Electron paramagnetic resonance identification of a highly reactive thiol group in the proximity of the catalytic site of human placenta glutathione transferase.

The reaction of the glutathione transferase from human placenta with a maleimide spin label derivative has been followed by EPR. Incubation of the enzyme at pH 7.0 with 50-fold molar excess of the spin label reagent gives rise to an immobilized nitroxyl EPR spectrum indicative of two reacting thiol groups per dimer of enzyme as evaluated by double integration of the EPR spectrum; the activity is lost in parallel. The same type of spectrum can be obtained simply by adding 2 eq of the spin label reagent to the enzyme. The binding is completed after less than 1 min at pH 8.0; it requires 2 min at pH 7.0 and more than 10 min at pH 6.0. These data indicate that the maleimide derivative reacts, in each subunit, with a thiol group which plays a crucial role for the maintenance of the catalytic activity and is characterized by a low pK. Inactivation of the enzyme at pH 7.0 in the presence of 2 eq of spin label reagent per mol of enzyme requires 15 min, suggesting the occurrence of a structural rearrangement after the binding of the thiol blocking agent. The same binding in the presence of S-methylglutathione or protoporphyrin IX shows a decreased reaction rate with respect to the reaction in the absence of inhibitors, indicating that the thiols are in proximity of both the glutathione and the porphyrin binding sites. For this latter case, this is unambiguously demonstrated by the titration of spin-labeled enzyme with hemin, which produces a decrease of the EPR signal amplitude from which an interspin distance of about 10 A can be evaluated.     

Production and properties of the linamarase and amygdalase activities of Penicillium aurantiogriseum P35.

The effects of medium composition on the production of beta-glucosidase (amygdalase and linamarase) by Penicillium aurantiogriseum P35 were studied and the medium optimized as follows (g/l of deionized water): pectin, 10.0; (NH4)2SO4, 8.0; KH2PO4, 8.0; Na2HPO4, 2.8; MgSO4.7H2O, 0.5; yeast extract, 4.0; initial pH 6.0. When grown in a bench fermenter on this medium, the fungus produced 50.5 mU of amygdalase and 9.4 mU of linamarase per ml of culture broth. Two beta-glucosidases (PGI and PGII), each having amygdalase and linamarase activities, were recovered from the culture broth and purified; their relative molecular weights, as native enzymes, were estimated to be about 247,000 and 147,000, respectively. Both enzymes showed the same optimum pH (6.0) but different optimum temperatures (55 and 60 degrees C for PGI and PGII, respectively). Thermostability (10 min at 60 degrees C) and half-life of enzyme activity (7 hours at 60 degrees C) of PGII were higher than those of PGI (10 min at 50 degrees C and 2 hours at 55 degrees C, respectively). A wide range of cyanogenic glycosides (such as tetraphyllin B, epivolkenin, gynocardin, passibiflorin, prunasin, taxiphyllin, amygdalin, lucumin, sambunigrin, dhurrin, linamarin and cardiospermin sulfate) were hydrolyzed by both enzymes.     

Specialized Plant Metabolism Characteristics and Impact on Target Molecule Biotechnological Production

Plant secondary metabolism evolved in the context of highly organized and differentiated cells and tissues, featuring massive chemical complexity operating under tight environmental, developmental and genetic control. Biotechnological demand for natural products has been continuously increasing because of their significant value and new applications, mainly as pharmaceuticals. Aseptic production systems of plant secondary metabolites have improved considerably, constituting an attractive tool for increased, stable and large-scale supply of valuable molecules. Surprisingly, to date, only a few examples including taxol, shikonin, berberine and artemisinin have emerged as success cases of commercial production using this strategy. The present review focuses on the main characteristics of plant specialized metabolism and their implications for current strategies used to produce secondary compounds in axenic cultivation systems. The search for consonance between plant secondary metabolism unique features and various in vitro culture systems, including cell, tissue, organ, and engineered cultures, as well as heterologous expression in microbial platforms, is discussed. Data to date strongly suggest that attaining full potential of these biotechnology production strategies requires being able to take advantage of plant specialized metabolism singularities for improved target molecule yields and for bypassing inherent difficulties in its rational manipulation.     

Mercury Exposure: Protein Biomarkers of Mercury Exposure in Jaraqui Fish from the Amazon Region

Poly-trans-[(2-carboxyethyl)germasesquioxane] (Ge-132) is a water-soluble organogermanium compound that exerts various physiological effects, including anti-inflammatory activity and pain relief. In water, Ge-132 is hydrolyzed to 3-(trihydroxygermyl)propanoic acid (THGP), which in turn is capable of interacting with cis-diol compounds through its trihydroxy group, indicating that this compound could also interact with diol-containing nucleic acid constituents. In this study, we evaluated the ability of THGP to interact with nucleosides or nucleotides via nuclear magnetic resonance (NMR) analysis. In addition, we evaluated the effect of added THGP on the enzymatic activity of adenosine deaminase (ADA) when using adenosine or 2′-deoxyadenosine as a substrate. In solution, THGP indeed formed complexes with nucleotides or nucleosides through their cis-diol group. Moreover, the ability of THGP to form complexes with nucleotides was influenced by the number of phosphate groups present on the ribose moiety. Notably, THGP also inhibited the catalysis of adenosine by ADA in a concentration-dependent manner. Thus, interactions between THGP and important biological nucleic acid constituents might be implicated in the physiological effects of Ge-132.     

Fingerprinting trifoliate orange germ plasm accessions with isozymes, RFLPs, and inter-simple sequence repeat markers

 Trifoliate orange [Poncirus trifoliata (L.) Raf.] is frequently used as a parent in citrus rootstock breeding, but the origin and amount of genetic diversity in germ plasm collections are poorly understood. Most accessions are self-compatible, but produce a mixture of sexual and apomictic seedlings. Variation among 48 vegetatively propagated trifoliate orange accessions was assessed at seven isozyme loci, together with the restriction fragment length polymorphisms (RFLPs) detected by 38 probe-enzyme combinations and the inter-simple sequence repeat (ISSR) markers generated by 11 primers. Isozymes and RFLPs detected few polymorphisms among accessions, although genetic analysis has shown that the common phenotype is heterozygous for four isozyme and at least four RFLP loci. ISSR amplification generated multiple banding profiles with an average of 58 fragments/primer/accession. These fragments were repeatable across DNA samples extracted from different trees of the same accession or extracted at different times, and across separate PCR runs. Seventeen unique marker phenotypes were identified. The 48 trifoliate orange accessions were classified into four major groups based on polymorphic ISSR markers. All large-flowered accessions are in group 4, while small-flowered accessions are in group 3. Many ISSR markers segregated in progeny derived by open-pollination (probably mostly selfing) of a common accession, indicating that these ISSR markers are also heterozygous. Accessions having identical genotypes for a large number of heterozygous markers are unlikely to have diverged by recombination. Thus the limited divergence we detected among most accessions most likely originated by mutation. ‘Monoembryonic’ and ‘Simmons’ differed from other accessions only in the loss of specific markers, indicating that they originated as zygotic seedlings of individuals similar to the common genotype. Three accessions recently introduced from China have relatively different fingerprints with 3–14 unique ISSR markers, and probably represent a much more divergent germ plasm that may be a valuable breeding resource. Received: 8 August 1996 / Accepted: 21 March 1997     

Evidence for the presence of ferritin in plant mitochondria.

In this work, evidence for the presence of ferritins in plant mitochondria is supplied. Mitochondria were isolated from etiolated pea stems and Arabidopsis thaliana cell cultures. The proteins were separated by SDS/PAGE. A protein, with an apparent molecular mass of approximately 25-26 kDa (corresponding to that of ferritin), was cross-reacted with an antibody raised against pea seed ferritin. The mitochondrial ferritin from pea stems was also purified by immunoprecipitation. The purified protein was analyzed by MALDI-TOF mass spectrometry and the results of both mass finger print and peptide fragmentation by post source decay assign the polypeptide sequence to the pea ferritin (P < 0.05). The mitochondrial localization of ferritin was also confirmed by immunocytochemistry experiments on isolated mitochondria and cross-sections of pea stem cells. The possible role of ferritin in oxidative stress of plant mitochondria is discussed.     

Minireplicon from pBtoxis of Bacillus thuringiensis subsp. israelensis

A 2.2-kb fragment containing a replicon from pBtoxis, the large plasmid that encodes the insecticidal endotoxins of Bacillus thuringiensis subsp. israelensis, was identified, cloned, and sequenced. This fragment contains cis elements, including iterons, found in replication origins of other large plasmids and suggests that pBtoxis replicates by a type A theta mechanism. Two genes, pBt156 and pBt157, encoding proteins of 54.4 kDa and 11.8 kDa, respectively, were present in an operon within this minireplicon, and each was shown by deletion analysis to be essential for replication. The deduced amino acid sequences of the 54.4-kDa and 11.8-kDa proteins showed no substantial homology with known replication (Rep) proteins. However, the 54.4-kDa protein contained a conserved FtsZ domain, and the 11.8 kDa protein contained a helix-turn-helix motif. As FtsZ proteins have known functions in bacterial cell division and the helix-turn-helix motif is present in Rep proteins, it is likely that these proteins function in plasmid replication and partitioning. The minireplicon had a copy number of two or three per chromosome equivalent in B. thuringiensis subsp. israelensis but did not replicate in B. cereus, B. megaterium, or B. subtilis. A plasmid constructed to synthesize large quantities of the Cry11A and Cyt1A endotoxins demonstrated that this minireplicon can be used to engineer vectors for cry and cyt gene expression.