Active transport of α-methylglucoside by the photosynthetic bacterium Chromatium vinosum

The photosynthetic purple sulfur bacterium Chromatium vinosum possesses an active transport system for glucose that also transports the nonmetabolizable glucose analog, αmethylglucoside. Transport is not accompanied by phosphorylation nor does it appear to require ATP. Rather α-methylglucoside uptake appears to be driven directly by the electrochemical proton gradient produced by light-driven cyclic electron flow.     

The formation of bacteriochlorophyll · protein complexes of the photosynthetic apparatus of Rhodopseudomonas capsulata during early stages of development

Cells of Rhodopseudomonas capsulata cultivated at an oxygen partial pressure of 400 mmHg in the dark contained 0.1 nmol or less total bacteriochlorophyll per mg membrane protein. The bacteriochlorophyll was found in the reaction center (10 pmol bacteriochlorophyll/mg membrane protein) and in the light harvesting bacteriochlorophyll I but not in the light harvesting bacteriochlorophyll II. Formation of the photosynthetic apparatus in those cells was induced by incubation at a very low oxygen tension in the dark. Reaction center bacteriochlorophyll and light harvesting bacteriochlorophyll increased three fold after 60 min of incubation at 1–2 mmHg (pO₂). Light harvesting bacteriochlorophyll II increased strongly after 60 min and became dominating after 90 min of incubation. The total bacteriochlorophyll content doubled every 30 min, but synthesis of reaction center bacteriochlorophyll proceeded at much lower rates. Consequently the size of the photosynthetic unit (total bacteriochlorophyll/reaction center bacteriochlorophyll) increased from 15 to 52 during 150 min of incubation. The proteins of the photosynthetic apparatus were synthesized concomitantly with bacteriochlorophyll.Cells which were incubated at 0.5 mmHg (pO₂) do not grow but form the photosynthetic apparatus. During the first hours of incubation light harvesting bacteriochlorophyll I and reaction center bacteriochlorophyll were the dominant bacteriochlorophyll species, but light harvesting bacteriochlorophyll II was synthesized only in small amounts. Total bacteriochlorophyll and reaction center bacteriochlorophyll increased from 30 min up until 210 min of incubation more than 10 fold. The final concentrations of total bacteriochlorophyll and reaction center bacteriochlorophyll were 8.6 nmol and 0.26 nmol per mg membrane protein, respectively. The three protein components of the reaction centers (mol. wts. 28 000, 24 000 and 21 000) and the protein of the light harvesting I complex (mol. wt. 12 000) were incorporated simultaneously. The protein of band 1 (mol. wt. 14 000) which was present in the isolated light harvesting complex II, was synthesized only in very small amounts. The proteins of bands 3 and 4 (mol. wt. 10 000 and 8000) however, which were shown to be associated with light harvesting bacteriochlorophyll II, were synthesized in noticeable amounts as was light harvesting bacteriochlorophyll II. In addition a protein with an apparent molecular weight of 45 000 showed a strong incorporation of ¹⁴C-labeled amino acids. This protein comigrates with one protein which was found to be associated with a green pigment excreted during incubation at 0.5 Torr into the medium. The in vivo-absorption maxima of this pigment complex were 660, 590, 540, 417 and 400 nm. The succinate oxidase and the NADH oxidase seemed to be incorporated into the newly formed intracytoplasmic membrane only in very small amounts. Thus, reaction center and light harvesting bacteriochlorophyll and their associated proteins were simultaneously synthesized, whereas light harvesting complex II is the variable part of the photosynthetic apparatus.     

Configuration of the carotenoid in the reaction centers of photosynthetic bacteria. Comparison of the resonance Raman spectrum of the reaction center of Rhodopseudomonas sphaeroides G1C with those of cis-trans isomers of β-carotene

The resonance Raman spectrum of the reaction center of Rhodopseudomonas sphaeroides G1C as well as those of the cis-trans isomers of β-carotene (all-trans, 9-cis, 13-cis, 15-cis and 9-cis, 13-cis- (or 9-cis, 13′-cis)) have been recorded at liquid N₂ temperature by use of the 457.9, 488.0 and 514.5 nm excitation lines. Comparison of the spectra indicated that the carotenoid in the reaction center takes the 15-cis configuration.     

The use of cycloamylose to probe the “charge-relay” system

The effect of the combination of imidazolyl and carboxyl groups on the cleavage of m-t-butylphenyl acetate in the presence of α-cyclodextrin was examined to shed light on the role of the “charge-relay” system in serine esterases. 2-Benzimidazole-acetic acid, which has both the imidazolyl and carboxyl groups in the same molecule, accelerates the cleavage of m-t-butylphenyl acetate in the presence of α-cyclodextrin. On the other hand, neither benzimidazole (which has only an imidazolyl group) nor 2-naphthaleneacetic acid (which has only a carboxyl group) exhibited measurable acceleration. The cleavage of m-t-butylphenyl acetate by the α-cyclodextrin-2-benzimidazolecetic acid system takes place through inclusion complex formation between m-t-butylphenyl acetate and α-cyclodextrin, followed by catalysis associated with the combination of the carboxyl anion, the neutral imidazolyl group, and the alkoxide anion. The most probable explanation for the combination of the three groups in the catalysis involves nucleophilic attack by the imidazolyl group, assisted by the carboxyl and alkoxide anions. The mechanism of the combination of the imidazolyl, carboxyl, and hydroxyl groups is apparently different from those shown by the “charge-relay” system in enzymatic reactions.     

Biosynthetic conversion of α-methylbutyric acid to tiglic acid in Datura meteloides

The administration of RS-α-methylbutyric-[1-¹⁴C] acid to Datura meteloides plants resulted in the formation of radioactive meteloidine. A systematic degradation indicated that essentially all the activity was located at C-1 of the tiglic acid moiety of the alkaloid.     

The effect of the fluorescent probe, 3,3′-dipropylthiodicarbocyanine iodide,on the membrane potential of Ehrlich ascites tumor cells

The effects of the potential-sensitive fluorescent dye, 3,3′dipropylthiodicarbocyanine iodide, on factors establishing the membrane potential of Ehrlich ascites tumor cells have been tested. The dye itself induces membrane hyperpolarization as monitored by electrophysiological methods. In addition, the dye inhibits active (Na⁺+K⁺-transport and increases cell membrane permeability to K⁺ by about 65% in these cells.     

An improved synthesis of dansylated α-galactosylceramide and its use as a fluorescent probe for the monitoring of glycolipid uptake by cells

A highly efficient synthesis of the biologically important fluorescent probe dansyl α-GalCer is presented. Key in our strategy is the incorporation of the fluorescent dansyl group at an early stage in the synthesis to facilitate in the monitoring and purification of intermediates via TLC and flash column chromatography, respectively, and the use of a high yielding α-selective glycosylation reaction between the phytosphingosine lipid and a galactosyl iodide donor. The ability of dansyl α-GalCer to activate iNKT cells and to serve as a fluorescent marker for the uptake of glycolipid by dendritic cells is also presented.     

The structure and stereochemistry of lantanilic acid,the β,β-dimethylacryloyl ester of lantaninilic acid,isolated from Lantana camara

The structure of lantanilic acid, a new triterpene isolated from the leaves of Lantana camara, has been determined as the β,β-dimethylacryloyl ester of lantaninilic acid.     

A 4-vinylprotochlorophyllide complex accumulated by “phofil” mutant of Rhodopseudomonas spheroides. An authentic intermediate in the development of the photosynthetic apparatus

A photosynthetically competent mutant strain of Rhodopseudomonas spheroides was isolated. In addition to bacteriochlorophyll, this organism produced particle-bound precursor 4-vinylprotochlorophyllide. The spectral characteristics of the pigment complex(es) accumulated in the culture medium were very variable. The spectral form occurring within the bacteria was characterized from fluorescence data. Its particle weight, 130 000, was determined by Sephadex G200 filtration. The main components of the complex were protein, lipid and pigment (6.8 : 6 : 1, w/w). As indicated by qualitative analysis, the lipid components were characteristic constituents of the photosynthetic membrane.Kinetics of pigments synthesis showed that the total pigment synthesis was not affected by the mutation; bacteriochlorophyll content was always lower in the mutant than in the parent strain. The repigmentation process was followed by fluorescence emission. The results indicated that the mutation affected membrane component synthesis required for the bacteriochlorophyll(ide) incorporation.The pigment complex was concluded to be an authentic intermediate in photosynthetic apparatus morphogenesis. The reasons for its excretion are discussed.     

Distribution of α-amino-β-methylaminopropionic acid in Cycas

α-Amino-β-methylaminopropionic acid, previously isolated from seeds of Cycas circinalis, has now been identified either free or bound in all th     

The tip region of the MacA α-hairpin is important for the binding to TolC to the Escherichia coli MacAB-TolC pump

The tripartite efflux pump MacAB-TolC found in Gram-negative bacteria is involved in resistance to antibiotics. We previously reported the funnel-like hexameric structure of the adaptor protein MacA to be physiologically relevant. In this study, we investigated the role of the tip region of its α-hairpin, which forms a cogwheel structure in the funnel-like shape of the MacA hexamer. Mutational and biochemical analyses revealed that the conserved residues located at the tip region of the α-hairpin of MacA play an essential role in the binding of TolC. Our findings offer a molecular basis for understanding the drug resistance of pathogenic bacteria.     

Biosynthesis and in vitro enzymatic synthesis of the isoleucine conjugate of 12-oxo-phytodienoic acid from the isoleucine conjugate of α-linolenic acid

The isoleucine conjugate of 12-oxo-phytodienoic acid (OPDA-Ile), a new member of the jasmonate family, was recently identified in Arabidopsis thaliana and might be a signaling molecule in plants. However, the biosynthesis and function of OPDA-Ile remains elusive. This study reports an in vitro enzymatic method for synthesizing OPDA-Ile, which is catalyzed by reactions of lipoxygenase (LOX), allene oxide synthase (AOS), and allene oxide cyclase (AOC) using isoleucine conjugates of α -linolenic acid (LA-Ile) as the substrate. A. thaliana fed LA-Ile exhibited a marked increase in the OPDA-Ile concentration. LA-Ile was also detected in A. thaliana. Furthermore, stable isotope labelled LA-Ile was incorporated into OPDA-Ile. Thus, OPDA-Ile is biosynthesized via the cyclization of LA-Ile in A. thaliana.     

Membrane-protein interaction and the molten globule state: Interaction of α-lactalbumin with membranes

The insertion of soluble proteins into membranes has been a topic of considerable interest. We have studied the insertion of bovineα-lactalbumin into single-bilayer vesicles prepared from egg phosphatidylcholine (PC). Fluoresence studies indicated rapid and tight binding of apo-α-lactalbumin (apo-α-LA) to PC vesicles as a function of pH. The binding was maximal at pH values which favor the formation of the molten globule state. As an increase of hydrophobic surface is observed in the molten globule state, this conformational state can provide a molecular basis for insertion of soluble proteins into membranes. The membrane-bound complex formed at low pH (3.0) could be isolated and was found to be stable at neutral pH. The structural characterization of the apo-α-LA-PC complex was studied by fluorescence quenching using iodide, acrylamide, and 9,10-dibromostearic acid. The results obtained indicated that some of the tryptophans of apo-α-LA were buried in the membrane interior and some were exposed on the outer side. Fluorescence quenching and CD studies indicated the membrane-bound conformation of apo-α-LA was some conformational state that is between the soluble, fully folded conformation and the molten globule state.     

Biosynthesis of unnatural bacteriochlorophyll c derivatives esterified with α,ω-diols in the green sulfur photosynthetic bacterium Chlorobaculum tepidum

Unnatural bacteriochlorophyll (BChl) c derivatives possessing a hydroxy group at the terminus of a hydrocarbon chain at the 17-propionate were biosynthesized in the green sulfur photosynthetic bacterium Chlorobaculum tepidum. Addition of exogenous 1,8-octanediol, 1,12-dodecanediol, and 1,16-hexadecanediol in acetone to liquid cultures resulted in accumulation of BChl c monoesterified with the corresponding diols. The relative ratios of the novel BChl c derivatives esterified with 1,8-, 1,12-, and 1,16-diols to totally producing BChl c were 8.2, 50.2, and 57.6% in the cells grown with additive α,ω-diols at concentrations of 1.5, 0.06, and 0.06 mM, respectively, at the final concentration. The homologue composition of BChl c derivatives esterified with these α,ω-diols was similar to that of original, coexisting BChl c esterified with farnesol (BChl c(F)), suggesting that esterification of α,ω-diols occurred at the last step of the BChl c biosynthetic pathway by BChl c synthase, BchK, in the same manner as in BChl c(F). Chlorosomes, which were isolated from cells grown in the presence of exogenous α,ω-diols, contained a ratio and a composition of BChl c derivatives esterified with the diols similar to those in the whole cells, indicating that these BChl c derivatives were actually present in chlorosomes. Q(y) absorption bands of C. tepidum cells containing the novel BChl c derivatives were shifted to a shorter wavelength, although their bandwidths were analogous to those of cells obtained by normal cultivation. Circular dichroism spectra of cells that had BChl c derivatives esterified with α,ω-diols exhibited S-shaped signals in the Q(y) region, whose polarities were the reverse of those of cells grown in the normal medium and by supplementation with neat acetone as a control experiment. These spectral features of C. tepidum possessing BChl c derivatives esterified with α,ω-diols imply that the novel BChl c derivatives possessing a hydroxy group at the terminus of a hydrocarbon chain affect their self-assembly in chlorosomes.     

Occurrence of α-ecdysone in the developing embryos of the silkworm, Bombyx mori

From an acetone-ethanol extract of the developing embryos of the silkworm, Bombyx mori, the presence of α-ecdysone, but not of β-ecdysone, was shown by high pressure liquid chromatography and bioassay. The amount of α-ecdysone was estimated to be 0.74 μg per gram of eggs. The absence of a hydroxylating system at C-20 in the embryos is suggested.