Characterization of a new sulfhydryl group reagent: 6,6′-Diselenobis-(3-Nitrobenzoic acid), a selenium analog of Ellman's reagent

A new reagent, 6,6′-diselenobis-(3-nitrobenzoic acid) (DSNB) has been synthesized and is shown to be useful for quantitative estimation of sulfhydryl groups in proteins. This reagent, which is a selenium analog of Ellman's reagent, reacts specifically and quantitatively with thiol groups of proteins to yield a selenenyl sulfide and the dianion of 3-nitro-6-selenobenzoic acid. The molar absorption coefficient of the chromophoric dianion is 10,000 at 432 nm in dilute aqueous solutions. The titration can best be performed at pH 8.20 where >98% of 3-nitro-6-selenobenzoic acid is in the form of the intensely colored dianion. Sulfhydryl content determinations by this reagent of reduced and denatured ribonuclease, reduced and denatured lysozyme, native papain, and native and denatured thymidylate synthetase are compared with those from corresponding 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) titrations. The reagent was found to inactivate thymidylate synthetase, an enzyme with essential sulfhydryl groups. Unlike DTNB which undergoes alkaline decomposition of pH values greater than 9, DSNB was found to be stable to hydrolysis, even in 0.05 m NaOH.     

In situ isolation of allelochemicals released from soft corals (Coelenterata : Octocorallia): A totally submersible sampling apparatus

A submersible apparatus has been developed which permits in situ sampling of allelochemical substances released from sessile marine organisms. Concentration of the allomones from sea water is achieved by adsorption on SEP-PAK C-18, reverse-phase cartridges. The procedure is performed under water and causes minimal disturbance to the organism while in its natural habitat. Design and application of this apparatus are detailed.

The isolation and identification of toxins released by two species of alcyonarian corals (order Alcyonacea) are described. This represents the first direct in situ isolation of water-borne allelochemicals released from a marine organism.     

Acylhomoserine lactone synthase activity of the Vibrio fischeri AinS protein.

Acylhomoserine lactones, which serve as quorum-sensing signals in gram-negative bacteria, are produced by members of the LuxI family of synthases. LuxI is a Vibrio fischeri enzyme that catalyzes the synthesis of N-(3-oxohexanoyl)-L-homoserine lactone from an acyl-acyl carrier protein and S-adenosylmethionine. Another V. fischeri gene, ainS, directs the synthesis of N-octanoylhomoserine lactone. The AinS protein shows no significant sequence similarity with LuxI family members, but it does show sequence similarity with the Vibrio harveyi LuxM protein. The luxM gene is required for the synthesis of N-(3-hydroxybutyryl)-L-homoserine lactone. To gain insights about whether AinS and LuxM represent a second family of acylhomoserine lactone synthases, we have purified AinS as a maltose-binding protein (MBP) fusion protein. The purified MBP-AinS fusion protein catalyzed the synthesis of N-octanoylhomoserine lactone from S-adenosylmethionine and either octanoyl-acyl carrier protein or, to a lesser extent, octanoyl coenzyme A. With the exception that octanoyl coenzyme A served as an acyl substrate for the MBP-AinS fusion protein, the substrates for and reaction kinetics of the MBP-AinS fusion protein were similar to those of the several LuxI family members previously studied. We conclude that AinS is an acylhomoserine lactone synthase and that it represents a second family of such enzymes.     

An Inexpensive Apparatus Facilitating the Storage of Oxygen Sensitive Compounds

The design, construction, and use of an inexpensive apparatus which facilitates the storage of oxygen sensitive compounds in serum bottles is described.     

Nutrient salts promote light-induced degradation of indole-3-acetic Acid in tissue culture media

The disappearance of indole-3-acetic acid (IAA) from cell-free liquid culture medium was followed in response to nutrient salts found in Murashige-Skoog salt base, light, and pH range of 4 to 7. The loss of IAA was accelerated by light or Murashige-Skoog salts. However, the combination of both light and Murashige-Skoog salts acted synergistically to catalyze the destruction of over 80% of the original IAA within 7 days of continuous incubation. Under these same conditions, the loss of IAA was decreased to approximately 50% by adjusting the initial pH of the medium to 7. Iron was identified as the single major contributor to light-catalyzed destruction of IAA. Removal of nitrates, which represented 87% of the molar salt composition, also reduced the light-catalyzed loss of IAA. Treatments that protected IAA from degradation, such as darkness or removal of iron from the medium, suppressed the growth of muskmelon (Cucumis melo. Naud., var. reticulatus) callus tissue cultured for 30 days. Treatments in the light that rapidly degraded IAA resulted in maximum growth. Consequently, the brief exposure to IAA prior to degradation was apparently sufficient to initiate physiological changes required for growth. Possible approaches to the preservation of IAA during incubation are discussed.     

Isolation of the covalent binary complex of 5-fluorodeoxyuridylate and thymidylate synthetase by trichloroacetic acid precipitation

Strong chemical evidence for the existence of a covalent binary complex between 5-fluorodeoxyuridylate and thymidylate synthetase was provided by the isolation of the complex by trichloroacetic acid precipitation. This result together with that of a control experiment with N-ethymaleimide inactivated thymidylate synthetase demonstrated that only nucleotide covalently bound to the protein survived repeated washings of the precipitate. Under the conditions used, a maximum binding stoichiometry of about 0.9 was obtained for the covalent binary complex, Kd = 1.1 X 10(-5) M. Also, a binding ratio of 1.7 was obtained for the methylenetetrahydrofolate-5-fluorodeoxyuridylate-thymidylate synthetase ternary complex.     

Evidence for the existence of covalent nucleotide-thymidylate synthase complexes, identification of site of attachment, and enhancement by folates

The formation of covalent binary complexes of thymidylate synthase and its nucleotide substrate dUMP, product dTMP, and inhibitor, 5-fluorodeoxyuridylate (FdUMP) was investigated using the trichloroacetic acid precipitation method. It was observed that, in addition to FdUMP, both dUMP and dTMP were capable of covalent interactions with the enzyme in the absence of added folates. The presence of folate, dihydrofolate, or tetrahydrofolate (H4folate) was found to produce substantial enhancements in the covalent binding of both FdUMP and dUMP to the enzyme with H4folate being the most effective agent. Further, covalent binary complexes of the enzyme with the three radiolabeled nucleotides were isolated by trichloroacetic acid precipitation and subjected to CNBr cleavage. The active-site CNBr peptide was isolated by reverse phase high performance liquid chromatography, and the first five N-terminal amino acid residues were sequenced by the dansyl-Edman procedure. Each active site peptide obtained from the covalent binary complexes as well as that from the covalent inhibitory ternary complex formed from enzyme, FdUMP, and 5,10-methylene-H4folate exhibited an identical sequence of Ala-Leu-Pro-Pro-(X)-, and the 5th amino acid was found to be associated with radiolabeled nucleotide ligand. Dansyl-Edman sequence analysis of the active site CNBr peptide, derived from enzyme which had been treated with iodoacetic acid, gave a sequence of Ala-Leu-Pro-Pro-CmCys (where CmCys is carboxymethylcysteine), thus confirming the fact that the fifth residue from the N terminus is Cys-198. In all the cases, the active site Cys-198 residue was found to be covalently linked to the nucleotides. These results provide unequivocal proof that the covalent binary complexes of enzyme with dUMP and dTMP predicted in the catalytic reaction mechanism actually exist.     

Kinetic studies on drug-resistant variants of Escherichia coli thymidylate synthase: functional effects of amino acid substitutions at residue 4.

A naturally occurring mutant of human thymidylate synthase (hTS) that contains a Tyr to His mutation at residue 33 was found to confer 4-fold resistance to 5-fluoro-2'-deoxyuridine (FdUrd), a prodrug of 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP). The crystal structure of hTS implicated this Tyr residue in a drug resistance mechanistic role that may include both substrate binding and catalysis (Schiffer et al., Biochemistry, 34, 16279-16287, 1995). Because of the existence of a defined kinetic scheme and the development of a bacterial expression vector for the overproduction of Escherichia coli TS (ecTS), we chose to initially study the corresponding residue in the bacterial enzyme, Tyr 4 of ecTS. Nine mutant ecTS enzymes that differed in sequence at position 4 were generated. Mutants with a charged or polar side chain (Ser, Cys, Asp, and Arg) and Gly precipitated in the cell paste, resulting in no catalytic activity in cell-free extracts. Although most of the His 4 mutant precipitated, sufficient amounts remained in the cell-free extract to permit isolation to near homogeneity. Wild-type ecTS and mutants with a hydrophobic side chain (Phe, Ile, and Val) were expressed at nearly 30% of the total cellular protein. The k(cat) values for the isolatable mutants were 2- to 10-fold lower than that of the wild-type enzyme, while the K(m) values for 2'-deoxyuridylate (dUMP) and 5,10-methylenetetrahydrofolate (CH(2)H(4)folate) were similar for all the mutants. Dissociation constants for binary complex formation determined by stopped-flow spectroscopy were similar for the wild-type and mutant enzymes for both dUMP and 2'-deoxythymidylate, indicating that this mutation does not significantly alter the binding of the natural nucleotide ligands. However, each mutant enzyme had three- to 5-fold lower affinity for FdUMP in the binary complex compared with the wild-type enzyme, and only His 4 showed a lower affinity for FdUMP in the ternary complex. Analysis of k(burst) showed that the initial binding of CH(2)H(4)folate is weaker for each mutant compared to the wild-type enzyme and that lower k(cat) values were due to compromised rates that govern the chemical transformation of bound substrates to bound products.     

Reducing production of fumonisin mycotoxins in <Emphasis Type="Italic">Fusarium verticillioides</Emphasis> by RNA interference

The fungus Fusarium verticillioides is a maize pathogen that can produce fumonisin mycotoxins in ears under certain environmental conditions. Because fumonisins pose health risks to humans and livestock, control strategies with minimal risk to the environment are needed to reduce fumonisin contamination. Host-induced gene silencing is a promising technique in which double-stranded RNA expressed in the plant host is absorbed by an invading fungus and down-regulates genes critical for pathogenicity or mycotoxin production in the fungus. A key preliminary step of this technique is identification of DNA segments within the targeted fungal gene that can effectively silence the gene. Here, we used segments of the fumonisin biosynthetic gene FUM1 to generate double-stranded RNA in F. verticillioides. Several of the resulting transformants exhibited reduced FUM1 gene expression and fumonisin production (24- to 3675-fold reduction in fumonisin FB₁). Similar reductions in fumonisin production resulted from double-stranded RNA constructs with segments of FUM8, another fumonisin biosynthetic gene (3.5- to 2240-fold reduction in fumonisin FB₁). FUM1 or FUM8 silencing constructs were transformed into three isolates of F. verticillioides. Whole genome sequence analysis of seven transformants revealed that reductions in fumonisin production were not due to mutation of the fumonisin biosynthetic gene cluster and revealed a complex pattern of plasmid integration. These results suggest the cloned FUM1 or FUM8 gene segments could be expressed in maize for host-induced gene silencing of fumonisin production.