V. Ullrich - Academia.edu (original) (raw)

Papers by V. Ullrich

Concepts in Biochemical Pharmacology, 1975

European Journal of Biochemistry, 1987

The metabolism of [1-14C]arachidonic acid by microsomal and purified prostaglandin (PG) H synthas... more The metabolism of [1-14C]arachidonic acid by microsomal and purified prostaglandin (PG) H synthase was investigated. HPLC analysis confirmed that arachidonic acid (20:4) was extensively converted into prostaglandin G2 (PGG2) and/or prostaglandin H2 (PGH2) but several minor labelled products were formed in addition. Their formation, mediated by PGH synthase was established by inhibition with aspirin and indomethacin [Hecker, M., Hatzelmann, A. & Ullrich, V. (1987) Biochem. Pharmacol. 36, 851-855]. Upon comparison with authentic reference material these unknown PGH synthase metabolites were identified with respect to chromatographic properties, ultraviolet spectroscopy and mass spectrometry as 11 (R)-hydroperoxy-5Z,8Z,12E,14Z-eicosatetraenoic acid (11-OOH-20:4), 12(S)-hydroperoxy-5Z,8E,10E-heptadecatrienoic acid (OOH-17:3), 12(S)-hydroxy-5Z,8E,10E-heptadecatrienoic acid (OH-17:3), 15(RS)-hydroperoxy-5Z,8Z,11Z,13E-eicosatetraenoi c acid (15-OOH-20:4), 15(RS)-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid (15-OH-20:4), 13-hydroxy-5Z,14Z-prostaglandin H2, 15(S)-hydroxy-8-iso-5Z,13E-prostaglandin H2 and 15-oxo-prostaglandin H2. Unlike PGG2 and PGH2, 8-iso-PGH2, 13-hydroxy-PGH2 and 15-oxo-PGH2 failed to induce aggregation of washed human platelets and to form thromboxane upon incubation with homogeneous human platelet thromboxane synthase. In contrast to the formation of OOH-17:3, 15-oxo-PGH2 and OH-17:3 which can be attributed to the heme-catalyzed decomposition of PGG2 and PGH2, 11-OOH-20:4,15-(O)OH-20:4-,8 iso-PGH2 and 13-hydroxy-PGH2 represent potential side products of arachidonic acid conversion into PG endoperoxides. Their formation allows to conclude on PGH synthase mechanism and its intermediates for which an extended reaction scheme is proposed.

Biochemical and Biophysical Research Communications, 1976

Abstract In the presence of iodosobenzene, liver microsomes catalyze the O-dealkylation of 7-etho... more Abstract In the presence of iodosobenzene, liver microsomes catalyze the O-dealkylation of 7-ethoxycoumarin. The reaction proceeds in the absence of NADPH and O 2 and is dependent on cytochrome P450. The results indicate that cytochrome P450 acts as an oxene transferase probably involving [FeO] 3+ as the transient intermediate of active oxygen.

Biochemical Pharmacology, 1974

The O-dealkylation activity for 7-ethoxycoumarin of mouse small intestine was determined by recor... more The O-dealkylation activity for 7-ethoxycoumarin of mouse small intestine was determined by recording the surface fluorescence of the umbelliferone formed in everted intestinal segments. A simple holding device suitable for commercially available cuvettes has been designed. After hypotonic shocking of the mucosal cells the substrate was added in 0.1 M Tris buffer pH 7.9 causing a linear increase in fluorescence for more than 15 min. Pretreatment of mice with phenobarbital increased the activity several fold, the induction following a biphasic course. The maximal rate of O-dealkylation by the entire small intestine was calculated as 19 nmoles min-1. In agreement with previous results obtained with homogenates of small intestine the highest activity was located at a distance of 8-12 cm from the pylorus. Inhibition experiments indicate the involvement of cytochrome P-450.

FEBS Letters, 1993

Human thromboxane (TX) synthase (EC 5.3.99.5) was produced by the baculovirus expression system u... more Human thromboxane (TX) synthase (EC 5.3.99.5) was produced by the baculovirus expression system using cDNA encoding human TX synthase [(1991) Biochem. Biophys. Res. Commun. 78, 1479-14841. A recombinant baculovirus TXS7 was expressed in Spodopterafrugiperda St9 insect cells. The expressed protein was recognized by monoclonal antibody, Kon 7 raised against human TX synthase [(1990) Blood 76, SCrSS]. The recombinant TX synthase catalyzed the conversion of prostaglandin (PC) Hz to TXA, and 12-hydroxy-heptadecatrienoic acid (HHT). Both conversions of PGHz to TXAz and HHT by the expressed TX synthase were completely inhibited by a specific TX synthase inhibitor, OKY-046 (5 PM).

European Journal of Biochemistry, 1990

Human platelet soluble guanylate cyclase activity was studied with respect to the function of its... more Human platelet soluble guanylate cyclase activity was studied with respect to the function of its hemecontaining regulatory subunit. As an enzyme source, the 10000 x g supernatant was used and, since its specific activity proved to be too low for inhibition studies, also a partially purified preparation was employed. The partially purified enzyme was stimulated about 2.5-fold by carbon monoxide and this effect was abolished by illumination with visible light. Sodium nitroprusside also increased the basal activity about fourfold, which, however, is much less than the > 100-fold stimulation seen with the supernatant. Superoxide anions generated by the xanthine/xanthine-oxidase system were strongly inhibitory in the enriched preparation as well as in the COstimulated platelet supernatant (median effector concentration = 0.1 mUjml). Unlike CO and NO, the effect of superoxide cannot be mediated through the heme-containing regulatory subunit, since heme-free enzyme, which could not be activated by NO or CO, was inhibited to the same extent as the heme-containing enzyme. Superoxide dismutase did not influence the basal activity, but resulted in a synergistic stimulation in the presence of CO. When Mn2+ replaced MgZf as a cofactor, the basal activity was higher but superoxide could not inhibit the enzyme, possibly due to the superoxide-dismutase-like activity of Mn2+. Superoxide turned out to be a potent and reversible inhibitor of soluble guanylate cyclase which, together with endothelium-derived relaxing factor, recently identified as NO, could form a physiologically relevant regulatory effector system. Guanosine 3',5'-(cyc1ic)phosphate (cGMP) is emerging as an important cellular second messenger, although its mode of action and the regulation of soluble guanylate cyclase and particulate guanylate cyclase are only poorly understood (for a review see [l, 21). Soluble guanylate cyclase contains a heme regulatory subunit and represents the dominant enzyme in platelets and smooth muscle. Its activation is associated with inhibition of platelet aggregation [3] and with smooth muscle relaxation [4, 51. Hence its stimulation under conditions of coronary heart diseases is a preferred pharmacological target. Likewise, the potent vasodilatory properties of organic nitrates seem to be mediated by the release of nitric oxide [6, 71. Recently, the endothelium-derived relaxing factor (EDRF) was identified as NO which has therefore gained not only a pharmacological but also a physiological role in the relaxation of vascular tissue [8]. When studying platelet aggregation, we obtained evidence that carbon monoxide prevents aggregation of human platelets by stimulation of soluble guanylate cyclase [9]. From the reversibility of this effect by visible light, we concluded

Molecular pharmacology, 1993

Activation of human platelets is associated with an increased level of cGMP, when total cGMP in i... more Activation of human platelets is associated with an increased level of cGMP, when total cGMP in individual samples is measured. However, by discriminating between intracellular and extracellular cGMP we were able to demonstrate that cGMP accumulates in the extracellular space only, whereas the level of intraplatelet cGMP actually decreases. Therefore, during the first minutes of platelet aggregation cGMP is released from the cell, and it thereby escapes hydrolysis by intracellular phosphodiesterases. In contrast, during direct activation of soluble guanylyl cyclase by nitrovasodilators, such as sodium nitroprusside, the newly synthesized cGMP remains mainly inside the cells. Elevation of intracellular calcium and activation of protein kinase C are likely to be involved in promoting cGMP efflux. Our results are discussed in contrast to the general hypothesis that the cGMP increase associated with platelet aggregation may represent a feedback mechanism designed to terminate early even...

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Die Medizinische Welt, Jan 20, 1969

Advances in prostaglandin, thromboxane, and leukotriene research, 1990

European Journal of Biochemistry, 1973

Properties of a 4-Methoxybenzoate Monooxygenese of Pseudomonas putida Bur. J. Biochem.

Biochemical Pharmacology, 1991

The reported relaxing effect of CO on various smooth muscle tissues could also be found in guinea... more The reported relaxing effect of CO on various smooth muscle tissues could also be found in guinea pig ileal strips. The effect was pronounced after precontraction with 10-100 nM acetylcholine and rather small with KCI. Based on the photoreversibility of the CO-dependent relaxation, a photochemical action spectrum was established which showed a maximum at around 422 nm. This definitely rules out the participation of a cytochrome P450 dependent process as postulated for the CO induced relaxation of lamb ductus arteriosus. With regard to the potency of KCN and antimycin A to relax ileal smooth muscle, the involvement of respiratory chain inhibition was reinvestigated, but no indication for such a mechanism could be obtained. In analogy to the mechanism of CO-inhibition of platelet activation we found that CO about doubles cGMP levels in guinea pig ileal strips. This is similar to NO which also leads to effective relaxation. We propose that CO can be considered and experimentally used as a convenient activator of soluble G-cyclase in smooth muscle and platelets.

Biochimica et Biophysica Acta (BBA) - Biomembranes, 1976

The temperature dependence of drug monooxygenation in phenobarbitalinduced rat liver microsomes h... more The temperature dependence of drug monooxygenation in phenobarbitalinduced rat liver microsomes has been investigated. With 7-ethoxycoumarin as a substrate the activity of the microsomes could be measured down to 0 °C by the increase in fluorescence of the dealkylated reaction product 7-hydroxycoumarin (umbelliferone). Arrhenius plots of the activities at various temperatures between 0 °C and 45 °C showed a break in the activation energy around 20 °C. Addition of deoxycholate or high concentrations of glycerol, known to solubilize membrane-bound enzymes, abolished the break of the activation energy. Cholesterol, incorporated into the microsomal membrane in amounts equimolar to the microsomal phospholipid content led to a decrease of the activation energy at low temperatures and to an increase at higher temperatures, resulting in a loss of the break. The activity of microsomal NADPH-cytochrome c reductase with the watersoluble electron acceptor dichlorophenolindophenol showed no discontinuity in the Arrhenius plot. In addition the cumene hydroperoxide-mediated and cytochrome P-450-dependent O-dealkylation of 7-ethoxycoumarin proceeded without a break in the activation energy. It is concluded that phospholipid phase transitions affect the electron transfer from the reductase to cytochrome P-450.

Hoppe-Seyler´s Zeitschrift für physiologische Chemie, 1968

FEBS Letters, 1996

The possible active site Cys 441 in the Cys-pocket and Glu 347 and Arg ~s° of the EXXR motif of t... more The possible active site Cys 441 in the Cys-pocket and Glu 347 and Arg ~s° of the EXXR motif of the human prostacyclin synthase, which catalyzes the conversion of prostaglandin H2 to prostacyclin, were subjected to site-directed mutagenesis in order to understand the role of these residues in expressing the enzymatic activity. Five expression vectors encoding the mutant enzymes with a single replacement, Cys441Ala, Cys441Ser, Cys441His, Glu347Ala and Arg3S°Ala, as well as the wild-type enzyme were expressed in 293 cells. The microsomal fraction of the cells expressing the wild-type enzyme showed a specific activity of 96 nmol 6-keto-PGF1Jmin per mg protein. All of the mutant enzymes examined showed no detectable enzyme activity, although immunoblot analysis demonstrated that levels of all the expressed mutant enzymes were similar to that of the wild-type enzyme. These results indicated that the Cys 441 in the Cyspocket, and Glu 347 and Arg 35° of the EXXR motif of human prostacyclin synthase are important for expressing the enzymatic activity.

Journal of Biological Chemistry, 1989

The present research describes studies which address the mechanism of prostacyclin (PGI2) and thr... more The present research describes studies which address the mechanism of prostacyclin (PGI2) and thromboxane A2 (TXA2) biosynthesis. In addition to prostaglandin H1 (PGH1), PGG2, PGH2, and PGH3, also 8-iso-PGH2, 13(S)-hydroxy-PGH2, and 15-keto-PGH2 were applied to determine the substrate specificities and kinetics of prostacyclin and thromboxane synthase in more detail. Human platelet thromboxane synthase converted PGH1, 8-iso-PGH2, 13(S)-hydroxy-PGH2 and 15-keto-PGH2 into the corresponding heptadecanoic acid (C17) plus malondialdehyde, whereas the thromboxane derivative was formed only from PGG2, PGH2, and PGH3 together with the corresponding C17 metabolite and malondialdehyde in a 1:1:1 ratio. In contrast, PGG2, PGH2, 13(S)-hydroxy-PGH2, 15-keto-PGH2 and PGH3 were almost completely isomerized to the corresponding prostacyclin derivative by bovine aortic prostacyclin synthase, whereas PGH1 and 8-iso-PGH2 only produced the corresponding C17 hydroxy acid plus malondialdehyde. Isotope-labeling experiments with [5,6,8,9,11,12,14,15-2H]PGH2 revealed complete retention of label and no isotope effect in the course of thromboxane biosynthesis, but the loss of one 2H atom at C-6 with an isotope effect of 1.20 during PGI2 formation. Prostacyclin and thromboxane synthase bind both 9,11-epoxymethano-PGF2 alpha and 11,9-epoxymethano-PGF2 alpha at the heme iron, but according to their difference spectra in opposite ways with respect to the 9- and 11-position. In agreement with published model studies, a cage radical mechanism is proposed for both enzymes according to which the initial radical process is terminated through oxidation of carbon-centered radicals by the iron-sulfur catalytic site, followed by ionic rearrangement to PGI2 or TXA2. Various Fe(III) model compounds as well as liver microsomes or cytochrome P-450CAM can also form small amounts of PGI2 and TXA2, but mainly yield 12(S)-hydroxy-5,8,10-heptadecatrienoic acid plus malondialdehyde probably by a radical fragmentation pathway.

European Journal of Biochemistry, 1982

The rates of the NADPH-dependent formation of superoxide radicals and hydrogen peroxide have been... more The rates of the NADPH-dependent formation of superoxide radicals and hydrogen peroxide have been measured in liver microsomes from phenobarbital-pretreated rats. Correcting a quenching of O2(-) radicals by microsomes, a stoichiometry of O2(-) to H2O2 close to 2:1 was obtained. This, and the fact that pseudo-substrates of microsomal cytochrome P450 like perfluoro-n-hexane and perfluorinated cyclohexane did not increase H2O2 formation in a catalase-inhibited assay, rules out a two-electron reduced oxygen species as the source of H2O2. The rates of O2(-) as well as H2O2 generation in the presence of 7-ethoxycoumarin were equally inhibited by carbon monoxide (75%) and resulted in photochemical action spectra with a maximum reactivation at 450 nm. Using the same conditions the monooxygenation was inhibited to a high degree (83%) but without exogenous substrate the inhibition of H2O2 formation dropped to 55%. It was concluded that most of the O2(-) originated from the oxycomplex of cytochrome P450 and that substrates can modify the rates of its decomposition and sensitivity to carbon monoxide. No correlation of H2O2 formation or of substrate monooxygenation with the optical substrate binding spectra could be observed. From the pH dependence a proton-assisted decomposition of oxy-cytochrome P450 appears likely. H2O2 formation was only slightly decreased at 20 microM dioxygen suggesting that H2O2 formation via cytochrome P450 should also occur in vivo.

European Journal of Biochemistry, 1992

The relationship between agonist-sensitive calcium compartments and those discharged by the Ca2+-... more The relationship between agonist-sensitive calcium compartments and those discharged by the Ca2+-ATPase inhibitor thapsigargin were studied in human platelets. In this context, calcium mobilization from intracellular pools and manganese influx was investigated in relation to the effect of altered cyclic-nucleotide levels. For maximal calcium release from intracellular stores, thapsigargin, compared to a receptor agonist like thrombin, requires the platelet's self-amplification mechanism, known to generate thromboxane A2. With this lipid mediator formed, thapsigargin released calcium and stimulated manganese influx in a manner similar to thrombin. Blocking the thromboxane receptor by addition of sulotroban (BM13.177) or, alternatively, increasing platelet cAMP or cGMP using prostacyclin or sodium nitroprusside, dramatically reduced the ability of thapsigargin to release calcium from intracellular compartments. The same experimental conditions significantly reduced the rate of manganese influx initiated by thapsigargin compared to thrombin. The experiments indicate that thapsigargin-sensitive compartments play only a minor role in inducing manganese influx compared to the receptor-sensitive compartment. Cyclic nucleotides accelerate the redistribution of an agonist-elevated platelet calcium into the thapsigargin-sensitive compartment, from which calcium can be released by inhibition of the Ca2+-ATPase. In human platelets, thapsigargin-induced calcium increase and influx were responsible for only part the calcium release resulting from inhibition of the corresponding ATPase; another part results from the indirect effect of thapsigargin acting via thromboxane-A2-receptor activation. Cyclic nucleotides are therefore an interesting regulatory device which can modify the thapsigargin response by not allowing the self-amplification mechanism of platelets to operate. Human platelets are stimulated by various agonists which act on specific receptors [l]. Among several activating compounds, one also finds thrombin and thromboxane Az; these initiate an ordered sequence of events associated with an activation of phospholipase C [2] during which the hydrolysis of phosphatidylinositol 4,5-bisphosphate produces at least two second messengers: inositol 1,4,5-trisphosphate, which accounts for a cytosolic-calcium increase, and 1,2-diacylglycerol known to activate protein kinase C [3, 41. These compounds act synergistically, inducing further platelet responses such as platelet aggregation and secretion. An increased level of cytosolic calcium activates phospholipase Az, which concomitantly releases arachidonic acid from membrane phospholipids. Free arachidonate is metabolized via prostaglandin endoperoxides to generate thromboxane AZ. Endoperoxides and thromboxane A2 are potent activators by themselves and serve to amplify the response to weak agonists [5-71. The release of calcium from intracellular stores by the action of inositol 1,4,5-trisphosphate has been demonstrated [3], as well as the central role of calcium during platelet activation [S, 91. Recently, we reported [lo, 111 the existence of two intracellular calcium pools in human platelets, which can be discriminated either by receptor activation or Ca2 +-ATPase inhi

Journal of Inorganic Biochemistry, 1979

Hemin coordinated with mercaptide sulfur as fifth ligand and various sixth ligands were investiga... more Hemin coordinated with mercaptide sulfur as fifth ligand and various sixth ligands were investigated as models for cytochrome P450 in its native ferric low-spin state and its ligand complexes. Mixing the hemin with its ligands below -60 degrees C prevented the reduction of the hemin by mercaptide and made it possible to characterize each sample both by electronic and ESR spectra. Excess of mercaptide formed hemin-dimercaptide complexes with hyperporphyrin spectra with two Soret bands around 380 and 370 nm. The second mercaptide could be exchanged by other ligands with hydroxyl, phosphine, thioether, isocyanide, amine, imidazole, and pyridine groups. The comparison of these spectral data with cytochrome P450 substantiates mercaptide as the fifth ligand and makes a hydroxyl group a more likely candidate for the native sixth ligand than an imidazole group.