Biomedical subjects

W Junge

Publications and source records attributed to W Junge.

At least 55 records · Page 3Linked to original sources

PubMed1995

ATP synthase of chloroplasts: selective action of agents binding to F1 on partial reactions of proton transfer in F0.

We studied the basic steps of proton transfer through ATP synthase of chloroplasts, CF0CF1, under conditions of proton slip, a conducting stage in the absence of added nucleotides. On the background of a steady transmembrane pH difference, voltage steps were induced by flashing light. Proton intake, transfer, and release by CF0CF1 were kinetically resolved by spectrophotometric probes. Kinetic disparities between these three steps were observed. Rapid but limited proton intake from the thylakoid lumen and proton release into the medium (tau 1/2 approximately 5 ms) preceded charge transfer across the dielectric barrier in the enzyme (tau 1/2 approximately 30 ms). The saturation behavior under multiple flashes suggested a sequential reaction mechanism. ADP and dequalinium, when bound to subunit beta of the catalytic portion, CF1, blocked different partial reactions involving protons. ADP in the catalytic cleft blocked the electrogenic transfer step, and dequalinium at the adjacent DELSEED sequence on the same subunit blocked the release of protons. Both effects prove a long-range conformational transmission between remote (approximately 10 nm) domains on F1 and on F0. For the normal sequence of events from protons to ATP they suggest a specific action of certain proton-transfer steps on different domains of the catalytic portion.

Binding Sites↗

PubMed1995

ATP synthase: activating versus catalytic proton transfer.

ATP synthase (F-ATPase) of chloroplasts, CF0CF1, is both activated and driven by transmembrane protonmotive force. We dichotomized between activating and driving proton transfer by specific inhibitors, tentoxin and venturicidin. Thylakoids membranes were submitted to voltage steps (by flashing light) superimposed to a steady pH-difference. Transient proton intake, transfer and release by CF0CF1 was monitored by spectroscopic probes. Both activities, activation and catalysis, required all three partial reactions of the proton, however, activating proton transfer rose first (monophasically, tau 1/2 approximately 15 ms) followed by another phase of equal magnitude with a time lag of about 15 ms. Both types of consecutive proton transfer reactions contribute free energy for ATP synthesis.

Catalysis↗

PubMed1995

Endocrine effects in Asian postmenopausal women, treated with SH D 461 M and Prempak-C.

We reported the results of a randomized cross-over study comparing SH D 461 M (Climen) and Prempak-C in 38 postmenopausal women who were established users of hormone replacement therapy (HRT). Climen contains 11 tablets of 2 mg estradiol valerate (EV), and 10 tablets with 2 mg EV plus 1 mg of cyproterone acetate. Prempak-C, on the other hand, is a regimen consisting of 28 tablets of 0.625 mg conjugated equine estrogens (CEE); the last 12 tablets are taken together with 0.15 mg of norgestrel (NG) tablets. Patients in Sequence I started with Climen for 6 months and then crossed-over to Prempak-C, for the next 6 months. Patients in Sequence II followed the reverse order. Following Climen treatment, significantly higher levels (P < 0.05, t-test) of sex hormone binding globulin (SHBG) and estradiol, when compared to Prempak-C treated subjects, were noted. No significant differences in follicle stimulating hormone (FSH), corticosteroid binding globulin (CBG), renin angiotensinogen, angiotensin-I and aldosterone levels between the two treatment regimens were noted. While both regimens were effective in reducing menopausal symptoms, none of the regimens could eliminate all symptoms completely. Treatment with Climen appeared to result in less frequent occurrences of some symptoms. During periods of no estrogen (only true for Climen) as well as periods of maximum P and E, subjects on Climen had significantly lower incidence of some of the symptoms (backache, lack of concentration, lethargy and swelling) when compared to those on Prempak-C.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1994

Purification and characterization of the inhibitory subunit (delta) of the ATP-synthase from Micrococcus luteus.

Subunit delta was isolated from the ATP-synthase from Micrococcus luteus strain (ATCC 4698). delta, in the case of M. luteus F0F1-ATPase, acts as an inhibitor of ATP hydrolysis and thus resembles subunits in E. coli and chloroplast ATP-synthase. After treatment with 1.5 M LiCl the ATP-synthase dissociated, and subsequently subunit delta (27 kDa) was purified by hydrophobic interaction chromatography. Inhibition of ATP-synthase lacking delta by addition of delta showed non-competitive kinetics with a Ki of approximately 5.9 nM. Subunit epsilon from chloroplast F1, which corresponds functionally to the M. luteus F0F1-delta, and chloroplast delta were tested for ATPase inhibitory activity by addition to the partially delta-depleted ATP-synthase from M. luteus. CF1-epsilon inhibited M. luteus ATP-synthase up to 80%, whereas CF1-delta did not show any influence.

Adenosine Triphosphate↗

PubMed1994

Photosynthetic oxygen evolution: net charge transients as inferred from electrochromic bandshifts are independent of proton release into the medium.

The manganese containing center of the oxygen evolving complex accumulates four oxidizing equivalents in the four stepped water oxidation cycle. Based on experiments on electrochromic absorption transients and the reduction rate of the primary electron donor, P680, it has been speculated that the oscillations of these variables reflect the net charge of the center as calculated from the difference between electron abstraction and proton release into the medium. We compared proton release with electrochromism in thylakoids and core particles, and under variation of the rate of proton release. We found no equivalent of the variations of the extents and the rates of proton release in electrochromism. The oscillatory pattern of the latter reflects the topological properties of the stepped charge storage relative to the position and orientation of electrochromically responsive pigments rather than responding to proton release from the periphery.

Chloroplasts↗

PubMed1994

Calibration and time resolution of lumenal pH-transients in chromatophores of Rhodobacter capsulatus following a single turnover flash of light: proton release by the cytochrome bc1-complex is strongly electrogenic.

The flash-induced proton release into the lumen of chromatophores from Rhodobacter capsulatus was studied with Neutral red as pH-indicator. Calibration of the acidification jump after a single flash yielded a much larger figure, at least 0.8 units, than previously thought. A slow kinetic phase of proton release (85-90% of total) was sensitive to inhibitors of the cytochrome bc1-complex. Its half-rise time, about 10 ms, was the same as the rise time of the electrogenic reaction in the cytochrome bc1-complex that was recorded by electrochromism of carotenoids. The oxidoreduction of the two b-hemes was significantly faster (t1/2 approximately equal to 3 ms). Thus the major electrogenic event in the cytochome bc1-complex is proton and not electron transfer.

Bacterial Chromatophores↗

PubMed1994

Over-production, renaturation and reconstitution of delta and epsilon subunits from chloroplast and cyanobacterial F1.

We studied the functioning of chimeric F0F1-ATPases by replacing subunits delta and epsilon of spinach CF1 with their counterparts from Synechocystis sp. PCC 6803. The sequence identities between these subunits are 26 and 41%, respectively. For a systematic approach to such studies and later extension to genetically modified subunits recombinant proteins are required. The genes coding for spinach and Synechocystis delta and epsilon were cloned into pET3 expression vectors and expressed in Escherichia coli. Upon expression at 37 degrees C the recombinant subunits formed inclusion bodies within the host cells except for spinach delta, which was soluble. Synechocystis delta and epsilon could be obtained in soluble form upon expression at 20 degrees C. After purification (and refolding of spinach epsilon) both epsilon subunits inhibited the Ca(2+)-ATPase activity of soluble CF1(- epsilon). Subunits delta and epsilon from both species raised the rate of ATP synthesis in partially CF1-depleted spinach thylakoids when added together with CF1(- delta) or CF1(- delta, epsilon). This showed the functionality of recombinant Synechocystis and spinach delta and epsilon together with spinach alpha 3 beta 3 gamma. The molar excess of epsilon necessary for saturation was higher for Ca(2+)-ATPase inhibition than for reconstitution of photophosphorylation thus pointing to a direct interaction between epsilon and both CF1 and CF0.

Amino Acid Sequence↗

PubMed1994

The rates of proton uptake and electron transfer at the reducing side of photosystem II in thylakoids.

Proton and electron transfer at the reducing side of photosystem II of green plants was studied under flashing light, the former at improved time resolution by using Neutral red. The rates of electron transfer within QAFeQB were determined by pump-probe flashes through electrochromic transients. The extent of proton binding was about 1 H+/e-. The rates of proton transfer were proportional to the concentration of Neutral red (collisional transfer), whereas the rates of electron transfer out of QA- and from QAFeQB- to the cytochrome b6f complex were constant. The half-rise times of electron transfer (tau e) and the apparent times of proton binding (tau h) at 30 microM Neutral red were: QA- --> FeIIIQB (tau c < or = 100 microseconds, tau h = 230 microseconds); QA- --> FeIIQB (tau c = 150 microseconds, tau h = 760 microseconds); and QA- --> FeIIQB (tau c = 150 microseconds, tau h = 760 microseconds); and QA- --> FeIIQB (tau c = 620 microseconds, tau h = 310 microseconds).

Adaptation, Physiological↗

PubMed1994

Extent and rate of proton release by photosynthetic water oxidation in thylakoids: electrostatic relaxation versus chemical production.

The detailed chemical mechanism of the four steps of photosynthetic oxidation of two molecules of water to yield molecular oxygen plus four protons is under contention. The observed release of protons is a composite of the chemical production and more indirect reactions such as electrostatically induced shifts of acid/base equilibria of peripheral amino acids. In thylakoids we studied the extent and the rate (at microsecond time resolution) of proton release and uptake by each of the four oxidation steps. The pattern of net proton release in thylakoids varied drastically (between 0.3 and 2 H+/e-) as a function of pH. It differed substantially from the pH-dependent patterns of PSII-enriched membrane fragments and core particles, but the stepped progression toward release of dioxygen (the Kok parameter triple) was about the same. This implied an electrostatic origin of this variation and, within the observed limits, a lack of (inhibitory) feedback of the uncompensated charge on the electron transfer from the catalytic Mn cluster to TyrZ+. The rate of rapid proton transfer to the amphiphilic, surface-adsorbed indicator neutral red was proportional to its concentration. The shortest half-transfer time was 12 microseconds, substantially shorter than the time for electron transfer from Mn to TyrZ+ at any oxidation step. Rapid deprotonation thus occurred at the level of TyrZ+. By rapid deprotonation acts the four light-driven oxidation steps S0-->S1-->S2-->S3-->S4 created between 3.4 (at pH 7.4) and 4.5 (pH 6.3) bases per photosystem II.(ABSTRACT TRUNCATED AT 250 WORDS)

Electricity↗

PubMed1994

Endocrine effects in Asian postmenopausal women treated with SH D 461 M and Prempak-C.

We reported the results of a randomized cross-over study comparing SH D 461 M (Climen) and Prempak-C in 38 postmenopausal women who were established users of hormone replacement therapy (HRT). Climen contains 11 tablets of 2 mg estradiol valerate (EV), and 10 tablets with 2 mg EV plus 1 mg of cyproterone acetate. Prempak-C, on the other hand, is a regime consisting of 28 tablets of 0.625 mg conjugated equine estrogens (CEE); the last 12 tablets are taken together with 0.15 mg of norgestrel (NG) tablets. Patients in Sequence I started with Climen for 6 months and then crossed-over to Prempak-C, for the next 6 months; patients in Sequence II, followed the reverse order. Following Climen treatment, significantly higher levels (P < 0.05, t-test) of sex hormone binding globulin (SHBG) and estradiol, when compared to Prempak-C treated subjects, were noted. No significant differences in follicle stimulating hormone (FSH), corticosteroid binding globulin (CBG), renin, angiotensinogen, angiotensin-I and aldosterone levels between the two treatment regimes were noted. While both regimes were effective in reducing menopausal symptoms, none of the regimes could eliminate all symptoms completely. Treatment with Climen appeared to result in less frequent occurrences of some symptoms. During periods of no estrogen (only true for Climen) as well as periods of maximum progestagen and estrogen (P and E), subjects on Climen had significantly lower incidence of some of the symptoms (backache, lack of concentration, lethargy and swelling) when compared to those on Prempak-C.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1993

Chloride-depletion of photosynthetic water oxidase. No proton release during the second oxidation step, S2==>S3, and a transmembrane radical pair recombination from the third on.

Chloride depletion blocks the normal four-step progress of photosynthetic water oxidation. We studied proton release in chloride-depleted thylakoids which were dark-adapted and excited by flashing light. Proton release was blocked from the second flash on, possibly leaving an uncompensated positive charge in the catalytic centre. The reduction of P+680 by Tyrz was still very rapid (<< 10 microseconds). From the third flash on, P+680 was reduced more slowly (70 microseconds/200 microseconds), and by an electrogenic back-reaction. The uncompensated positive charge may be the reason why the rapid reduction of P+680 by Tyrz is prevented and the transmembrane charge-pair recombination is facilitated.

Chlorides↗

PubMed1993

Complementation of Escherichia coli unc mutant strains by chloroplast and cyanobacterial F1-ATPase subunits.

The genes encoding the five subunits of the F1 portion of the ATPases from both spinach chloroplasts and the cyanobacterium Synechocystis sp. PCC 6803 were cloned into expression vectors and expressed in Escherichia coli. The recombinant subunits formed inclusion bodies within the cells. Each particular subunit was expressed in the respective unc mutant, each unable to grow on non-fermentable carbon sources. The following subunits restored growth under conditions of oxidative phosphorylation: alpha (both sources, cyanobacterial subunit more than spinach subunit), beta (cyanobacterial subunit only), delta (both spinach and Synechocystis), and epsilon (both sources), whereas no growth was achieved with the gamma subunits from both sources. Despite a high degree of sequence homology the large subunits alpha and beta of spinach and cyanobacterial F1 were not as effective in the substitution of their E. coli counterparts. On the other hand, the two smallest subunits of the E. coli ATPase could be more effectively replaced by their cyanobacterial or chloroplast counterparts, although the sequence identity or even similarity is very low. We attribute these findings to the different roles of these subunits in F1: The large alpha and beta subunits contribute to the catalytic centers of the enzyme, a function rendering them very sensitive to even minor changes. For the smaller delta and epsilon subunits it was sufficient to maintain a certain tertiary structure during evolution, with little emphasis on the conservation of particular amino acids.

Amino Acid Sequence↗

PubMed1993

Proton slip of the chloroplast ATPase: its nucleotide dependence, energetic threshold, and relation to an alternating site mechanism of catalysis.

The F-ATPase of chloroplasts couples proton flow to ATP synthesis, but is leaky to protons in the absence of nucleotides. This "proton slip" can be blocked by small concentrations of ADP or by inhibitors of the channel portion, CF0. We studied charge flow through the ATPase by flash spectrophotometry and analyzed the inhibition of proton slip by nucleotides, phosphate/arsenate, and insufficient proton motive force. The following inhibition constants (at given background concentrations) were observed: ADP, 0.2 microM (0.5 mM P(i)); ADP, 13.4 microM (no P(i)); P(i), 43 microM (1 microM ADP); GDP, 2.5 microM (0.5 mM P(i)); ATP, 2 microM. ADP and P(i) mutually lowered their respective inhibition constants. Phosphate could be replaced by arsenate. Proton slip occurred only if the proton motive force exceeded a certain threshold, similar to that for ATP synthesis. The inhibition of proton slip by ADP and GDP qualified the respective nucleotide binding sites as belonging to the subset of two (or three) potentially catalytic sites out of the total of six. We interpreted the ADP-induced transition between different conduction states of the ATPase from "slipping" to "closed" to "coupled" as a consequence of the alternating site mechanism of catalysis. Whereas the proton translocator idles in the absence of nucleotides, the high-affinity binding of the first ADP/P(i) couple to one site clutches proton flow to some (conformational) change that can only be executed after the binding of another ADP/P(i) couple to a second site. From there on these sites alternate in the catalytic cycle. An entropic machine is presented which likewise models proton slip, unisite, and multisite ATP synthesis and hydrolysis.

Adenosine Diphosphate↗

PubMed1992

Added subunit beta of CF1 as well as gamma/delta/epsilon restore photophosphorylation in partially CF1-depleted thylakoids.

We investigated the ability of subunits beta, gamma, delta, and epsilon of CF1, the F1-ATPase of chloroplasts, to interact with exposed CF0 in EDTA-treated, partially CF1-depleted thylakoid membranes. We measured the ability of subunits beta, gamma, delta, and epsilon to stimulate the rate of photophosphorylation under continuous light and, for subunit beta, also the ability to diminish the proton leakage through exposed CF0 by deceleration of the decay of electrochromic absorption transients under flashing light. The greatest effect was caused by subunit beta, followed by gamma/delta/epsilon. Pairwise combinations of gamma, delta, and epsilon or each of these subunits alone were only marginally effective. Subunit gamma from the thermophilic bacterium PS 3 in combination with chloroplast delta and epsilon was as effective as chloroplast gamma. The finding that the small CF1 subunits in concert and the beta subunit by itself specifically interacted with the exposed proton channel CF0, qualifies the previous concept of subunit delta acting particularly as a plug to the open CF0 channel. The interactions between the channel and the catalytic portion of the enzyme seem to involve most of the small, and at least beta of the large subunits.

Amino Acid Sequence↗

PubMed1992

THE CHLOROPLAST H+-ATPase: PARTIAL REACTIONS OF THE PROTON.

This article reviews proton intake, charge transfer and proton release by F-ATPases, based in part on flash spectrophotometric studies on the chloroplast ATPase in thylakoid membranes, CF1Fo. The synthesis-coupled translocation of charges by CF1Fo (maximum rate <1500 s-1) and the dissipative flow through its exposed channel portion, CFo (rate >10 000 s-1), are extremely proton-specific (selectivity H+:K+>10(7):1). The proton-specific filter is located in CFo. Proton flow through exposed CFo can be throttled by adding subunit (&dgr;) or subunit &bgr; of CF1. These subunits thus may provide energy-transducing contacts between CF1 and CFo. Recently, we characterized two conditions where, in contrast to the above situation, proton intake by CF1Fo was decoupled from proton transfer across the main dielectric barrier: (a) CF1Fo structurally distorted by low ionic strength transiently trapped protons in a highly cooperative manner, but remained proton tight. This result has been interpreted in terms of Mitchell's proton well. (b) In the absence of nucleotides there is a proton slip. Addition of nucleotides (100 nmol l-1 ADP) abolished proton conduction but not proton intake by CF1Fo. These experiments functionally tag proton binding groups on CF1Fo that are located before the main dielectric barrier.

Journal Article↗

PubMed1992

Thylakoids from pea seedlings grown under intermittent light: biochemical and flash-spectrophotometric properties.

Thylakoid membranes were isolated from pea seedlings grown under intermittent light (2-min light/118-min dark cycles). These preparations differed from controls (thylakoids from plants grown under 16-h light/8-h dark cycles) in the following respects: 15 times smaller chlorophyll/protein ratio, 10 times greater chlorophyll a/b ratio, absence of light-harvesting chlorophyll a/b binding proteins, and 2-3-fold greater ratio of photosystem II over photosystem I. In addition we found the following: (1) Electrogenic electron transfer around cytochrome b6/f under flashing light was greatly enhanced, probably as a consequence of the greater photosystem II/photosystem I ratio. (2) The rate of proton uptake from the medium at the acceptor side of photosystem II was enhanced, probably by unshielding of the quinone binding domain. (3) The N,N'-dicyclohexylcarbodiimide sensitivity of the proton-pumping activity of photosystem II was absent, which was consistent with the attribution of a N,N'-dicyclohexylcarbodiimide-induced protonic short circuit to chlorophyll a/b binding proteins. (4) The sensitivity of oxygen evolution under continuous light to variations of pH or the concentration of Ca2+ was altered. Chlorophyll a/b binding proteins serve as light-harvesting antennas. We found in addition that they modulated the activity of water oxidation and, in particular, the proteolytic reactions around photosystem II.

Cell Fractionation↗

PubMed1992

Proton release during the four steps of photosynthetic water oxidation: induction of 1:1:1:1 pattern due to lack of chlorophyll a/b binding proteins.

In photosynthesis of green plants water is oxidized to dioxygen. This four-step process is accompanied by the release of four protons (per molecule of dioxygen) into the lumen of thylakoids. In dark-adapted thylakoids which are excited with a series of short flashes of light, the extent of proton release oscillates with period four as a function of flash number. Noninteger and pH-dependent proton/electron ratios (e.g., 1.1, 0.25, 1.0, and 1.65 at pH 7) have been attributed to a superposition of two reactions: chemical production of protons and transient electrostatic response of peripheral amino acid side chains. Aiming at the true pattern of proton production, we investigated the relative contribution of peripheral proteins. Thylakoids with and without chlorophyll a/b binding proteins were compared. Thylakoids lacking chlorophyll a/b binding proteins were prepared from pea seedlings grown under intermittent light [Jahns, P., & Junge, W. (1992) Biochemistry (preceding paper in this issue)]. We found no oscillation of proton release in the pH range from 6 to 7.5. These and other results showed that chlorophyll a/b binding proteins, which primarily serve as light-harvesting antennas, modulate proton release by water oxidation. A nonoscillating pattern of proton release, with proton/electron ratios of 1:1:1:1 more closely represents the events in the catalytic center proper. This implies hydrogen abstraction rather than electron abstraction from water during the oxygen-evolving step S3----S0.

Fabaceae↗

PubMed1992

Dimerization constant and single-channel conductance of gramicidin in thylakoid membranes.

The effect of the pore-forming antibiotic gramicidin on pure lipid membranes is well characterized. We studied its action in protein-rich thylakoid membranes that contain less than 25% (wt/wt) acyl lipids. A transmembrane voltage was induced by flashing light, and its decay was measured and interpreted to yield the distribution of gramicidin over thylakoids, its dimerization constant and its single-channel conductance in this membrane. The distribution of gramicidin over the ensemble of thylakoids was immediately homogeneous when the antibiotic was added under stirring, while it became homogeneous only after 20 min in a stirred suspension that was initially heterogeneous. The dimerization constant, 5 x 10(14) cm2/mol, was about 10 times larger than in pure lipid membranes. This was attributed to the up-concentration of gramicidin in the small fractional area of protein-free lipid bilayer and further by a preference of gramicidin for stacked portions of the membrane. The latter bears important consequences with regard to bioenergetic studies with this ionophore. As gramicidin was largely dimerized from a concentration of 1 nM (in the suspension) on, the membrane's conductance then increased linearly as a function of added gramicidin. When the negative surface potential at the thylakoid membrane was screened, the conductance of a single gramicidin dimer agreed well with figures reported for bilayers from neutral lipid (about 0.5 pS at 10 mM NaCl). The modulation of the conductance by the surface potential in spinach versus pea thylakoids and between different preparations is discussed in detail.

Chloroplasts↗