Identification of the functional site in the mosquito larvicidal binary toxin of Bacillus sphaericus 1593M by site-directed mutagenesis

To study the mode of action of the binary toxin (51- and 42-kDa) of Bacillus sphaericus, amino acid residues were substituted at selected sites of the N- and C-terminal regions of both peptides. Bioassay results of the mutant binary toxins tested against mosquito larvae, Culex quinquefasciatus, revealed that most of the substitutions made on both peptides led to either decrease or total loss of the activity. Furthermore, receptor binding studies carried out for some of the mutants of the 42-kDa peptide showed mutations in N- and C-terminal regions of the 42-kDa peptide did not affect the binding of the binary toxin to brush border membrane vesicles of mosquito larvae. One of the mutants having a single amino acid substitution at the C-terminal region ((312)R) of the 42-kDa peptide completely abolished the biological activity, implicating the role of this residue in membrane pore formation. These results indicate the importance of the C-terminal region of the 42-kDa of binary toxin, in general, and particularly the residue (312)R for biological activity against mosquito larvae.     

Aminopeptidase-A. I. CDNA cloning and expression and localization in rat tissues

Aminopeptidase-A (APA) is an ectoenzyme that selectively hydrolyzes acidic residues from the amino terminus of oligopeptides, including biologically active [Asp(1)]ANG II and [Asp(1)]CCK-8. We sought to characterize rat APA by cDNA cloning and expression and to determine its tissue distribution by in situ hybridization and immunohistochemistry. Sequence analysis of overlapping cDNA clones isolated from rat kidney cDNA libraries indicated that the full-length cDNA encoded a 945-amino acid protein with a predicted molecular mass of 108 kDa; the size was confirmed by in vitro translation of a full-length cDNA construct. Transient transfection of the full-length cDNA construct in mammalian cells yielded a protein approximately 140 kDa in size, a size that agrees with the immunoblots of APA from rat tissue and is consistent with APA being known as a glycosylated protein. Tissue APA activity and mRNA expression were highest in the kidney and ileum. Localization of APA by in situ hybridization and immunohistochemistry indicated that, with the exception of the kidney and ileum, where APA was localized to the luminal brush border of proximal tubules and enterocytes, respectively, APA was associated with either capillaries or the lining of sinusoids. Areas known to be physiological targets for ANG II, including glomeruli, the zona glomerulosa, and anterior pituitary, had high levels of APA. The localization pattern suggests that APA may subserve multiple functions, i.e., a generalized role in peptide scavenging and perhaps a more specific role in metabolism of circulating or locally produced ANG II or CCK-8.     

Release of the neocarzinostatin chromophore from the holoprotein does not require major conformational change of the tertiary and secondary structures induced by trifluoroethanol

Neocarzinostatin is a potent enediyne antitumor antibiotic complex in which a chromophore is noncovalently bound to a carrier protein. The protein regulates availability of the drug by proper release of the biologically active chromophore. To understand the physiological mechanism of the drug delivery system, we have examined the trifluoroethanol (TFE)-induced conformational changes of the protein with special emphasis on their relation to the release of the chromophore from holoneocarzinostatin. The effect of the alpha helix-inducing agent, TFE, on all the beta-sheet neocarzinostatin proteins was studied by circular dichroism, fluorescence, and (1)H NMR studies. By using binding of anilinonaphthalene sulfonic acid as a probe, we observed that the protein exists in a stable, partially structured intermediate state around 45-50% TFE, which is consistent with the results from tryptophan fluorescence and circular dichroism studies. The native state is stable until 20% TFE and is half-converted into the intermediate state at 30% TFE, which starts to collapse beyond 50%. High pressure liquid chromatographic analysis of the release of the chromophore caused by TFE treatment at 0 degrees C suggests that the release process, which occurs below 20% TFE, does not result from an observable conformational change in the protein. Kinetic measurements of the release of chromophore at 25 degrees C reveal that TFE does stimulate the rate of release, which increases sharply at 15% and reaches a maximum at 20% TFE, although no major secondary or tertiary structural change of the carrier protein is observed under these same conditions. Our data suggest that chromophore release results from a fluctuation of the protein structure that is stimulated by TFE. Complete release of the chromophore occurs at TFE concentrations where no overall observable unfolding of the apoprotein is seen. Thus, the results suggest that denaturation of the protein by TFE is not a necessary step for release of the tightly bound chromophore.     

Elucidation of the solution structure of cardiotoxin analogue V from the Taiwan cobra (Naja naja atra)--identification of structural features important for the lethal action of snake venom cardiotoxins

The aim of the present study is to understand the structural features responsible for the lethal activity of snake venom cardiotoxins. Comparison of the lethal potency of the five cardiotoxin isoforms isolated from the venom of Taiwan cobra (Naja naja atra) reveals that the lethal potency of CTX I and CTX V are about twice of that exhibited by CTX II, CTX III, and CTX IV. In the present study, the solution structure of CTX V has been determined at high resolution using multidimensional proton NMR spectroscopy and dynamical simulated annealing techniques. Comparison of the high resolution solution structures of CTX V with that of CTX IV reveals that the secondary structural elements in both the toxin isoforms consist of a triple and double-stranded antiparallel beta-sheet domains. Critical examination of the three-dimensional structure of CTX V shows that the residues at the tip of Loop III form a distinct "finger-shaped" projection comprising of nonpolar residues. The occurrence of the nonpolar "finger-shaped" projection leads to the formation of a prominent cleft between the residues located at the tip of Loops II and III. Interestingly, the occurrence of a backbone hydrogen bonding (Val27CO to Leu48NH) in CTX IV is found to distort the "finger-shaped" projection and consequently diminish the cleft formation at the tip of Loops II and III. Comparison of the solution structures and lethal potencies of other cardiotoxin isoforms isolated from the Taiwan cobra (Naja naja atra) venom shows that a strong correlation exists between the lethal potency and occurrence of the nonpolar "finger-shaped" projection at the tip of Loop III. Critical analysis of the structures of the various CTX isoforms from the Taiwan cobra suggest that the degree of exposure of the cationic charge (to the solvent) contributed by the invariant lysine residue at position 44 on the convex side of the CTX molecules could be another crucial factor governing their lethal potency.     

Functional Complementation of Nontoxic Mutant Binary Toxins of Bacillus sphaericus 1593M Generated by Site-Directed Mutagenesis

Alanine residues were substituted by site-directed mutagenesis at selected sites of the N- and C-terminal regions of the binary toxin (51- and 42-kDa peptides) of B. sphaericus 1593M, and the mutant toxins were cloned and expressed in Escherichia coli. Bioassays with mosquito larvae, using binary toxins derived from individual mutants, showed that the substitution of alanine at some sites in both the 51-kDa and the 42-kDa peptides resulted in a total loss of activity. Surprisingly, after mixing two nontoxic derivatives of the same peptide, i.e., one mutated at the N-terminal end and the other mutated at the C-terminal end of either the 51-kDa or the 42-kDa peptide, the toxicity was restored. This result indicates that the altered binary toxins can functionally complement each other by forming oligomers.     

Transgenic carrot plants accumulating ketocarotenoids show tolerance to UV and oxidative stresses

Ketocarotenoids are strong antioxidant compounds which accumulate in salmon, shrimp, crustaceans and algae, but are rarely found naturally in higher plants. In this study, we engineered constitutive expression of an algal beta-carotene ketolase gene (bkt) in carrot plants to produce a number of ketocarotenoids from beta-carotene. These included astaxanthin, adonirubin, canthaxanthin, echinenone, adonixanthin and beta-cryptoxanthin. Leaves accumulated up to 56mug/g total ketocarotenoids and contained higher beta-carotene levels but lower levels of alpha-carotene and lutein. The photosynthetic capacity of transgenic plants was not significantly altered by these changes. However, when high-expressing transgenic plants were exposed to UV-B irradiation, they grew significantly better than the wild-type controls. Similarly, leaf tissues exposed to various oxidative stresses including treatment with H(2)O(2) and methyl viologen showed less injury and retained higher levels of chlorophyll a+b. Total carotenoid extracts from transgenic leaves had higher antioxidant and free-radical scavenging activity in vitro compared to control leaves. Transgenic tissues also accumulated lower amounts of H(2)O(2) following exposure to oxidative stresses, suggesting that free radical and reactive oxygen species were quenched by the ketocarotenoids.