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J Messinger

Publications and source records attributed to J Messinger.

17 recordsLinked to original sources

Evaluation of inhibitors for 17beta-hydroxysteroid dehydrogenase type 1 in vivo in immunodeficient mice inoculated with MCF-7 cells stably expressing the recombinant human enzyme.

17Beta-hydroxysteroid dehydrogenase (17HSD1) is an enzyme activating estrone (E1) to estradiol (E2). In the present study, a mechanistic animal model was set up for evaluating putative inhibitors for the human enzyme in vivo. Estrogen-dependent MCF-7 human breast carcinoma cells were stably transfected with a plasmid expressing human 17HSD1. These cells formed estrogen-dependent tumors in immunodeficient mice. In the optimized model, tumor sizes were decreased in both ovariectomized and intact vehicle-treated mice, whereas they were maintained or slightly increased in mice supplemented 2 weeks with an appropriate dose of the 17HSD1-substrate E1. Tumor sizes in mice treated with 0.1 micromol/kg/d of E1 were reduced by administering 5 micromol/kg/d of different 17HSD1-inhibitors and a 86% reduction in size was detected with the most potent inhibitor. A dose-response relationship in the inhibitory effect of this compound further confirmed the validity of the model for testing the drug candidates in vivo.

17-Hydroxysteroid Dehydrogenases↗

Electron spin-lattice relaxation of the S0 state of the oxygen-evolving complex in photosystem II and of dinuclear manganese model complexes.

The temperature dependence of the electron spin-lattice relaxation time T1 was measured for the S0 state of the oxygen-evolving complex (OEC) in photosystem II and for two dinuclear manganese model complexes by pulse EPR using the inversion-recovery method. For [Mn(III)Mn(IV)(mu-O)2 bipy4]ClO4, the Raman relaxation process dominates at temperatures below 50 K. In contrast, Orbach type relaxation was found for [Mn(II)Mn(III)(mu-OH)(mu-piv)2(Me3 tacn)2](ClO4)2 between 4.3 and 9 K. For the latter complex, an energy separation of 24.7-28.0 cm(-1) between the ground and the first excited electronic state was determined. In the S0 state of photosystem II, the T1 relaxation times were measured in the range of 4.3-6.5 K. A comparison with the relaxation data (rate and pre-exponential factor) of the two model complexes and of the S2 state of photosystem II indicates that the Orbach relaxation process is dominant for the S0 state and that its first excited state lies 21.7 +/- 0.4 cm(-1) above its ground state. The results are discussed with respect to the structure of the OEC in photosystem II.

Electron Spin Resonance Spectroscopy↗

Absence of Mn-centered oxidation in the S(2) --> S(3) transition: implications for the mechanism of photosynthetic water oxidation.

A key question for the understanding of photosynthetic water oxidation is whether the four oxidizing equivalents necessary to oxidize water to dioxygen are accumulated on the four Mn ions of the oxygen-evolving complex (OEC), or whether some ligand-centered oxidations take place before the formation and release of dioxygen during the S(3) --> [S(4)] --> S(0) transition. Progress in instrumentation and flash sample preparation allowed us to apply Mn Kbeta X-ray emission spectroscopy (Kbeta XES) to this problem for the first time. The Kbeta XES results, in combination with Mn X-ray absorption near-edge structure (XANES) and electron paramagnetic resonance (EPR) data obtained from the same set of samples, show that the S(2) --> S(3) transition, in contrast to the S(0) --> S(1) and S(1) --> S(2) transitions, does not involve a Mn-centered oxidation. On the basis of new structural data from the S(3)-state, manganese mu-oxo bridge radical formation is proposed for the S(2) --> S(3) transition, and three possible mechanisms for the O-O bond formation are presented.

Electron Spin Resonance Spectroscopy↗

High-resolution X-ray spectroscopy of rare events: a different look at local structure and chemistry.

The combination of large-acceptance high-resolution X-ray optics with bright synchrotron sources permits quantitative analysis of rare events such as X-ray fluorescence from very dilute systems, weak fluorescence transitions or X-ray Raman scattering. Transition-metal Kbeta fluorescence contains information about spin and oxidation state; examples of the characterization of the Mn oxidation states in the oxygen-evolving complex of photosystem II and Mn-consuming spores from the marine bacillus SG- are presented. Weaker features of the Kbeta spectrum resulting from valence-level and 'interatomic' ligand to metal transitions contain detailed information on the ligand- atom type, distance and orientation. Applications of this spectral region to characterize the local structure of model compounds are presented. X-ray Raman scattering (XRS) is an extremely rare event, but also represents a unique technique to obtain bulk-sensitive low-energy (<600 eV) X-ray absorption fine structure (XAFS) spectra using hard (approximately 10 keV) X-rays. A photon is inelastically scattered, losing part of its energy to promote an electron into an unoccupied level. In many cases, the cross section is proportional to that of the corresponding absorption process yielding the same X-ray absorption near-edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) features. XRS finds application for systems that defy XAFS analysis at low energies, e.g. liquids or highly concentrated complex systems, reactive compounds and samples under extreme conditions (pressure, temperature). Recent results are discussed.

Bacillus↗

Towards understanding the chemistry of photosynthetic oxygen evolution: dynamic structural changes, redox states and substrate water binding of the Mn cluster in photosystem II.

This mini-review summarizes my postdoctoral research in the labs of T. Wydrzynski/C.B. Osmond, J.H.A. Nugent/M.C.W. Evans and V.K. Yachandra/K. Sauer/M.P. Klein. The results are reported in the context of selected data from the literature. Special emphasis is given to the mode of substrate water binding, Mn oxidation states and the structures of the Mn cluster in the four (meta)stable redox states of the oxygen evolving complex. The paper concludes with a working model for the mechanism of photosynthetic water oxidation that combines mu-oxo bridge oxidation in the S(3) state (V.K. Yachandra, K. Sauer, M.P. Klein, Chem. Rev. 96 (1996) 2927-2950) with O-O bond formation between two terminal Mn-hydroxo ligands during the S(3)-->(S(4))-->S(0) transition.

Manganese↗

Kinetic determination of the fast exchanging substrate water molecule in the S3 state of photosystem II.

In a previous communication we showed from rapid isotopic exchange measurements that the exchangeability of the substrate water at the water oxidation catalytic site in the S3 state undergoes biphasic kinetics although the fast phase could not be fully resolved at that time [Messinger, J., Badger, M., and Wydrzynski, T. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 3209-3213]. We have since improved the time resolution for these measurements by a further factor of 3 and report here the first detailed kinetics for the fast phase of exchange. First-order exchange kinetics were determined from mass spectrometric measurements of photogenerated O2 as a function of time after injection of H218O into spinach thylakoid samples preset in the S3 state at 10 degreesC. For measurements made at m/e = 34 (i. e., for the mixed labeled 16,18O2 product), the two kinetic components are observed: a slow component with k1 = 2.2 +/- 0.1 s-1 (t1/2 approximately 315 ms) and a fast component with k2 = 38 +/- 4 s-1 (t1/2 approximately 18 ms). When the isotopic exchange is measured at m/e = 36 (i.e., for the double labeled 18,18O2 product), only the slow component (k1) is observed, clearly indicating that the substrate water undergoing slow isotopic exchange provides the rate-limiting step in the formation of the double labeled 18,18O2 product. When the isotopic exchange is measured as a function of temperature, the two kinetic components reveal different temperature dependencies in which k1 increases by a factor of 10 over the range 0-20 degreesC while k2 increases by only a factor of 3. Assuming simple Arrhenius behavior, the activation energies are estimated to be 78 +/- 10 kJ mol-1 for the slow component and 39 +/- 5 kJ mol-1 for the fast component. The different kinetic components in the 18O isotopic exchange provide firm evidence that the two substrate water molecules undergo separate exchange processes at two different chemical sites in the S3 state, prior to the O2 release step (t1/2 approximately 1 ms at 20 degreesC). The results are discussed in terms of how the substrate water may be bound at two separate metal sites.

Catalytic Domain↗

Synthesis, pharmacological characterization, and quantitative structure-activity relationship analyses of 3,7,9,9-tetraalkylbispidines: derivatives with specific bradycardic activity.

A series of 3,7,9,9-tetraalkyl-3,7-diazabicyclo[3.3.1]nonane derivatives (bispidines) was synthesized and identified as potential antiischemic agents. Pharmacological experiments in vitro as well as in vivo are described, and the results are listed. For selection of those compounds fitting best to the desired profile of a specific bradycardic antianginal agent--decrease in heart rate without affecting contractility and blood pressure--these results were scored and ranked. Quantitative structure--activity relationship (QSAR) analyses were performed and discussed a posteriori by means of Hansch, nonelementary discriminant and factor analysis to get insight into the molecular features determining the biological profile. Highly significant equations were obtained, indicating hydrophobic and steric effects. Both pharmacological ranking and QSAR considerations showed compound 6 as the optimum within the structural class under investigation. Compound 6 (tedisamil, KC8857) has been selected as the most promising compound and was chosen for further pharmacological and clinical investigations as an antiischemic drug.

Animals↗

Detection of an EPR multiline signal for the S0* state in photosystem II.

The S0* state was generated by incubation of dark-adapted (S1 state) photosystem II membranes either with the exogenous two electron reductant hydrazine and subsequent 273 K illumination in the presence of DCMU or by dark incubation with low amounts of the one electron reductant hydroxylamine. In agreement with earlier reports, the S1 and S-1 states were found to be electron paramagnetic resonance (EPR) silent. However, in the presence of 0.5-1.5% methanol, a weak EPR multiline signal centered around g = 2.0 was observed at 7 K for the S0* states generated by both procedures. This signal has a similar average line splitting to the well-characterized S2 state multiline EPR signal, but can be clearly distinguished from that and other modified S2 multiline signals by differences in line position and intensities. In addition, at 4 K it can be seen that the S0* multiline has a greater spectral breadth than the S2 multilines and is composed of up to 26 peaks. The S0* signal is not seen in the absence of methanol and is not affected by 1 mM EDTA in the buffer medium. We assign the S0* multiline signal to the manganese cluster of the oxygen evolving complex in a mixed valence state of the form MnIIMnIIIMnIIIMnIII,MnIIMnIIIMnIVMnIV, or MnIIIMnIIIMnIIIMnIV. Addition of methanol may be helpful in future to find an EPR signal originating form the natural S0 state.

Electron Spin Resonance Spectroscopy↗

S(-3) state of the water oxidase in photosystem II.

The effect of the reductant hydrazine on the flash-induced oxygen oscillation patterns of spinach thylakoids was used to characterize a new super-reduced redox state of the water oxidase in photosystem II. The formation of a discrete S(-3) state is evident from the shift of the first maximum of oxygen evolution from the 3rd flash through the 5th flash to the 7th flash during a 90 min incubation of dark-adapted thylakoids with 10 mM hydrazine sulfate at pH 6.8 on ice. A distinct period four oscillation with further maxima on the 11th and 15th flashes is still observed at this stage of the incubation. The data analysis within the framework of an extended Kok model reveals that a S(-3) state population of almost 50% can be achieved by this treatment. A prolonged incubation of the S(-3) sample with 10 mM hydrazine (and even 100 mM) does not lead to a further shift of the first maximum toward the 9th flash that could reflect the formation of the S(-5) state. Instead, a slow oxidation of S(-3) to S(-2) takes place by an as yet unidentified electron acceptor. A consistent simulation of all the measured oxygen oscillation patterns of this study could, however, only be achieved by including the formal redox states S(-4) and S(-5) in the fits (S(-4) + S(-5) up to 35%). The implications of these findings for the oxidation states of the manganese in the tetranuclear cluster of the water oxidase are discussed.

Hydrazines↗

Structural changes in the water-oxidizing complex monitored via the pH dependence of the reduction rate of redox state S1 by hydrazine and hydroxylamine in isolated spinach thylakoids.

A detailed kinetic analysis is presented for the pH dependence of the reduction of the water-oxidizing complex (WOC) in redox state S1 by hydrophilic amines NH2R (R = NH2, OH) in suspensions of isolated thylakoids. Measurements of patterns of the oxygen yield induced by a train of single-turnover flashes and evaluation of the data within the framework of an extended Kok model [Messinger, J., Wacker, U., & Renger, G. (1991) Biochemistry 30, 7852-7862] led to the following results: (a) the rate constants kS1(NH2R) exhibit strikingly similar pH dependencies for NH2OH and NH2NH2 with "titration waves" at pH 5.3-5.6; 6.2-6.5, and above a critical pH value of about 7.4; (b) the differences in the reaction mechanism between NH2OH (1-electron reduction) and NH2NH2 (2-electron reduction) are almost pH-independent; (c) the ratio of the rate constants, kS1(NH2OH)/kS1(NH2NH2), decreases by a factor of about 9 within the range 5 < pH < 8.5. A detailed analysis reveals that these data cannot be consistently explained by the assumption that the unprotonated forms NH2OH and NH2NH2 are the active species while the protonated cations [NH3OH]+ and [N2H5]+ are nonreactive. A quantitative description is achieved by the additional postulate that pH-dependent structural changes take place in the WOC, thereby modulating the reactivity toward exogenous redox active amines of the type NH2R. On the basis of the results of this study and a recent report [Messinger, J., & Renger, G. (1994) Biochemistry 33, 10896-10905], it is inferred that the WOC undergoes three specific structural changes, with characteristic pH values of 5.3-5.5, 6.2-6.5, and above 7.4.

Hydrazines↗

Detection of one slowly exchanging substrate water molecule in the S3 state of photosystem II.

The exchangeability of the substrate water molecules at the catalytic site of water oxidation in photosystem II has been probed by isotope-exchange measurements using mass spectrometric detection of flash-induced oxygen evolution. A stirred sample chamber was constructed to reduce the lag time between injection of H2(18)O and the detecting flash by a factor of more than 1000 compared to the original experiments by R. Radmer and O. Ollinger [(1986) FEBS Lett. 195, 285-289]. Our data show that there is a slow (t1/2 approximately 500 ms, 10 degrees C) and a fast (t1/2 <25 ms, 10 degrees C) exchanging substrate water molecule in the S3 state of photosystem II. The slow exchange is coupled with an activation energy of about 75 kJ/mol and is discussed in terms of a terminal manganese oxo ligand, while the faster exchanging substrate molecule may represent a water molecule not directly bound to the manganese center.

Journal Article↗

Analyses of pH-induced modifications of the period four oscillation of flash-induced oxygen evolution reveal distinct structural changes of the photosystem II donor side at characteristic pH values.

This study presents a thorough analysis of the reaction pattern of flash-induced oxygen evolution in spinach thylakoids as a function of pH (4.5 < or = pH < or = 9) and the redox state of tyrosine YD in polypeptide D2. Evaluation of the experimental data within the conventional Kok model [Kok, B., Forbush, B., & McGloin, M. (1970) Photochem. Photobiol. 11, 457-475] led to the following results: (1) the probability of the miss factor is strongly pH dependent (with a pronounced minimum near neutral pH) while the double hit factor is less affected; (2) a marked increase of the apparent S0 population arises at alkaline pH in dark-adapted samples where most of the YD is reduced, but this effect is absent if the percentage of PS II containing the oxidized form YDox is high; and (3) the lifetimes of S2 and S3 exhibit a characteristic pH dependence that is indicative of conformational changes of functional relevance within the water-oxidizing complex and its environment; (4) the kinetic interaction of redox states S2 and S3 with YD is characterized by a change of its behavior at a threshold pH of 6.5-7.0; and (5) at acidic pH values the extent of S2 and S3 reduction by YD decreases concomitant with the occurrence of a very fast decay kinetics. On the basis of a detailed discussion of these results and data from the literature, the water oxidase is inferred to undergo structural changes at pH values of 5-5.5 and 6.5-7.0. These transitions are almost independent of the redox state Si and modify the reaction coordinates of the water oxidase toward endogenous reductants.

Hydrogen-Ion Concentration↗

Generation, oxidation by the oxidized form of the tyrosine of polypeptide D2, and possible electronic configuration of the redox states S0, S-1, and S-2 of the water oxidase in isolated spinach thylakoids.

Suitable treatment of thylakoids with hydrazine permits a high population of the redox states S0, S-1, and S-2 in the water oxidase. Experiments performed with dark-adapted samples enriched either in the oxidized or reduced form of the redox-active tyrosine, YD, of polypeptide D2 reveal that YoxD is a unique endogenous oxidant within the PS II complex which causes a one-electron abstraction from the water oxidase in states S0, S-1, and S-2, respectively. A kinetic analysis of the period four oscillation of oxygen yield induced by a train of short flashes in dark-adapted samples permits the determination of the rate constants of electron abstraction from the reduced water oxidase by YoxD. A value of 9 x 10(-4) s-1 was found for the oxidation of S0 and S-2, while S-1 becomes oxidized with a rate constant of 4 x 10(-4) s-1 at 20 degrees C and pH 7.2. The redox state S0 generated either from S1 via the three-flash-induced oxidative pathway through S4 or from a one-flash oxidation of the S-1 state obtained by S1 reduction with NH2NH2 exhibits the same kinetics as S0 oxidation by YoxD. On the basis of these findings and data taken from the literature, the electronic configuration of the manganese atoms in the tetranuclear cluster is discussed. It is assumed that the dimer model of two binuclear manganese groups within the tetranuclear cluster is discussed.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrochemistry↗

Structure-function relations in photosystem II. Effects of temperature and chaotropic agents on the period four oscillation of flash-induced oxygen evolution.

The characteristic period four oscillation patterns of oxygen evolution induced by a train of single-turnover flashes were measured in dark-adapted samples as a function of temperature and upon addition of chaotropic agents. The following results were obtained: (a) Within the range of 0 < theta < 35 degrees C, the ratio of the oxygen yield induced by the 4th and 3rd flashes of the train, Y4/Y3, and the oxygen yield induced by the 2nd flash, Y2, exhibit similar dependencies on the temperature in isolated thylakoids, PS II membrane fragments, and inside-out vesicles. (b) Below a characteristic temperature theta c of 20-25 degrees C, the values of Y4/Y3 and Y2, which reflect (at constant S0 dark population) the probabilities of misses and double hits, respectively, remain virtually independent of temperature, whereas above theta c these parameters increase. (c) The dark decays of S2 and S3 via fast and slow kinetics due to reduction of the water oxidase by YD and other endogenous electron donor(s), respectively, exhibit comparatively strong temperature dependencies in thylakoids with the following activation energies: EA(S2fast) = 55 kJ/mol, EA(S3fast) = 50 kJ/mol, EA(S2slow) = 85 kJ/mol, and EA(S3slow) = 75 kJ/mol. The activation energy of S0 oxidation to S1 by YDox was found to be markedly smaller with a value of EA(S0) = 30 kJ/mol. (d) Incubation with chaotropic agents at concentrations which do not significantly impair the oxygen evolution capacity leads to modifications of the oscillation pattern with remarkable differences for various types of agents: Tris and urea are practically without effect; guanidine hydrochloride affects Y4/Y3 in a similar way as elevated temperature but without significant changes of Y2 and the decay kinetics of S2 and S3; and anions of the Hofmeister series (SCN-, ClO4-, I-) cause a drastic destabilization of YDox. Possible structure-function relations of the PS II complex are discussed on the basis of these findings.

Guanidine↗

Unusual low reactivity of the water oxidase in redox state S3 toward exogenous reductants. Analysis of the NH2OH- and NH2NH2-induced modifications of flash-induced oxygen evolution in isolated spinach thylakoids.

The effect of redox-active amines NH2R (R = OH or NH2) on the period-four oscillation pattern of oxygen evolution has been analyzed in isolated spinach thylakoids as a function of the redox state Si (i = 0, ..., 3) of the water oxidase. The following results were obtained: (a) In dark-adapted samples with a highly populated S1 state, NH2R leads via a dark reaction sequence to the formal redox state "S-1"; (b) the reaction mechanism is different between the NH2R species; NH2OH acts as a one-electron donor, whereas NH2NH2 mainly functions as a two-electron donor, regardless of the interacting redox state Si (i = 0, ..., 3). For NH2NH2, the modified oxygen oscillation patterns strictly depend upon the initial ratio [S0(0)]/[S1(0)] before the addition of the reductant; while due to kinetic reasons, for NH2OH this dependence largely disappears after a short transient period. (c) The existence of the recently postulated formal redox state "S-2" is confirmed not only in the presence of NH2NH2 [Renger, G., Messinger, J., & Hanssum, B. (1990) in Current Research in Photosynthesis (Baltscheffsky, M., Ed.) Vol. 1, pp 845-848, Kluwer, Dordrecht] but also in the presence of NH2OH. (d) Activation energies, EA, of 50 kJ/mol were determined for the NH2R-induced reduction processes that alter the oxygen oscillation pattern from dark-adapted thylakoids. (e) Although marked differences exist between NH2OH and NH2NH2 in terms of the reduction mechanism and efficiency (which is about 20-fold in favor of NH2OH), both NH2R species exhibit the same order of rate constants as a function of the redox state Si in the nonperturbed water oxidase: kNH2R(S0) greater than kNH2R(S1) much less than kNH2R(S2) much greater than kNH2R(S3) The large difference between S2 and S3 in their reactivity toward NH2R is interpreted to indicate that a significant change in the electronic configuration and nuclear geometry occurs during the S2----S3 transition that makes the S3 state much less susceptible to NH2R. The implications of these findings are discussed with special emphasis on the possibility of complexed peroxide formation in redox state S3 postulated previously on the basis of theoretical considerations [Renger, G. (1978) in Photosynthetic Water Oxidation (Metzner, H., Ed.) pp 229-248, Academic Press, London].

Hydrazines↗

The reactivity of hydrazine with photosystem II strongly depends on the redox state of the water oxidizing system.

The decay kinetics of the redox states S2 and S3 of the water-oxidizing enzyme have been analyzed in isolated spinach thylakoids in the absence and presence of the exogenous reductant hydrazine. In control samples without NH2NH2 a biphasic decay is observed. The rapid decline of S2 and S3 with YD as reductant exhibits practically the same kinetics with t1/2 = 6-7 s at pH = 7.2 and 7 degrees C. The slow reduction (order of 5-10 min at 7 degrees C) of S2 and S3 with endogenous electron donors other than YD is about twice as fast for S2 as for S3 under these conditions. In contrast, the hydrazine-induced reductive shifts of the formal redox states Si (i = 0...3) are characterized by a totally different kinetic pattern: (a) at 1 mM NH2NH2 and incubation on ice the decay of S2 is estimated to be at least 25 times faster (t1/2 less than or equal to 0.4 min) than the corresponding reaction of S3 (t1/2 approximately 13 min); (b) the NH2NH2-induced decay of S3 is even slower (about twice) than the transformation of S1 into the formal redox state 'S-1' (t1/2 approximately 6 min), which gives rise to the two-digit phase shift of the oxygen-yield pattern induced by a flash train in dark adapted thylakoids. (c) the NH2NH2-induced transformation S0----'S-2' [Renger, Messinger and Hanssum (1990) in: Curr.' Res. Photosynth. (Baltscheffsky, M., ed), Vol. 1, pp. 845-848, Kluwer, Dordrecht] is about three times faster (t1/2 approximately 2 min) than the reaction [see text]. Based on these results, the following dependence on the redox state Si of the reactivity towards NH2NH2 is obtained: S3 less than S1 less than S0 much less than S2. The implications of this surprising order of reactivity are discussed.

Chloroplasts↗