Ultrastructure ofCatharanthus roseus cells culturedin vitro and exposed to conditions for alkaloid accumulation

Summary Periwinkle (Catharanthus roseus) cells cultured in 1-B 5 medium display the ultrastructure of parenchyma cells. The parenchyma character remained unchanged when cells were exposed to any one of three different conditions effecting alkaloid accumulation. Transfer of cells to alkaloid production medium for 2 weeks (condition 1) accorded two special features,i.e., unusually big lipid droplets in the cytoplasm and, upon fixation, one or several electron-dense droplets of spongy precipitate in vacuoles. Among hormone-autotrophic cultures (condition 2) some cells showed a fine electron-dense vacuolar precipitate. Addition ofPhythium homogenate (fungal elicitor) to cells cultured in 1-B 5-medium for 10 days (condition 3), cells showed a frequent appearance of singular big lipid droplets in the cytoplasm, whereas vacuoles remained devoid of precipitate. The appearance of big lipid droplets and of vacuolar precipitate is interpreted as progressing cytodifferentiation, but is coincidental with alkaloid accumulation.NRCC no. 24524.     

Adenosine deaminase: viscosity studies and the mechanism of binding of substrate and of ground- and transition-state analogue inhibitors

We have studied the effects of viscosogenic agents, sucrose and ficoll, on (1) the hydrolysis of adenosine and of 6-methoxypurine riboside catalyzed by adenosine deaminase and (2) the rates of association and dissociation of ground-state and transition-state analogue inhibitors. For adenosine, Vmax/Km is found to be inversely proportional to the relative viscosity with sucrose, an agent affecting the microscopic viscosity, while no effect is found with ficoll, an agent affecting the macroscopic viscosity. Viscosogenic agents have no effect on the kinetic constants for 6-methoxypurine riboside. Thus, the bimolecular rate constant, Vmax/Km = 11.2 +/- 0.8 microM-1 s-1, for the reaction with adenosine is found to be at the encounter-controlled limit while that for the reaction with the poor substrate 6-methoxypurine riboside, 0.040 +/- 0.004 microM-1 s-1, is limited by some other process. Viscosity-dependent processes do not make a significant (less than 10%) contribution to Vmax. The dissociation constants for inhibitors are unaffected by viscosity. The ground-state analogue inhibitor purine riboside appears to bind at a rate comparable to that of adenosine. However, the slower rates of association (0.16-2.5 microM-1 s-1) and dissociation (5 X 10(-6) to 12 s-1) of transition-state analogue inhibitors are affected by the viscosity of the medium to approximately the same extent as the encounter-controlled rates of association and dissociation of adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased accumulation of indole alkaloids by some cell lines of Catharanthus roseus in response to addition of vanadyl sulphate

Vanadyl sulphate (10–500 mg/l), when added to cell suspension cultures of Catharanthus roseus stimulated increased intracellular accumulation of catharanthine and ajmalicine. This response was demonstrated in both flask and fermenter (30 litre) systems. The response varied, and depended upon cell line, concentration of vanadyl sulphate and the stage of the growth phase at which the cells were treated. This process has the potential to increase the yield and reduce the production time for commercially useful secondary plant metabolites.Abbreviations Ajm ajmalicine - Cath catharanthine - CAS ceric ammonium sulphate - VOSO₄ vanadyl sulphate - FW fresh weight - n.d. not detected     

Immunological Detection and Quantitation of Tryptophan Decarboxylase in Developing Catharanthus roseus Seedlings

l-Tryptophan decarboxylase (TDC) (EC 4.2.1.27) enzyme activity was induced in cell suspension cultures of Catharanthus roseus after treatment with a Pythium aphanidermatum elicitor preparation. The enzyme was extracted from lyophilized cells containing high levels of TDC and the protein was purified to homogeneity. The pure protein was used to produce highly specific polyclonal antibodies, and an enzyme-linked immunosorbent assay (ELISA) was developed to quantitate the level of TDC antigen during seedling development and in leaves of the mature plant. Western immunoblotting of proteins after SDS-PAGE with anti-TDC antibodies detected several immunoreactive proteins (40, 44, 54.8, 55, and 67 kilodaltons) which appeared at different stages during seedling development and in leaves of the mature plant. The major 54.8 and 55 kilodalton antigenic proteins in immunoblots appeared transiently between days 1 to 5 and 5 to 8 of seedling development, respectively. The 54.8 kilodalton protein was devoid of TDC enzyme activity, whereas the appearance of the 55 kilodalton protein coincided with the appearance of this decarboxylase activity. The minor immunoreactive proteins (40, 44, and 67 kilodaltons) appeared after day 5 of seedling development and in older leaves of the mature plant, and their relationship, if any, to TDC is presently unknown. Results suggest that the synthesis and degradation of TDC protein is highly regulated in Catharanthus roseus and that this regulation follows a preset developmental program.     

Homologous plasmid recombination is elevated in immortally transformed cells.

The levels of intramolecular plasmid recombination, following transfection of a plasmid substrate for homologous recombination into normal and immortally transformed cells, have been examined by two independent assays. In the first assay, recovered plasmid was tested for DNA rearrangements which regenerate a functional neomycin resistance gene from two overlapping fragments. Following transformation of bacteria, frequencies of recombinationlike events were determined from the ratio of neomycin-resistant (recombinant) colonies to ampicillin-resistant colonies (indicating total plasmid recovery). Such events, yielding predominantly deletions between the directly repeated sequences, were substantially more frequent in five immortal cell lines than in any of three normal diploid cell strains tested. Effects of plasmid replication or interaction with T antigen and of bacterially mediated rejoining of linear molecules generated in mammalian cells were excluded by appropriate controls. The second assay used limited coamplification of a control segment of plasmid DNA, and of the predicted recombinant DNA region, primed by two sets of flanking oligonucleotides. Each amplified band was quantitated by reference to a near-linear standard curve generated concurrently, and recombination frequencies were determined from the ratio of recombinant/control DNA regions. The results confirmed that recombinant DNA structures were generated within human cells at direct repeats in the transfected plasmid and were markedly more abundant in an immortal cell line than in the diploid normal cells from which that line was derived.     

Protein structural changes accompanying formation of enzymatic transition states: tryptophan environment in ground-state and transition-state analogue complexes of adenosine deaminase

The accessibility of protein tryptophan fluorescence to the quenching agent acrylamide has been studied in adenosine deaminase and in binary complexes of the enzyme with ground-state or transition-state analogues. Although the enzyme contains three tryptophan residues, Stern-Volmer plots are linear with all the fluorescence quenchable at high acrylamide concentrations. Tryptophan fluorescence is less easily quenched in the binary complexes than in the free enzyme, indicating a decrease in the accessibility of these residues. The greatest decrease in accessibility is found for the transition-state analogue complexes. Although the affinities of the transition-state analogues studied span a range of 10(6), the Stern-Volmer constants of the complexes are the same within experimental error. Thus, as measured by this technique, changes in enzyme conformation accompanying formation of these complexes are similar for all transition-state analogues. Resonance energy transfer from tryptophan as donor to ligand as acceptor successfully explains the differing abilities of ligands to quench the enzyme's intrinsic fluorescence upon formation of complexes in the absence of acrylamide. On the basis of Forster distance calculations, it is likely that the residues partially quenched upon formation of transition-state analogue complexes are distant from the active site.     

Evidence from 13C NMR for polarization of the carbonyl of oxaloacetate in the active site of citrate synthase

The carbon-13 NMR spectrum of oxaloacetate bound in the active site of citrate synthase has been obtained at 90.56 MHz. In the binary complex with enzyme, the positions of the resonances of oxaloacetate are shifted relative to those of the free ligand as follows: C-1 (carboxylate), -2.5 ppm; C-2 (carbonyl), +4.3 ppm; C-3 (methylene), -0.6 ppm; C-4 (carboxylate), +1.3 ppm. The change observed in the carbonyl chemical shift is successively increased in ternary complexes with the product [coenzyme A (CoA)], a substrate analogue (S-acetonyl-CoA), and an acetyl-CoA enolate analogue (carboxymethyl-CoA), reaching a value of +6.8 ppm from the free carbonyl resonance. Binary complexes are in intermediate to fast exchange on the NMR time scale with free oxaloacetate; ternary complexes are in slow exchange. Line widths of the methylene resonance in the ternary complexes suggest complete immobilization of oxaloacetate in the active site. Analysis of line widths in the binary complex suggests the existence of a dynamic equilibrium between two or more forms of bound oxaloacetate, primarily involving C-4. The changes in chemical shifts of the carbonyl carbon indicate strong polarization of the carbonyl bond or protonation of the carbonyl oxygen. Some of this carbonyl polarization occurs even in the binary complex. Development of positive charge on the carbonyl carbon enhances reactivity toward condensation with the carbanion/enolate of acetyl-CoA in the mechanism which has been postulated for this enzyme. The very large change in the chemical shift of the reacting carbonyl in the presence of an analogue of the enolate of acetyl-CoA supports this interpretation.     

Acetyl Coenzyme A: Deacetylvindoline O-Acetyltransferase,A Novel Enzyme from Catharanthus

A new enzyme, Acetyl Coenzyme A: deacetylvindoline 0-acetyl transferase (EC 2.3.1. -) which catalyses the synthesis of vindoline from acetyl coenzyme A and deacetylvindoline was isolated from the soluble protein extract of Catharanthus roseus leaves and purified approximately 365-fold. The enzyme had an apparent pI of 4.6 upon chromatofocusing, an apparent molecular weight of 45,000 daltons and a pH optimum between 8.0 to 9.0. Dithiothreitol was essential to maintain enzyme activity.Substrate saturation studies of this enzyme resulted in Michaelis Menton kinetics giving Km values of 5.4 and 0.7µM respectively for acetyl coenzyme A and deacetylvindoline. Studies of the forward reaction demonstrated an absolute requirement for acetyl coenzyme A and deacetylvindoline derivatives containing a double bond at positions 6, 7, whereas the reverse reaction occurred only in the presence of free coenzyme A and vindoline derivatives containing the same double bond. The forward reaction was subject to product inhibition by coenzyme A with an apparent Ki of 8 µM, but was not inhibited by up to 2 mM vindoline. The rate of reaction could therefore be regulated by the level of free coenzyme A in the cell, unaffected by the accumulation of indole alkaloid product.It was suggested that this enzyme catalyses a late step in the biosynthesis of vindoline.     

Photolabile derivatives of bile salts. Synthesis and suitability for photoaffinity labeling

In an approach to the identification of bile salt-binding carriers, the photoactivable bile acid derivatives A) 3 beta-azido, 7 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oic acid, B) 7,7-azo-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oic acid, and C) 11 xi-azido-12-oxo-3 alpha,7 alpha-dihydroxy-5 beta-cholan-24-oic acid were synthesized in unconjugated and taurine-conjugated form. Photolysis of the 3 beta-azido derivatives was studied using a light source with a maximum emission at 300 nm and established a half-life time of 18.5 min. The photochemistry of the 7,7-azo derivatives was investigated using light with a maximum at 350 nm and had a half-life time of 2.2 min. The 11 xi-azido-12-oxo derivatives were photolyzed with light having a maximum at 300 nm resulting in a half-life time of 8.5 min. The suitability of the 7,7-azo derivatives for photoaffinity labeling was demonstrated by photolyses in 14C-labeled methanol and acetonitrile. The generated carbene reacted with the solvents under covalent bond formation of 6 to 12%. The efficiency of all synthesized photolabile derivatives for photoaffinity labeling of bile salt binding proteins was demonstrated.