Functional interaction between the epidermal growth factor receptor and c-Src kinase activity

AbstractTo study the relationship between the tyrosine kinase c-Src and the epidermal growth factor receptor (EGF-R), we used the breast cancer cell line ZR75-1, which was transfected with the EGF-R. The EGF-R transfected cell line expressed 60 times more EGF-R than a control cell line transfected with the empty vector. In the presence of EGF, the EGF-R over-expressing cell line grew much faster than the control cell line. Both cell lines expressed approximately equal amounts of c-Src. However, the cell line over-expressing the EGF-R showed a twofold enhancement of c-Src kinase activity after EGF stimulation. The activation of c-Src kinase by EGF was confirmed in other EGF-R expressing cell types

Functional changes associated with the sequential transformation of L′4 into L4 pyruvate kinase

The functional changes, associated with the sequential transformation of L′4 into L4 pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) were studied. L′4 enzyme from human erythrocytes shows strong hysteretic behaviour: the initial rate of the enzyme preincubated with an unsaturating concentration of phosphoenolpyruvate is much higher than of the enzyme preincubated with ADP, at the same phosphoenolpyruvate concentration, although the ‘final activity’ (the activity of the linear part of the reaction progress curve) was the same in both cases. This phenomenon was observed both in the presence and absence of fructose 1,6-diphosphate. High concentrations of both Mg2+free and MgATP2- diminish the difference in initial rate, between the ADP and phosphoenolpyruvate preincubated enzymes: Mg2+free by stabilizing the phosphoenolpyruvate-induced form; ATPMg2- by stabilizing the ADP-induced form. The magnitude of the difference in initial rates of the ADP- or phosphoenolpyruvate-preincubated enzyme is a function of both substrates. L4 pyruvate kinase(either from human liver or trypsin treated L′4 enzyme) does not, or to a very slight extent, show such behaviour. L′2L2 pyruvate kinase shows behaviour intermediate between L′4 and L4 enzymes. A model is proposed to describe the kinetic behaviour of L′4 and L4 enzymes

The effect of a varying Mg2+free concentration upon the kinetic behaviour of human liver L-type pyruvate kinase

We have proposed negative cooperativity for human liver L-type pyruvate kinase, a phenomenon also observed in: Lineweaver-Burk plots V⁻¹ versus [phosphoenolpyruvatel]⁻¹ were straight lines at low phosphoenolpyruvate concentrations, and bent downward near they axis (high phosphoenolpyruvate concentrations). Our conclusions were drawn from experiments performed at Mg²⁺total = 12 mM or 20 mM. However, phosphoenolpyruvate has been proposed to bind Mg²⁺free with a Keq = 5.5 X 1O⁻³ M⁻¹ at 25°C. So, at high phosphoenolpyruvate concentrations Mg²⁺free would be removed from the reaction mixture. We investigated the hypothesis that the phenomenon interpreted as negative cooperativity is in fact due to removing inhibitory Mg²⁺free from the reaction mixture at high phosphoenolpyruvate concentrations. Our results are in favour of this hypothesis

Acquired pyruvate kinase deficiency. The effect of maleic acid upon human erythrocyte pyruvate kinase

1. 1. Maleic acid is shown to be able to bind the thiol compound 2-mercaptoethanol. This is fully consistent with the data of Morgan and Friedman (1938). 2. 2. Human erythrocyte pyruvate kinase dissolved and quantitated in Trismaleate shows a loss of positive homotropic interactions, as compared to the same preparation in Tris-HCl. Hill coefficients (n) of n = 1.0−1.2 and n = 1,6−1.8 are obtained in Tris-maleate and Tris-HCl respectively. Half saturation [S]0.5 and Vmax remain unchanged. Pyruvate kinase in Tris-maleate is slightly more stable to heating at 60°C than in Tris-HCl. Incubation of the enzyme in Tris-maleate for one h with high concentrations of dithiotreitol restores the positive homotropic interactions. 3. 3. It is proposed, that the abnormalities of the pyruvate kinase of some patients with acquired pyruvate kinase deficiency, obtained from a study in Tris-maleate, may partly be induced by the buffer itself

The reaction mechanism of glutathione reductase from human erythrocytes

1. 1. The reaction mechanism of glutathione reductase (NAD(P)H:oxidized glutathione oxidoreductase, EC 1.6.4.2) was studied. By varying the GSSG concentration, a series of parallel lines was obtained in the 1/vvs 1/[NADPH] plots. NADP+ is a competitive inhibitor towards NADPH and a noncompetitive one towards GSSG. Both GSSG and NADPH were inhibitory at high concentrations. In the 1/v vs. 1/[GSSG] plots only parallel lines were obtained at low NADPH concentrations. The maximum velocity was dependent on the Na+ concentration. 2. 2. With NADH as electron donor, a series of converging lines was obtained in the 1/v vs. 1/[NADH] plots; at high [GSSG] activation was observed. The maximum velocity was not affected by the Na+ concentration. NADH+ did not inhibit these reactions. 3. 3. The results were interpreted in terms of a combination of simplified ordered bi-bi and ping-pong bi-bi mechanism and individual rateconstants were calculated.Opposite effects of the Na+ concentration on the reaction rate of GSSG with the NADPH- and the NADH-reduced enzyme were found. 4. 4. The influence of the temperature on the velocity of the NADPH dependent reaction in relation to different concentrations of buffer and GSSG led to either linear or nonlinear Arrhenius plots

The effect of copper on human erythrocyte glutathione reductase

1. 1. The influence of copper on purified human erythrocyte glutathione reductase (E.C. 1.6.4.2) was studied. The holoenzyme was inhibited at low oxidized glutathione (GSSG) concentrations. At a glutathione concentration of 1 mM and higher no inhibition at all was found. The inhibition was independent of the concentration of NADPH. 2. 2. When the holoenzyme was preincubated with copper much more inhibition was found. This inhibition could be prevented by adding oxidized glutathione to the incubation mixture. 3. 3. The recombination of the apoenzyme with FAD was inhibited by copper. This inhibition by copper was competitive with respect to FAD

Purification and properties of enolase of human erythrocytes

1. 1. Human erythrocyte enolase (2-phospho-D-glycerate hydrolyase, EC 4.2.1.11) was purified I000-fold. 2. 2. The pH-optimum was at pH 6.5. The molecular weight, estimated by gel filtration, was found to be 95,000 ± 5,000. 3. 3. Electrophoresis on agar-agarose at pH 8.5 and 6.4 showed only one band in hemolysate as well as in purified preparations. 4. 4. The enzyme was stabilized by magnesium and ammonium ions. 5. 5. The Km values for 2-phosphoglyceric acid at pH 6.5 and pH 7.2 were found to be the same: Km, (2-phosphoglyceric acid) = 28 μM. The optimal magnesium concentration was between 1 and 5 mM. 6. 6. Ca2+, at physiological concentrations, inhibited the enzyme. This inhibition depends on the Mg2+ concentration. 7. 7. The enzyme was inhibited by fluoride. This inhibition depends on magnesium, fluoride and phosphate concentration. Moreover the F− inhibition was dependent on 2-phosphoglyceric acid concentration in a competitive way

Purification and properties of glutathione reductase of human erythrocytes

1. 1. Human erythrocyte glutathione reductase (NAD(P)H: oxidized glutathione oxidoreductase, EC 1.6.4.2) was purified 47 000-fold by column chromatography. The enzyme contained FAD as the prosthetic group. From the flavin content a minimum mol. wt. of 56 600 was calculated. 2. 2. The mol. wt. was determined by gel filtration with Sephadex G-200 and was found to be 115 000 ± 4000. Blue dextran-2000 was not usable to determine the void volume of the column as it interacted with glutathione reductase. 3. 3. The Km value for FAD binding to be apoenzyme was temperature dependent. The Arrhenius plot of the rate of recombination was nonlinear. Restoration of full activity occurred at least through several steps. 4. 4. Preincubation of the apoenzyme with FMN retarded the restoration of the activity with FAD. Full restoration of the activity did not guarantee that the original enzyme conformation had been acquired

Hexokinase isozyme distribution and regulatory properties in lymphoid cells

The glycolytic enzyme hexokinase is studied in cultured leukemic lymphoblasts, in normal lymphocytes and in lymphoblasts obtained by stimulation of normal lymphocytes with phytohaemagglutinin. Hexokinase activity levels in cultured lymphoblasts and in normal lymphocytes are identical, but somewhat higher levels are found in stimulated lymphocytes. Cultured leukemic lymphoblasts differ in isozyme content in comparison to the other lymphoid cells. Besides hexokinase I, which is detected in all the lymphoid cells, they are characterized by the presence of hexokinase II. The concentration of type II increases during cell growth. Another difference between leukemic lymphoblasts and mature and stimulated lymphocytes is found in the regulatory properties of hexokinase I. Hexokinase I from both normal and stimulated lymphocytes is inhibited by glucose-1,6-diphosphate. This inhibition is decreased in part by addition of inorganic phosphate. Hexokinase I from leukemic lymphocytes, however, is inhibited to a lesser extent by glucose-1,6-diphosphate. Inorganic phosphate has no effect at all on this inhibition. In accordance with these findings a different pattern in the hexokinase I region was detected in electrophoresis with several cell types. The subisozyme hexokinase Ib, which appears to be the phosphate-regulated form, is predominant in lymphocytes, whereas it is present in a minor fraction in the cultured leukemic lymphoblasts. In these cells hexokinase Ic predominates