Kinetics of activation of the tyrosine kinase of a deletion mutant of epidermal growth factor receptor missing the sites of self-phosphorylation.

The addition of epidermal growth factor (EGF) to epidermal growth factor receptor (EGF receptor) dissolved in a solution of the detergent Triton X-100 results in the activation of its protein tyrosine kinase. To investigate the importance of the sites for self-phosphorylation on the enzyme in this process, the kinetics of activation of a deletion mutant missing the last 195 amino acids of the protein, including all of the sites for self-phosphorylation, were followed by monitoring the initial velocity at which the enzyme catalyzes the phosphorylation of the exogenous substrate RRKGSTAENAEYLRV. The activation of the enzymatic activity of this deletion mutant of EGF receptor displays kinetics that are second-order with respect to the concentration of the enzyme as does wild-type EGF receptor. The second-order rate constant for its activation is 36 +/- 10 microM-1 min-1, which is only 3-fold greater than the second-order rate constant for the activation of wild-type EGF receptor under the same conditions (13 +/- 2 microM-1 min-1). These results suggest that the mechanism by which the protein tyrosine kinase of the deletion mutant is activated is the same as that for the activation of the wild-type receptor and that the sites of self-phosphorylation in the wild-type EGF receptor do not participate in the mechanism of activation of the enzyme.

Self-phosphorylation of epidermal growth factor receptor is an intermolecular reaction.

The binding of epidermal growth factor (EGF) to epidermal growth factor receptor (EGF receptor) results in the dimerization and self-phosphorylation of the receptor. Both of these responses were followed as a function of time and the concentration of EGF receptor. Dimerization of EGF receptor was monitored by immunoblotting the protein after it had been cross-linked with glutaraldehyde. The capacity for self-phosphorylation was followed by measuring the relative level of incorporation of [32P]phosphate into EGF receptor on autoradiograms of the same immunoblots used for the assay of its dimerization. When these two properties were followed as a function of time, it was found that dimerization preceded the appearance of the capacity for self-phosphorylation. Both dimeric and monomeric forms of EGF receptor were self-phosphorylated in the presence of EGF, but the dimeric form was phosphorylated preferentially to the monomeric form. When the dimerization and the capacity for self-phosphorylation were followed as a function of the concentration of dimeric EGF receptor, it was observed that the self-phosphorylation of dimeric EGF receptor increased as the concentration of dimeric EGF receptor increased. An equation including terms representing both intramolecular and intermolecular rates of self-phosphorylation was fit to the plots of self-phosphorylation as a function of concentration of EGF receptor. These fits demonstrate that intramolecular self-phosphorylation within dimers of EGF receptor is insignificant and that self-phosphorylation is an intermolecular process between dimers of EGF receptor.

Insufficiency of self-phosphorylation for the activation of epidermal growth factor receptor.

Polyclonal immunoglobulins were produced against the carboxy terminus, -SEFIGA, of the receptor for epidermal growth factor (EGF). The addition of these immunoglobulins to a solution containing EGF receptor resulted in the activation of its protein tyrosine kinase. The levels of activation were assessed by measuring the initial velocities of the phosphorylation of the tyrosine in angiotensin II. The enzymatic activity induced by the immunoglobulins was significant, usually 50-70% of the maximum activity induced by EGF, and the induction occurred over a narrow range of concentration of the immunoglobulins. In order to achieve the activation, the immunoglobulins had to be bivalent; the addition of monovalent Fab fragments to EGF receptor did not produce any activation of the protein tyrosine kinase. The activation produced by the immunoglobulins was found to be reversible upon the addition of the synthetic peptide SEFIGA against which the immunoglobulins had been produced. Self-phosphorylation of the EGF receptor also occurred as the enzyme was activated by the immunoglobulins. Tryptic peptide maps demonstrated that the self-phosphorylation caused by the immunoglobulins had the same signature as that produced by EGF. When the synthetic peptide that had been used as the hapten was added to EGF receptor that had been self-phosphorylated in the presence of the immunoglobulins, the stimulated enzymatic activity was lost even though the protein remained phosphorylated. It follows from the results of deletion mutation [Walton, G. M., Chen, W. S., Rosenfeld, M. G., & Gill, G. N. (1990) J. Biol. Chem. 265, 1750-1754] and the results reported here that self-phosphorylation is neither necessary nor sufficient for the activation of EGF receptor.

Activation of epidermal growth factor receptor by epidermal growth factor.

The binding of epidermal growth factor (EGF) to epidermal growth factor receptor (EGF receptor) induces dimerization of the receptor and activation of its protein tyrosine kinase. Each of these three steps was followed as a function of the concentrations of EGF and of EGF receptor. Binding of EGF was followed by sedimentation of the complex between [3H]EGF and EGF receptor, dimerization was measured by quantitative cross-linking with glutaraldehyde, and the activation of the protein tyrosine kinase was monitored under the same conditions by following the initial velocity of the phosphorylation of peptides containing tyrosine. The binding of epidermal growth factor to its receptor was measured as a function of the concentration of epidermal growth factor, and the relationship was sigmoid with an average value of 1.7 for the Hill coefficient. Both dimerization and the activation of the tyrosine kinase displayed saturation as a function of the concentration of EGF. The ranges of the concentrations of EGF where dimerization and activation of the tyrosine kinase activity were half-maximal were 15-30 and 50-200 nM, respectively, but the value for the concentration of EGF at the half-maximum for the activation of the tyrosine kinase was a complex function of the concentration of EGF receptor. The observed behavior of the binding of EGF, the dimerization of EGF receptor, and the activation of the tyrosine kinase were used as criteria against which to test mechanisms for the process of activation. Equations were derived for various reversible and irreversible mechanisms and used to calculate the theoretical behaviors of the three properties. In direct comparisons of the experimental and the theoretical data, several of the previously proposed reversible and irreversible mechanisms for the activation of EGF receptor were found to be inadequate, but a reasonable mechanism was formulated that was compatible with the experimental data. In this mechanism, dimeric EGF receptor must be occupied by two molecules of EGF for enzymatic activity to be expressed.

Influence of dietary pectin on intestinal microfloral metabolism and toxicity of nitrobenzene.

Intestinal microfloral metabolism of nitrobenzene is essential for the production of methemoglobin. Since dietary pectin alters intestinal microflora, these studies were designed to examine the effects of dietary pectin on nitrobenzene-induced methemoglobinemia. Male Fischer-344 rats were fed either AIN-76A (purified diet containing 5% cellulose), AIN-76A with 5% pectin replacing the cellulose, or NIH-07 (cereal-based diet containing 8.4% pectin) for 28 days. Following this period, nitrobenzene (200 mg/kg) was administered by gastric intubation, and methemoglobin concentrations were determined after 1, 2, 4, 8, and 24 hr. Nitrobenzene-induced methemoglobinemia was evident as early as 1 hr, peaked at 4 hr, and diminished thereafter in rats fed NIH-07 diet. In contrast, nitrobenzene-induced methemoglobinemia was not detectable in rats fed AIN-76A; however, inclusion of 5% pectin in this diet resulted in methemoglobinemia comparable to that of NIH-07-fed animals at 4, 8, and 24 hr. Administration of 400 or 600 mg/kg nitrobenzene resulted in significant diet-related differences in methemoglobinemia. Administration of 600 mg/kg nitrobenzene to animals fed NIH-07 resulted in the highest methemoglobin concentrations (64 +/- 1%); those fed AIN-76A had the lowest (20 +/- 5%), and those fed AIN-76A containing pectin had intermediate methemoglobin concentrations (44 +/- 6%). No diet-related differences in the microbial population of the stomach or small intestine were observed. However, the number of anaerobes present in the ceca of rats fed AIN-76A containing pectin was 2 to 2.5 times greater than that of rats fed AIN-76A. In vitro reductive metabolism of [14C]nitrobenzene was significantly greater in the cecal contents of rats fed NIH-07 than that in the cecal contents of either of the groups fed the AIN-76A-based diets. These studies indicate that intestinal microfloral metabolism and red blood cell toxicity of nitrobenzene is markedly different in animals fed cereal-based versus purified diets. Furthermore, since inclusion of pectin into the purified diet diminishes the magnitude of these effects, differences in dietary composition of fermentable carbohydrates in cereal-based and purified diets may mediate differences in metabolism and toxicity of nitrobenzene.

An evaluation of a water purification system for use in animal facilities.

A commercially available water purification system was evaluated for its ability to minimize chemical and microbial contaminants. The reduction or removal of these impurities from the drinking water of experimental animals would reduce experimental variability. 3 strains of bacteria were collected from the processed water. An increase in the total number of bacteria was observed the longer the filters remained in use. Determinations of heavy metals in water samples before and after processing were made for lead, zinc, copper, nickel, manganese, iron, arsenic and mercury. Calcium and magnesium levels were also determined. The concentrations of these inorganic chemicals were reduced by the purification process except at 2 time points in which desorption of the chemical could have occurred. Bacterial colonization and desorption of these chemicals were controlled by installing new filter cartridges. Volatile halocarbon concentrations were determined for water samples before and after purification. All volatile halocarbons analyzed were less than 10 ppb before and after purification at all time points. Other organic chemicals were greatly reduced by the purification process. In a study of contaminants associated with installation of the unit, it was found that flushing the unit for 8 days reduced lead and methyl ethyl ketone concentrations to insignificant levels. The purification system was found to be effective in providing high quality drinking water as verified by a microbial and chemical testing program.

Effects of pectin-containing diets on the hepatic macromolecular covalent binding of 2,6-dinitro-[3H]toluene in Fischer-344 rats.

The influence of diets varying in pectin content on intestinal microfloral metabolic capacity of rats has been investigated as a possible mechanism for the alteration of toxicity of 2,6-dinitrotoluene (2,6-DNT) produced by these diets. Male F-344 rats were fed a purified diet (AIN-76A), AIN-76A plus 5% or 10% citrus pectin, or either of two cereal-based diets that vary in pectin content, NIH-07 or Purina Chow 5002. After 28 days, rats were given tritium-labeled 2,6-DNT (10 or 75 mg/kg po) and killed 12 hr later. Total hepatic macromolecular covalent binding (CVB) was determined by exhaustive extraction. The CVB of 2,6-DNT was found to be independent of diet at 10 mg/kg. However, at 75 mg/kg CVB was increased 40% by feeding 5% pectin in the purified diet and 90% by feeding 10% pectin in the purified diet. Animals fed Purina 5002 and NIH-07 had 135 and 150% higher CVB, respectively, than animals fed the purified diet alone and significantly greater CVB than animals fed the pectin supplemented diets. Elevated (two- to threefold) beta-glucuronidase and nitroreductase activities, microfloral enzymes proposed to be involved in the activation of 2,6-DNT to a toxicant, were found in the cecal contents of animals fed the pectin-containing diets which correlated with a two- to threefold increase in total number of cecal anaerobes. These results suggest that pectin-induced changes in microflora may enhance hepatoxicity after high doses of 2,6-DNT.

Influence of intestinal bacteria, sex of the animal, and position of the nitro group on the hepatic genotoxicity of nitrotoluene isomers in vivo.

The nitrotoluenes failed to induce unscheduled DNA synthesis in in vitro cultures of rat hepatocytes. Because intestinal bacteria are known to be involved in the metabolic activation of other nitroaromatic compounds, the genotoxicity of the nitrotoluenes was evaluated using an in vivo-in vitro hepatocyte DNA repair assay. 2-Nitrotoluene (2NT), 3-nitrotoluene, or 4-nitrotoluene was administered by gavage to male F344 rats. At selected times after treatment, primary hepatocyte cultures were prepared and incubated with [3H]thymidine, and unscheduled DNA synthesis was assessed by quantitative autoradiography. Corn oil controls ranged from -6 to -3 net grains/nucleus (NG). Only 2NT at 12 hr after treatment induced DNA repair (200 mg/kg: 15.4 NG). Twenty-four hr following treatment with 2NT, a 50-fold increase in the number of hepatocytes in S phase was observed and indicated that 2NT induces cell division in addition to DNA repair. To examine the influence of intestinal bacteria on the hepatic genotoxicity of 2NT, germ-free rats and germ-free rats inoculated with Charles River Altered Schaedler Flora were gavaged with 2NT. The cecal bacterial status was confirmed at sacrifice. 2NT did not induce DNA repair in germ-free animals (200 mg/kg: -3.8 NG), whereas DNA repair was induced in Charles River Altered Schaedler Flora-associated animals (200 mg/kg: 5.4 NG). When F344 females with conventional intestinal microflora were gavaged with 2NT and primary hepatocyte cultures were prepared, no unscheduled DNA synthesis was observed (200 mg/kg: -2.6 NG). Male and female F344 rats were shown to have similar populations of intestinal bacteria. These results demonstrate that the mononitrotoluenes display marked isomeric differences in their genotoxic potential, indicate the obligatory role of intestinal bacteria in the metabolic activation of 2NT, and show that the genotoxic potential of 2NT is dependent upon the sex of the animal under study.