L. Y. Yatsu

Association of lysosomal activity with aleurone grains in plant seeds

Abstract Cotyledonary tissue of ungerminated cottonseed has been found to contain alcohol dehydrogenase, acid proteinase, and acid phosphatase activities. A large part of the acid phosphatase activity observed in situ was associated with aleurone grains (protein bodies). These subcellular organelles have been isolated in glycerol from homogenized cotyledons by differential centrifugation, and were found to contain 77% of the acid phosphatase activity, 100% of the acid proteinase activity, and 75% of the protein which were recovered from the homogenized tissue. Alcohol dehydrogenase, a “soluble” cytoplasmic marker, was not associated with aleurone grains. Thus aleurone grains are principal sites for at least two types of acid hydrolases.

Association of lipase activity with the spherosomes of Ricinus communis

Abstract The fatty layer obtained after centrifuging a macerate of ungerminated castor beans, Ricinus communis , contains an active lipase. The fatty layer has been examined by a combination of biochemical and electron microscopic techniques before and after periods of active lipolysis with Pb ++ in the reaction mixture. The results confirm the presence of spherosomes, the fat storage organelles of the cells, concentrated in the fatty layer. Lipase activity is localized in the spherosomes derived from endosperm tissue of ungerminated seeds.

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.

Isolation of edestin from aleurone grains of Cannabis sativa.

Abstract Aleurone grains, protein bodies, have been isolated from a homogenate of viable hempseeds by employing a density gradient of carbon tetrachloride and refined cottonseed oil. Electron microscopic monitoring of a cross-section of aleurone grains showed them to be composed of at least three different components: large crystalloid substructures, globoids, and some other minor constituents. All are enveloped by a membrane. The crystalloids were extracted from the isolated aleurone grains by repeated washings with solutions of sodium chloride and sodium dodecyl sulfate. Sedimentation coefficients and electrophoretic analysis of solutions of crystalloids indicated that the crystalloids were comprised of pure edestin, and therefore established edestin as an aleurin. Further monitoring of the crystalloids by electron microscopy suggested that they have a pebbly surface and are composed of repeating polygonal-shaped units even down to the 80 A level.

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.

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.

Extraction of lipids from cottonseed tissue: IV. Use of hexane-acetic acid

Hexane and mixtures of hexane and 2–25% acetic acid (v/v) were used to prepare oil and protein from glanded cottonseed by solvent extraction. As the amount of acetic acid in the solvent increased, the amounts of total lipid, phospholipid, neutral oil, and gossypol in each miscella increased, but the amount of free fatty acids did not change significantly. However, the solubility of protein in 0.02N NaOH decreased as the amount of acetic acid in the solvent used to prepare each meal increased. Other aspects of using acidified hexane are described.

Extraction of lipids from cottonseed tissue: V. Ultrastructural effects of extraction with hexane-acetic acid

Cottonseed tissue was extracted with acidified hexane (hexane containing 2–25% acetic acid, v/v) and then examined with an electron microscope. In all cases, the contents of the oil-rich spherosomes were emptied and cytoplasm remained intact after lipid extraction. However, membranous elements of the cytoplasm appeared diffuse and disorganized. The possible effect of this disorganization of membranes in accounting for the greater amount of lipid extracted by acidified hexane than by hexane is discussed.

Phosphate-inhibition of lipase activity in peanuts.

Competitive inhibition of lipase activity in extracts of germinating peanuts occurred in media containing inorganic phosphate in concentrations above 10 mM. Lipase activity in germinating castor, cotton, and pumkin seeds and in quiescent castor seed was not similarly affected. Applications of this finding to peanut processing are suggested.