T. J. Jacks

Isolation and physicochemical characterization of the half-unit membranes of oilseed lipid bodies

The lipid-free residue of lipid body membranes was isolated from quiescent peanuts and was physicochemically characterized. The preponderant component of the residue was proteinaceous and consisted of at least two polypeptides according to ultracentrifugation, gel filtration, gel electrophoresis and HPLC. The molecular weight of the principal polypeptide was between 10,000 and 16,000 daltons. Only one antigen, immunochemically unique with respect to other peanut components, was detected. Spectral analyses indicated the presence of a protoheme and revealed conformational modes of β-sheet and unordered structure but no α-helix. The amino acid composition was similar to that of an integral membrane polypeptide rather than to those of peripheral membranes or other plant polypeptides. The hydrophobicity, conformation and quantitative content of polypeptides were suitable for the existence of a monolayer at the lipid body-cytoplasm interface. The results indicated that lipid body coatings physicochemically resemble membranes of intracellular organelles and supported the morphological concept that the coatings are half-unit biological membranes. Reutilization of lipid body membranes appeared possible after lipid depletion during seed germination.

SUPEROXIDE, HYDROGEN PEROXIDE, AND THE RESPIRATORY BURST OF FUNGALLY INFECTED PLANT CELLS

The principal route of oxygen utilization in the respiratory burst of fungally infected plants was determined from stoichiometries of the uptake and electronic reduction of oxygen in cotton cells exposed to Aspergillus favus walls. Using 2,2′-azino-di-(3-ethyl-benzothiazoline-6-sulfonic acid) and epinephrine as redox reagents to manipulate oxygen transitions, we found that oxygen consumption doubled when superoxide disproportionation was abolished and was abolished when disproportionation doubled. Of four possible pathways for oxygen consumption, only monovalent reduction of molecular oxygen to superoxide was consistent with this inversely proportional relationship. According to the observed rate of oxygen consumption in this pathway and in the absence of competition to disproportionation of superoxide, infected cells are capable of generating intracellular concentrations of 1 M hydrogen peroxide in 13 min.

Effects of chloroperoxidase and hydrogen peroxide on the viabilities of Aspergillus flavus conidiospores.

The effects of chloroperoxidase [EC 1.1.1.10] and hydrogen peroxide on the viabilities of quiescent and germinating conidiospores of an aflatoxigenic fungus, Aspergillus flavus, were determined. Hydrogen peroxide was found moderately lethal and chloroperoxidase produced a 30-fold increase in the lethality of hydrogen peroxide to germinating conidia, which were 75-fold more susceptible to chloroperoxidase than were quiescent conidia. According to infrared examinations of fungal corpses, mortality occurred by oxidation rather than peroxidative chlorination.

Fungicidal and binding properties of three plant peptides

The fungicidal properties of plant seed peptides from Heuchera sanginea (Hs-AFP1), Raphanus sativus (EA-AFP2), and Impatiens balsamina (Ib-AMP3) were determined for the nongerminated and germinated conidia of Aspergillus flavus and Fusarium moniliforme . These peptides were weakly lethal for germinated but not for nongerminated conidia of A. flavus . Both nongerminated and germinated conidia of F. moniliforme were susceptible to these peptides. Overall, F. moniliforme was more susceptible than A. flavus to the peptides. The peptides bound strongly to chitin, mannan, galactocerebrosides, and sphingomyelin. Binding results varied for ergosterol, cholesterol, and β1,3-glucan.The fungicidal properties of plant seed peptides from Heuchera sanginea (Hs-AFP1), Raphanus sativus (EA-AFP2), and Impatiens balsamina (Ib-AMP3) were determined for the nongerminated and germinated conidia of Aspergillus flavus and Fusarium moniliforme . These peptides were weakly lethal for germinated but not for nongerminated conidia of A. flavus . Both nongerminated and germinated conidia of F. moniliforme were susceptible to these peptides. Overall, F. moniliforme was more susceptible than A. flavus to the peptides. The peptides bound strongly to chitin, mannan, galactocerebrosides, and sphingomyelin. Binding results varied for ergosterol, cholesterol, and β1,3-glucan.

Superoxide radicals in intact tissues and in dimethyl sulphoxide-based extracts

Abstract Steady state contents of free radicals were investigated in intact living tissues and in tissue extracts prepared with dimethyl sulphoxide-tetrabutylammonium perchlorate (DMSO-TBAP). DMSO-TBAP was suitable for extraction because free radical artifacts were not generated and hydrous superoxide was both stable and readily spin-trapped for electron paramagnetic resonance (EPR) spectrometry. Intact leaves and roots contained an unresolved radical and paramagnetic manganese. Superoxide was EPR-silent in whole tissues but was spin-trapped in extracts of roots where its steady-state concentration in vivo was approximately 10 −5 M. Ascorbyl radicals were generated upon alkalization of whole tissues and by comminution of tissues in DMSO-TBAP. Aprotic sites for the storage of superoxide in vivo are considered and a role for storage is discussed in regards to cytochemical defence mechanisms of plant protection.

Extraction of lipids from cotronseed tissue: I. Comparison of hexane-acetone-water, its nonqueous components and chloroform-methanol

Flaked cottonseed was extracted with chloroform-methanol-water, chloroform-methanol, hexane-acetone-water, hexane-acetone, hexane and acetone. Amounts of total material in the miscellae were greatest with chloroform-methanol-water and decreased to acetone in the order given above. The first three solvents extracted 6% more neutral oil and over 100% more lipophilic phosphorus than the latter three solvents. All solvents showed similar rates of extraction, each removed over 70% of extractables with the first of four passes.

Extraction of lipids from cottonseed tissue: II. Ultrastructural effects of lipid extraction

Cottonseed tissue was extracted with chloroform-methanol-water, hexane-acetone-water, cyloroform-methanol, hexane-acetone, hexane and acetone, and then examine with an electron microscope. In all cases, contents of oil-rich spherosomes were emptied and cell walls remained intact after lipid extraction. In addition, the two water-containing solvents obtained disruption of intracellular structures. Severalford greater amounts of water-soluble phophorus compounds were extracted with the water-containing solvents than with their nonaqueous counterparts.

Aqueous solvents for extracting glanded cottonseed protein without gland rupture

The presence of pigment glands has thwarted attempts to extract edible cottonseed protein aqueously from glanded seeds or gland-rich meals, probably because of the widely held belief that glands rupture on contact with aqueous media. We found several aqueous salt solutions in which glands did not rupture. Glands remained intact in saturated (2m) sodium sulfate, but not in saturated 2m or 4m solutions of sulfates, chlorides, and nitrates of other Group IA elements as well as sodium chloride and sodium nitrate. Glands also remained intact in saturated solutions of sulfates of aluminum, ammonium, cadmium, copper, magnesium, nickel, and zinc, and chlorides of calcium, iron, and magnesium. Some of these solutions were diluted to <50% saturation before glands started rupturing. Cottonseed protein in the liquid cyclone underflow fraction (gland-rich fraction) was soluble in sodium sulfate and magnesium sulfate, but not in calcium chloride or sodium phosphate. Its solubility in sodium sulfate was investigated further with the following results: Alkalinity of sodium sulfate solution had no effect on solubility; ratio of solid to solvent had no effect in the range of 1:3.5–1:60 (wt:vol); 80% saturated sodium sulfate was optimal for solubility without gland rupture; the period of contact of meal and solvent had no effect on protein solubility in the range of a few minutes to 2 hr. These results indicate that the extraction of cottonseed protein with aqueous solvents in the presence of pigment glands appears technically feasible.

Extraction of lipids from cottonseed tissue: VI. Ultrastructural morphology of isolated pigment glands

Cottonseed pigment glands, isolated from the underflow fraction of the liquid cyclone process, were examined with an electron microscope. The glands were circumscribed by a layer of tangentially flattened cells. Subjacent to the flattened cells were partially lysed cells containing recognizable remnants of cell walls. Within the lumens or matrices of the glands were myriads of pigment spherules measuring 0.1–1.5 µ in diameter. These spherules remained within the glands, even though gland walls were ruptured mechanically. Since aggregates of pigment spherules devoid of gland walls were observed in the gland-rich fraction, it was concluded that maintenance of intact pigment glands was not a requisite for successful separation of gossypol from other cottonseed components with the liquid cyclone process. Avoiding dispersion of aggregated spherules from the glandular matrix was probably just as important as maintaining intact gland walls during mechanical separation of gossypol from other cottonseed components.

Evaluation of kojic acid for determining heme and nonheme haloperoxidase activities spectrofluorometrically

Abstract Heme‐containing haloperoxidases (heme haloperoxidases) catalyze peroxide‐dependent oxidation of halide ions to hypohalite ions. Nonheme haloperoxidases catalyze peroxidation of alkyl acids to peroxyacids that subsequently oxidize halide ions to hypohalite ions. Hypohalite ions react with kojic acid spontaneously with concomitant production of fluorescence. This reaction was examined for suitability in assaying heme and nonheme haloperoxidase activities spectrofluorometrically. Heme haloperoxidases were assayed directly from hypohalite generation whereas nonheme enzymes were assayed by coupling peroxyacid formation to bromide ion oxidation. Peroxidation of kojic acid by chloroperoxidase and myeloperoxidase did not require halide ions and was not increased upon their addition, demonstrating that haloperoxidase activity was not distinguished from the classical peroxidase‐like activity of these enzymes. Addition of bromide ions to specimens of nonheme haloperoxidase, however, resulted in increased rates of fluorescence generation, indicative of the presence of distinct peroxidase and haloperoxidase enzymes. The heme poison sodium azide inhibited chloroperoxidase, myeloperoxidase, and horseradish peroxidase activities totally but inhibited only a portion of nonheme haloperoxidase activity, verifying that the last specimen was contaminated with heme‐containing peroxidase. Results show the utility of kojic acid for fluorometrically measuring haloperoxidase activity, particularly that of the nonheme enzyme.