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W Schoner

Publications and source records attributed to W Schoner.

At least 91 records · Page 5Linked 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↗
PubMed1985

Demonstration of an Mg2+-induced conformational change by photoaffinity labelling of the high-affinity ATP-binding site of (Na+ + K+)-ATPase with 8-azido-ATP.

8-Azido-ATP (8-N3ATP) is a substrate of (Na+ + K+)-ATPase from pork kidney and photoinactivates it by binding to the Mr = 100 000 alpha-subunit. The photoinactivation requires the presence of Mg2+ even though 8-azido-ATP is recognized by the high-affinity ATP binding site (Kd = 3.1 microM). K+ ions protect the enzyme against photoinactivation as does excess ATP. To see whether the Mg2+-requirement of the photoinactivation is due to the action of free Mg2+ or to the existence of an Mg X 8-azido-ATP complex, the action of the stable Mg X ATP complex analogue, chromium X 8-N3ATP (Cr X 8-N3ATP), was studied. Cr X 8-N3ATP photoinactivates (Na+ + K+)-ATPase in the absence of Mg2+, but the photoinactivation is enhanced by Mg2+, indicating that the formation of a Mg X ATP complex is an absolute requirement for photoinactivation. However, the interaction of Mg2+ with a low-affinity site also enhances the photoinactivation. It is therefore concluded that interactions with MgATP and free Mg induce conformational changes in the purine subsite of the high-affinity ATP binding site. Controlled trypsinolysis of the [alpha-32P]8-N3ATP-photolabelled enzyme in the presence of K+ results in the formation of an Mr = 56 000 radioactive peptide, whereas trypsinolysis of a [gamma-32P]Cr X ATP-labelled enzyme under identical conditions forms an Mr = 41 000 radioactive peptide. Extensive trypsinolysis of the [alpha-32P] 8-N3ATP-photolabelled alpha-subunit leads to the formation of a radioactive peptide of Mr = 1800.

Adenosine Triphosphate↗
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

Imidazole chloride and tris-chloride substitute for sodium chloride in inducing high-affinity AdoPP[NH]P binding to (Na+ + K+)-ATPase.

Optimal binding of [2,8-3H]AdoPP[NH]P to (Na+ + K+)-ATPase requires 25 mM Na+ (Cl-), 50 mM imidazole+ (Cl-) or 50 mM Tris+ (Cl-). Chloride is essential as counterion. We conclude that imidazole+ and Tris+ are able to bind to the Na+ site, and recommend the use of dilute buffers for studying the partial reactions of (Na+ + K+)-ATPase. In NaCl or the substituting buffers the dissociation constant for the enzyme-AdoPP[NH]P complex at 0 degrees C and pH 7.25 is 0.4 microM, whereas in millimolar MgCl2 it is about 2 microM. These distinct levels in affinity with MgCl2 as compared to NaCl, together with the MgCl2-dependence of photolabelling of the enzyme with ATP analogues (Rempeters, G. and Schoner, W. (1981) Eur. J. Biochem. 121, 131-137), suggest significant changes within the substrate site of (Na+ + K+)-ATPase upon binding of Mg2+ (Cl-)2.

Adenosine Triphosphate↗
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↗
PubMed1981

Evidence for a Mg2+-induced conformational change at the ATP-binding site of (Na+ + K+)-ATPase demonstrated with a photoreactive ATP-analogue.

1. The 3'-ribosyl ester of ATP with 2-nitro-4-azidophenyl propionic acid has been prepared and its ability to act as a photoaffinity label of (Na+ + K+)-ATPase has been tested. 2. In the dark 3'-O-[3-(2-nitro-4-azidophenyl)-propionyl]adenosine triphosphate (N3-ATP) is a substrate of (Na+ + K+)-ATPase and a competitive inhibitor of ATP hydrolysis. 3. Upon irradiation by ultraviolet light, N3-ATP photolabels the high-affinity ATP-binding site and is covalently attached to the alpha-subunit and an approximately 12000-Mr component. 4. Photolabeling of the alpha-subunit by N3-ATP irreversibly inactivates (Na+ + K+)-ATPase. 5. Photoinactivation is strictly Mg2+-dependent. Na+ enhances the inactivation. ATP or ADP and K+ protect the enzyme against inactivation. 6. Mg2+, in concentrations required for photoinactivation, protects (Na+ + K+)-ATPase against inactivation by tryptic digestion under controlled conditions. 7. It is assumed that a conformational change of the ATP-binding site of (Na+ + K+)-ATPase occurs upon binding of Mg2+ to a low-affinity site.

Adenosine Triphosphate↗
PubMed1980

Demonstration of two different reactive sulfhydryl groups in the ATP-binding sites of Ca2+-ATPase of sarcoplasmic reticulum by disulfides of thioinosine triphosphates.

1. The disulfide of thioinosine triphosphate, (SnoPPP)2, is a substrate of the Ca2+-pump and the Ca2+-ATPase of sarcoplasmic reticulum (Km = 400 microM). 2. Inactivation of Ca2+-ATPase by the beta,gamma-methylene diphosphonate analogue of the disulfide of thioinosine triphosphate, (SnoPP[CH2]P)2, in the presence of (Ca2+ + Mg2+ + K+) is preceeded by a dissociable enzyme inhibitor complex with a dissociation constant of 130 microM for a low-affinity binding site. ATP protected Ca2+-ATPase against the inactivation under these conditions with a dissociation constant of 140 microM. 3. Kinetic analysis of the inactivations of Ca2+-ATPase by (SnoPP[CH2]P)2 in the absence of Ca2+ and Mg2+ but the presence of K+ and EGTA led to the appearance of two nucleotide binding sites with two different inactivation velocities. Inactivation rate constants k2 were found for the rapid inactivating part (k2' = 1.44 X 10(-2) s-1) and the slow inactivating part (k2" = 1.15 X 10(-3) s-1). From the protective effect of ATP under these conditions a high-affinity (Kd = 48.78 microM) and a low-affinity ATP binding site (Kd = 114 microM) were apparent. 4. The affinity of the analogues to the enzyme is decreased in the sequence: (SnoPPP)2 > (SnoPP[NH]P)2 > (SnoPP[CH2]P)2 > (SnoP)2. 5. (SnoPPP)2-inactivated Ca2+-ATPase was reactivated by incubation with dithiothreitol. 6. Inactivation of Ca2+-ATPase by [gamma-32P](SnoPPP)2 in the presence of (Mg2+ + K+ + Ca2+) or (EGTA + K+) was accompanied by the incorporation of hydroxylamine-insensitive radioactivity into the acid-precipitable protein. The enzyme-bound [gamma-32P]SnoPPP was cleaved by dithiothreitol. 7. It is concluded that (SnoPPP)2 and its non-hydrolyzable analogues (SnoPP[NH]P)2 and (SnoPP[CH2]P)2 act as ATP affinity labels and form mixed disulfides with a sulfhydryl group within the active site.

Adenosine Triphosphate↗
PubMed1980

Inactivation of (Na+ + K+)-ATPase by chromium(III) complexes of nucleotide triphosphates.

(Na+ + K+)-ATPase from beef brain and pig kidney are slowly inactivated by chromium(III) complexes of nucleotide triphosphates in the absence of added univalent and divalent cations. The inactivation of (Na+ + K+)-ATPase activity was accompanied by a parallel decrease of the associated K+-activated p-nitrophenylphosphatase and a parallel loss of the capacity to form, Na+-dependently, a phosphointermediate from [gamma-32P]ATP. The kinetics of inactivation and of phosphorylation with [gamma-32P]CrATP and [alpha-32P]CrATP are consistent with the assumption of the formation of a dissociable complex of CrATP with the enzyme (E) followed by phosphorylation of the enzyme: formula: (see text). The dissociation constant of the CrATP complex of the pig kidney enzyme at 37 degrees C was 43 microM. The inactivation rate constant (k + 2 = 0.033 min-1) was in the range of the dissociation rate constant kd of ADP from the enzyme of 0.011 min-1. The phosphoenzyme was unreactive towards ADP as well as to K+. No hydrolysis of the native isolated phosphoenzyme was observed within 6 h under a variety of conditions, but high concentrations of Na+ reactivated it slowly. The capacity of the Cr-phosphoenzyme of 121 +/- 18 pmol/unit enzyme is identical with the capacity of the unmodified enzyme to form, Na+-dependently, a phosphointermediate. The Cr-phosphoenzyme behaved after acid denaturation like an acylphosphate towards hydroxylamine, but the native phosphoenzyme was not affected by it. ATP protected the enzyme against the inactivation by CrATP (dissociation constant of the enzyme ATP complex = 2.5 microM) as well as low concentrations of K+. CrATP was a competitive inhibitor of (Na+ + K+)-ATPase. It is concluded that CrATP is slowly hydrolyzed at the ATP-binding site of (Na+ + K+)-ATPase and inactivates the enzyme by forming an almost non-reactive phosphoprotein at the site otherwise needed for the Na+-dependent proteinkinase reaction as the phosphate acceptor site.

Adenosine Triphosphate↗
PubMed1979

Immunohistochemical localization of pyruvate kinase isoenzymes in chicken tissues.

A method for the localization of pyruvate kinase isoenzymes type L, M2 and M1 in tissue sections is described. Mono-specific antibodies directed against isoenzymes of pyruvate kinase from chicken and the peroxidase antiperoxidase method were used. The following preferential localizations of the isoenzymes in chicken tissues were observed: Pyruvate kinase M1 was found in skeletal muscle. The white muscle fibers were more intensely stained than the red. Some dark muscles (e.g., anterior latissimus dorsi) and the heart muscle showed no reaction with antiserum against pyruvate kinase M1. Pyruvate kinase type L was found in the hepatocytes and in kidney cortex. Pyruvate kinase type M2 was seen in the distal tubules of kidney, in hepatocytes and sinusoidal cells in liver, in lung, adipose tissue, and in the spleen mainly in the bursa dependent areas. Pyruvate kinase type M2 was detected in high concentrations in the granulation tissue of regenerating liver after partial hepatectomy. Liver sections of a hen bearing a pancreatic tumor showed an unusually high content of pyruvate kinase type M2 in some hepatocytes, which were each clustered to spots in the liver parenchyma. Thus, contrary to previous reports, the tissue distribution of isoenzymes in chicken is similar to that of other vertebrates.

Animals↗
PubMed1979

Modification of pyruvate kinase activity by proteins from chicken liver.

The partial purification of a protein fraction inhibiting pyruvate kinase isoenzymes is described. The fraction was isolated from the (NH4)2SO4 step of the purification procedure for pyruvate kinase isoenzymes from chicken liver (Eigenbrodt, E. & Schoner, W. (1977) Hoppe-Seyler's Z. Physiol. Chem. 358, 1033-1046) by extraction with 1N NaOH, acidification to pH 3, ethanol precipitation and chromatography of the supernatant on DEAE-cellulose. The inhibitor fraction was further purified by disc gel electrophoresis using a gel gradient from 10 to 25%; this procedure separated activating proteins from the inhibitor fraction. The inhibitor fraction inhibited the pyruvate kinase isoenzymes from chicken in the sequence of decreasing effect: M2 greater than L greater than M1. The inhibition was due to a decrease in the affinity for phosphoenolpyruvate. The inhibitor is stable against heating for 5 min in 1% sodium dodecyl sulfate at 100 degrees C; it is destroyed by pepsin digestion. The inhibitor fraction could be purified further only by dodecyl sulfate gel electrophoresis. This resulted in the separation of 2 inhibitors (Mr = 33,500 +/- 8500 and ca. 5000), an activator (Mr = 15,100 +/- 5200), and an unidentified protein (Mr = 27,000).

Animals↗