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R Grosse

Publications and source records attributed to R Grosse.

At least 73 records · Page 4Linked to original sources
PubMed1984

Initiation of cultured rat hepatocyte proliferation does not involve Na+-dependent plasma membrane Ca2+ fluxes.

Adult rat hepatocytes in primary culture were examined to determine if Na+-dependent transmembrane Ca2+ fluxes precede reinitiation of DNA synthesis. Studies with 45Ca2+ and atomic absorption measurements of 40Ca2+ showed that hepatocytes lack plasma membrane Na+-Ca2+ exchange activity. Under chemically defined conditions, combinations of mitogens - EGF, insulin, and glucagon - failed to induce transmembrane Ca2+ fluxes early in the prereplicative phase. In addition, a Ca2+ ionophore, A23187, was non-mitogenic. Thus, plasma membrane Na+-Ca2+ exchange is not a mitogenic signal for hepatocytes. Elevated intracellular Ca2+ levels are thought to mediate early prereplicative events required for animal cell proliferation. These conclusions stem partly from findings that A23187, a Ca2+ ionophore, stimulates transmembrane Ca2+ fluxes and proliferation in several cell systems (reviewed in Boynton et al., 1982). Sodium ion fluxes also are implicated as "initiating" mitogenic signals (Koch and Leffert, 1979). In particular, amiloride-sensitive Na+ influxes, stimulated by growth factors, may be necessary to initiate DNA synthesis in rat hepatocytes, mouse and human fibroblasts, rat liver derived cell lines, mouse sympathetic neurons, human lymphocytes, and monkey kidney epithelial cells (reviewed in Leffert, 1982). Several investigators, using cells from electrically excitable tissues (Schellenberg and Swanson, 1981; Eckert and Grosse, 1982), have reported that plasma membrane Na+-Ca2+ exchange carriers regulate intracellular Na+ and Ca2+ concentration. It is unclear if this exchange system exists in non-electrically excitable membranes, especially with regard to hepatocytes (Judah and Ahmed, 1964; van Rossum, 1970). We have here investigated the possible association of Na+ influxes with transmembrane Ca2+ movement following reinitiation of hepatocyte growth.

Animals↗
PubMed1984

Is ribonucleotide reductase in Ehrlich ascites mammary tumour cells the target of a growth inhibitor purified from bovine mammary gland?

2'-deoxycytidine (dCyd), 2'-deoxyuridine (dUrd) and cytidine (Cyd) were found to relieve the inhibitory action by a factor, highly purified from bovine mammary gland, on the resumption of growth in vitro of stationary Ehrlich ascites mammary tumour cells. As described earlier, this effect is also achieved by insulin, proinsulin and epidermal growth factor and fetal calf serum. The effect of purine-2'-deoxyribonucleosides could not be properly assessed, because they were inhibitory themselves. Purine ribonucleosides as well as uridine and uracil were inactive. Because of the inactivity of the latter two compounds, the effect is attributed to an influence of the factor on the ribonucleotide reductase (RR) (and not on the de novo pyrimidine synthesis). The active nucleosides need not be present for the whole incubation time of 24 h; when treated for 4 h with the nucleosides, which are then removed before the addition of the factor, cells become insensitive against the latter. Again a similar effect has been described earlier for serum and insulin. The inhibitor of RR, hydroxyurea, prevents this specific effects of serum and (partially) insulin as well that of dCyd, also suggesting the involvement of RR in the factor action.

Animals↗
PubMed1983

Identification of Ca2+-pump-related phosphoprotein in plasma membrane vesicles of Ehrlich ascites carcinoma cells.

Plasma membrane vesicles of Ehrlich ascites carcinoma cells have been isolated to a high degree of purity. In the presence of Mg2+, the plasma membrane preparation exhibits a Ca2+-dependent ATPase activity of 2 mumol Pi per h per mg protein. It is suggested that this (Ca2+ + Mg2+)-ATPase activity is related to the measured Ca2+ transport which was characterized by Km values for ATP and Ca2+ of 44 +/- 9 microM and 0.25 +/- 0.10 microM, respectively. Phosphorylation of plasma membranes with [gamma-32P]ATP and analysis of the radioactive species by polyacrylamide gel electrophoresis revealed a Ca2+-dependent hydroxylamine-sensitive phosphoprotein with a molecular mass of 135 kDa. Molecular mass and other data differentiate this phosphoprotein from the catalytic subunit of (Na+ + K+)-ATPase and from the catalytic subunit of (Ca2+ + Mg2+)-ATPase of endoplasmic reticulum. It is suggested that the 135 kDa phosphoprotein represents the phosphorylated catalytic subunit of the (Ca2+ + Mg2+)-ATPase of the plasma membrane of Ehrlich ascites carcinoma cells. This finding is discussed in relation to previous attempts to identify a Ca2+-pump in plasma membranes isolated from nucleated cells.

Animals↗
PubMed1983

Identification of the insulin receptor in plasma membranes of Ehrlich ascites carcinoma cells by photoaffinity labeling.

Binding of 125I-insulin to Ehrlich ascites carcinoma cells revealed the presence of high affinity binding sites characterized by a dissociation constant of 6.1 . 10(-9) M and a number of sites per cell of 2.6 . 10(3). These values are in line with respective data for other cell types reported in the literature. The receptor was identified by photoaffinity labeling of highly purified plasma membranes with 4-azido-benzoyl-125I-insulin as a probe. The only receptor protein detectable has a molecular mass of 120-122 kD. This finding is discussed in terms of metabolic and cell proliferation effects of insulin.

Affinity Labels↗
PubMed1982

Incorporation of Na+ - Ca2+ antiporter and of (Na+ + K+)-ATPase into liposomes and demonstration of their non-identity.

(Na+ + K+)-ATPase was isolated from the grey matter of brain and incorporated into liposomes. Most of the reconstituted enzyme was oriented 'inside-out' with respect to its in vivo orientation and externally added ATP promoted Na+ uptake that was inhibitable by internally trapped ouabain. Using the same proteoliposomes, an Na+ - Ca2+ exchange system was observed as indicated by the following pieces of evidence. (1) The Na+ gradient provided the only readily apparent driving force for acceleration of Ca2+ accumulation into proteoliposomes. (2) The antiporter was specific for Ca2+, high Mg2+ excess did not inhibit Ca2+ antiport. (3) The Na+ efflux was dependent on the extravesicular Ca2+ concentration. (4) The Na+ efflux was not inhibited by tetrodotoxin. The demonstrated Na+ - Ca2+ exchange could not be related to (Na+ + K+)-ATPase protein, since it was not purified with (Na+ + K+)-ATPase, as followed from transport studies with liposomes containing (Na+ + K+)-ATPase of different specific activity. The results strongly indicate that plasma membranes isolated from the grey matter of brain contain an Na+ - Ca2+ exchange system and that the proteoliposomes are suitable for further purification of the carrier molecule.

Animals↗
PubMed1982

Is the sarcolemmal Na+ -K+ ATPase involved in active calcium transport?

Sarcolemmal preparations from rat and guinea pig cardiac muscle consisting mostly of inside-out vesicles were found to accumulate Ca2+ in the presence of ATP. The normalized rate of calcium uptake (the rate of calcium accumulation divided by its initial content in vesicles) correlated with the Na+ -K+ ATPase activity of the preparation. ATP-dependent calcium uptake by sarcolemmal vesicles was inhibited by 40-50% by cardenolids, digitoxigenin and ouabain, when the latter was included inside the vesicles. The Ca2+ gradient formed in the presence of ATP was dissipated by the addition of external sodium; Li+ was found to be ineffective in this process. External sodium also caused calcium release from vesicles pre-equilibrated with calcium in the medium. In the energy-dependent calcium uptake, the membrane-bound creatine kinase ATP-regenerating system was found to be the most effective energy source for active calcium transport. The results obtained may be interpreted to show that either the cardiac sarcolemma contains an energy-dependent calcium transport system or the sarcolemmal Na+ -K+ ATPase is able to transport calcium instead of sodium.

Adenosine Triphosphate↗
PubMed1982

[Effect of cardenolids and sodium ion gradient on ATP-dependent Ca2+ accumulation in cardiac sarcolemmal vesicles].

Sarcolemmal preparations isolated from rat and guinea pig hearts mostly consisting of inside-out membrane vesicles catalyzed ATP-dependent Ca2+ accumulation. The creatine kinase ATP-regenerating system containing exogenous creatine kinase and phosphocreatine was most effective in supporting Ca2+ accumulation. The normalized rate of Ca2+ accumulation obtained by dividing the Ca2+ uptake rate by its initial equilibrium vesicular content was correlated with the (Na+ K+)-ATPase activity. The ATP-dependent Ca2+ uptake in sarcolemmal vesicles was inhibited by cardenolids--digitoxigenin and ouabain (40-50%), when the latter acted from inside the vesicles. The Ca2+ gradient formed in sarcolemmal vesicles at the expense of ATP was dissipated by Na+ but not by Li+ added into the external medium. The external sodium ions also caused Ca2+ efflux from sarcolemmal vesicles equilibrated with Ca2+. The described effects of Na+ are taken to show the existence of a Na-Ca exchange system in cardiac sarcolemma. Ca2+-ATPase of sarcoplasmic reticulum (SR) vesicles from guinea pig heart as well as ATP-dependent Ca2+ influx in these preparations were found to be partially suppressed by digitoxigenin. External NaCl led to be a rapid (during 5-10 sec) fall in vesicular Ca2+ content (by about 40%) accumulated at the expense of ATP followed by a return of Ca2+ content to its initial level. LiCl had no effect on Ca2+ content in SR. NaCl gradient directed inside the SR vesicles did not influence the distribution of Ca2+ between internal and external vesicular volumes in equilibrium (without ATP). The differences in properties of the Ca-pump of sarcolemma and sarcoplasmic reticulum found in this work support the idea on the existence of sarcolemmal system of ATP-dependent transport of Ca2+.

Animals↗
PubMed1979

Mathematical modelling of ATP, K+ and Na+ interactions with (Na+ + K+)-ATPase occurring under equilibrium conditions.

The controlling effect of ATP, K+ and Na+ on the rate of (Na+ + K+)-ATPase inactivation by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-C1) is used for the mathematical modelling of the interaction of the effectors with the enzyme under equilibrium conditions. 1. Of a series of conceivable interaction models, designed without conceptual restrictions to describe the effector control of inactivation kinetics, only one fits the experimental data described in a preceding paper. 2. The model is characterized by the coexistence of two binding sites for ATP and the coexistence of two separate binding sites for K+ and Na+ on the enzyme-ATP complex. On the basis of this model, the effector parameters fitting the experimental data most closely are estimated by means of nonlinear least-squares fits. 3. The apparent dissociation constants for ATP fo the enzyme-ATP complex or of the enzyme-(ATP)2 complex are computed to lie near 0.0024 mM and 0.34 mM, respectively, irrespective of whether K+ and Na+ were absent or K+ and K+ plus Na+, respectively, were present in the experiments. 4. The origin of the high and the low affinity site for binding of ATP to the (Na+ + K+)-ATPase molecule is traced back to the coexistence of two catalytic centres which, although primarily equivalent as to the reactivity of their thiol groups with NBD-C1, are induced into anticooperative communication by ATP binding and thus show an induced geometric asymmetry. 5. On the basis of the interaction model outlined under item 2 the apparent dissociation constant for K+ or Na+ in the (K+ + Na+)-liganded enzyme-ATP complex are computed to be 1.7 mM and 3.5 mM, respectively. 6. The conclusions concerning the coexistence of two primarily equivalent but anticooperatively interacting catalytic centres and the coexistence of two separate ionophoric centres for Na+ and K+ correspond to the appropriate basic postulates of the flip-flop concept of (Na+ + K+)-ATPase mechanism.

4-Chloro-7-nitrobenzofurazan↗
PubMed1979

[Treatment of acute schizophrenic stupor: the effect of biperiden (author's transl)].

The study reports about the intravenous application of Biperiden at patients with acute hypokinetic reaction suffering from schizophrenia of the paranoid-hallucinatory type. In all of the six cases examined a fast abolition of the stupor could be observed. The pathophysiological mechanisms deriving from our clinical experiences are discussed. Furthermore, a therapeutic procedure is suggested to treat acute schizophrenic stupor merely with drugs.

Acute Disease↗
PubMed1978

Analysis of function-related interactions of ATP, sodium and potassium ions with Na+- and K+-transporting ATPase studied with a thiol reagent as tool.

The paper describes the interaction of ATP, Na+ and K+ with (NaK)-ATPase exploiting the inactivation by reaction with NBD-chloride as an analytical tool for the evaluation of enzyme ligandation with the various effectors. 1. The inactivation of (NaK)-ATPase by reaction with NBD-chloride showing under all conditions studied a pseudo first-order rate rests on the alkylation of thiol groups in or near catalytic centre. ATP bound to catalytic centre prevents from enzyme inactivation by NDD-chloride through protection of these thiol groups from alkylation. Na+ and K+ affect the reactivity of the thiol groups towards NBD-chloride either indirectly via influencing ATP binding or more directly via changing the conformation of catalytic centre. Proceeding from these interrelations, the interaction of the various effectors with the enzyme was analyzed. 2. The K'D-values of various nucleotides determined by our approach correspond to the values obtained by independent methods. As shown for the first time, two catalytic centres per enzyme molecule exist. They exhibit high or low affinity to both ATP and ADP apparently caused by anticooperative interaction of the half-units of the enzyme through intersubunit communication ("half-of-the-sites reactivity"). 3. In the absence of ATP, Na+ or K+ ligandation of (NaK)-ATPase produce opposite effects on the reactivity of the thiol groups of catalytic centres reflecting different changes of their conformation. This corresponds to the well-known antagonistic effect of Na+ and K+ on some partial reactions of (NaK)-ATPase. The Na+ and K+ concentrations required to change thiol reactivity are rather high, i.e. the ionophoric centres for both Na+ and K+ are not readily accessible for cation complexation in the absence of enzyme complexation with ATP. 4. Na+ being without effect on ATP binding to the enzyme also does not influence the inactivating reaction with NBD-chloride while K+ by decreasing ATP binding dramatically decreases the protective effect of ATP. The K+ affinity of the enzyme-ATP complex is by more than two orders of magnitude higher than that of free enzyme. Na+ ligandation of the K+-liganded enzyme-ATP complex reverses the effect of K+ ligandation and produces a protective effect which distinctly surpasses that of the complexation of free enzyme with ATP. Hence, the enzyme molecule carries simultaneously ionophoric centres for both Na+ and K+. 5. The findings that per enzyme molecule ionophoric centres for Na+ and K+, and two catalytic centres with anticooperative interaction coexist corroborate the corresponding basic predictions of the flip-flop concept of (NaK)-ATPase pump mechanism, and explain some peculiar kinetic features of transport and enzyme activities of (NaK)-ATPase.

4-Chloro-7-nitrobenzofurazan↗
PubMed1977

Electron microscopic visualization of the arrangement of the two protein components of (Na+ + K+)-ATPase.

The information obtained by electron microscopic examination of highly purified membrane preparations of (Na+ + K+)-ATPase after freeze-fracturing or negative staining suggests the following conclusions. The catalytic 100 000 dalton protein component penetrates with its greater 'globular' mass the plasma membrane and protudes with its smaller mass from the protoplasmic surface by a stalked knob carrying the catalytic centre. The 40 000 dalton glycoprotein component is anchored in the membrane interior by a non-pom the outer membrane surface forming a surface coat of ill-definable substructure.

Adenosine Triphosphatases↗
PubMed1977

Spectroscopic studies on effector-induced and substrate-induced conformation changes of phosphofructokinase.

The interaction of phosphofructokinase with NH4+, AMP, ATP, citrate, MgATP or fructose 6-phosphate, and in part with their mixtures forming either binary or ternary complexes has been studied by means of ultraviolet difference spectroscopy and circular dichroism spectroscopy in the wavelength range 265-300 nm with the aim of characterizing the conformational corollaries of the ligand effects on phosphofructokinase. The positive as well as the negative effectors change phosphofructokinase conformation in different ways, not easily interpretable in terms of one active and one inactive enzyme conformation. The spectroscopic equivalents of phosphofructokinase conformation changes resulting from catalytic activity are similar to those produced by the reaction products. The ligand concentration-dependent changes of absorption differences in the tryptophyl, tyrosyl and phenylalanyl region parallel each other, i.e. the interactions of the ligands with phosphofructokinase are not confined to specific aromatic side chains, but involve conformation changes of the large domains of the protein. ATP affinity to the enzyme shows temperature-dependent biphasic changes so that ATP binding appears to be either an entropy-driven or enthalpy-driven process. The dissociation constants of the ligands derived from spectroscopic titration of binary complex formation are comparable to those calculated from kinetic experiments. MgATP and fructose 6-phosphate each alone change phosphofructokinase conformation by binary complex formation in keeping with a random order of reaction sequence.

Adenosine Monophosphate↗
PubMed1977

The localization of the MM isozyme of creatine phosphokinase on the surface membrane of myocardial cells and its functional coupling to ouabain-inhibited (Na+, K+)-ATPase.

A rat heart plasma membrane preparation isolated in a sucrose medium and some of its enzymatic properties have been investigated. It has been shown that a rat heart plasma membrane fraction contains high creatine phosphokinase activity which can not be diminished by repeated washing with sucrose solution. Creatine phosphokinase extracted from a plasma membrane fraction with potassium chloride and 0.01% deoxycholate solution is electrophoretically identical to MM isoenzyme of creatine phosphokinase. Under the conditions where (Na+,K+)-ATPase is activated by addition of Na+, K+ and MgATP, creatine phosphokinase of plasma membrane fraction is able to maintain a low ADP concentration in the medium if creatine phosphate is present. The rate of creatine release is dependent upon MgATP concentration in accordance with the kinetic parameters of the (Na+,K+)-ATPase and is significantly inhibited by ouabain (0.5 mM). The rate of creatine release is also dependent on creatine phosphate concentration in conformance with the kinetic parameters of MM isozyme of creatine phosphokinase. It is concluded that in intact heart cells the plasma membrane creatine phosphokinase may ensure effective utilization of creatine phosphate for immediate rephosphorylation of ADP produced in the (Na+,K+)-ATPase reaction.

Adenosine Diphosphate↗