low levels PK-P inhibits the autophosphorylation, at intermediate levels it stimulates the autophosphorylation as well as the EGF-dependent phosphorylation of numerous other membrane proteins, and at high levels it inhibits the phosphorylation of these proteins.

Epidermal Growth Factor↗
PubMed1987

Polypeptide-dependent protein kinase from bakers' yeast.

The purification and properties of a protein serine kinase (PK-P) extracted with Triton X-100 from membranes of bakers' yeast are described. The enzyme is virtually inactive unless either a histone or a heat-stable polypeptide from yeast membranes and Mg2+ are added. Other divalent cations substitute for Mg2+ poorly or not at all; most of them, including Mn2+, inhibit when added in the presence of 5 mM Mg2+. The enzyme is unstable but can be stabilized by addition of 0.1% Triton X-100 and 20% glycerol. The final preparation shows, on silver-stained electrophoresis gels, two major bands (Mr 41,000 and 35,000). According to gel filtration the molecular weight of the active protein is about 75,000. Of the two subunits, only the smaller one appears to be autophosphorylated. In addition to casein, the enzyme phosphorylates several proteins including the H+-ATPase (Mr 100,000) in the yeast plasma membrane. In order to demonstrate the phosphorylation of the ATPase (up to 0.9 equivalents), exposure of the latter to an acid phosphatase was required. Other phosphorylated proteins include mRNA cap-binding protein from mammalian erythrocytes and yeast, a glucocorticoid receptor protein, and a preparation of the guanine nucleotide-binding proteins Gi and Go from brain. A partial purification of a natural activator from yeast plasma membranes is described.

Chemical Phenomena↗
PubMed1987

Opposing effects of a ras oncogene on growth factor-stimulated phosphoinositide hydrolysis: desensitization to platelet-derived growth factor and enhanced sensitivity to bradykinin.

Expression of a transforming Harvey or Kirsten ras gene caused opposing effects in the ability of platelet-derived growth factor (PDGF) and bradykinin to activate phospholipase C-mediated phosphoinositide hydrolysis. In [3H]inositol-labeled rat-1 fibroblasts, PDGF (5 ng/ml) resulted in a 2-fold increase in the level of [3H]inositol trisphosphate (InsP3) after 2 min and, in the presence of LiCl, a 3- to 8-fold increase in the level of [3H]inositol monophosphate (InsP1) after 30 min. However, in EJ-ras-transfected rat-1 cells, which exhibit near normal levels of PDGF receptors, PDGF resulted in little or no accumulation of either [3H]InsP3 or [3H]InsP1. Similarly, marked stimulations by PDGF were observed in NIH 3T3 cells, as well as in v-src-transformed 3T3 cells, but not in 3T3 cells transformed by Kirsten sarcoma virus or by transfection with v-Ha-ras DNA. This diminished phosphoinositide response in ras-transformed cells was associated with a markedly attenuated mitogenic response to PDGF. On the other hand, both phosphoinositide metabolism and DNA synthesis in ras-transformed fibroblasts were stimulated several-fold by serum. In NIH 3T3 cells carrying a glucocorticoid-inducible v-Ha-ras gene, a close correlation was found between the expression of p21ras and the loss of PDGF-stimulated [3H]InsP1 accumulation. In contrast to this ras-induced desensitization to PDGF, ras-transformed NIH 3T3 cells exhibited an enhanced sensitivity to bradykinin; this effect was associated with an elevated level of high-affinity [3H]bradykinin binding. We propose that a ras gene product (p21) can, directly or indirectly, influence growth factor-stimulated phosphoinositide hydrolysis, as well as DNA synthesis, via alterations in the properties of specific growth factor receptors.

Animals↗
PubMed1986

Partial purification and characterization of protein tyrosine kinases from normal tissues.

Three membranous protein tyrosine kinases (PTKs) have been partially purified from human placenta and pig brain. The two placental enzymes (PTK-1 and -2) are distinct with respect to solubility in detergents, molecular weight, and enzymatic properties. The brain protein tyrosine kinase resembles placental PTK-1 with respect to molecular weight and some kinetic properties. However, stimulation of brain PTK is greater with Mn2+ than with Mg2+ whereas placental PTK-1 gives higher rates with Mg2+ than with Mn2+. All three enzymes are inhibited about 50% by 0.1 M NaCl. A monoclonal antibody raised in vitro against the brain enzyme inhibits brain PTK as well as placental PTK-2, but has no effect against PTK-1 or pp60src. It thus appears that these three enzymes are distinct entities that differ from each other both kinetically and immunologically. With synthetic tyrosine-glutamic acid polymers as a substrate, protein tyrosine kinase activity can be detected in crude extracts of membranes.

Animals↗
PubMed1986

Use of slow Ca2+ channel blockers to enhance inhibition by taxol of growth of drug-sensitive and -resistant Chinese hamster ovary cells.

The toxicity of taxol for drug-sensitive and -resistant Chinese hamster ovary cells has been examined in tissue cultures in the presence and absence of drugs that are being used as slow Ca2+ channel blockers. Enhancement of toxicity was found to be greater in the case of taxol than of daunorubicin. Several blockers including pimozide and nimodipine were more effective as toxicity enhancers than verapamil. Studies of uptake of 14C-labeled daunorubicin revealed a poor correlation between the effectiveness of various drugs to enhance intracellular daunorubicin concentration and their toxicity to cells grown in tissue cultures.

Alkaloids↗