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1.
A group of 12 alkaloids were tested as inhibitors of photophosphorylation in spinach chloroplasts. Ajmaline, a dihydroindole alkaloid, was found to be the strongest inhibitor of both cyclic and non-cyclic photophosphorylation. Low concentrations of ajmaline also inhibited the dark and light ATPases, and the coupled electron flow from water to ferricyanide, measured either as ferrocyanide formed or as oxygen evolved, but not the uncoupled electron transport or the pH rise of illuminated unbuffered suspensions of chloroplasts. Higher concentrations of ajmaline stimulated, instead of inhibiting, photosynthetic electron transport or oxygen evolution and decreased the pH rise, thus behaving as an uncoupler, such as ammonia.Photophosphorylation was partially inhibited by 100 μM dihydrosanguinarine, 100 μM dihydrochelerythrine (benzophenanthridine alkaloids); 500 μM O,O'-dimethylmagnoflorine, 500 μM N-methylcorydine (aporphine alkaloids) and 1 mM julocrotine. They also inhibited coupled oxygen evolution and only partially (dihydrosanguinarine and dihydrochelerythrine) or not at all (the other alkaloids) uncoupled oxygen evolution.Spegazzinine (dihydroindole alkaloid), magnoflorine, N-methylisocorydine, coryneine (aporphine alkaloids), candicine and ribalinium chloride were without effect on photophosphorylation at 500 μM. 相似文献
2.
Monoterpenoid indole alkaloids are a vast and structurally complex group of plant secondary compounds. In contrast to other groups of plant products which produce many glycosides, indole alkaloids rarely occur as glucosides. Plants of Rauvolfia serpentina accumulate ajmaline as a major alkaloid, whereas cell suspension cultures of Rauvolfia mainly accumulate the glucoalkaloid raucaffricine at levels of 1.6 g/l. Cell cultures do contain a specific glucosidase. known as raucaffricine-O-beta-D-glucosidase (RG), which catalyzes the in vitro formation of vomilenine, a direct intermediate in ajmaline biosynthesis. Here, we describe the molecular cloning and functional expression of this enzyme in Escherichia coli. RG shows up to 60% amino acid identity with other glucosidases of plant origin and it shares several sequence motifs with family 1 glucosidases which have been characterized. The best substrate specificity for recombinant RG was raucaffricine (KM 1.3 mM, Vmax 0.5 nkat/microg protein) and only a few closely related structural derivatives were also hydrolyzed. Moreover, an early intermediate of ajmaline biosynthesis, strictosidine, is a substrate for recombinant RG (KM 1.8 mM, Vmax 2.6 pkat/microg protein) which was not observed for the low amounts of enzyme isolated from Rauvolfia cells. 相似文献
3.
L D Zaborovskaia B I Khodorov 《Biulleten' eksperimental'no? biologii i meditsiny》1985,100(9):297-299
The effects of external or internal application of quaternary derivatives of ajmaline and N-propyl ajmaline (NMA) to the frog nodes of Ranvier were studied by the voltage clamp method. It was established that external application of NMA is much less effective than that of the tertiary amine ajmaline or its quaternary analog N-ethyl ajmaline at comparable concentration. Even at a concentration of 1 mM NMA when applied externally caused only relatively insignificant tonic and use-dependent inhibition of sodium currents. In contrast, internal application of NMA (1 mM) through the cut ends of the fiber led to pronounced use-dependent inhibition of sodium currents. The conclusion is drawn that the binding site for NMA is located in the inner mouth of the Na channels which becomes available for the charged blocker only after opening of the activation gate. 相似文献
4.
Natural products and enzymes from plant cell cultures 总被引:3,自引:0,他引:3
J. Stöckigt P. Obitz H. Falkenhagen R. Lutterbach S. Endreß 《Plant Cell, Tissue and Organ Culture》1995,43(2):97-109
Plants represent an unlimited source of natural products. Many of the recently detected phytochemicals exhibit remarkable bioactivities, ranging from anticancer activity, phosphodiesterase inhibition to cytotoxicity against HIV-infected cells. Cultivated plant cells produce at their unorganized, dedifferentiated stage secondary metabolites, but in very different amounts in so far as new compounds are concerned. In fact, more than 140 novel natural products are presently known from plant cell cultures, which also include new metabolites formed by biotransformation. The biotransformation capacity of suspended cells is described and recent high yielding transformations, like the formation of arbutin by hydroquinone-transformation withRauwolfia cells are discussed. As an example of alkaloid production by cell suspensions, the pattern of monoterpenoid indole alkaloids of the Indian medicinal plantRauwolfia serpentina Benth. is described and the so far 30 identified compounds are divided into eight groups which are biosynthetically closely related. Some of the key biosynthetic reactions leading to theRauwolfia alkaloids are discussed and an overview of the enzymes involved in the formation of the alkaloid ajmaline and proteins catalyzing side reactions of the ajmaline pathway are given. 相似文献
5.
Carbon-13 nuclear magnetic resonance studies of myocardial glycogen metabolism in live guinea pigs 总被引:3,自引:0,他引:3
Myocardial glycogen metabolism was studied in live guinea pigs by 13C NMR at 20.19 MHz. Open-chest surgery was used to expose the heart, which was then positioned within a solenoidal radio frequency coil for NMR measurements. The time course of myocardial glycogen synthesis during 1-h infusions of 0.5 g of D-[1-13C]glucose (and insulin) into the jugular vein was investigated. The possible turnover of the 13C-labeled glycogen was also studied in vivo by following the labeled glucose infusion with a similar infusion of unlabeled glucose. The degree of 13C enrichment of the C-1 glycogen carbons during these infusions was measured in heart extracts by 1H NMR at 360 MHz. High-quality proton-decoupled 13C NMR spectra of the labeled C-1 carbons of myocardial glycogen in vivo were obtained in 1 min of data accumulation. This time resolution allowed measurement of the time course of glycogenolysis of the 13C-labeled glycogen during anoxia by 13C NMR in vivo. With the solenoidal coil used for 13C NMR, the spin-lattice relaxation time of the labeled C-1 carbons of myocardial glycogen could be measured in vivo. For a comparison, spin-lattice relaxation times of heart glycogen were measured in vitro at 90.55 MHz. Natural abundance 13C NMR studies of the quantitative hydrolysis of extracted heart glycogen in vitro at 90.55 MHz showed that virtually all the carbons in heart glycogen contribute to the 13C NMR signals. The same result was obtained in 13C NMR studies of glycogen hydrolysis in excised guinea pig heart. 相似文献
6.
We have used a coaxial superfusion system to obtain physiologically interpretable in vivo 1H NMR spectra at 500 MHz of carrot roots, maize roots, and rice shoots in water (no 2H2O). The superfusion system was constructed from common laboratory parts, required no modification of the probe and sample loading procedure, and was inherently leak resistant. The assignment and quantitation of the in vivo 1H NMR resonances were achieved by performing two-dimensional NMR experiments in vivo, and by in vitro analysis including NMR and gas chromatography-mass spectrometry. The in vivo spectra were dominated by resonances arising from sugars, organic acids, amino acids, and ethanol. In vivo measurements of spin-lattice relaxation times and chemical shifts of beta protons of malate in carrot roots suggested that malate was located in a relatively viscous and acidic compartment. In rice shoots, the hypoxic time courses of 9 metabolites were established in vivo, and 23 in vitro. In both cases, accumulation of lactate, ethanol, Ala, and gamma-aminobutyrate as well as a decrease in Gln and Asn concentrations were observed. These findings are consistent with accelerated glycolysis and decreased tricarboxylic acid cycle activity. 相似文献
7.
1-Deoxynojirimycin is a glycosidase-inhibitory alkaloid obtained from several plants and microorganisms. Administration experiments using [1-(13C)] glucose in the higher plant Commelina communis and 13C NMR spectroscopic analyses of products suggested that 1-deoxynojirimycin was biosynthesized through a different route compared with that in Streptomyces and Bacilli microorganisms. 相似文献
8.
Sequential isolation of superoxide dismutase and ajmaline from a tissue culture of Rauwolfia serpentina Benth 总被引:1,自引:0,他引:1
Two biologically active compounds, the enzyme superoxide dismutase (EC 1.15.1.1) and the antiarrhythmic indole alkaloid ajmaline, were isolated from a callus culture of Rauwolfia serpentina Benth. Sequential isolation of biologically active compounds by metal-chelate affinity chromatography followed by azoadsorbent affinity chromatography allowed us to obtain highly purified products. The yields of superoxide dismutase and ajmaline were 180 mg/kg biomass and 16.5 g/kg dry weight, respectively. 相似文献
9.
Delineation of the biochemical pathway leading to the antiarrhythmic Rauvolfia alkaloid ajmaline has been an important target in biosynthetic research for many years. The biosynthetic sequence starting with tryptamine and the monoterpene secologanin consists of about 10 different steps. Most of the participating enzymes have been detected and characterized previously, except those catalyzing the reduction of the intermediate vomilenine. A novel NADPH-dependent enzyme that reduces the intermediate has been isolated from Rauvolfia serpentina cell suspension cultures. Vomilenine reductase (M(r )43 kDa, temp opt 30 degrees C, pH opt 5.7-6.2), saturates the indolenine double bond of vomilenine with stereospecific formation of 2beta(R)-1,2-dihydrovomilenine. The described detection, enrichment and properties of the reductase not only closes a gap in ajmaline biosynthesis but is also a prerequisite for overexpressing the protein heterologously for final clarification of its molecular properties. 相似文献
10.
Ni da Q Zook J Klewer DA Nieman RA Soll J Fromme P 《Protein expression and purification》2011,80(2):157-168
Membrane proteins compose more than 30% of all proteins in the living cell. However, many membrane proteins have low abundance in the cell and cannot be isolated from natural sources in concentrations suitable for structure analysis. The overexpression, reconstitution, and stabilization of membrane proteins are complex and remain a formidable challenge in membrane protein characterization. Here we describe a novel, in vitro folding procedure for a cation-selective channel protein, the outer envelope membrane protein 16 (OEP16) of pea chloroplast, overexpressed in Escherichia coli in the form of inclusion bodies. The protein is purified and then folded with detergent on a Ni–NTA affinity column. Final concentrations of reconstituted OEP16 of up to 24 mg/ml have been achieved, which provides samples that are sufficient for structural studies by NMR and crystallography. Reconstitution of OEP16 in detergent micelles was monitored by circular dichroism, fluorescence, and NMR spectroscopy. Tryptophan fluorescence spectra of heterologous expressed OEP16 in micelles are similar to spectra of functionally active OEP16 in liposomes, which indicates folding of the membrane protein in detergent micelles. CD spectroscopy studies demonstrate a folded protein consisting primarily of α-helices. 15N-HSQC NMR spectra also provide evidence for a folded protein. We present here a convenient, effective and quantitative method to screen large numbers of conditions for optimal protein stability by using microdialysis chambers in combination with fluorescence spectroscopy. Recent collection of multidimensional NMR data at 500, 600 and 800 MHz demonstrated that the protein is suitable for structure determination by NMR and stable for weeks during data collection. 相似文献
11.
Joachim Stöckigt Santosh Panjikar Martin Ruppert Leif Barleben Xueyan Ma Elke Loris Marco Hill 《Phytochemistry Reviews》2007,6(1):15-34
The biosynthetic pathway leading to the monoterpenoid indole alkaloid ajmaline in Rauvolfia serpentiin serpentina is one of the most studied in the field of natural product biosynthesis. Ajmaline has a complex structure which is based
on a six-membered ring system harbouring nine chiral carbon atoms. There are about fifteen enzymes involved, including some
involving the side reactions of the ajmaline biosynthetic pathway. All enzymes exhibit pronounced substrate specificity. In
the recent years isolation and sequencing of their cDNAs has allowed a detailed sequence analysis and comparison with functionally
related and occasionally un-related enzymes. Site-directed mutations of several of the ajmaline-synthesizing enzymes have
been performed and their catalytic residues have been identified. Success with over-expression of the enzymes was an important
step for their crystallization and structural analysis by X-ray crystallography. Crystals with sufficient resolution were
obtained from the major enzymes of the pathway. Strictosidine synthase has a 3D-structure with a six-bladed β-propeller fold
the first time such a fold found in the plant kingdom. Its ligand complexes with tryptamine and secologanin, as well as structure-based
sequence alignment, indicate a possible evolutionary relationship to several primary sequence-unrelated structures with this
fold. The structure of strictosidine glucosidase was determined and its structure has as a (β/α)8 barrel fold. Vinorine synthase provides the first 3D structure of a member of BAHD enzyme super-family. Raucaffricine glucosidase
involved in a side-route of ajmaline biosynthesis has been crystallized. The ajmaline biosynthetic pathway is an outstanding
example where many enzymes 3D-structure have been known and where there is a real potential for protein engineering to yield
new alkaloid. 相似文献
12.
E Dogru H Warzecha F Seibel S Haebel F Lottspeich J St?ckigt 《European journal of biochemistry》2000,267(5):1397-1406
The biosynthesis of the anti-arrhythmic alkaloid ajmaline is catalysed by more than 10 specific enzymes. In this multistep process polyneuridine aldehyde esterase (PNAE) catalyses a central reaction by transforming polyneuridine aldehyde into epi-vellosimine, which is the immediate precursor for the synthesis of the ajmalane skeleton. PNAE was purified from cell suspension cultures of Rauvolfia serpentina. The N-terminal sequence and endoproteinase LysC fragments of the purified protein were used for primer design and for the amplification of specific PCR products leading to the isolation of PNAE-encoding cDNA from a R. serpentina library. The PNAE cDNA was fused with a C-terminal His-tag, expressed in Escherichia coli and purified to homogeneity using Ni-affinity chromatography. The pure enzyme shows extraordinary substrate specificity, completely different to other esterases. Sequence alignments indicate that PNAE is a new member of the alpha/beta hydrolase super family. 相似文献
13.
N. V. Kirillova M. G. Smirnova V. P. Komov 《Applied Biochemistry and Microbiology》2001,37(2):160-163
Two biologically active compounds, the enzyme superoxide dismutase (EC 1.15.1.1) and the anti-arhythmic indole alkaloid ajmaline, were isolated from a callus culture of Rauwolfia serpentinaBenth. Sequential isolation of biologically active compounds by metal–chelate affinity chromatography followed by azoadsorbent affinity chromatography allowed us to obtain highly purified products. The yields of superoxide dismutase and ajmaline were 180 mg/kg biomass and 16.5 g/kg dry weight, respectively. 相似文献
14.
DNA structure in which an adenine-cytosine mismatch pair forms an integral part of the double helix 总被引:1,自引:0,他引:1
M H Sarma G Gupta R H Sarma R Bald U Engelke S L Oei R Gessner V A Erdmann 《Biochemistry》1987,26(24):7707-7715
Extensive studies using one- and two-dimensional 1H NMR at 500 MHz revealed that the oligonucleotide d(CGCCGCAGC) in solution at 5 degrees C forms a double helix under conditions of high salt (500 mM in NaCl, 1 mM sodium phosphate), low pH (pH 4.5), and high DNA concentration (4 mM in duplex). The presence of very strong nuclear Overhauser effects (NOEs) from base H8/H6 to sugar H2',H2" and the absence of NOE from base H8/H6 to sugar H3' suggested that the oligomer under these solution conditions forms a right-handed B-DNA double helix. The following lines of experimental evidence were used to conclude that C4 and A7 form an integral part of the duplex: (i) the presence of a NOESY cross-peak involving H8 of A7 and H8 of G8, (ii) the presence of a two-dimensional NOE (NOESY) cross-peak between H6 of C3 and H6 of C4, (iii) base protons belonging to C4 and A7 forming a part of the H8/H6---H1' cross-connectivity route, and (iv) the pattern of H8/H6---H2',H2" NOESY cross-connectivity based upon a B-DNA model requiring that both C4 and A7 form an integral part of the duplex. The possibility of an A-C pair involving H bonds was also examined. Two possible structural models of the duplex at pH 4.5 are proposed: in one model A-C pairing involves two H bonds, and in the other A-C pairing involves a single H bond. 相似文献
15.
Raucaffricine (vomilenine-galactoside) was shown to be the major indole alkaloid of Rauwolfia serpentina Benth. cell suspension cultures grown in AP-medium (alkaloid production medium). Several grams of this glycoalkaloid can conveniently be isolated by RLCC (Rotation Locular Countercurrent Chromatography). A newly discovered enzyme efficiently converts the glycoalkaloid to its aglycon, vomilenine, which occupies a key function in the biosynthesis of ajmaline. This is the first demonstration of the occurrence of raucaffricine in Rauwolfia serpentina. 相似文献
16.
Recent in vivo NMR studies have raised interest in the structural changes of cellular lipids during proliferative activity. We investigated the changes in plasma membrane lipid and total cell lipid during mitogenically-stimulated proliferation of human peripheral blood lymphocytes by extraction of lipids and assay by 500 MHz 1H-NMR. Resonances were assigned using one- and two-dimensional spectroscopic techniques, and signals unique to certain species of lipid were identified. Choline and ethanolamine-containing lipids, glycerophospholipid backbones, sphingolipids, cholesterol, plasmalogens and triacylglycerols were readily detected. Resolution of a number of lipid species was not possible, despite the use of high-resolution techniques. NMR values for proliferation-induced changes in the most easily determined parameters, namely the total cholesterol to total phospholipid molar ratio, and phosphatidylcholine, phosphatidylethanolamine and sphingolipid composition, were found to agree with traditional methods. Differences in phospholipid and fatty acid profiles were found between plasma membranes and total cell lipid for resting values and for response to mitogen. 相似文献
17.
Proton line narrowing in biomembranes spun at the magic angle, for spinning speeds greater than 7 kHz, was investigated in two ways: increasing the field strength from 200 to 800 MHz and changing the membrane fluidity. The resolution that one can obtain on natural lipid membranes under the form of liposomes is 0.019 ppm at 800 MHz. On the other hand, spinning bicelles (disk-like model membranes made of synthetic long and short chain lipids) at the magic angle decreases the line width by an additional factor of 3 provided the bicelle is subjected to large orientational disorder. This leads to proton line widths of the order of 6 Hz at 500 MHz. The conjunction of high field, magic angle spinning and use of bicelle membranes should prove to be useful to solve membrane protein structure in a membrane environment. 相似文献
18.
Proton line narrowing in biomembranes spun at the magic angle, for spinning speeds greater than 7 kHz, was investigated in two ways: increasing the field strength from 200 to 800 MHz and changing the membrane fluidity. The resolution that one can obtain on natural lipid membranes under the form of liposomes is 0.019 ppm at 800 MHz. On the other hand, spinning bicelles (disk-like model membranes made of synthetic long and short chain lipids) at the magic angle decreases the line width by an additional factor of 3 provided the bicelle is subjected to large orientational disorder. This leads to proton line widths of the order of 6 Hz at 500 MHz. The conjunction of high field, magic angle spinning and use of bicelle membranes should prove to be useful to solve membrane protein structure in a membrane environment. 相似文献
19.
McNally DJ Jarrell HC Li J Khieu NH Vinogradov E Szymanski CM Brisson JR 《The FEBS journal》2005,272(17):4407-4422
Recently, the CPS biosynthetic loci for several strains of Campylobacter jejuni were sequenced and revealed evidence for multiple mechanisms of structural variation. In this study, the CPS structure for the HS:1 serostrain of C. jejuni was determined using mass spectrometry and NMR at 600 MHz equipped with an ultra-sensitive cryogenically cooled probe. Analysis of CPS purified using a mild enzymatic method revealed a teichoic acid-like [-4)-alpha-d-Galp-(1-2)-(R)-Gro-(1-P](n), repeating unit, where Gro is glycerol. Two branches at C-2 and C-3 of galactose were identified as beta-d-fructofuranoses substituted at C-3 with CH(3)OP(O)(NH(2))(OR) groups. Structural heterogeneity was due to nonstoichiometric glycosylation at C-3 of galactose and variable phosphoramidate groups. Identical structural features were found for cell-bound CPS on intact cells using proton homonuclear and (31)P heteronuclear two-dimensional HR-MAS NMR at 500 MHz. In contrast, spectroscopic data acquired for hot water/phenol purified CPS was complicated by the hydrolysis and subsequent loss of labile groups during extraction. Collectively, the results of this study established the importance of using sensitive isolation techniques and HR-MAS NMR to examine CPS structures in vivo when labile groups are present. This study uncovered how incorporation of variable O-methyl phosphoramidate groups on nonstoichiometric fructose branches is used in C. jejuni HS:1 as a strategy to produce a highly complex polysaccharide from its small CPS biosynthetic locus and a limited number of sugars. 相似文献
20.
This work investigated the use of nuclear magnetic resonance (NMR) spectroscopy in combination with a mathematical model of an encapsulated cell system as a method for rapidly assessing the status of a pancreatic substitute. To validate this method, an in vitro experiment was performed in which the encapsulated cells were perfused in an NMR-compatible system and the dissolved oxygen (DO) concentration of the perfusing medium was lowered from 0.20 to 0.05 mM, then returned to 0.20 mM in a stepwise fashion. The cellular metabolic activity and bioenergetics were evaluated by measuring the oxygen consumption rate (via DO sensors) and nucleotide triphosphate levels (via (31)P NMR). By incorporating a perfluorocarbon emulsion into the alginate beads, the cellular oxygenation state was monitored by measuring the average intrabead DO (AIDO) concentration by (19)F NMR. The in vitro measurements were then compared with model predictions based on the measured external DO concentration and time. Model-predicted cell growth and AIDO closely matched the experimentally acquired data. As the DO concentrations both external to and within the pancreatic substitute are needed to apply this methodology in vivo, the feasibility of measuring the DO concentration from two distinct bead populations implanted in the peritoneal cavity of mice was established. It is concluded that PFC incorporation and (19)F NMR measurements, in combination with a mechanistic model of the encapsulated system, allow the tracking of the state of a pancreatic substitute in vitro and potentially in vivo. 相似文献