Host-Pathogen Interactions : XXIII. The Mechanism of the Antibacterial Action of Glycinol, a Pterocarpan Phytoalexin Synthesized by Soybeans.

The biochemical basis for the ability of the pterocarpan phytoalexin glycinol (3,6a,9-trihydroxypterocarpan) to inhibit the growth of bacteria was examined. Glycinol at bacteriostatic concentrations (e.g. 50 micrograms per milliliter) inhibits the ability of Erwinia carotovora to incorporate [(3)H]leucine, [(3)H]thymidine, or [(3)H]uridine into biopolymers. Exposure of Escherichia coli membrane vesicles to glycinol at 20 micrograms per milliliter results in inhibition of respiration-linked transport of [(14)C]lactose and [(14)C]glycine into the vesicles when either d-lactate or succinate is supplied as the energy source. The ability of E. coli membrane vesicles to transport [(14)C]alpha-methyl glucoside, a vectorial phosphorylation-mediated process, is also inhibited by glycinol at 20 micrograms per milliliter. Furthermore, exposure of membrane vesicles to glycinol (50 micrograms per milliliter) at 20 degrees C results in the leakage of accumulated [(14)C]alpha-methyl glucoside-6-phosphate. The effects of the phytoalexins glyceollin, capsidiol, and coumestrol, and daidzein, a compound structurally related to glycinol but without antibiotic activity, upon the E. coli membrane vesicle respiration-linked transport of [(14)C]glycine and of [(14)C]alpha-methyl glucoside was also examined. Glyceollin and coumestrol (50 micrograms per milliliter), but not daidzein, inhibit both membrane-associated transport processes. These data imply that the antimicrobial activity of glycinol, glyceollin, and coumestrol are due to a general interaction with the bacterial membrane. Capsidiol (50 micrograms per milliliter) inhibits d-lactate-dependent transport of [(14)C]glycine but not vectorial phosphorylation-mediated transport of [(14)C]alpha-methyl glucoside. Thus, capsidiol's mechanism of antimicrobial action seems to differ from that of the other phytoalexins examined.

Host-Pathogen Interactions : XVIII. ISOLATION AND BIOLOGICAL ACTIVITY OF GLYCINOL, A PTEROCARPAN PHYTOALEXIN SYNTHESIZED BY SOYBEANS.

A previously unrecognized phytoalexin has been isolated from soybean cotyledons that had been infected with bacteria or exposed to ultraviolet light. The phytoalexin has been purified to homogeneity by silica gel flash chromatography and high pressure liquid chromatography. It has been structurally characterized by its ultraviolet, circular dichroism and nuclear magnetic resonance spectra, polarimetry, and its mass spectrometric fragmentation pattern. The phytoalexin, (6aS,11aS)-3,6a,9-trihydroxypterocarpan, is a compound that had previously been detected in CuCl(2)-treated soybeans and is structurally related to the previously identified soybean phytoalexins glycerollins I to IV. It is proposed that the trivial name glycinol be used for this phytoalexin. Glycinol is a broad spectrum antibiotic capable of prolonging the lag phase of growth of all six bacteria examined, namely Erwinia carotovora, Pseudomonas glycinea (races 1 and 3), Escherichia coli, Xanthomonas phaseoli, and Bacillus subtilis. Glycinol also inhibits the growth of the fungi Phytophthora megasperma f. sp. glycinea (race 1), Saccharomyces cerevisiae, and Cladosporium cucumerinum. Glycinol is a static agent against the six bacterial species listed above and against S. cerevisiae, and appears to be static against the other fungi examined. As with other phytoalexins, there is no correlation between the pathogenicity of a microorganism and its sensitivity to glycinol.

Study of protein topography with flash photolytically generated nonspecific surface-labeling reagents: surface labeling of ribonuclease A.

A method for nonspecifically labeling essentially all exposed residues of a protein is described. A reactive aryl nitrene is generated from N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate (NAP-Taurine), within 500 mus by flash photolysis in the presence of protein. The reactive nitrene is inserted in about 2 ms into those carbon-hydrogen bonds of the protein that are exposed to the solvent. The method is applied here to ribonuclease A to demonstrate the different degree of labeling of the native and denatured protein. On the basis of amino acid analysis, it appears that residues of the native protein that are buried in the interior of the molecule (as judged from the x-ray structure) do not react with the nitrene. However, when these residues (even nonreactive ones such as valine and proline) are exposed by denaturation of the protein, they do react with the nitrene. It is shown that native ribonuclease A retains 90% of its enzymatic activity when flashed in the absence of NAP-Taurine. This small loss in activity arises from the disruption of a limited portion of the native enzyme structure, as judged by circular dichroism, ultraviolet, and Raman spectra. The site of this limited disruption may be a portion of the enzyme surface near the Cys-26-Cys-84 disulfide bond. The utility of this surface labeling technique for studying the pathways of protein folding or unfolding is discussed.

Immobilized carboxypeptidase A as a probe for studying the thermally induced unfolding of bovine pancreatic ribonuclease.

A method for the preparation of Sephadex-immobilized carboxypeptidase A is presented. This form of the enzyme has the same specific activity as the soluble enzyme at room temperature, but retains its activity at higher temperatures (60-70 degrees). This preparation of immobilized carboxypeptidase A was used, as a proteolytic probe, to investigate the thermally induced unfolding of the C-terminus of ribonuclease A. This technique indicates that the C-terminal residues of ribonuclease A do not unfold until the high-temperature region of the thermal transition (as determined by ultraviolet difference spectrophotometry and optical rotation).