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Biomedical subjects

E Eigenbrodt

Publications and source records attributed to E Eigenbrodt.

At least 55 records · Page 3Linked to original sources
PubMed1986

Purification of pyruvate kinase isoenzymes type M1 and M2 from dog (Canis familiaris) and comparison of their properties with those from chicken and rat.

Pyruvate kinase isoenzymes type M1 and M2 from dog muscle, lung and tumor have been isolated. The K0.5 for phosphoenol pyruvate have been determined to be 0.04 mM for dog muscle type M1, 0.24 mM for lung type M2 and 0.28 mM for tumor type M2 isoenzymes. The activator constant ka of L-serine is 240 nM from lung and 70 nM from tumor isoenzyme. Consistent with the assumption of a special form of pyruvate kinase M2 in dog tumor cells different isoelectric points and amino acid compositions have been found for the isoenzymes of lung and tumor.

Amino Acids↗
PubMed1986

Pyruvate kinase isoenzymes in altered foci and carcinoma of rat liver.

Pyruvate kinase (PK) isoenzymes, rate limiting for the last steps of glycolysis, were studied in normal rat liver, putative preneoplastic foci, neoplastic nodules and hepatocellular carcinoma. These lesions were produced by an initiation-promotion protocol: treatment with a single dose of N-nitrosomorpholine (NNM) was followed by feeding diets containing phenobarbital (PB) or alpha-hexachlorocyclohexane (alpha-HCH), or basal diet. PK was demonstrated (i) by immunocytochemistry on histological sections with antibodies specifically directed against the L and M2 isoenzymes, (ii) by electrophoretic separation of isoenzymes in homogenates from liver and larger tumors, and (iii) by electrophoretic separation of isoenzymes in parenchymal and stromal cells isolated from liver and tumors. Immunocytochemistry showed decreases of L-PK (L-PK-) in hepatocytes of most of the foci, nodules and carcinomas. Most L-PK- foci showed increases in gamma-glutamyltransferase (gamma-GT) and epoxide hydrolase (EH). PB or alpha-HCH treatment further decreased expression of L-PK in foci, but not in normal liver. Cells and foci with enhanced L-PK (L-PK+) were also found after carcinogen treatment. These did not show increases of gamma-GT or EH or any distinct morphological alterations with the exception of some which were basophilic ('tigroid') in H and E stained sections. No L-PK+ tumors were found. We could not demonstrate the M2-type PK in parenchymal cells of liver or any of the lesions described above. This isoenzyme was restricted to stromal cells in normal rat liver and in all stages of carcinogenesis as shown by immunohistology and by electrophoresis of preparations from isolated cell populations. However, stromal cells from hepatocellular carcinomas exhibited a 3-fold increase of M2-PK compared with stromal cells from normal liver. These results do not support an isoenzyme shift from L to M2-PK in the course of malignant transformation of hepatocytes as suggested previously.

Animals↗
PubMed1986

Carbohydrate metabolism in neoplastic tissue.

The kinetic and immunological properties of purified, homogeneous pyruvate kinase type M2 from chicken lung and tumors, including that of Rous sarcoma virus transformed chicken fibroblasts, have been compared. The type M2 enzymes from both lung and tumors have immunologically distinct structures. The enzyme isolated from tumors is characterized by a low affinity for phosphoenol pyruvate, pronounced L-serine activation and a strong inhibition by L-alanine, L-proline and several other amino acids. The chicken lung enzyme is only slightly affected by serine. The cellular amino acid concentration is in such a range that the tumor enzyme is strongly inhibited whereas the lung enzyme is only partially inhibited. The characteristics of the tumor enzyme allow the optimal adaptation to lactic acid and production of energy from glucose or amino acids dependent on substrate and oxygen supply.

Amino Acids↗
PubMed1985

False-positive results in the radioimmunoassay detection of stilbene derivatives after administration of fluorinated corticosteroids to animals.

During routine inspections, indications were found that the therapeutic use of fluocortolone or dexamethasone can cause false-positive results in the detection of stilbene derivatives by radioimmunoassay (RIA) in samples of animal origin. After experimental application of the fluorinated corticosteroids to rabbits or calves, the assumed false-positive RIA results were confirmed. The kinetics after application of dexamethasone were concomitant with the elimination of radioactive [3H]dexamethasone. Furthermore, it could be demonstrated that [3H]dexamethasone was transformed in the faeces of rabbits and calves. The metabolites formed were not completely separated by the extraction procedure used for the RIA and seemed to be responsible for the false-positive results.

Animals↗
PubMed1984

Fructose 1,6-bisphosphatase in the diagnosis of chronic hepatitis. I. Activity measurements of fructose 1,6-bisphosphatase in human serum.

In this communication, we propose a method for the determination in human serum of fructose 1,6-bisphosphatase based on parallel measurements of enzyme activities in presence of 1-p-bromotetramisole oxalate and adenosine 5'-monophosphate. The employment of these specific inhibitors renders the discrimination between specific and non-specific activities feasible. A regression analysis identifies fructose 1,6-bisphosphatase (EC 3.1.3.11) as the origin of the specific and alkaline phosphatase (EC 3.1.3.1) as the source of the non-specific fructose 1,6-bisphosphate dephosphorylating activities. This procedure lends itself to the diagnosis using serum samples of 'piecemeal' necrosis in liver disease.

Adenosine Monophosphate↗
PubMed1984

Fructose 1,6-bisphosphatase in the diagnosis of chronic hepatitis. II. Classification of chronic hepatitis based on fructose 1,6-bisphosphatase and other laboratory data.

The histomorphology of typical liver cell necroses are here correlated with heterotope distributions of enzymes in liver parenchyma. A variety of findings indicate a congruence between gluconeogenetic areas of the liver and the typical pattern of 'piecemeal' necrosis. We therefore propose a diagnostic index based on fructose 1,6-bisphosphatase activity and the data from the clinical laboratory. This index makes it possible to distinguish between chronic persistent and chronic aggressive hepatitis.

Acute Disease↗
PubMed1983

Pyruvate kinase type M2 is phosphorylated in the intact chicken liver cell.

Isolated hepatocytes from 24 h starved chicks were depleted of phosphate and incubated in the presence of 32P-orthophosphate. Pyruvate kinase type M2 from crude cell extracts was partially purified by chromatography on DEAE-Sephacel and hydroxylapatite. SDS slab gel electrophoresis of the fractions containing the enzyme and immunoprecipitation with antisera showed the phosphorylation of pyruvate kinase type M2. Phosphoamino acid analysis identified serine as phosphate acceptor.

Amino Acids↗
PubMed1983

Influence of transformation by Rous sarcoma virus on the amount, phosphorylation and enzyme kinetic properties of enolase.

Using chicken embryo fibroblasts infected with the NY68 transformation-defective temperature-sensitive mutant of Rous sarcoma virus, the phosphorylation and enzyme kinetic properties of enolase have been studied before, and at different stages after, the onset of transformation. A method for purification of enolase was developed, which minimized dephosphorylation. Two enolase (EC 4.2.1.11) isoenzymes were separated by isoelectric focussing revealing that it was the gammagamma form (pI 5.2-6.7) which had become phosphorylated at tyrosine residues after transformation. The phosphorylation of enolase in tyrosine occurred slowly after shift to the permissive temperature, rising from undetectable levels in phenotypically normal cells, to < 10% of the total phosphoamino acid after 3 h, and reaching 30-50% of the total phosphoamino acid by 16 h. Interestingly, the fraction of phosphorylated enolase molecules declined during transformation from 8% in normal cells to 5% by 16 h after temperature shift, due to a 3- to 5-fold increase in the total amount of enolase present in the transformed cultures. Although transformation had no apparent effect on the K0.5 of enolase (26 +/- 4 microM for 2-phosphoglycerate), its specific activity was reduced by about one third.

Animals↗
PubMed1983

Glucagon induced inactivation of phosphofructokinase and its counteraction by insulin in isolated hepatocytes from the domestic fowl (Gallus domesticus).

The effects of glucagon and insulin on phosphofructokinase activity in isolated chicken hepatocytes were studied. Phosphofructokinase activity was decreased in extracts of hepatocytes exposed to glucagon both at subsaturating (0.2 mM) and saturating (5 mM) concentrations of fructose 6-phosphate. Both effects were still present after Sephadex G-25 gel filtration and subsequent ammonium sulfate precipitation. Half-maximal effects of glucagon were found between 10(-11) and 10(-10) M glucagon. Insulin alone had no effect but decreased the action of glucagon.

Animals↗
PubMed1983

Structural and kinetic differences between the M2 type pyruvate kinases from lung and various tumors.

The kinetic and structural properties of purified, homogeneous pyruvate kinase type M2 from chicken lung and tumors, including that from Rous sarcoma virus-transformed chicken fibroblasts, have been compared. The "tumor enzyme" is characterized by a low affinity for phosphoenolpyruvate, pronounced serine activation, and strong alanine inhibition as compared to the "lung type". In contrast to the rat lung enzyme, which is not affected by serine, the chicken lung enzyme is slightly activated by serine. The serine metabolites phosphoserine and glycine do not activate the "tumor type M2 pyruvate kinase", but L-alpha-glycerophosphorylcholine and phosphatidylserine do slightly activate at physiological concentrations. Studies with substances structurally related to serine reveal that the hydroxyl group of serine is a prerequisite for the activation and that the amino and carboxyl groups determine the affinity of the "tumor type M2 pyruvate kinase" for serine. Two different fragmentation methods (CNBr-cleavage and V-8 proteolysis) and two different methods for separation of the resulting peptide fragments (polyacrylamide gel isoelectric focussing and SDS-polyacrylamide electrophoresis) indicated a high degree of homology between the type M2 pyruvate kinases from lung and tumors as well the type M1 from muscle. Each type of pyruvate kinase, however, contains one or two unique protein fragments which are characteristic for its type. We have termed those fragments L (lung), M (muscle), and T (tumor).

Animals↗
PubMed1982

Purification and properties of pyruvate kinase type M2 from rat lung.

(1) Pyruvate kinase type M2 from rat lung has been purified 840-fold with an overall yield of 20%. The enzyme gave a single band upon SDS-electrophoresis and isoelectrofocusing and had a specific activity of 1340 U/mg protein. The homotetramer of Mr = 224000 and an isoelectric point of pH 5.8 had an amino acid composition closely resembling that of other pyruvate kinase isoenzymes type M2, except that of the chicken liver. The enzyme was crystallized. (2) The enzyme has its pH optimum at pH 6.5. The K0.5 value for phosphoenolpyruvate is 0.26 mM (nH = 1.81) which decreases in the presence of 0.2 mM fructose 1,6-bisphosphate to 0.056 mM (nH = 1.06). 1 microM fructose 1,6-bisphosphate activates the enzyme at 0.1 mM phosphoenolpyruvate half-maximally. The Km value for ADP at 1 mM phosphoenolpyruvate is 0.4 mM. The Km value for other nucleoside diphosphates increases in the order ADP less than GDP less than IDP less than UDP. (3) No evidence for an interconversion of pyruvate kinase type M2 from rat or chicken lung was found. The enzyme was neither a substrate for the cAMP-dependent protein kinase from rabbit muscle nor for the cAMP-independent protein kinase from chicken liver. Since pyruvate kinase type M2 from chicken liver is inactivated by phosphorylation catalyzed by a cAMP-independent protein kinase (Eigenbrodt, E., Abdel-Fattah Mostafa, M. and Schoner, W. (1977) Hoppe-Seyler's Z. Physiol. Chem. 358, 1047-1055) we suggest that the interconvertible form of pyruvate kinase type M2 may represent a separate form of the pyruvate kinase type M2 family.

Amino Acids↗
PubMed1982

Inactivation of phosphofructokinase in chicken hepatocytes by epinephrine.

The effects of epinephrine on phosphofructokinase activity in hepatocytes from fed chickens were studied in crude extracts and after partial purification. The mechanism of epinephrine action was elucidated by use of phentolamine and penbutulol. Phosphofructokinase activity was decreased in extracts of cells exposed to epinephrine when measured at sub-saturating as well as saturating concentrations of fructose 6-phosphate. This effect was still present after filtration through Sephadex G25 gel and a subsequent precipitation with ammonium sulfate. Half-maximal effects of epinephrine were found in a concentration range between 10(-8) and 10(-7) M epinephrine. The effect of epinephrine was unaffected by Ca2+ or the alpha-adrenergic antagonist phentolamine, and was abolished by the beta-adrenergic antagonist penbutulol. We assume that the effects of epinephrine on the activity of chicken liver phosphofructokinase are mediated by a cAMP-dependent mechanism which cannot be explained by concentration shifts of low-molecular-mass substances such as fructose 2,6-bisphosphate.

Animals↗
PubMed1982

Cytosolic malic dehydrogenase activity is associated with a putative substrate for the transforming gene product of Rous sarcoma virus.

A cellular protein of apparent Mr 34,000--36,000 was suggested as a possible physiological substrate for the protein kinase (EC 2.7.1.37) activity associated with the transforming gene product of Rous sarcoma virus. We find this protein to migrate with an apparent Mr of 38,000 in NaDodSO4/polyacrylamide gels. It was not separable from cytosolic malic dehydrogenase activity when purified by chromatography on DEAE-Sephacel, hydroxylapatite, poly(A)-Sepharose, and blue Sepharose, by gel filtration, and by isoelectric focusing. The Mr 38,000 protein as well as cytosolic malic dehydrogenase activity focused with a pI of 7.5. In gel filtration experiments, both displayed an apparent native Mr of 68,000. The male dehydrogenase activity contained in homogeneous preparations of the Mr 38,000 protein had a specific activity of up to 130 units/mg of protein. The recovery of the enzyme was 5--10% of the activity in the extract. Antiserum against the Mr 38,000 protein inactivated the malic dehydrogenase activity associated with the Mr 38,000 protein.

Animals↗
PubMed1981

Quercetin inhibits tyrosine phosphorylation by the cyclic nucleotide-independent, transforming protein kinase, pp60src.

The bioflavonoid quercetin is a potent inhibitor of a cyclic nucleotide-independent, tumor virus-coded protein kinase which phosphorylates tyrosine residues and acts as a cellular transforming protein. Half-maximal inhibition of the protein kinase occurred at 3-4 microM quercetin whereas rutin was much less effective. The finding, that quercetin inhibits a cyclic nucleotide-independent protein kinase activity, may provide clues to the diverse pharmacological effects of the bioflavonoids.

Cyclic AMP↗