Function and properties of a soluble c-type cytochrome c-551 in secondary photosynthetic electron transport in whole cells of Chromatium vinosum as studied with flash spectroscopy

Changes in the absorption spectrum induced by 10-μs flashes and continuous light of various intensities were studied in whole cells of Chromatium vinosum.This paper describes the role and function of a soluble c-type cytochrome, c-551, which was surprisingly found to act in many ways similar to the cytochrome c-420 in Rhodospirillum rubrum, described in a previous paper [1].After the photooxidation of the membrane bound high potential cytochrome c-555 by a 10-μs flash, (the low potential cytochrome c-552 was kept permanently in the oxidized state) the oxidation of c-551 is observed ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 0.3}\text{ms}$). From a careful analysis of the absorbance difference spectrum and the kinetics it is concluded that there is approximately 0.6–0.7 c-551 per reaction center and that essentially all the c⁺-555 is reduced via the cytochrome c-551. The oxidized-reduced difference spectrum of c-551 shows peaks at 551 and 421.5 nm. The reduction of c⁺-551 following the flash-induced oxidation is strongly inhibited by HOQNO, but only slightly by antimycin A.Cytochrome c-551 reduces only the oxidized high potential cytochrome c-555, which is probably located on the outside of the membrane, on the opposite side of the primary acceptor. The low potential cytochrome c-552 does not show any detectable interaction with cytochrome c-551. After the cells have been sonicated, no c-551 is photooxidized and at least part of the cytochrome occurs in the solution.Analysis of the reduction kinetics of c⁺-551 in the absence and presence of external donors suggests that c⁺-551 is partly reduced via a cyclic pathway, which is blocked by addition of o-phenanthroline, and partly via a non-cyclic pathway. The non-cyclic reduction rate of c⁺-551 (k = 6 s^?1) is increased approximately 5–10 times upon thiosulphate addition, suggesting a role for c-551 between the final donor pool and the oxidized membrane bound c-type cytochromes.     

Redox properties of b-type cytochromes in Escherichia coli and rat liver mitochondria and techniques for their analysis

We describe here apparatus and procedures for conducting potentiometric titrations and for analyzing the collected data in terms of the number of components present, their amounts and their midpoint potentials. Using these procedures we have determined the presence of three forms of cytochrome b₁ in Escherichia coli with midpoint potentials at pH 7.1 of about ?50, +110 and +220 mV. We were not able to demonstrate a change in any of these potentials by the addition of phosphate, ATP, or 2,4-dinitrophenol. We have been able to confirm the presence of two forms of cytochrome b in non-energized mitochondria and the apparent conversion of the low-potential component to a new high potential component upon energization of the mitochondria. However we cite further experimental data that question the actual conversion of one form of cytochrome b to another. An alternative interpretation based on our analysis suggests that the high voltage component may be present in a masked form in the non-energized mitochondria.     

Evidence for involvement of the electron transport system at a late step of anaerobic microbial heme synthesis

The penultimate step in heme biosynthesis, the oxidation of protoporphyrinogen to protoporphyrin, can be anaerobically coupled to the reduction of fumarate in extracts of anaerobically-grown Escherichia coli. This coupling is approximately 90% inhibited by 2-heptyl-4-hydroxy quinoline-N-oxide (HQNO), a known inhibitor of the electron transport chain. This observation suggests that the mechanism of the anaerobic oxidation of protoporphyrinogen in E. coli involves a coupling into the anaerobic electron transport system. In contrast, the aerobic oxidation of protoporphyrinogen, which occurs in mammalian and yeast mitochondria, is known to be linked directly to oxygen without the mediation of an electron transport system.     

Evidence for involvement of the electron transport system at a late step of anaerobic microbial heme synthesis.

The penultimate step in heme biosynthesis, the oxidation of protoporphyrinogen to protoporphyrin, can be anaerobically coupled to the reduction of fumarate in extracts of anaerobically-grown Escherichia coli. This coupling is approximately 90% inhibied by 2-heptyl-4-hydroxy quinoline-N-oxide (HQNO), a known inhibitor of the electron transport chain. This observation suggests that the mechanism of the anaerobic oxidation of protoporphyrinogen in E. coli involves a coupling into the anaerobic electron transport system. In contrast, the aerobic oxidation of protoporphyrinogen, which occurs in mammalian and yeast mitochondria, is known to be linked directly to oxygen without the mediation of an electron transport system.     

Cytoplasmic changes in level and distribution of glucose-6-phosphatase activities from rat liver during diethylnitrosamine-induced carcinogenesis.

Glucose-6-phosphatase (EC 3.1.3.9) activities were determined in isolated microsomes, cytoplasmic smooth and rough membranes, ribosomes and free cytosol from rat liver undergoing carcinogenesis by diethylnitrosamine (DENA) and compared with cytoplasmic fractions isolated in parallel from healthy animals from the same age.With continuous administration of a low dose of DENA (2.6 mg/kg rat per day for 20 weeks in the drinking water) livers of carcinogen treated rats became heavier than the control livers but the body weight decreased. About 70% of total glucose-6-phosphatase activity could be detected in the microsomal fraction. While there was no significant difference in this activity in both animal groups up to the 4th week, glucose-6-phosphatase of cancerous liver showed a distinct decrease of activity compared with normal liver.During cancer induction this enzyme became more soluble, confirmed by the observation that it was detached from firmer structures of cytoplasm as rough membranes and polysomes and translocated to smooth membranes and the soluble cytoplasmic fraction successively. The corresponding increase in glucose-6-phosphatase activity in the 105 000 g supernatant appears to be due to the loss of enzyme activity in a distinct cytoplasmic membrane fraction. These data strongly suggest that in parallel with alteration of cytoplasmic membrane structures during carcinogen feeding glucose-6-phosphatase is detached from heavier components of the cytoplasm while total activity decreased. Possible mechanisms of these findings are discussed.     

Diversity of anaerobic microbial processes in chlorobenzoate degradation: Nitrate,iron, sulfate and carbonate as electron acceptors

 The utilization of monochlorobenzoate isomers (2-, 3- and 4-chlorobenzoate) by anaerobic microbial consortia in River Nile sediments was systematically evaluated under denitrifying, Fe-reducing, sulfidogenic and methanogenic conditions. Loss of all three chlorobenzoates was noted in denitrifying cultures; furthermore, the initial utilization of chlorobenzoates was fastest under denitrifying conditions. Loss of 3-chlorobenzoate was seen under all four reducing conditions and the degradation of chlorobenzoates was coupled stoichiometrically to NO^- ₃ loss, Fe²⁺ production, SO^2- ₄ loss or CH₄ production, indicating that the chlorobenzoates were oxidized to CO₂. To our knowledge, this is the first observation of halogenated aromatic degradation coupled to Fe reduction. Received: 29 July 1994/Received revision: 22 November 1994/Accepted 16 December 1994     

Localization of the basic protein and lipophilin in the myelin membrane with a non-penetrating reagent

The localization of proteins in myelin was studied by the use of a non-penetrating penetrating reagent. Tritiated 4,4′-diisothiocyano-2,2′-ditritiostilbene disulfonic acid was used to label the isolated myelin membrane. The membrane was labelled, the basic protein and the hydrophobic protein, lipophilin, were isolated. After 10 min of exposure to the reagent, the specific activity of lipophilin was found to be 10 times greater than that of the basic protein. Water shock did not alter the specific activities. However, sonication increased the specific activity of lipophilin but not that of basic protein. When the isolated proteins were labelled with ³H-labelled, 4,4′-diisothiocyano-2,2′-ditritiostilbene disulfonic acid, the specific activity of the basic protein was 10 times that of lipophilin. We concluded that the low specific activity of basic protein isolated from the labelled membrane was due to the inaccessible position of this protein in the membrane bilayer.     

Unfolding and refolding of juvenile hormone binding protein

Juvenile hormone (JH) regulates insect development. JH present in the hemolymph is bound to a specific glycoprotein, juvenile hormone binding protein (JHBP), which serves as a carrier to deploy the hormone to target tissues. In this report structural changes of JHBP from Galleria mellonella induced by guanidine hydrochloride have been investigated by a combination of size-exclusion chromatography, protein activity measurements, and spectroscopic methods. Molecules of JHBP change their conformation from a native state via two unstable intermediates to a denatured state. The first intermediate appears in a compact state, because it slightly changes its molecular size and preserves most of the JHBP secondary structure of the native state. Although the second intermediate also preserves a substantial part of the secondary structure, it undergoes a change into a noncompact state changing its Stokes radius from approximately 30 to 39 A. Refolding experiments showed that JHBP molecules recover their full protein structure, as judged from the CD spectrum, fluorescence experiments, and JH binding activity measurements. The free energy of unfolding in the absence of the denaturant, DeltaG(D-N), is calculated to be 4.1 kcal mol(-1).     

Measurement and interpretation of fluorescence polarisations in phospholipid dispersions

An instrument that measures the temperature dependence of fluorescence polarisation and intensity directly and continuously is described. The behaviour of four fluorescent probes bound to a number of well characterised model systems was then examined. The motional properties of the probes were determined from the polarisation and intensity data and were found to be sensitive to the crystallineliquid crystalline phase transitions in phospholipid vesicles of dimyristoyl and dipalmitoyl phosphatidylcholine. Binary mixture of dilauroyl and dipalmitoyl phosphatidycholine show lateral phase separation and in this system the probes partition preferentially into the more ‘fluid’ phase. In systems that have been reported to contain ‘short range order’ or ‘liquid clustering’, such as dioleoyl phosphatidylcholine and liquid paraffin, the motion of the probes was found to have anomalous Arrhenius behaviour consistent with the idea that homogeneous phases were not being sampled. The significance of these findings for the interpretation of the behaviour of fluorescent probes bound to natural membranes is discussed.     

Heterologous expression and purification of a biologically active legume lectin from Cratylia mollis seeds (CRAMOLL 1)

CRAMOLL 1 is a mannose/glucose isolectin isolated from Cratylia mollis seeds. This lectin has 82% sequence identity with Con A and essentially the same quaternary structure. As with Con A, CRAMOLL 1 seems to undergo complex post-translational processing which makes it difficult to the use of traditional molecular cloning for heterologous expression. Here we report the expression and purification of functional recombinant CRAMOLL 1 (rCRAMOLL 1) in Escherichia coli. This was accomplished by introducing a chemically synthesized DNA encoding the mature CRAMOLL 1 amino acid sequence into a bacterial expression vector under T7 promoter control. Most of the recombinant lectin was found in insoluble aggregates (inclusion bodies), but we were able to recover reasonable amounts of soluble lectin in the active form by decreasing the protein induction temperature. The recombinant lectin was purified to homogeneity with one-step affinity chromatography. The plant CRAMOLL 1 (pCRAMOLL 1) and rCRAMOLL 1 share several physicochemical properties such as molecular mass, charge density and secondary and tertiary structures. However, pCRAMOLL 1 has a lower thermodynamic stability than rCRAMOLL 1 when probed by acidification, high temperature or high hydrostatic pressure, and this is probably caused by the presence of tetramers composed of fragmented monomers, which are formed in the plant cotyledon but absent from the recombinant protein. rCRAMOLL 1 behaves identically to its plant counterpart with respect to its specificity for monosaccharides, and to its agglutinating activities against rabbit erythrocytes and Trypanosoma cruzi epimastigote cells.     

Protoporphyrinogen oxidation in chloroplasts and plant mitochondria,a step in heme and chlorophyll synthesis

The oxidation of protoporphyrinogen to protoporphyrin was demonstrated in greening plastids and mitochondria from greening barley shoots. The plastids, purified by sucrose gradient centrifugation, were essentially free of a mitochondrial marker enzyme. The plastid activity was destroyed by mild heating and was proportional to plastid concentration suggesting, an enzymatic reaction. Uroporphyrinogen I was not oxidized at an appreciable rate. Activity was also demonstrated in etioplasts and mitochondria from dark-grown barley, and in chloroplasts from commercial spinach leaves. The chelating agent 1,10-phenanthroline partially decreased activity in plant organelles, but cyanide did not. The plastid activity, like the activity in liver mitochondria, was readily demonstrable at pH 8.4 in the presence of glutathione as reducing agent. However, the plastid activity was markedly enhanced by assay at pH 7.0 and the absence of reducing agents. These properties distinguish the activity in plants from that previously described in mammalian mitochondria and photosynthetic bacteria.     

The acidification of rat liver lysosomes in vitro: a role for the membranous ATPase as a proton pump.

Lysosomes were purified from the livers of rats which had been treated with Triton WR-1339. The ATPase activity of these lysosomes was stimulated by preincubation with NaCl or KCl, conditions which diminish the proton gradient due to Donnan equilibrium. Subsequent to this preincubation measurements of methylamine uptake by lysosomes showed an ATP-dependent enhancement. Simultaneous measurements of the internal volumes of lysosomes confirmed that ATP-dependent methylamine uptake is due to acidification of lysosomes by 0.3 to 0.5 pH units. Because the conditions which stimulated ATP-dependent methylamine uptake also stimulated the ATPase activity it is concluded that acidification of lysosomes requires an ATPase which functions as a proton pump.     

p-nitroacetophenoxime N-methylcarbamate,a new electron acceptor in isolated thylakoids

p-Nitroacetophenoxime N-methylcarbamate (MCPNA) is a rather potent inhibitor of the electron transfer in spinach class A chloroplasts. In isolated thylakoids, MCPNA is an electron acceptor at the level of photosystem I (PS I). It inhibits O₂ evolution in the presence of NADP and ferredoxin but not the reduction of ferricyanide. MCPNA is active as an acceptor between 3 μM and 100 μM. At concentrations higher than 300 μM, inhibition of photosystem II (PS II) occurs. MCPNA has no uncoupling effect on photophosphorylation. Reduction of MCPNA by thylakoids in the presence of light is in accordance with the E′_o of this compound (??0.57 V) and is followed by an electron transfer to O₂. This reaction probably explains the inhibitory effect of MCPNA on class A chloroplasts.     

The stimulatory effect of the organic sulfur supplement, mercaptopropane sulfonic acid on cellulolytic rumen microorganisms and microbial protein synthesis in cattle fed low sulfur roughages

Two metabolism trials (experiments 1 and 2) were conducted to examine the effect of the organic S compound, sodium 3-mercapto-1-propane sulfonic acid (MPS) on feed intake, fiber digestibility, rumen fermentation and abundance of cellulolytic rumen microorganisms in cattle fed low S (<0.11%) roughages. Urea was provided in all treatments to compensate for the N deficiency (<0.6%) in the roughages. In experiment 1, steers (333 ± 9.5 kg liveweight) were fed Angleton grass (Dicanthium aristatum) supplemented with S in equivalent amounts as either MPS (6.0 g/day) or sodium sulfate (9.56 g/day). Supplementation of Angelton grass with either sulfate or MPS resulted in an apparent increase in flow of rumen microbial protein from the rumen. Sulfur supplementation did not significantly change whole tract dry matter digestibility or intake, even though sulfate and MPS supplementation was associated with an increase in the relative abundance of the fibrolytic bacteria Fibrobacter succinogenes and anaerobic rumen fungi. Ruminal sulfide levels were significantly higher in the sulfate treatment, which indicated that the bioavailability of the two S atoms in the MPS molecule may be low in the rumen. Based on this observation, experiment 2 was conducted in which twice the amount of S was provided in the form of MPS (8.0 g/day) compared with sodium sulfate (6.6 g/day) to heifers (275 ± 9 kg liveweight) fed rice straw. Supplementation with MPS compared with sulfate in experiment 2 resulted in an increase in concentration of total volatile fatty acids, and ammonia utilization without a change in feed intake or whole tract fiber digestibility even though S and N were above requirement for growing cattle in both these treatment groups. In conclusion, supplementation of an S deficient low-quality roughage diet with either MPS or sodium sulfate, in conjunction with urea N, improved rumen fermentation, which was reflected in an increase in urinary purine excretion. However, MPS appeared to have a greater effect on stimulating short-chain fatty acid production and ammonia utilization when provided at higher concentrations than sulfate. Thus, the metabolism of MPS in the rumen needs to be investigated further in comparison with inorganic forms of S as it may prove to be more effective in stimulating fermentation of roughage diets.     

Protoporphyrinogen oxidation, a step in heme synthesis in soybean root nodules and free-living rhizobia.

Extracts of the crude bacteroid fraction of symbiotically grown Bradyrhizobium japonicum were much more active in oxidizing protoporphyrinogen to protoporphyrin than were extracts of cells grown under free-living conditions, especially when assayed in atmospheres containing only traces of oxygen. This correlates with the higher heme content of the microaerophilic nodules. Furthermore, the high level of oxidative activity in the crude bacteroid fraction was associated with an uncharacterized membrane fraction, probably of plant origin, that was separable from the bacteroids by Percoll gradient centrifugation.