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S Klumpp

Publications and source records attributed to S Klumpp.

53 records · Page 3Linked to original sources
PubMed1990

Cyclic GMP levels and guanylate cyclase activity in pheromone-sensitive antennae of the silkmoths Antheraea polyphemus and Bombyx mori.

Female sex pheromones applied to freshly isolated, living antennae of male Antheraea polyphemus and Bombyx mori led to an increase of cGMP. A 1:1 mixture of 2 pheromone components of Antheraea polyphemus blown for 10 sec in physiological concentrations over their antennal branches raised cGMP levels about 1.34-fold (+/- 0.08 SEM, n = 23) from a basal level of 3.0 +/- 0.6 (SEM, n = 20) pmol/mg protein. Similarly, bombykol elicited a 1.29-fold (+/- 0.13 SEM, n = 23) cGMP increase in antennae of male Bombyx mori from a basal level of 2.7 +/- 0.5 (SEM, n = 24) pmol/mg protein. No cross-sensitivity was found with respect to pheromones from either species. In antennae of female silkmoths, the cGMP response was missing upon stimulation with their own respective pheromones according to the known lack of pheromone receptor cells in the female. cAMP levels in the male antennae of 14.2 +/- 2.9 (SEM, n = 4) pmol/mg protein in A. polyphemus and 15.0 +/- 3.0 (SEM, n = 5) pmol/mg protein in B. mori were not affected by pheromone stimulation. Within 1-60 sec, the extent of cGMP increase in B. mori was independent of the duration of pheromone exposure. The levels of cGMP in pheromone-stimulated antennae of both species remained elevated for at least 10 min, i.e., much longer than the duration of the receptor potential measured in single-cell recordings. Guanylate cyclase activity was identified in homogenates of male and female antennae from both species. The Km of the guanylate cyclase from male B. mori for the preferential substrate MnGTP was 175 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

An improved procedure for identifying and quantitating protein phosphatases in mammalian tissues.

The type 2A protein phosphatases in mammalian tissue extracts are inhibited completely and specifically by 1-2 nM okadaic acid. In contrast, type 1 protein phosphatases are hardly affected at these concentrations, complete inhibition requiring 1 microM okadaic acid. These observations have been exploited to develop an improved procedure for the identification and quantitation of type 1, type 2A and type 2C protein phosphatases in tissue extracts.

Adrenal Medulla↗
PubMed1989

Pyrrolo[2,3-d]pyrimidines as inhibitors of cAMP-phosphodiesterase. Structure-activity relationship.

The effects of pyrrolo[2,3-d]pyrimidine compounds (7-desazapurines) on cAMP hydrolyzing, calmodulin dependent and calmodulin independent phosphodiesterase were studied. Phosphodiesterase inhibition depended on the chemical nature of substituents attached to the pyrrolo-pyrimidine-nucleus at positions 2, 4, 5, 6 and 7. Among a total of 28 compounds tested, the 4-amino-7-phenyl-7H-pyrrolo[2,3-d]pyrimidine-5,6-dicarbaldehyde (9) was the most potent inhibitor of phosphodiesterase activity (IC50 = 16 microM). In addition to the 5,6-disubstitution, position 2 of the pyrrolo-pyrimidine derivatives had to be unsubstituted and position 4 had to bear an amino-group for an optimal inhibitory effect. Calmodulin dependent and calmodulin independent isozymes were affected to the same extent. Inhibition of PDE activity was reversible upon removal of the pyrrolo-pyrimidine derivative 9 and non-competitive with respect to cAMP (Ki = 27 microM).

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1989

A possible role of plasma glutathione in glucose-mediated insulin secretion: in vitro and in vivo studies in rats.

In isolated rat pancreatic islets exogenous glutathione which is not able to penetrate into cells, augmented glucose (11.1 mmol/l)-mediated insulin release. In the presence of a non-stimulatory glucose concentration (2.8 mmol/l) glutathione had no effect. The half-maximal synergistic action of glutathione on insulin secretion was observed at approximately 8.0 mumol/l. This concentration of glutathione is similar to that found in the plasma of non-fasted anaesthetised rats (6.5 mumol/l). Oral ingestion of glucose increased the arterial plasma glutathione in rats. Our data provide for the first time indirect evidence for a modulating effect of plasma glutathione in postprandial glucose-mediated insulin secretion which appears to be located at the extracellular site of islet cells.

Animals↗
PubMed1987

Ionic regulation of adenylate cyclase from the cilia of Paramecium tetraurelia.

The kinetics of the ionic regulation of an adenylate cyclase associated with the excitable ciliary membrane from Paramecium tetraurelia was examined. Glycerol (30%, v/v) stabilized the enzyme, and activated by an increase in Vmax. (3-fold) and a decrease in the apparent Km for MgATP (6-fold). Kinetic analysis of Mg2+ effects showed a stimulation via a single metal-binding site separate from the substrate site, with a dissociation constant, Ks, of 0.27 mM. Analysis of Ca2+ effects showed (i) an uncompetitive inhibition with respect to substrate MgATP, and (ii) dependence of the extent of inhibition on the free Mg2+ concentration. Ki values ranged from 4 to 130 microM-Ca2+ in the presence of 0.55-2 mM-Mg2+ respectively. This indicates competition between Mg2+ and Ca2+ at the metal-binding site. The Ca2+ effect was specific; Sr2+ and Ba2+ were almost without effect, and 100 microM-Ba2+ did not interfere with the Ca2+ inhibition. The actions of Ca2+ were readily reversible after addition of EGTA. K+ activated the adenylate cyclase at concentrations around 20 mM. The stimulatory potency of K+ was dependent on the free Mg2+ concentration. At 1 mM free Mg2+, 20 mM-K+ doubled the adenylate cyclase activity. The inhibitory Ca2+ and stimulatory K+ inputs were independent of each other.

Adenosine Triphosphate↗
PubMed1987

Effect of tryptic calmodulin fragments on guanylate cyclase activity from Paramecium tetraurelia.

Tryptic bovine brain calmodulin fragments 1-77 or 1-106 reactivated La-inactivated ciliary guanylate cyclase from Paramecium dose-dependently up to 60%. They were 20-fold less potent compared to bovine brain calmodulin. Fragment 78-148 was even less active. Concomitant addition of fragments 1-77 and 78-148 had no additive effect. Genetically engineered calmodulin lacking a blocked amino terminus and trimethyllysine at position 115 reactivated La-treated guanylate cyclase as good as bovine brain calmodulin. After detergent solubilization of La-inactivated guanylate cyclase intact bovine brain calmodulin and calmodulin fragments 1-77 and 78-148 were equipotent. 80% Reactivation was obtained with 40 microM of either fragment.

Animals↗
PubMed1983

Calcium/calmodulin-regulated guanylate cyclase and calcium-permeability in the ciliary membrane from Tetrahymena.

A guanylate cyclase of high specific activity was localized in the ciliary membrane from Tetrahymena pyriformis. Purity of cilia was checked by electron microscopy and purity of membrane fractions isolated by a sucrose density gradient by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Enzyme activity was due to the presence of endogenous calmodulin as evidenced by the inhibition of guanylate cyclase by addition of antiserum against calmodulin from Tetrahymena or soybean. Removal of endogenous calmodulin by La3+-treatment of ciliary membranes resulted in loss of guanylate cyclase activity. In addition to protozoan calmodulins, the original activity could also be restored by the nonhomologous calmodulins from soybean and pig brain but not by calcium-binding proteins like Dictyostelium calmodulin, parvalbumin, and troponin C, lacking the trimethyllysine characteristic for mammalian calmodulins. However, only calmodulins from the protozoans Tetrahymena and Paramecium stimulated guanylate cyclase activity in excess of the initial activity. This indicates that the guanylate cyclase either contains two binding sites for calmodulin with different specificities or that a single, but only partially occupied binding site is modified possibly by hydrolytic exo-proteases during membrane preparation. The ciliary membrane from Tetrahymena contains a discrete calcium-permeability as demonstrated by calcium-flux measurements using the calcium indicator dye arsenazo III. In analogy to the excitable ciliary membrane of the larger relative Paramecium, the ciliary membrane of Tetrahymena may thus carry the voltage-sensitive calcium-channels known from electrophysiological studies.

Animals↗
PubMed1983

Calcium/calmodulin-regulated guanylate cyclase of the excitable ciliary membrane from Paramecium. Dissociation of calmodulin by La3+: calmodulin specificity and properties of the reconstituted guanylate cyclase.

Ca2+-regulated guanylate cyclase in ciliary membranes from Paramecium contained tightly bound calmodulin. Antisera against calmodulin from Tetrahymena and soybean inhibited enzyme activity. EGTA did not easily release calmodulin; however, La3+ inhibited guanylate cyclase by dissociation of calmodulin. While La could not replace Ca in the activation of guanylate cyclase, it substituted for Ca2+ in the activation of calmodulin-dependent phosphodiesterase from pig brain independently of whether homologous or Paramecium calmodulin was used. After removal of endogenous calmodulin from guanylate cyclase, reconstitution was achieved with calmodulin from Paramecium, Tetrahymena, pig brain, and soybean. Ca2+-binding proteins lacking trimethyllysine like calmodulin from Dictyostelium, parvalbumin, and troponin C failed to restore enzyme activity. The properties of the native and reconstituted guanylate cyclase/calmodulin complex were compared. Reassociation of calmodulin with its target enzyme was weak since all calmodulin remained in the supernatant after a single centrifugation. While most enzyme characteristics remained unchanged in the reconstituted complex, the inhibition by Ca greater than 100 microM was of a mixed-type compared to noncompetitive inhibition in the native enzyme. The regulation of the enzyme by cations was also altered. Whereas Ca was the most potent and specific activator of the native enzyme, in the reconstituted system Sr was far more effective.

Animals↗
PubMed1983

Immunocytochemical localization of cyclic GMP, cGMP-dependent protein kinase, calmodulin and calcineurin in Paramecium tetraurelia.

The localization of cGMP, cGMP-dependent protein kinase, calmodulin and the calmodulin-binding protein calcineurin in Paramecium tetrauelia cells has been examined with immunocytochemical methods. These molecules appeared to be localized to a large extent in the cilia of this protozoan. To ascertain that antibodies had access to all cellular compartments we have used three different preparations for immunocytochemistry: (i) with 'whole cell' preparations immunofluorescent staining for the four molecules was mainly visible in the cilia; (ii) in 'deciliated' Paramecium, staining for cGMP and calmodulin was found in regular patterns on the cell surface most likely representing kinetosomes; (iii) using 'sectioned cells', additional cytoplasmic calmodulin appeared to be associated with glycogen particles as evidenced by the disappearance of the granular staining pattern after preincubation with alpha-amylase. In contrast, cGMP, cGMP-dependent protein kinase and calcineurin fluorescence was only very weak and diffuse in cell bodies. No nuclear fluorescence was detectable after staining with any of the antibodies. Because of the colocalization of cGMP, cGMP-dependent protein kinase, a guanylate cyclase-calmodulin-complex, and calcineurin in cilia from Paramecium, an involvement of these components in the regulation of ciliary activity is discussed.

Animals↗
PubMed1982

Characterization of a Ca2+-dependent guanylate cyclase in the excitable ciliary membrane from Paramecium.

The membraneous guanylate cyclase of cilia from Paramecium tetraurelia used MgGTP and MnGTP as substrate with Michaelis constants for GTP of 71.5 microM and 36 microM, respectively. A linear Arrhenius plot indicated that a single enzyme entity exists not sensitive to possible phase transitions of membrane lipids. Guanylate cyclase is activated by low concentrations (less than 100 microM) and inhibited by high concentrations (greater than 100 microM) of calcium, half-maximal effects were obtained with 8 microM and 500 microM Ca2+, respectively. Only strontium ions displayed partial activating and inhibiting potency, all other divalent cations tested, Ba2+, Fe2+, Co2+, Mn2+, Sn2+ and Ni2+ had no effect on guanylate cyclase activity. Ca2+ activation increased V; Km remained identical. The Ca2+ stimulated activity was not inhibited by trifluoperazine, tentatively suggesting that the stimulation may not be mediated by calmodulin. Ca2 inhibition was due to a single binding site of Ca2+ at the guanylate cyclase as evidence by a Hill coefficient h = -1 and was noncompetitive. The lanthanides La3+, Ce3+ and Tb3+ were powerful inhibitors of guanylate cyclase, with La3+ the half-maximal effect was obtained with 0.6 microM, it was kinetically a mixed-type inhibition. La3+ and CA2+ competed for the same binding site on the guanylate cyclase as determined by detailed kinetic analysis. Addition of EDTA reversed the activation and inhibition by Ca2+ and the inhibition by La3+. It is discussed that guanylate cyclase may be the initial target enzyme in the cilia for the calcium transient of the calcium-potassium action potential of Paramecium.

Animals↗
PubMed1982

Differential distribution of voltage-dependent calcium channels and guanylate cyclase in the excitable ciliary membrane from Paramecium tetraurelia.

A novel method for isolation of cilia and ciliary membrane vesicles from Paramecium tetraurelia has been developed. Using a continuous Percoll gradient of low osmolarity after fragmentation of purified cilia by French Press treatment two membrane fractions with different buoyant densities were obtained. These fractions were further purified by conventional discontinuous sucrose density gradients and characterized biochemically and by electron microscopy. Guanylate cyclase, a membrane bound enzyme, was found almost exclusively in membrane vesicles of high buoyant density while the voltage-sensitive calcium-channel of the ciliary membrane was predominantly localized in low density vesicles. Examination of both fractions by SDS polyacrylamide gel electrophoresis revealed only minor differences in protein pattern in the 34 and 64 kilodaltons range. Morphologically both membrane vesicle fractions had a diameter of about 300 nm, however, the high density vesicle fraction contained a considerably larger amount of multilamellar structures with a multishell, onion-like appearance. Freeze-fracture analysis failed to detect differences in intramembrane particle content between low and high density vesicles. The possible biological relevance of the spatial separation of the calcium-sensor enzyme guanylate cyclase and the voltage-sensitive calcium-channels in the ciliary membrane is discussed in terms of a diffusion controlled mechanism for graded signal transmission.

Animals↗
PubMed1979

Human erythrocyte thiol methyltransferase: radiochemical microassay and biochemical properties.

A radiochemical microassay for the measurement of thiol methyltransferase (TMT) activity in human red blood cell (RBC) membranes has been developed. Both 2-mercaptoethanol and dithiothreitol were used as substrates for the enzyme. The pH optimum of the reaction was approximately 9.0 when glycine NaOH was used as a buffer. The apparent Michaelis-Menten (KM) value for the methyl donor for the reaction, S-adenosyl-L-methionine, was 43 mumol/l. Human RBC TMT activity was neither activated nor inhibited by Ca2+, Mg2+, or tropolone, but the enzyme was inhibited by SKF 525A and by reagents that react with sulfhydryl groups. The mean TMT activity in blood from 289 randomly selected adult white subjects was 10.93 +/- 3.22 units per mg protein (mean +/- S.D.). The activity was the same in samples from men and women. The results of experiments in which TMT activity was measured in mextures of RBC membranes with relatively "low" and relatively "high" activities provided no evidence that individual variations in the enzyme activity were due to variations in endogenous TMT activators or inhibitors.

Chromatography, High Pressure Liquid↗