Nonradioactive assays of protein-tyrosine kinase activity using anti-phosphotyrosine antibodies.
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Antibodies↗
Biomedical subjects
G E Staal
Publications and source records attributed to G E Staal.
Partial characterization of protein tyrosine kinase activity in normal and leukemic human myeloid cells.
We have examined the expression of the protein tyrosine kinase (PTK) encoding oncogenes fes and abl in normal and malignant human myeloid cells in immunoblotting experiments. fes was markedly present in all cytosolic and most membrane fractions of normal and malignant cells. abl was only visible in normal cells, and occurred mostly in the cytosolic fractions. Molecular weights of identified proteins were different from the known products of fes and abl, possibly by alternative splicing at the mRNA level or by proteolysis. PTKs in myeloid cells were further purified by fast liquid protein chromatography (FPLC). PTK-activities of column fractions were assayed using a solid-phase non-radioactive dot-blot assay. Cytosolic and membrane fractions showed a FPLC pattern with a constant as well as a variable part in both normal and malignant cells, possibly indicative for PTKs with specialized functions in normal cell growth and transformation. Partial characterization of PTKs from different eluted peaks of AML-M4 blast cells demonstrated that PTKs from these peaks are kinetically distinct from each other.
Cell Membrane↗
Phosphorylation of pyruvate kinase and glycolytic metabolism in three human glioma cell lines.
Three cell lines established from human gliomas were found to differ in the capacity to phosphorylate the glycolytic enzyme pyruvate kinase in vitro. Phosphorylation in the glioblastoma cell line U-138 was more pronounced than in the glioma cell line Hs 683 and in the glioblastoma cell line A-172. All 3 cell lines showed similar pyruvate kinase isozyme patterns and expressed about 90% K-type and 10% M-type subunits. So, differences in pyruvate kinase phosphorylation could not be explained by differences in the availability of the appropriate substrate, being pyruvate kinase type K. As in gliomas, phosphorylation could specifically and almost completely be inhibited by fructose-1,6-bisphosphate. In order to investigate a potential physiological significance of the phosphorylation of pyruvate kinase, we have characterized these cell lines for several glycolytic parameters. In U-138 cells, the production of lactate appeared to be 2 times higher as compared with A-172 and Hs 683 cells under normal growth conditions and even 4 times higher under low glucose culture regime. The efflux of lactate correlated with the pyruvate kinase phosphorylation pattern in the cell lines. In none of the cell lines could the lactate production be stimulated by glutamine as additional energy source under low glucose culture conditions. The higher glycolytic flux in U-138 cells was not accompanied by higher glycolytic enzyme activities. The isozyme patterns of hexokinase, pyruvate kinase, aldolase, enolase and lactate dehydrogenase in the cell lines were nearly identical and resembled the patterns previously described for solid gliomas. However, the isozyme composition of phosphofructokinase in the cell lines differed from the situation in gliomas. While in gliomas the expression of L-type phosphofructokinase is favored, in the glioma cell lines, we found an increase in the expression of C-type subunits.
Adenosine Triphosphate↗
Phosphorylation of pyruvate kinase type K in human gliomas by a cyclic adenosine 5'-monophosphate-independent protein kinase.
In recent years, we reported the isozyme shift of pyruvate kinase from the M- toward the K-type in human neuroectodermal tumors. To investigate whether this shift enables phosphorylation of pyruvate kinase in these tumors, we studied 29 different specimens of human brain tumors for endogenous pyruvate kinase phosphorylation. While in normal human brain no phosphorylation of pyruvate kinase was detected, in all brain tumors pyruvate kinase became phosphorylated. There was no correlation between the extent of the pyruvate kinase phosphorylation and the histological classification and grading or the pyruvate kinase isozyme composition of the tumors. Only pyruvate kinase type K, and not type M, served as a substrate in the phosphorylation reaction. The phosphorylation of pyruvate kinase could be completely inhibited by addition of fructose 1,6-bisphosphate, a positive effector of pyruvate kinase type K; alanine, however, a negative effector, and phospho-enol-pyruvate, a substrate in the pyruvate kinase reaction, had no effect. While pyruvate kinase type L in liver is phosphorylated by a cyclic AMP-dependent protein kinase, the incorporation of phosphate into pyruvate kinase in human brain tumors appeared to be cyclic AMP independent and occurred exclusively on serine residues.
Biopsy↗
Diagnosis of pyruvate kinase deficiency in a transfusion-dependent patient with severe hemolytic anemia.
In a 2-yr old girl a hemolytic anemia was present since birth requiring multiple blood transfusions. Pyruvate kinase deficiency was suspected on the basis of a marginal enzyme activity, but could not be established due to the presence of massive numbers of donor cells in her peripheral blood. However, by density fractionation we succeeded in the isolation of a small fraction of the patient's own cells, in which a severe pyruvate kinase deficiency could be detected. In contrast hexokinase and glucose-6-phosphate dehydrogenase activities were extremely high, which is indicative that a very immature cell population is present in this fraction. In immunofluorescence studies a clear crossreaction was apparent with anti M2-type pyruvate kinase antibodies, whereas only a faint reaction with anti L-type could be detected. Despite the presence of a slight amount of L-type immunoreactive material, the residual activity in the patient's cell fraction could only be attributed to M2-type pyruvate kinase as was shown by cellulose acetate electrophoresis.
Anemia, Hemolytic↗
Interference of deoxyadenosine with transmembrane signaling events in human T lymphocytes.
Deoxyadenosine (dAdo) has been recognized as the toxic metabolite in the immunodeficiency disease associated with adenosine deaminase (ADA) deficiency. Under ADA deficient conditions, dAdo accumulates intracellularly as deoxyadenosine triphosphate (dATP) which by interference with ribonucleotide reductase, prevents DNA synthesis. Recently, we and others have demonstrated that in cells rendered ADA deficient by treatment with deoxycoformycin, dAdo affects T-cell activation events which precede DNA synthesis, such as interleukin 2 receptor (IL-2R) expression and IL-2 production. Here we have analyzed interference of dAdo with the early events of T-cell activation. It is shown that dAdo affects the mitogen induced phosphatidyl inositol turnover. Furthermore dAdo interferes with increase of intracellular calcium. Deoxycytidine, although capable of preventing intracellular accumulation of dATP, cannot reverse the functional consequences of dAdo treatment. The ability of a cell to increase its cytoplasmic free Ca2+, as induced by ionomycin, is not affected by dAdo. The exact target for this novel effect of dAdo is at the present unknown.
Adenosine Deaminase↗
Phosphofructokinase and pyruvate kinase in mouse embryonal carcinoma P19 cells in relation to growth and differentiation.
Two key enzymes of glycolysis, phosphofructokinase and pyruvate kinase, were studied in embryonal carcinoma cells (P19 EC cells) and three differentiated derivatives in relation to growth rate and differentiation state. The growth rates of P19 EC cells and its differentiated derivatives are positively correlated with both the specific activity of phosphofructokinase and the expression of the L-subunit of this enzyme. The specific activity of pyruvate kinase and its isozyme composition is not correlated with growth rate but seems to be correlated with the differentiation state of these cells. The decrease in specific activity of pyruvate kinase during differentiation of P19 EC cells induced by retinoic acid or dimethylsulfoxide preceded the shift from K- to M-type pyruvate kinase. In contrast to aggregates that were treated with dimethylsulfoxide, the specific activity of pyruvate kinase was reduced after aggregation in the presence of retinoic acid. Only after plating dimethylsulfoxide-treated aggregates again in the presence of dimethylsulfoxide, was a decrease in specific activity obtained. Both retinoic acid and dimethylsulfoxide are able to induce a K- to -M shift of pyruvate kinase.
Animals↗
Activity of glycolytic enzymes and glucose-6-phosphate dehydrogenase in smooth muscle proliferation.
The specific activity of hexokinase, phosphofructokinase, aldolase, enolase, pyruvate kinase and glucose-6-phosphate dehydrogenase was measured in 41 smooth muscle cell tumors: 20 leiomyomas and 21 cases of leiomyosarcoma. Statistical analysis revealed no significant differences in specific activity between normal smooth muscle tissue and the benign and malignant tumors originating from it. Quantification of the isozyme composition of pyruvate kinase showed a significant shift in isozyme pattern towards K-type subunits in leiomyosarcomas as compared to leiomyomas.
Fructose-Bisphosphate Aldolase↗
Activity of glycolytic enzymes and glucose-6-phosphate dehydrogenase in lipoblastic and neurogenic proliferations.
The alterations in specific activity and/or isozyme pattern of hexokinase, phosphofructokinase, aldolase, enolase, pyruvate kinase and glucose-6-phosphate dehydrogenase were studied in the tissue specimens of 26 patients with lipoblastic tumors and 28 patients with tumors of neurogenic origin. Although the biochemical data demonstrated that the activities of most enzymes studied were elevated in the specimens of the malignant tumors, only the differences in activity of hexokinase, pyruvate kinase and glucose-6-phosphate dehydrogenase measured between benign and malignant neurogenic tumors were significant. In malignant tumors, especially those of neurogenic origin, the isozyme pattern of pyruvate kinase showed a shift towards K-type subunits.
Adolescent↗
Hydrops fetalis associated with red cell pyruvate kinase deficiency.
A hydrops fetalis and multicystic encephalomalacia were diagnosed in a neonate who was one of twins. The co-twin had died 5 weeks prior to delivery. The most likely explantation for both hydrops and multicystic encephalomalacia was fetal anemia caused by a red cell pyruvate kinase deficiency, and aggravated by an intrauterine disseminated intravascular coagulation.
Erythrocytes↗
Pyruvate kinase deficiency and delayed clinical response to recombinant human erythropoietin treatment.
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Adult↗
Phosphofructokinase in alveolar type II cells isolated from fetal and adult rat lung.
The glycolytic enzyme 6-phosphofructokinase (EC 2.7.1.11) was studied in adult and fetal type II pneumocytes which had been isolated from rat lung at different days of development. In addition, the activities of the enzymes hexokinase (EC 2.7.1.1), enolase (EC 4.2.1.11) and pyruvate kinase (EC 2.7.1.40) were assayed. The specific activities of the latter enzymes decrease during perinatal development and reach about adult values shortly after birth. In contrast, 6-phosphofructokinase activity increases slightly until 2 days before birth, and drops sharply afterwards. The 6-phosphofructokinase subunit composition was determined in fetal and adult type II cells. The ratio of the three subunits of 6-phosphofructokinase in type II cells isolated on fetal days 19 and 21 (term is at day 22) and in adult type II cells was identical: the three subunits were present in a ratio of 68: 14: 18 for types L, M and C, respectively. In addition, we investigated some regulatory properties of 6-phosphofructokinase from alveolar type II cells. 6-Phosphofructokinase from alveolar type II cells is strongly inhibited by increasing MgATP concentrations. This inhibition is reflected by an increase in the S0.5 for fructose 6-phosphate. Fructose 2,6-bisphosphate stimulates alveolar type II 6-phosphofructokinase. Half-maximal stimulation occurs at 1.6 and 2.0 microM fructose 2,6-bisphosphate for fetal and adult type II cells, respectively. The level of the most potent positive effector of 6-phosphofructokinase, fructose 2,6-bisphosphate, was also determined. The level of the hexose bisphosphate decreases during prenatal development; however, the level in the adult type II cells is considerably lower. The concentration of fructose 2,6-bisphosphate appears to be sufficient to fully activate 6-phosphofructokinase both in fetal and adult type II cells.
Adenosine Triphosphate↗
Intracellular free magnesium and phosphorylated metabolites in hexokinase- and pyruvate kinase-deficient red cells measured using 31P-NMR spectroscopy.
The erythrocyte metabolism of two patients with nonspherocytic hemolytic anemia caused by a hexokinase deficiency, and a pyruvate kinase deficiency, respectively, were studied with NMR. The complexing of ATP and 2,3-diphosphoglycerate (2,3-DPG) with Mg2+ and hemoglobin (Hb) was determined using 31P-NMR on oxygenated and deoxygenated cells to investigate the influences of these enzyme defects on intracellular magnesium distribution and on Hb oxygen dissociation. In the pyruvate kinase-deficient red blood cells, the 2,3-DPG concentration was almost twice the normal value and the ATP concentration was near the lower limit of the normal range. In the hexokinase-deficient red cell population, the predominance of young cells masked the deficiency. Therefore, reticulocyte control cells were included in this study. In the oxygenated pyruvate kinase-deficient cells, the fraction of ATP that is complexed to magnesium as well as the free Mg2+ concentration were normal, despite the abnormal concentration of 2,3-DPG. In the deoxygenated cells the free Mg2+ concentration was lower than in normal cells. The fraction of Hb complexed with 2,3-DPG was higher than normal in both oxygenated and deoxygenated pyruvate kinase-deficient cells, in accordance with the high p50 of the oxygen-hemoglobin dissociation curve. In hexokinase-deficient cells, two major abnormalities are found: when the cells were deoxygenated, the concentration of ATP and 2,3-DPG fell. This was not observed for any other sample and could, therefore, be a consequence of the hexokinase deficiency. Despite almost normal levels of magnesium-binding metabolites, the free Mg2+ concentration in oxygenated and deoxygenated cels is much lower than in normal cells. This could be a cell-age-related phenomenon, since lower free Mg2+ concentrations were also found in reticulocyte control cells.
2,3-Diphosphoglycerate↗
Characterization of pyruvate kinase from human rhabdomyosarcoma in relation to immunohistochemical and morphological criteria.
We have compared the pyruvate kinase (PK) isoenzyme pattern of three rhabdomyosarcomas with foetal skeletal muscle tissue of 19 and 23 weeks of gestation, together with adult muscle in relation to immunohistochemical and morphological criteria. In foetal tissue of 19 weeks of gestation a focal immunopositivity for desmin and myoglobin was observed, whereas in tissue of 23 weeks an overall positivity for these proteins was present. Two of the three neoplasms were poorly differentiated and of the alveolar subtype. They were desmin immunoreactive. Some large spindle-shaped cells expressed myoglobin. The third one was more differentiated in microscopic characteristics and all cells showed immunoreactivity for desmin and myoglobin. In the foetal tissues five forms of pyruvate kinase isoenzymes were present with K2M2 as the predominant form. In adult muscle tissue only M4 was present. The tumors were characterized by a profound shift to the K-type, whereas the M4-type was not expressed at all. A difference in isoenzyme composition of pyruvate kinase was found between the morphologically less differentiated tumors and the more differentiated tumor; in the latter more M-subunits were expressed.
Adolescent↗
Tyrosine kinase activity in breast cancer, benign breast disease, and normal breast tissue.
Tyrosine specific protein kinase activity was determined in 70 specimens of the human mammary gland. These included 28 cancers of the breast, 21 benign breast diseases, and 21 normal breast tissues. We measured tyrosine kinase activity in the cytosol fraction and in the membrane fraction of the homogenates. In addition cytosolic aldolase activity was measured. Tyrosine kinase activity was determined using poly(glutamic acid:tyrosine = 4:1) as an artificial substrate. Cancers of the breast exhibited considerable higher tyrosine kinase activities in both cytosol and membrane fractions, compared to benign breast tumors (P less than or equal to 0.001). Benign tumors demonstrated increased activities in cytosol in comparison to normal breast tissues (P less than 0.001). Furthermore, there appears to be a strong association of an enhanced expression of activity of tyrosine kinase in cytosol of primary carcinomas and early systemic relapse. In combination with aldolase activity a nearly complete discrimination is achieved between malignant specimens on one hand and benign and normal tissues on the other.
Adult↗
Erythrocyte Na+/K+ ATPase activity measured with 23Na NMR.
A 23Na NMR assay for measurement of erythrocyte Na+/K+ ATPase activity is presented. Using the nonpermeant shift reagent dysprosium tripolyphosphate the signals of intra- and extracellular sodium are separated, enabling measurement of sodium fluxes nondestructively, without the need to physically separate the cells from their environment. By increasing membrane permeability with nystatin we have shown that the assay allows the detection of differences in membrane permeability. With low doses of nystatin the ouabain-sensitive sodium flux increased more than twofold. With high doses of nystatin the Na+/K+ pump could not prevent an almost total equilibration of intra- and extracellular sodium. All sodium that entered the cells remained NMR visible, proving that sodium influx can be measured quantitatively. 31P NMR spectra taken before and after the assay revealed a slight acidification of the cells and no significant change in ATP concentration. No evidence of Dy3+ entering the cell was observed.
Cell Membrane Permeability↗
The mechanism of deoxyguanosine mediated toxicity in proliferating human peripheral blood T lymphocytes.
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Binding Sites↗
A nonradioactive dot-blot assay for protein tyrosine kinase activity.
A new procedure for the assay of protein tyrosine kinase, based on the detection of phosphorylated tyrosyl residues by using monoclonal antibodies to phosphotyrosine, is described. After incubation of a protein tyrosine kinase sample with the substrates poly-(GluNa,Tyr)4:1 and unlabeled ATP an aliquot of the reaction mixture is transferred to a polyvinylidene difluoride membrane. The extent of tyrosine phosphorylation is measured by probing the membrane with antiphosphotyrosine antibody followed by detection by the immunogold silver staining procedure. The signal is quantified by densitometry. The assay is linear with time and is quantitative in a wide range of sample protein concentrations. Its sensitivity allows the kinetic characterization of protein tyrosine kinases at low substrate concentrations, whereas on the other hand the avoidance of radioactivity enables the use of high ATP concentrations as well. Protein tyrosine kinase activities of human breast carcinomas and normal breast tissues measured with this method correlated well with the conventional assay, in which the incorporation of [32P]phosphate is measured by TCA precipitation and liquid scintillation counting. Compared to the latter, the new assay is at least as sensitive and accurate and harbors the advantage of the avoidance of radioactivity, thus enabling one to perform a large number of protein tyrosine kinase assays simultaneously.
Adenosine Triphosphate↗