The use of non-denaturing Deriphat-polyacrylamide gel electrophoresis to fractionate pigment-protein complexes of purple bacteria

The suitability of Deriphat-polyacrylamide gel electrophoresis as a method for separating purple bacterial pigment-protein complexes has been tested. When appropriate non-denaturing detergents are used to solubilize chromatophores, this method provides a rapid, easy and microscale procedure for analyzing the composition of the bacterial photosynthetic apparatus with minimal disruption of individual pigment-proteins. Its usefulness is further illustrated by employing it to test for suitable detergents with which to solubilize purple bacterial chromatophores, and as an assay to study variation in the composition of the photosynthetic unit of bacterial cultures grown under different conditions.     

Cardiolipin increases in chromatophores isolated from Rhodobacter sphaeroides after osmotic stress: structural and functional roles

Chromatophores isolated from cells of Rhodobacter sphaeroides exposed to hypertonic solutions were enriched in cardiolipin (CL). Because CL levels are raised by increasing the incubation time of R. sphaeroides in hypertonic solutions, it was possible to isolate chromatophores containing different CL amounts by starting from cells incubated in hypertonic solutions for different times. The functionality and stability of the photosynthetic proteins in chromatophore membranes having different CL levels were investigated. Reaction center (RC) stabilization with respect to thermal denaturation and photoxidative damage was observed by flash photolysis and fluorescence emission experiments in CL-enriched chromatophores. To gain detailed information about the structures of endogenous CLs, this lipid family was isolated and purified by preparative TLC, and characterized by high-resolution mass spectrometry. We conclude that osmotic shock can be used as a tool to modulate CL levels in isolated chromatophores and to change the composition of the RC lipid annulus, avoiding membrane artifacts introduced by the use of detergents.     

Light-induced oxygen uptake by chromatophores and subchromatophore pigment-protein complexes of Rhodospirillum rubrum]

Chromatophores of R. rubrum incubated with electron donors, e. g. reduced diaminodurene, TMPD, phenazine methosulphate, cytochrome c or ferrocyanide, are able to catalyze O2 uptake upon illumination. This process is inhibited by o-phenanthroline as well as upon extraction of quinones from chromatophores, but not by antimycin A, rotenone or CN-. The O2 uptake sensitive to the action of o-phenanthroline is also observed in the illuminated subchromatophore P870 reaction center complexes and reaction center plus light-harvesting antenna complexes incubated with electron donors, quinones and detergents. The data obtained are in agreement with a suggestion that the photooxidase activity of chromatophores and subchromatophore pigment-protein complexes is due to the interaction of photoreduced ubiquinone with O2.     

A large photoreactive particle from Chromatium vinosum chromatophores

Large photoreactive particles from Chromatium vinosum are obtained pure and in high yield by using a mixture of detergents at high ionic strength to dissociate the chromatophore membrane. The particles contain all of the secondary electron acceptor of the chromatophores and about half of the cytochrome. Their content of ubiquinone is greatly enriched as compared with chromatophores. The individual particles have an estimated molecular weight of between 650 000 and 810 000.Gel electrophoresis of the preparation in sodium dodecylsulfate shows polypeptides with molecular weights of 50–45 000, 30 000, 27 000, 22 000 and 12 000. The 50–45 000 components are cytochromes. The 30 000, 27 000 and 22 000 components may be analogous to the triad of polypeptides present in Rhodopseudomonas spheroides reaction centers. The non-cytochrome components are partly soluble in chloroform/methanol.Aggregates of particles appear in these preparations. Electron microscopy of the aggregates demonstrates rectilinear lattices of isodiametric particles, 120 Å in diameter. These sheet-like structures are one unit thick and typically contain 9–16 members. They appear to arise by aggregation during isolation but are probably similar to native aggregates apparent within chromatophores after treatment with detergents at low salt concentration.     

Comparison of permeant ion uptake and carotenoid band shift as methods for determining the membrane potential in chromatophores from Rhodopseudomonas sphaeroides Ga.

1. A comparison was made of two methods for estimating the membrane potential in chromatophores from Rhodopseudomonas sphaeroides Ga. Illuminated chromatophores generated a potential that is apparently much larger when estimated on the basis of the red-band shift of carotenoids rather than from the extent of uptake of the permeant SCN- ion. 2. In contrast, when the chromatophores were oxidizing NADH or succinate the uptake of SCN- indicated a larger membrane potential than was estimated from the carotenoid band shift. 3. The extent of SCN- uptake and the carotenoid-band shift respond differently to changes in the ionic composition of the reaction medium. 4. The effects of antimycin on the carotenoid band shift and SCN- uptake are reported. 5. It is concluded that the carotenoid band shift and the uptake of SCN- are responding to different aspects of the energized state.     

Resolution and reconstitution of Rhodospirillum rubrum pyridine dinucleotide transhydrogenase

The Rhodospirillum rubrum pyridine dinucleotide transhydrogenase system is comprised of a membrane-bound component and an easily dissociable soluble factor. Active transhydrogenase complex was solubilized by extraction of chromatophores with lysolecithin. The membrane component was also extracted from membranes depleted of soluble factor. The solubilized membrane component reconstituted transhydrogenase activity upon addition of soluble factor. Various other ionic and non-ionic detergents, including Triton X-100, Lubrol WX, deoxycholate, and digitonin, were ineffectual for solubilization and/or inhibited the enzyme at higher concentrations. The solubilized membrane component was significantly less thermal stable than the membrane-bound component. None of the pyridine dinucleotide substrate affected the thermostability of the solubilized membrane-bound component, whereas NADP⁺ and NADPH afforded protection to membrane-bound component. NADPH stimulated trypsin inactivation of membrane-bound component to a greater extent than NADP⁺, but inactivation of solubilized membrane component was stimulated to the same extent by both pyridine dinucleotides. The solubilized membrane component appears to have a slightly higher affinity for soluble factor than does the membrane-bound component.Abbreviations AcPyAD⁺ oxidized 3-acetylpyridine adenine dinucleotide - BChl bacteriochlorophyll - C_T-particles chromatophores depleted of soluble transhydrogenase factor and devoid of transhydrogenase activity This work was supported by Grant GM 22070 from the National Institutes of Health, United States Public Health Service. Paper I of this series is R. R. Fisher et al. (1975)     

Heterogeneity of photosynthetic membranes from Rhodobacter capsulatus: size dispersion and ATP synthase distribution

The density distribution of photosynthetic membrane vesicles (chromatophores) from Rhodobacter capsulatus has been studied by isopicnic centrifugation. The average vesicle diameters, examined by electron microscopy, varied between 61 and 72 nm in different density fractions (70 nm in unfractionated chromatophores). The ATP synthase catalytic activities showed maxima displaced toward the higher density fractions relative to bacteriochlorophyll, resulting in higher specific activities in those fractions (about threefold). The amount of ATP synthase, measured by quantitative Western blotting, paralleled the catalytic activities. The average number of ATP synthases per chromatophore, evaluated on the basis of the Western blotting data and of vesicle density analysis, ranged between 8 and 13 (10 in unfractionated chromatophores). Poisson distribution analysis indicated that the probability of chromatophores devoid of ATP synthase was negligible. The effects of ATP synthase inhibition by efrapeptin on the time course of the transmembrane electric potential (evaluated as carotenoid electrochromic response) and on ATP synthesis were studied comparatively. The ATP produced after a flash and the total charge associated with the proton flow coupled to ATP synthesis were more resistant to efrapeptin than the initial value of the phosphorylating currents, indicating that several ATP synthases are fed by protons from the same vesicle.     

Heterogeneity of photosynthetic membranes from Rhodobacter capsulatus: Size dispersion and ATP synthase distribution

The density distribution of photosynthetic membrane vesicles (chromatophores) from Rhodobacter capsulatus has been studied by isopicnic centrifugation. The average vesicle diameters, examined by electron microscopy, varied between 61 and 72 nm in different density fractions (70 nm in unfractionated chromatophores). The ATP synthase catalytic activities showed maxima displaced toward the higher density fractions relative to bacteriochlorophyll, resulting in higher specific activities in those fractions (about threefold). The amount of ATP synthase, measured by quantitative Western blotting, paralleled the catalytic activities. The average number of ATP synthases per chromatophore, evaluated on the basis of the Western blotting data and of vesicle density analysis, ranged between 8 and 13 (10 in unfractionated chromatophores). Poisson distribution analysis indicated that the probability of chromatophores devoid of ATP synthase was negligible. The effects of ATP synthase inhibition by efrapeptin on the time course of the transmembrane electric potential (evaluated as carotenoid electrochromic response) and on ATP synthesis were studied comparatively. The ATP produced after a flash and the total charge associated with the proton flow coupled to ATP synthesis were more resistant to efrapeptin than the initial value of the phosphorylating currents, indicating that several ATP synthases are fed by protons from the same vesicle.     

Chromatophore distribution and inferior performance of albino Japanese flounder Paralichthys olivaceus with special reference to different chromatophore expression between albinism and pseudo-albinism

Albinism with a large variation in body color was found in a hatchery population of Japanese flounder. In addition to albinism, ambicoloration and pseudo-albinism were simultaneously observed in some individuals. Albinos had a remarkably lower number of melanophores on the scales of ocular side than wild-type individuals did, although no significant difference was observed in the numbers of xanthophores and iridophores. The intensity of body color significantly correlated with the number of melanophores among the albinos. No significant differences were observed in the intensity of body color and the number of melanophores between the ocular side and the ambicoloration area. Pseudo-albinism was accompanied by the reductions of melanophores and xanthophores, indicating the different expression patterns of chromatophores between albinism and pseudo-albinism. The combined effects of albinism and pseudo-albinism caused the disappearances of melanophores and xanthophores in the pseudo-albinism area of albinos. In addition to chromatophores, the different characteristics of several phenotypic traits were observed between albinos and wild-type individuals. Growth-related traits of the albinos were inferior to those of the wild-type individuals. Furthermore, the albinos had a larger pseudo-albinism area and a higher vertebral deformed rate than the wild-type individuals did. Individual multilocus heterozygosity and inbreeding coefficient measured by microsatellite loci did not show any indication that the albinos had higher inbreeding coefficient than the wild-type individuals did. This study demonstrated the expression patterns of chromatophores in the body color abnormalities of a flatfish species and the potential pleiotropic effects of an albinism gene on some phenotypic traits.     

The effects of two physiological salines, Locke's and van Harreveld's, on the midgut red chromatophores of the freshwater shrimp, Caridina denticulata

It was reported previously that the red chromatophores on the midgut of a freshwater shrimp, Caridina denticulata, are affected by Locke's and van Harreveld's solutions differently, i.e., the pigment disperses in Locke's solution and concentrates with the addition of crude eyestalk extract, but in Harreveld's solution the chromatophores do not change in the saline alone nor do they respond to eyestalk extract. The differences were probably due to the osmotic pressure and Mg ion concentrations of the two solutions not being the same. Harreveld's solution is commonly used as a physiological saline for freshwater crustaceans such as crayfish. Consequently, this solution was employed at first in a previous study (Miyawaki and Tsuruda, 1985). But this solution completely inhibited pigment migration in the chromatophores. But when Locke's solution was subsequently tried, migration of the pigment in the midgut chromatophores occurred. It seemed worthwhile to examine further the effects of both solutions on these chromatophores. The results of this study are presented below.     

Orientation of chromatophores and spheroplast-derived membrane vesicles of Rhodopseudomonas sphaeroides: analysis by localization of enzyme activities.

Intact spheroplasts, vesicles obtained from French-press lysates (chromatophores), and spheroplast-derived vesicles were isolated from photosynthetically grown cells of Rhodopseudomonas sphaeroides. Lysed spheroplasts showed specific activities of succinate, NADH, and l-lactate dehydrogenase which were eight-, six-, and seven-fold higher, respectively, than those of intact spheroplasts when ferricyanide was used as electron acceptor. Mg²⁺-ATPase activity of lysed spheroplasts, measured using an assay system coupled to the oxidation of NADH, was seven-fold higher than the activity of intact sheroplasts. Toluene-treated spheroplast-derived vesicles displayed higher succinate dehydrogenase (ferricyanide reduction) and Mg²⁺-ATPase activities than untreated vesicles whereas no differences were measured between untreated and toluene-treated chromatophores. However, NADH dehydrogenase (ferricyanide reduction) activities of both toluene-treated vesicles and chromatophores were higher than the activities of untreated vesicles and chromatophores. When chromatophores and spheroplast-derived vesicles were preincubated with trypsin, the l-lactate and succinate dehydrogenase activities of chromatophores were preferentially inactivated when phenazine methosulfate was used as electron acceptor. The data indicate that chromatophores are oriented in an opposite direction to the spheroplast-derived vesicles. At least 80% of the latter are oriented in a direction equivalent to the cytoplasmic membrane of intact cells and spheroplasts. Spheroplast-derived vesicles from cells grown with higher light intensities seem to be more uniformly oriented than those obtained from cells grown with lower light intensities.     

Freeze fracture studies of reaction centers from Rhodospirillum rubrum in chromatophores and liposomes

In freeze-fractures of chromatophores of Rhodospirillum rubrum the reaction centers are seen as hexagonal arranged particles of 13 nm diameter with a density of around 5,500 particles per m². Similar regions on the cytoplasmic membrane suggest that these parts are the prospective invagination sites.Isolated reaction centers are easily incorporated into liposomes. In freeze fractures of liposomes particles similar in shape and size, although less dense as in chromatophores are observed. In negative staining much smaller units of only 5 nm in diameter are found indicating that reaction centers occur in the membrane as tri- or tetramers. There is a strong correlation between particle density in chromatophores and titratable reaction centers remaining in these membranes after extraction of reaction centers by detergents; both values are in good agreement with the yield of reaction centers at a given detergent concentration.Abbreviations LDAO Lauryldimethylamine oxide - PF protoplasmic fracture face - EF exoplasmic fracture face     

A new inhibitor of the CoQ-dependent redox reactions in mitochondria and chromatophores

The effects of 3,4-dimethoxyphenyl-1-amylketone (DPK) on the CoQ-dependent stages of the electron transport systems in mitochondria and Rhodobacter sphaeroides chromatophores were studied. The two systems contain the complete Q-cycle. The sensitivities of the Q-cycles of two electron transport systems to antimycin, myxothiazole, and other inhibitors are virtually indistinguishable from one another, but these systems have different CoQ reduction processes. The dependence of the inhibition extent of the mitochondrial succinate oxidase on the DPK concentration was studied. The effective concentration of DPK is 0.5-2.5 mM. The presence of the point of inflection in the titration curve indicates that there are two mechanisms of inhibition. The effects of DPK on the extent of reduction of cytochromes b and c1 + c in mitochondria as well as on the electrogenic stages of the Q-cycle in chromatophores were examined. The experiments showed that DPK prevents three CoQ-dependent reactions related to the Q-cycle: electron transport between succinate dehydrogenase and the Q-cycle in mitochondria and functioning of the Z (o) and C (i) sites of the Q-cycle in chromatophores. DPK does not affect the electrogenic reaction associated with protonation of the secondary quinone acceptor QB in the reaction center of chromatophores. The mitochondrial NADH-dehydrogenase is inhibited by DPK at lower but comparable concentrations (C50 = 0.2 mM).     

Photooxidase activity of Rhodospirillum rubrum chromatophores and reaction center complexes. The role of non-cyclic electron transfer in generation of the membrane potential

The mechanism of light-induced O₂ uptake by chromatophores and isolated P-870 reaction center complexes from Rhodospirillum rubrum has been investigated.The process is inhibited by o-phenanthroline and also by an extraction of loosely bound quinones from chromatophores. Vitamin K-3 restored the o-phenanthroline-sensitive light-induced O₂ uptake by the extracted chromatophores and stimulated the O₂ uptake by the reaction center complexes. It is believed that photooxidase activity of native chromatophores is due to an interaction of loosely bound photoreduced ubiquinone with O₂. Another component distinguishable from the loosely bound ubiquinone is also oxidized by O₂ upon the addition of detergents (lauryldimethylamine oxide or Triton X-100) to the illuminated reaction center complexes and to the extracted or native chromatophores treated by o-phenanthroline. Two types of photooxidase activity are distinguished by their dependence on pH.The oxidation of chromatophore redox chain components due to photooxidase activity as well as the over-reduction of these components in chromatophores, incubated with 2,3,5,6-tetramethyl-p-phenylenediamine (Me₄Ph(NH₂)₂) or N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) (plus ascorbate) in the absence of exogenous electron acceptors, leads to an inhibition of the membrane potential generation, as measured by the light-induced uptake of penetrating phenyldicarbaundecaborane anions (PCB^?) and tetraphenylborate anions. The inhibition of the penetrating anion responses observed under reducing conditions is removed by oxygen, 1,4-naphthoquinone, fumarate, vitamin K-3 and methylviologen, but not by NAD⁺ or benzylviologen. Since methylviologen does not act as an electron acceptor with the extracted chromatophores, it is believed that this compound, together with fumarate and O₂, gains electrons at the level of the loosely bound ubiquinone. Data on the relationship between photooxidase activity and membrane potential generation by the chromatophores show that non-cyclic electron transfer from reduced Me₄Ph(NH₂)₂ to the exogenous acceptors is an electrogenic process, whereas non-cyclic electron transfer from reduced TMPD is non-electrogenic.Being oxidized, Me₄Ph(NH₂)₂ and TMPD are capable of the shunting of the cyclic redox chain of the chromatophores. Experiments with extracted chromatophores show that the mechanisms of the shunting by Me₄Ph(NH₂)₂ and TMPD are different.     

α-肾上能受体兴奋剂及阻断剂对米虾棕色载色细胞内色素聚散的效应

萘唑啉或肾上腺素能使米虾离体棕色胞内色素集中,此作用被酚妥拉明短时所阻断。异丙肾上腺素对棕色胞无影响。作者认为棕色胞上只具有α受体,可以作为筛选α受体兴奋剂及阻断剂的模型。     

Destabilization of collagen structure by amides and detergents in solution

The effects of amides and detergents on collagen to gelatin transition have been studied at neutral pH. Simple amides denature the protein. The substitution of H-atoms by the alkyl groups at the nonpolar end of amide increases the effectiveness of the compounds in destabilizing the collagen structure whereas substitution of the H-atom at the polar amide end shows marginal effects on the collagen transition. The capabilities of these reagents to denature collagen are much less pronounced than their effects on denaturing globular proteins. Anionic detergents are found to destabilize collagen at very low concentrations (below their cmc values). In this respect, the effects of the detergents on collagen are comparable to the denaturing effects of the detergents on globular proteins. The effect of detergents increases with the increase in the length of the alkyl chain. The structure of the anion in the detergent is also important as seen from the lower potency of the sulfonate containing detergent compared to the sulfate containing detergent in denaturing collagen. Cationic and nonionic detergents do not denature collagen.     

Reversible conversion from Ca(2)+-ATPase activity to Mg(2)+- and Mn(2)+-ATPase activities of coupling factor purified from acetone powder of Rhodospirillum rubrum chromatophores.

It is known that the coupling factor purified from the acetone powder of chromatophores from Rhodospirillum rubrum shows ATPase activity in the presence of Ca(2)+, but not in the presence of Mg(2)+ or Mn(2)+. The present study deals with conditions, under which the Ca(2)+-ATPase activity is reversibly converted into Mg(2)+- and Mn(2)+-ATPase activites with the purified coupling factor. 1. Of the pH indicators tested, 6 kinds coverted the Ca(2)+-ATPase activity into Mg(2)+- and Mn(2)+-ATPase activities in the order, ethyl orange greater than tropaeolin 000 greater than or equal to metanil yellow greater than tropaeolin 00 greater than ethyl red greater than or equal to bromthymol blue. 2. Of the detergents tested, those other than Triton X-100 and Brij 58 caused the conversion described above; dodecylsulfonate was most effective, whereas dodecylpyridinium chloride was moderately effective. 3. 2,4-Dinitrophenol stimulated approximately two-fold the Ca(2)+-ATPase activity, but not the Mg(2)+- or Mn(2)+-ATPase activity at all. However, in the presence of dodecylpyridinium chloride, the pH indicator remarkably stimulated the Mg(2)+- and Mn(2)+-ATPase activities, accompanied with a partial inhibition of the Ca(2)+-ATPase activity. Methyl red and ethyl red showed similar effects. 4. All the nucleoside triphosphates tested can serve as the substrate. ATP was most effective for the Ca(2)+-ATPase activity, whereas dATP was most effective for the Mg(2)+- and Mn(2)+-ATPase activities induced by ethyl orange. 5. In the presence of ethyl orange, the ATPase activity was induced by various divalent cations in the following order of effectiveness, Mg(2)+ greater than Zn(2)+ greater than CO(2)+ greater than Mn(2)+ greater than Ni(2)+. 6. The mechanism of the reversible conversion from the Ca(2)+-ATPase activity to the Mg(2)+- and Mn(2)+-ATPase activities by pH indicators and detergents is discussed.     

Examination of the putative Ca<Superscript>2+</Superscript>-binding site in the light-harvesting complex 1 of thermophilic purple sulfur bacterium <Emphasis Type="Italic">Thermochromatium</Emphasis><Emphasis Type="Italic">tepidum</Emphasis>

The core light-harvesting complex (LH1) of purple sulfur photosynthetic bacterium Thermochromatium tepidum exhibits an unusual absorption maximum at 915 nm for the Q _y transition, and is highly stable when copurified with reaction center (RC) in a LH1–RC complex form. In previous studies, we demonstrated that the calcium ions are involved in both the large red shift and the enhanced thermal stability, and possible Ca²⁺-binding sites were proposed. In this study, we further examine the putative binding sites in the LH1 polypeptides using purified chromatophores. Incubation of the chromatophores in the presence of EDTA revealed no substantial change in the absorption maximum of LH1 Q _y transition, whereas further addition of detergents to the chromatophores-EDTA solution resulted in a blue-shift for the LH1 Q _y peak with the final position at 892 nm. The change of the LH1 Q _y peak to shorter wavelengths was relatively slow compared to that of the purified LH1–RC complex. The blue-shifted LH1 Q _y transition in chromatophores can be restored to its original position by addition of Ca²⁺ ions. The results suggest that the Ca²⁺-binding site is exposed on the inner surface of chromatophores, corresponding to the C-terminal region of LH1. An Asp-rich fragment in the LH1 α-polypeptide is considered to form a crucial part of the binding network. The slow response of LH1 Q _y transition upon exposure to EDTA is discussed in terms of the membrane environment in the chromatophores.     

Structure and Photochemical Activity of Chlorophyll-Containing Particles from Rhodospirillum rubrum

Comparative studies on isolated chromatophores and on sectioned cells of the photosynthetic bacterium Rhodospirillum rubrum confirm the assumption expressed in earlier investigations that the photochemically active chromatophores isolated from disrupted cells represent structural chlorophyll-bearing components of the protoplast. Actively growing cells from light-grown cultures about 12 hours old do not release chromatophores when disrupted in dilute buffers, but do release smaller, chlorophyll-containing structures about 25 mµ in diameter. Sections of such cells do not reveal chromatophores, but contain in the ground cytoplasm numerous particles somewhat smaller in size than the 25 mµ chlorophyll-containing particles released from disrupted cells. Similar particles are obtained by the sonication of isolated chromatophores obtained from cells of 1-day-old cultures. The small, subchromatophore particles described here appear to be functionally complete units which are photochemically active in photo-oxidation, photoreduction, and photophosphorylation, and it is postulated that they represent the basic biochemical and structural components of the chromatophore.     

Morphological and Biochemical Characterization of the Cream Markings in the Integument of the Female Isopod,Armadillidium vulgare

Cream markings aligned along the dorsal region of the female isopod, A. vulgare, were investigated with light and a fluorescence microscope and an electron microscope. Biochemical studies were also carried out. The cream markings were observed in the dorsal integument as a group of cream-colored chromatophores that emit a yellow fluorescence. These chromatophores, which are distinguishable from ommochrome chromatophores, contained numerous granules in the cytoplasm, and these granules (0.6–3.0 μm in length by 0.4–1.5 μm in width) were electron-lucent and spheroidal in shape with a concentric arrangement of membranes. Based on various biochemical analyses, the principal component of the yellow pigment isolated from the cream markings was identified as sepiapterin. These facts revealed that the cream markings are the chromatophores that contain pteridine granules. The males have no cream markings like those of the females, since the cream-colored chromatophores are externally hidden by the ommochrome chromatophore layer. The content of sepiapterin in the males was about two times greater than that in the females. This quantitative difference in sepiapterin content between males and females suggests that the pteridine formation in this pigment cell may be regulated by hormones associated with sex determination.