Carotenoids of an Antarctic psychrotolerant bacterium, Sphingobacterium antarcticus, and a mesophilic bacterium, Sphingobacterium multivorum

The major carotenoid pigments of an Antarctic psychrotolerant bacterium, Sphingobacterium antarcticus, and a mesophilic bacterium, Sphingobacterium multivorum, were identified as zeaxanthin, beta-cryptoxanthin, and beta-carotene. Analysis was based on ultraviolet-visible spectroscopy, mass spectroscopy, and reversed-phase HPLC. Photoacoustic spectroscopy of intact bacterial cells revealed that the bulk of the pigments in S. antarcticus and S. multivorum was associated with the cell membrane. In vitro studies with synthetic membranes of phosphatidylcholine demonstrated that the major pigment was bound to the membranes and decreased their fluidity. The relative amounts of polar pigments were higher in cells grown at 5 degrees C than in cells grown at 25 degrees C. In the mesophilic strain, the synthesis of polar carotenoids was quantitatively less than that of the psychrotolerant strain.     

Isolation and Identification of Canthaxanthin from Micrococcus roseus

Cooney, J. J. (University of Dayton, Dayton, Ohio), H. W. Marks, Jr., and Anne M. Smith. Isolation and identification of canthaxanthin from Micrococcus roseus. J. Bacteriol. 92:342-345. 1966.-The principal colored carotenoid of Micrococcus roseus was purified by solvent partitioning followed by column and thin-layer chromatography. Absorption spectra, partition coefficients, and infrared spectra suggested that the pigment was a diketo derivative of beta-carotene. The pigment was subjected to reduction, and the reduced pigment was subsequently dehydrated. Spectral data and partition coefficients of these derivatives indicated that the original pigment was canthaxanthin (4',4'-diketo-beta-carotene). The pigment was an all-trans isomer; it does not exist as an ester in M. roseus. Canthaxanthin has not previously been identified as a bacterial pigment.     

Isolation and Identification of Echinenone from Micrococcus roseus

An orange carotenoid from Micrococcus roseus was purified by solvent partitioning followed by column and thin-layer chromatography. Absorption spectra, chromatographic mobility, and partition coefficient suggested that the pigment was echinenone (4-keto-beta-carotene). Reduction yielded a pigment with the spectral and polar properties of isocryptoxanthin (4-hydroxy-beta-carotene), the expected product. The orange pigment and its reduction product co-chromatographed with the respective authentic pigments, confirming the original pigment as echinenone. To our knowledge echinenone has not been identified previously as a bacterial pigment.     

Interaction of steroids with adrenal cytochrome P-450 (P-450(17)alpha,lyase) in liposome membranes

Purified cytochrome P-450(17)alpha,lyase from guinea-pig adrenal microsomes, which catalyzes progesterone 17 alpha-hydroxylation and sequentially C17-C20 bond cleavage of the 17 alpha-hydroxyprogesterone, was successfully incorporated into liposomal membranes composed of only phosphatidylcholine or of a phospholipid mixture of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine at a molar ratio of 5:3:1. Although the purified P-450(17)alpha,lyase was readily converted into P-420 in the detergent-solubilized system without substrates, the P-450 embedded in the liposomal membranes was found to be quite stable without the substrates. Using the P-450(17)alpha,lyase-proteoliposomes, the interaction of steroids with P-450(17)alpha,lyase was studied for progesterone, 17 alpha-hydroxyprogesterone and androstenedione in the liposomal system by optical difference spectroscopy and by equilibrium dialysis. The partition coefficients of steroids between the aqueous phase and the liposomal membranes were determined by the equilibrium dialysis. They were about 1.4-1.6-times higher in phosphatidylcholine liposomes than in the liposomes of the lipid mixture. The dissociation constants of the P-450-steroid complexes were calculated from the apparent dissociation constants using the partition coefficients for the situation where the substrate-binding site faces the lipid phase of the membranes or where it faces the aqueous phase. The dissociation constant in the former case was not affected by the lipid composition. These results suggest that P-450(17)alpha,lyase might interact only with the substrates in the lipid phase of the liposomal membranes.     

优美红游动菌类胡萝卜素的提取条件研究

研究了优美红游动菌(Rhodoplanes elegans)的生长以及类胡萝卜素的产生,通过比较4种广泛采用的细胞破碎方法,对优美红游动菌(Rhodoplanes elegans)类胡萝卜素的提取条件进行了研究,结果表明:类胡萝卜素的产生与菌种的生长曲线相符合,菌种活化后培养45h,类胡萝卜素的含量趋于稳定;取此时培养液,采用超声波法破碎细胞(破碎功率640W,时间10min),类胡萝卜素提取效果最好,初提液类胡萝卜素的提取率约为7.62mg·(g干菌体)^-1;酸热法对色素的破坏严重。这为进一步开发利用天然色素资源提供了一定的理论依据。     

Identification of the Carotenoid Pigment Canthaxanthin from Photosynthetic Bradyrhizobium Strains

Canthaxanthin (4,4(prm1)-diketo-(beta)-carotene) is produced as the major carotenoid pigment by orange- and dark-pink-pigmented bacteriochlorophyll-containing Bradyrhizobium strains isolated from stem nodules of Aeschynomene species. These two new pigmentation groups differ from the well-studied strain BTAi1, which accumulates spirilloxanthin as the sole carotenoid.     

Isolation and Characterization of the Thylakoid Membranes from the NaCl-Resistant (NaClr) Mutant Strain of the Cyanobacterium Anabaena variabilis

NaCl-induced changes in the thylakoid membrane of wild-type Anabaena variabilis and its NaCl^r mutant strain have been studied. Biochemical characterization of the thylakoid membrane was done by taking its absorption and fluorescence spectra at different wavelength. The thylakoid membranes of both strains were isolated by mechanical disruption of the freeze-dried and lysozyme-treated cells, followed by differential and density gradient centrifugation. The light absorption spectra of the thylakoid membrane showed three and two peaks in NaCl^r mutant strain and its wild-type counterpart respectively at wavelengths of 400–850 nm. These peaks revealed that the thylakoid membrane contains a large amount of carotenoid and chlorophyll a. Fluorescence emission spectra of thylakoid membrane of NaCl^r mutant and its wild-type strain at excitation wavelength of 335 nm showed two different peaks, one at 340 nm and the other at 663 nm respectively. The light absorption and fluorescence spectra of the thylakoid membrane also revealed that the membrane contained carotenoid pigment, chlorophyll (Chl) a, and a pigment with an emission peak at 335 nm. The HPLC analysis of the pigments of the thylakoid membrane indicates that the NaCl^r mutant strain under NaCl stress contained an additional peak for the carotenoid pigment, which was lacking in its wild-type counterpart. The major peak in thylakoid membrane was that of echinenone and β-carotene. Whereas the polypeptide composition of thylakoid membrane differed in the wild-type and its NaCl^r mutant strain, no difference in the cell wall protein pattern was observed in both strains. The thylakoid membrane of NaCl^r mutant strain contained two additional protein bands that were absent in its wild-type counterpart. The thylakoid membrane of the wild-type and its NaCl^r mutant strain also showed morphological variations under NaCl stress. Received: 14 April 2000 / Accepted: 23 May 2000     

Isolation and characterization of a cDNA clone from Catharanthus roseus encoding NADPH:cytochrome P-450 reductase, an enzyme essential for reactions catalysed by cytochrome P-450 mono-oxygenases in plants

The membrane-bound flavoprotein NADPH:cytochrome P-450 (cytochrome c) reductase, that functions in electron transfer to cytochrome P-450 mono-oxygenases, was purified from a cell suspension culture of the higher plant Catheranthus roseus . Anti-serum raised against the purified protein was found to inhibit NADPH:cytochrome c reductase activity as well as the activities of the cytochrome P-450 enzymes geraniol 10-hydroxylase and trans -cinnamate 4-hydroxylase, which are involved in alkaloid biosynthesis and phenylpropanoid biosynthesis, respectively. Immunoscreening of a C. roseus cDNA expression library resulted in the isolation of a partial NADPH: cytochrome P-450 reductase cDNA clone, which was identified on the basis of sequence homology with NADPH:cytochrome P-450 reductases from yeast and animal species. The identity of the cDNA was confirmed by expression in Escherichia coli as a functional protein capable of NADPH-dependent reduction of cytochrome c and neotetrazolium, two in vitro substrates for the reductase. The N-terminal sequence of the reductase, which was not present in the cDNA clone, was determined from a genomic NADPH: cytochrome P-450 reductase clone. It was demonstrated that the reductase probably is encoded by a single copy gene. A sequence comparison of this plant NADPH:cytochrome P-450 reductase with the corresponding enzymes from yeast and animal species showed that functional domains involved in binding of the cofactors FMN, FAD and NADPH are highly conserved between all kingdoms. In C. roseus cell cultures a rapid increase of the reductase steady state mRNA level was observed after the addition of fungal elicitor preparations that are known to induce cytochrome P-450-dependent biosynthetic pathways.     

Effects of substrates on the heat stability and on the reactivities of thiol groups of 3-phosphoglycerate kinase

The kinetics of the reduction of cytochrome P-450 LM2 mediated by NADPH-cytochrome P-450 reductase in reconstituted phospholipid vesicles was examined. An inefficient reduction of the hemoprotein in phosphatidylcholine vesicles was observed. However, by introducing negatively charged phospholipids into the membrane, the rate of reduction increased in a concomitant manner to the resulting net negative charge of the vesicles. In the presence of benzphetamine, the extent of cytochrome P-450 LM2 reduced 1 s after the addition of NADPH to the system was a linear function of the electrophoretic mobilities of the vesicles used. A similar relationship between the net negative charge of the vesicles, as measured electrophoretically, and the reduction rate was also attained in the absence of substrate. The enhanced reduction was mainly reflected in an altered phase distribution of the reduction; the extent of fast phase reduction in the absence or in the presence of added substrate was dependent upon the electrophoretic mobilities of the vesicles. A similar change in the distribution of the reduction phases was observed upon decreasing the phosphatidylcholine content of the vesicles; the fast phase reduction being more pronounced in membranes with higher relative amounts of the protein components. A decrease of the rate of O-demethylation of p-nitroanisole catalyzed by P-450 LM2 parallel to the extent of fast phase reduction was observed upon dilution of neutral phosphatidylcholine membranes with phospholipid. By contrast, no effect of lipid dilution was evident in negatively charged membranes. The results are consistent with the hypothesis that the extent of fast phase reduction is governed by the amount of complex formed between NADPH-cytochrome P-450 reductase and cytochrome P-450 in the membranes; negative membranes appear to favor the formation of such complexes, whereas similar complexes are less formed, or are not functional, in neutral membranes.     

产氢红杆菌类胡萝卜素突变株的诱变和筛选

用紫外线照射和氯化锂夹层平板培养法对产氢红杆菌(Rhodobacter sp. R7)进行复合诱变, 分离获得了一株产氢效率提高的类胡萝卜素突变株R726。该突变株在表观特征、光谱学特征、色谱特征、生长和产氢性能等方面与出发菌株有明显不同, 但16S rDNA序列一致。R726菌株有 550 nm类胡萝卜素特征性吸收峰, 类胡萝卜素组成上比出发菌株少一黄色类胡萝卜素组分, 生长和产氢性能均高于出发菌株, 产氢效率比出发菌株提高了33.3%, 类胡萝卜素含量比出发株提高了53.8%。     

Two modes of binding of adrenal NADPH-cytochrome P-450 reductase to liposomal membranes

NADPH-cytochrome P-450 reductase, purified from bovine adrenocortical microsomes, was shown to bind in two different modes to liposomal membranes composed of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine at a molar ratio of 5:3:1. As demonstrated by Ficoll density gradient centrifugation and HPLC gel filtration, the cholate dialysis method made the reductase bind tightly to the liposomal membranes, while the incubation with the preformed vesicles made the reductase bind loosely to the membranes. From the experiments of electron transfer to P-450C21 residing at the other vesicles, the loosely bound reductase was found to be transferable between the vesicles, whereas the tightly bound reductase was not readily transferred. The rates of the binding and the release of the loosely bound reductase to and from the membranes were measured with the stopped-flow method by observing the reduction of P-450C21 embedded in the vesicles. These kinetic studies showed that the rate-limiting step of the reductase transfer between the vesicles was the release of the reductase from the membranes. The reductase in both binding modes well supported the steroid 21-hydroxylase activity.     

Adrenal cytochrome P-45011 beta-proteoliposomes catalyzing aldosterone synthesis: preparation and characterization

Purified cytochrome P-45011 beta from bovine adrenocortical mitochondria was successfully incorporated into the liposome membranes composed of phosphatidylcholine, phosphatidylethanolamine and cardiolipin at a molar ratio of 2:2:1. The incorporation of P-45011 beta into the liposome membranes was ascertained by the Ficoll density gradient centrifugation and the protein refractoriness to trypsin digestion. The prepared proteoliposomes containing P-45011 beta and phospholipid at a molar ratio of 1:3000 were unilamellar vesicles of about 40 nm in average diameter. The P-45011 beta embedded in the liposome membranes was found to be more stable than the detergent-solubilized form. The reconstituted system containing the P-45011 beta-proteoliposomes, adrenodoxin and NADPH-adrenodoxin reductase showed catalytic activities not only for the hydroxylation of 11-deoxycorticosterone at 11 beta- and 18-positions but also for its conversion into aldosterone with a turnover number of 2.3 nmol/min per nmol of P-45011 beta. A successive reaction without the intermediates leaving from the enzyme was suggested for the P-45011 beta-mediated conversion of 11-deoxycorticosterone to aldosterone following the result that the formation of aldosterone was linear with respect to time without the lag phase; this was confirmed by the result that radioactivity in aldosterone from 3H-labeled 11-deoxycorticosterone was scarcely decreased by the addition of unlabeled intermediates to the reactions system.     

产氢红杆菌类胡萝卜素突变株的诱变和筛选

用紫外线照射和氯化锂夹层平板培养法对产氢红杆菌(Rhodobacter sp.R7)进行复合诱变,分离获得了一株产氢效率提高的类胡萝卜素突变株R726.该突变株在表观特征、光谱学特征、色谱特征、生长和产氢性能等方面与出发菌株有明显不同,但16S rDNA序列一致.R726菌株有550 nm类胡萝卜素特征性吸收峰,类胡萝卜素组成上比出发菌株少一黄色类胡萝卜素组分,生长和产氢性能均高于出发菌株,产氢效率比出发菌株提高了33.3%,类胡萝卜素含量比出发株提高了53.8%.     

光合细菌H3菌株色素分析

H3菌株系由盐田微生物层中分离获得的光合细菌株。具有丰富的天然色素。经活细胞色素光谱吸收峰值测定,色素经有机溶剂提取、硅胶薄板层析、SDS－PAGE电泳等,结果表明H3菌株的主要色素包括细菌叶绿素a、细菌脱镁叶绿素（Bacteriophaeophytin）和三种类胡萝卜素。总胡萝卜素含量占细胞于重的0．6％,胡萝卜素蛋白复合体的分子量约11,000.培养条件的差异对色素形成及相对含量有不同程度的影响。     

Phosphatidylcholine-induced repair of damaged hepatocyte membranes in heliotrine poisoning

Hepatocyte membranes destruction in experimental toxic hepatitis caused by heliotrine administration was accompanied by a 10-fold increase in blood serum activity of aldolase fructose-I-monophosphate, a decrease in cytochrome P-450 content, an increase in the rate of cytochrome P-450 inactivation, as well as a decrease in microsomal glucose-6-phosphatase activity. Administration of phosphatidylcholine liposomes decreased the activity of aldolase twofold, which indirectly shows partial reconstitution of liver cell membranes. Phosphatidylcholine protective action is also manifested in an increase in the activity of glucose-6-phosphatase, a microsomal marker enzyme, up to its control level and in a 20% reduced rate of cytochrome P-450 inactivation. It has been shown that destroyed liver cell membranes may be repaired by the introduction of phosphatidylcholine in the form of multilayer liposomes.     

Phosphatidate Kinase,a Novel Enzyme in Phospholipid Metabolism (Purification,Subcellular Localization,and Occurrence in the Plant Kingdom)

Microsomal membranes from suspension-cultured Catharanthus roseus cells possess an enzymic activity that catalyzes the ATP-dependent phosphorylation of phosphatidic acid (PA) to form diacylglycerol pyrophosphate (H. Behrbohm, J.B. Wissing [1993] FEBS Lett 315: 95-99). This enzyme activity, PA kinase, was purified and characterized. Plasma membranes, obtained from C. roseus microsomes by aqueous two-phase partitioning, were extracted, and PA kinase was purified 3200-fold by applying different chromatographic steps that resulted in a specific activity of about 10 [mu]mol min-1 mg-1. Sodium dodecyl sulfate-gel electrophoresis of the fractions obtained from the final chromatographic step revealed a 39-kD protein that correlated with the enzyme activity; PA kinase activity could be eluted from this protein band. Subcellular localization, investigated with C. roseus cells, showed that the activity was confined to membrane fractions, and at least 80% was associated with plasma membranes. The data revealed the same distribution within the cellular membranes of PA kinase as reported for diacylglycerol kinase, which is a typical plasma membrane-located enzyme. Furthermore, PA kinase activity was detected in the calli of 16 different plant species and in the different organs of C. roseus plants and obviously occurs ubiquitously in the plant kingdom.     

Carotenoids in Rhodoplanes Species: Variation of Compositions and Substrate Specificity of Predicted Carotenogenesis Enzymes

Phototrophic bacteria necessarily contain carotenoids for photosynthesis, and accumulate unusual carotenoids in some cases. The carotenoids in all established species of Rhodoplanes (Rpl.), a representative of phototrophic genera, were identified using spectroscopic methods. The major carotenoid was spirilloxanthin in Rpl. roseus and Rpl. serenus, and rhodopin in "Rpl. cryptolactis". Rpl. elegans contained rhodopin, anhydrorhodovibrin, and spirilloxanthin. Rpl. pokkaliisoli contained not only rhodopin but also 1,1'-dihydroxylycopene and 3,4,3',4'-tetrahydrospirilloxanthin. These variations in carotenoid composition suggested that Rpl. roseus and Rpl. serenus had normal substrate specificity of the carotenogenesis enzymes of CrtC (acyclic carotene 1,2-hydratase), CrtD (acyclic carotenoid 3,4-desaturase), and CrtF (acyclic 1-hydroxycarotenoid methyltransferase). On the other hand, CrtC of Rpl. elegans, CrtD of "Rpl. cryptolactis", and CrtC, CrtD, and CrtF of Rpl. pokkaliisoli might have different characteristics from the usual activity of these normal enzymes in the normal spirilloxanthin pathway. These results suggest that the variation of carotenoids among the species of Rhodoplanes results from modified substrate specificity of the carotenogenesis enzymes involved.     

ALLOXANTHIN IN DINOPHYSIS NORVEGICA (DINOPHYSIALES, DINOPHYCEAE) FROM THE BALTIC SEA

The pigment composition of Dinophysis norvegica Ehrenberg from the central Baltic Sea differs from the normal pigment pattern found in dinoflagellates, which contain peridinin as a typical marker pigment. In D. norvegica isolated by cell fractionation of field samples, the major carotenoid was alloxanthin, a typical cryptomonad pigment. No evidence was found that the presence of alloxanthin was due to a recent phagotrophic uptake of cryptomonads, so the presence of alloxanthin in D. norvegica may be a consistent feature of a permanent endosymbiosis.     

New carotenoids from the thermophilic green sulfur bacterium Chlorobium tepidum: 1′,2′-dihydro-γ-carotene, 1′,2′-dihydrochlorobactene, and OH-chlorobactene glucoside ester, and the carotenoid composition of different strains

The complete carotenoid composition of the thermophilic green sulfur bacterium Chlorobium tepidum strain TNO was determined by spectroscopic methods. Major carotenoids were four kinds of carotenes: γ-carotene, chlorobactene, and their 1′,2′-dihydro derivatives (1′,2′-dihydro-γ-carotene and 1′,2′-dihydrochlorobactene). In lesser amounts, hydroxyl γ-carotene, hydroxyl chlorobactene, and their glucoside fatty acid esters were found. The only esterified fatty acid present was laurate, and OH-chlorobactene glucoside laurate is a novel carotenoid. In other strains of C. tepidum, the same carotenoids were found, but the composition varied from strain to strain. The overall pigment composition in cells of strain TNO was 4 mol carotenoids and 40 mol bacteriochlorophyll c per mol bacteriochlorophyll a. The effects of nicotine on carotenoid biosynthesis in C. tepidum differed from those in the thermophilic green nonsulfur bacterium Chloroflexus aurantiacus. Received: 3 February 1997 / Accepted: 6 June 1997     

Organization of two-component monomolecular layers formed with dipalmitoylphosphatidylcholine and the carotenoid pigment,canthaxanthin

Canthaxanthin is a carotenoid pigment of physiological importance owing to potential modulation of the dynamic and structural properties of biomembranes. The effect of canthaxanthin on the organization of lipid membranes formed with dipalmitoylphosphatidylcholine (DPPC) was studied with application of monomolecular layer technique, FTIR spectroscopy and linear dichroism-FTIR. The specific molecular areas of the two-component monomolecular layers of canthaxanthin-DPPC show pronounced underadditivity in the concentration range below 2 mol% carotenoid with respect to the lipid, corresponding to the monomeric organization of the pigment. Additionally, the analysis of the FTIR spectra of the two-component monolayers deposited to the solid support shows that organization of the carotenoid in the lipid monolayer is governed primarily by van der Waals interactions between the pigment chromophore and lipid alkyl chains. This interaction is responsible for an ordering effect of canthaxanthin with respect to lipids. Analysis of FTIR spectra of two-component monolayers suggests the possibility of hydrogen bonding between the lipid polar headgroups and the keto groups of canthaxanthin via water bridges.