Fumarate reduction systems in members of the family Rhodospirillaceae with different quinone types

Nineteen established and one undesignated species of the Rhodospirillaceae were examined for fumarate reduction in connection with their quinone systems. The fumarate reductase activity with reduced methyl viologen (MVH) or FMNH₂ as electron donor was found in membrane (chromatophore) preparations from phototrophically grown cells of all species containing menaquinone (MK) and/or rhodoquinone. The species having ubiquinone as the sole quinone contained no fumarate reductase activity, except some Rhodobacter species showing the FMNH₂-dependent activity. The MVH-fumarate reductase activity of the MK-type species was not inhibited by Triton X-100 or acetone treatment, suggesting the presence of a fumarate reductase reacting directly with MVH, while such an enzyme was absent in the MK-lacking strains, with few exceptions. The FMNH₂-fumarate reduction system was abolished by a detergent or acetone extraction in all bacteria but differed much among species with different quinone types as to the response to respiratory inhibitors. These differences in fumarate-reducing properties and quinone systems among the phototrophic bacteria are discussed from evolutionary and taxonomic viewpoints.Non-standard abbreviations RQ rhodoquinone - MK menaquinone - MVH reduced methyl viologen - HOQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - TTFA 2-thenoyltrifluoroacetone     

Effects of pH on the peripheral light-harvesting antenna complex for Rhodopseudomonas palustris

In this work steady-state absorption spectroscopy, circular dichroism spectroscopy and sub-micro-second time-resolved absorption spectroscopy were used to investigate the effect of pH on the struc-tures and functions of LH2 complex for Rhodopseudomonas palustris. The results revealed that: (1) B800 Bchla was gradually transformed to free pigments absorbing around 760 nm on the minutes timescale upon the induction of strong acidic pH, and subsequently there disappeared the CD signal for Qy band of B800 in the absence of B800. In addition, Carotenoids changed with the similar tendency to B850 BChl. (2) The introduction of strong basic pH gave rise to no significant changes for B800 Bchla, while B850 BChla experienced remarkable spectral blue-shift from 852 to 837 nm. Similar phe-nomenon was seen for the CD signal for Qy band of B850. Carotenoids displayed strong and pH-independent CD signals in the visible range. (3) In the case of both physiological and basic pH, broad and asymmetrical positive Tn←T1 transient absorption appeared following the pulsed photo-excitation of Car at 532 nm. By contrast, the featureless and weak positive signal was observed on the sub-microsecond timescale in the acidic pH environment. The aforementioned experimental results indicated that acidic pH-induced removal of B800 Bchla prevented the generation of the caro-tenoid triplet state (3Car*), which is known to be essential for the photo-protection function. Neverthe-less, carotenoids can still perform this important physiological role under the basic pH condition, where the spectral blue shift of B850 exerts little effect on the overall structure of the cyclic aggregate, therefore favoring the formation of carotenoid triplet state.     

Nitrous oxide reduction by members of the family Rhodospirillaceae and the nitrous oxide reductase of Rhodopseudomonas capsulata.

After growth in the absence of nitrogenous oxides under anaerobic phototrophic conditions, several strains of Rhodopseudomonas capsulata were shown to possess a nitrous oxide reductase activity. The enzyme responsible for this activity had a periplasmic location and resembled a nitrous oxide reductase purified from Pseudomonas perfectomarinus. Electron flow to nitrous oxide reductase was coupled to generation of a membrane potential and inhibited by rotenone but not antimycin. It is suggested that electron flow to nitrous oxide reductase branches at the level of ubiquinone from the previously characterized electron transfer components of R. capsulata. This pathway of electron transport could include cytochrome c', a component hitherto without a recognized function. R. capsulata grew under dark anaerobic conditions in the presence of malate as carbon source and nitrous oxide as electron acceptor. This confirms that nitrous oxide respiration is linked to ATP synthesis. Phototrophically and anaerobically grown cultures of nondenitrifying strains of Rhodopseudomonas sphaeroides, Rhodopseudomonas palustris, and Rhodospirillum rubrum also possessed nitrous oxide reductase activity.     

The effect of pH, temperature, and D2O on the activity of porcine synovial collagenase and gelatinase

The pH dependence of Vmax and Vmax/Km for hydrolysis of Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 at the Gly-Leu bond by porcine synovial collagenase and gelatinase was determined in the pH range 5-10. Both enzymes exhibited bell-shaped dependencies on pH for these two kinetic parameters, indicating that activity is dependent on at least two ionizable groups, one of which must be unprotonated and the other protonated. For collagenase, Vmax/Km data indicate that in the substrate-free enzyme, these groups have apparent pK values of 7.0 and 9.5, while the Vmax profile indicates similar pK values of 6.8 and 10.1 for the enzyme-substrate complex. The corresponding pH profiles of gelatinase were similar to those of collagenase, indicating the importance of groups with apparent pK values of 5.9 and 10.0 for the free enzyme and 5.9 and 11.1 for the enzyme-substrate complex. When these kinetic constants were determined in D2O using the peptide substrate, there was no significant effect on Vmax or Km for collagenase or Km for gelatinase. However, there was a deuterium isotope effect of approximately 1.5 on Vmax for gelatinase. These results indicate that a proton transfer step is not involved in the rate-limiting step for collagenase, but may be limiting with gelatinase. The Arrhenius activation energies for peptide bond hydrolysis of the synthetic peptide as well as the natural substrates were also determined for both enzymes. The activation energy (81 kcal) for hydrolysis of collagen by collagenase was nine times greater than that determined for the synthetic substrate (9.2 kcal). In contrast, the activation energy for hydrolysis of gelatin by gelatinase (26.3 kcal) was only 2.4 times greater than that for the synthetic substrate (11 kcal).     

Effect of substrate concentration,inorganic nitrogen,O2 concentration,temperature and pH on dehydrogenase activity in soil

Summary Dehydrogenase activity was measured in a sandy loam soil under a variety of incubation conditions using the reduction of 2-(p-iodophenyl-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT) to iodonitrotetrazolium formazan (INT-formazan). There was a high positive correlation between dehydrogenase activity and substrate concentration, incubation temperature, and soil pH. Dehydrogenase activity also displayed a high negative correlation with O₂ concentrations. Ammonium sulfate at concentrations from 40 to 120 g/g soil had no significant effect on dehydrogenase activity. However, at concentrations of 160 and 200 g/g, dehydrogenase activity was significantly reduced. Potassium nitrate at concentrations ranging from 40 to 200 g/g had no significant effect on soil dehydrogenase activity, whereas sodium nitrite significantly inhibited activity at concentrations of 120 and 160 g/g soil.     

On the effects of PufX on the absorption properties of the light-harvesting complexes of Rhodobacter sphaeroides

Some species of purple bacteria as, e.g., Rhodobacter sphaeroides contain the protein PufX. Concurrently, the light harvesting complexes 1 (LH1) form dimers of open rings. In mutants without PufX, the LH1s are closed rings and photosynthesis breaks down, because the ubiquinone exchange at the reaction center is blocked. However, the main purpose of the LH1 is light harvesting. We therefore investigate the effects that the PufX-induced dimerization has on the absorption properties of the core complexes. Calculations with a dipole model, which compare the photosynthetic efficiency of various configurations of monomeric and dimeric core complexes, show that the dimer can absorb photons directly into the reaction centers more efficiently, but that the performance of the more sophisticated dimeric LH1 antenna degrades faster with structural perturbations. The calculations predict an optimal orientation of the reaction centers relative to the LH1 dimer, which agrees well with the experimentally found configuration. Based on experimental observations indicating that the dimeric core complexes are indeed rather rigid, we hypothesize that in PufX⁺ species the association between the LH1 and the reaction centers is enhanced. This mechanical stabilization of the core complexes would lead to the observed quinone blockage, when PufX is missing.     

A study of the content of pigments in the light-harvesting antenna of the green bacterium from the new family Oscillochloridaceae

The properties of the light-harvesting superantenna of the photosynthesizing bacteria from the new family of green filamentous bacteria Oscillochloridaceae were investigated by optical spectroscopy. The antenna of Oscillochloris trichoides consists of peripheral chlorosomal and membrane subantennas. A method of isolation of Osc. trichoides chlorosomal antenna was developed using the chaothropic agent sodium thiocyanate, which simultaneously acts to stabilize chlorosomal activity. An analysis of the second derivatives of the absorption spectra of isolated chlorosomes and their acetone-methanol extracts suggested that BChl c was a predominant light-harvesting pigment in Osc. trichoides chlorosomes. Besides, it was found that, in addition to the BChl c-antenna, chlorosomes contain a minor BChl a-antenna. It was shown that the membrane BChl a-subantenna is a light-harvesting complex with absorption maxima in the near infrared region at 805 and 860 nm. Analysis of the spectral data obtained suggested that the Osc. trichoides chlorosomal antenna resembles those from Chlorobiaceae species, whereas the membrane B805-860 BChl a antenna of Osc. trichoides is close to the membrane B808-866 BChl a antenna of Chloroflexaceae species.     

Bcl-2 family members and apoptosis, taken to heart

Loss of myocardial cells via apoptosis has been observed in many cardiovascular diseases and has been shown to contribute to the initiation and progression of heart failure. The Bcl-2 family members are important regulators of the mitochondrial pathway of apoptosis. These proteins decide whether the mitochondria should initiate the cell death program and release proapoptotic factors such as cytochrome c. The Bcl-2 proteins consist of anti- and proapoptotic members and play a key role in regulating apoptosis in the myocardium. The antiapoptotic proteins have been demonstrated to protect against various cardiac pathologies, whereas the antiapoptotic proteins have been reported to contribute to heart disease. This review summarizes the current understanding of the role of Bcl-2 proteins in the heart. cardiovascular disease; cytochrome c; protein; mitochondria     

The influence of pH and temperature on the properties of myosin

1. The rate of denaturation of myosin solutions at temperatures between 32 degrees and 45 degrees and at pH values between 5.3 and 6.2 has been studied, by using adenosine-triphosphatase activity and solubility in m-potassium chloride at pH6.1 as criteria. 2. Myosin, when heated, loses its adenosine-triphosphatase activity before it becomes insoluble. 3. The loss of adenosine-triphosphatase activity and solubility are both first-order and pH-dependent reactions. Myosin, however, becomes insoluble only when heated within a narrow range of pH values. 4. The thermodynamic functions found for the two processes of denaturation are compared and discussed. 5. The possibility is discussed that, in muscle undergoing rigor, conditions may obtain that would denature myosin.     

The structure, expression, and properties of additional members of the protein kinase C family

In rat brain three members of the protein kinase C family encoded by cDNAs termed delta, epsilon, and zeta were newly identified by molecular cloning and sequence analysis. The new members have a common structure that is closely related to but clearly distinct from the four members of the family previously isolated having alpha-, beta I-, beta II-, and gamma-sequences, although the zeta-cDNA available at present does not appear to contain a complete reading frame for protein kinase C. The delta-, epsilon-, and zeta-cDNAs all encode a characteristic cysteine-rich sequence and protein kinase domain sequence, both of which are highly homologous among the protein kinase C family. However, the new members lack one of the conserved regions that is present in alpha-, beta I-, beta II-, and gamma-sequences. An additional cDNA clone termed epsilon' was isolated, which is identical with epsilon-cDNA except for a short sequence at the 5'-terminal end region. The two members having delta- and epsilon-sequences were expressed in COS 7 cells, and partially purified and characterized. The enzymes having delta- and epsilon-sequences depend on phospholipid and diacylglycerol for the enzymatic activity, but their properties slightly differ from the previously known members of protein kinase C. Northern blot analysis suggests that the new members of protein kinase C exist in the brain and some other tissues.     

Influence of detergent concentration on aggregation and spectroscopic properties of light-harvesting complex II

Aggregation of photosynthetic light-harvesting complexes strongly influences their spectroscopic properties. Fluorescence yield and excited state lifetimes of the main light-harvesting complex (LHC II) of higher plants strongly depend on its aggregation state. Detergents are commonly used to solubilize membrane proteins and/or to circumvent their aggregation in aqueous environments. Nonlinear polarization spectroscopy in the frequency domain (NLPF) was performed with LHC II over a wide concentration range of the mild detergent n-dodecyl β-d-maltoside (β-DM). Additionally, conventional absorption-, fluorescence- and circular dichroism-spectra were measured. The results indicate that: (i) conventional spectroscopic techniques are not well suited to investigate aggregation effects. NLPF provides a novel approach to overcome this problem: NLPF spectra display dramatic alterations upon even minor β-DM concentration changes. (ii) Commonly used detergent concentrations (around or slightly above the critical micellar concentration) apparently do not lead to complete trimerization of LHC II. A long-wavelength species in the NLPF spectra (peaking at about 685 nm), indicative of residual aggregation, persists up to DM-concentrations of 0.06%. (iii) High-resolution NLPF spectra indicate the existence of a species with a considerably shortened excited state lifetime. (iv) No indication of denaturation was found even at the highest β-DM concentrations used. (v) A specific change in interaction between certain chlorophyll(s) b and a xanthophyll molecule, probably neoxanthin, was detected upon aggregation as well as at higher β-DM concentrations. The results are discussed with respect to the still elusive mechanism of nonradiative dissipation of excess excitation energy in the antenna system.     

Short- and long-term operation of the lutein-epoxide cycle in light-harvesting antenna complexes

The lutein-5,6-epoxide (Lx) cycle operates in some plants between lutein (L) and its monoepoxide, Lx. Whereas recent studies have established the photoprotective roles of the analogous violaxanthin cycle, physiological functions of the Lx cycle are still unknown. In this article, we investigated the operation of the Lx cycle in light-harvesting antenna complexes (Lhcs) of Inga sapindoides Willd, a tropical tree legume accumulating substantial Lx in shade leaves, to identify the xanthophyll-binding sites involved in short- and long-term responses of the Lx cycle and to analyze the effects on light-harvesting efficiency. In shade leaves, Lx was converted into L upon light exposure, which then replaced Lx in the peripheral V1 site in trimeric Lhcs and the internal L2 site in both monomeric and trimeric Lhcs, leading to xanthophyll composition resembling sun-type Lhcs. Similar to the violaxanthin cycle, the Lx cycle was operating in both photosystems, yet the light-induced Lx --> L conversion was not reversible overnight. Interestingly, the experiments using recombinant Lhcb5 reconstituted with different Lx and/or L levels showed that reconstitution with Lx results in a significantly higher fluorescence yield due to higher energy transfer efficiencies among chlorophyll (Chl) a molecules, as well as from xanthophylls to Chl a. Furthermore, the spectroscopic analyses of photosystem I-LHCI from I. sapindoides revealed prominent red-most Chl forms, having the lowest energy level thus far reported for higher plants, along with reduced energy transfer efficiency from antenna pigments to Chl a. These results are discussed in the context of photoacclimation and shade adaptation.     

Effects of glucose, growth temperature, and pH on listeriolysin O production in Listeria monocytogenes.

Expression of listeriolysin O of Listeria monocytogenes as a function of different growth conditions was studied by performing a direct hemolysin assay, immunoblotting experiments, and an enzyme-linked immunosorbent assay. Expression of listeriolysin O was reduced at a lower growth temperatures (26 degrees C) and at higher glucose concentrations (> or = 0.3%) in the growth media. The effect of glucose appeared to be due to a change in the pH of the growth media.