Involvement of glyoxysomal lipase in the hydrolysis of storage triacylglycerols in the cotyledons of soybean seedlings

The total cotyledon extract of soybean (Glycine max [L.] Merr. var. Coker 136) seedlings underwent lipolysis as measured by the release of fatty acids. The highest lipolytic activity occurred at pH 9. This lipolytic activity was absent in the dry seeds and increased after germination concomitant with the decrease in total lipids. Using spherosomes (lipid bodies) isolated from the cotyledons during the peak stage of lipolysis (5-7 days) as substrates, about 40% of the lipase activity was found in the glyoxysomes after organelle breakage had been accounted for; the remaining activity was distributed among other subcellular fractions but none was found in the spherosomal fraction. The glyoxysomal lipase had maximal activity at pH 9, and catalyzed the hydrolysis of tri-, di-, and monoacylglycerols of linoleic acid, the most abundant fatty acid in soybean. The spherosomes contained a neutral lipase that could hydrolyze monolinolein and N-methylindoxylmyristate, but not trilinolein. This spherosomal lipase activity dropped off rapidly during early seedling growth, preceding lipolysis. Spherosomes isolated from either dry or germinated seeds did not possess lipolytic activity, and spherosomes from germinated seeds but not from dry seeds could serve as substrates for the glyoxysomal lipase. It is concluded that the glyoxysomal lipase is the enzyme catalyzing the initial hydrolysis of storage triacylglycerols.     

Composition and Structure of Protein Bodies and Spherosomes Isolated from Ungerminated Seeds of Sorghum bicolor (Linn.) Moench

Protein bodies and spherosomes have been isolated from mature seeds of Sorghum bicolor (Linn.) Moench by a procedure which successfully disrupts the protein starch complex in the grain. Protein bodies from whole grain are 68% protein and have a distinct border and a monolithic appearance. Those from embryo are 95% protein and have diffuse borders, vacuoles, and appear very granular. Aleurone tissue protein bodies are 46% protein with a structure similar to those from embryo, but possibly are composed of a protein carbohydrate mixture. Spherosomes from all sources are quite similar in composition and structure. They have an average composition of 27% protein, 12% phosphorus, and 8.6% metals. Microscopically, they appear as small vesicles bounded by a wall which is probably composed of protein and the potassiummagnesium salt of phytic acid.     

Acid Hydrolases and Autolytic Properties of Protein Bodies and Spherosomes Isolated from Ungerminated Seeds of Sorghum bicolor (Linn.) Moench

Protein bodies and spherosomes isolated from mature seeds of Sorghum bicolor (Linn.) Moench have measurable activity of acid protease, α-glucosidase, β-glucosidase, β-galactosidase, phytase, acid pyrophosphatase, p-nitrophenyl phosphatase, and RNase. Protein bodies have largely insoluble activities, and produce soluble protein and soluble amino nitrogen during autolysis. They have the dual function of protein storage and protein catabolism. Spherosomes have considerable amounts of soluble enzymes and autolytically produce soluble amino nitrogen and inorganic phosphate but release little soluble protein. Spherosomes are similar to animal lysosomes but have an additional storage function for protein, phosphorus, and metals. Mature sorghum seed contains the necessary enzymes and substrates to generate two basic metabolites, amino acids and inorganic phosphate.     

STAINING AND ACID PHOSPHATASE REACTIONS OF SPHEROSOMES IN PLANT TISSUE CULTURE CELLS

Spherosomes in plant tissue culture cells from normal sunflower stems and sunflower crown gall tumors reacted similarly to several nonfluorescent and fluorescent lipid dyes. Sudan IV and black B were good selective spherosome stains. The lipid fluorochromes, Nile blue and 3, 4-benzpyrene were excellent selective spherosome stains and visualized the smallest particles more readily than did Sudan IV. Spherosomes could not be seen in tissues stained with Sudan IV or 3,4-benzpyrene after ether-alcohol extraction. Acid phosphatase was detected on the spherosomes in both normal and tumor tissues using the lead sulfide precipitation and the post-incubation coupling procedures.     

Lipid inclusions in the vacuoles of Saccharomyces cerevisiae

Summary Spherical or elongated phospholipid inclusions have been observed in vacuoles of Saccharomyces cerevisiae. The periodicity of these lipid inclusions is 56 Å and the material of which they are composed appears to be derived from spherosomes. The spherosomes arise in the cytoplasm, penetrate the tonoplast, and discharge their contents into the vacuole. It is postulated that the phospholipid matrix of the spherosome then aggregates to produce the myelin-like inclusions. Tween 80 and ergosterol together in the growth medium do not prevent the formation of the lipid inclusions.     

Spherosome membranes: half unit-membranes

Spherosomes are bounded by unusual single-line “membranes” which measure 2 to 3.5 nanometers in width, contrasted to the well known tripartite unit-membranes which measure 6 to 8.5 nanometers in over-all thickness. Juxtaposed externally (from the side addressing the hyaloplasm), two spherosomal membranes adjoin to form a thicker single line, but apposed internally (the sides that contact stored lipid) two single-line membranes touch to form a tripartite structure resembling a unit-membrane. Morphologically, we interpret the single-line membranes of spherosomes as half unit-membranes whose polar surfaces face the hyaloplasm and whose lipoidal nonpolar surfaces contact internal storage lipid.     

Membranous appendices of spherosomes (oleosomes)

Spherosomes (oleosomes) of cotyledons of rape (Brassica napus L.), sunflower (Helianthus annuus L.), and watermelon (Citrullus vulgaris, Schrad.) seedlings are delimited by a half unit membrane that appears to be continuous with each of the osmiophilic layers of a tripartite unit membrane forming a handlelike appendix of the spherosomes. Prior to any noticeable utilization of the spherosomal storage fat, ribosomes were found to be attached to these handles. At later stages appendices of the spherosomes are smooth, showing a diameter of about 22 nm that greatly exceeds the thickness of any other unit membrane profiles identical in structure and diameter osomes appears to be continuous with the thick lipid layer of the handles. In intermediate stages of fat depletion the spherosomal bodies become invaginated with cytoplasmic material. Finally vesicles with cytoplasmic contents surrounded by a membrane with a typically thick lipid layer are left in the cells. Membrane profiles indentical in structure and diameter to the spherosomal appendices were also present in electron micrographs of the lipolytic membrane fraction recovered from sucrose density gradients after centrifugation of a microsomal cell fraction. The ultrastructural observations are taken for evidence that the spherosomal appendices represent the lipase-carrying membranes isolated previously (Theimer and Rosnitschek, 1978). A novel hypothesis for development and utilization of fat-storing spherosomes is also proposed.     

Somatic Embryogenesis in Cell Suspension Culture of Saposhnikovia divaricata

Calli of Saposhnikovia divaricata (Turcz.) Schischk. were induced from the roots of test tube seedlings cultured on MS medium containing 0.5 mg/L 2,4-D. Cell suspension was established by shaking the caul in liquid medium of the same components supplemented with 10% coconut milk. After the formation of embryogenic clusters, 2,4-D was omitted promoting the transformation of the embryogenic clusters to somatic embryos. Micro and submicroscopic: structural changes during the single cell to globular embryonic stage were observed. It was noticed that cortical endoplasmic reticulum appeared in the cells at the stage of embryogenic clump formation but was absent in other stages. Perhaps this was related to the metabolic specification leading to embryo formation. Spherosomes were observed of embryogenesis but remarkably increased in number at proglobular embryo stage. Meanwhile, the central electric dense matrix became progressively smaller and paler, while the outer part became enlarged and more transparent. This implied that the spherosomes took part in the storage of proteins or lipidproteins in the early stages of embryogenesis and transformation to lipid dror)s when the proteins were exhausted in the development of embryo, vacuolar protein bodies could be risualized in many cells in the proglobular embryo stage. This together with the existance and changes of spherosomes was similar to that observed in Peucedanum terebmthaceum. Further studies are meritted to approach, whether these are general phenomena in Umbelliferae. This work also revealed that the aggregation of single cells in suspension culture stimulated the initiation of embryogenesis.     

Orientation and conformation of lipids in crystals of transmembrane proteins

Orientational order parameters and individual dihedral torsion angles are evaluated for phospholipid and glycolipid molecules that are resolved in X-ray structures of integral transmembrane proteins in crystals. The order parameters of the lipid chains and glycerol backbones in protein crystals are characterised by a much wider distribution of orientational order than is found in fluid lipid bilayers and reconstituted lipid–protein membranes. This indicates that the lipids that are resolved in crystals of membrane proteins are mostly not representative of the entire lipid–protein interface. Much of the chain configurational disorder of the membrane-bound lipids in crystals arises from C–C bonds in energetically disallowed skew conformations. This suggests configurational heterogeneity of the lipids at a single binding site: eclipsed conformations occur also in the glycerol backbone torsion angles and the C–C torsion angles of the lipid head groups. Conformations of the lipid glycerol backbone in protein crystals are not restricted to the gauche C1–C2 rotamers found invariably in phospholipid bilayer crystals. Lipid head-group conformations in the protein crystals also do not conform solely to the bent-down conformation, with gauche–gauche configuration of the phosphodiester, that is characteristic of phospholipid bilayer membranes. Stereochemical violations in the protein-bound lipids are evidenced by ester carboxyl groups in non-planar configurations, and even in the cis configuration. Some lipids have the incorrect enantiomeric configuration of the glycerol backbone, and many of the branched methyl groups in the phytanyl chains associated with bacteriorhodopsin have the incorrect S configuration.     

Protein-Associated Lipid of Bacillus stearothermophilus

The composition and patterns of metabolism of phospholipids isolated as part of a lipid-depleted membrane fragment (LDM fragment) and associated with the membrane adenosine triphosphatase complex have been compared with those of the bulk membrane phospholipid. The bulk lipid was extracted from washed membranes with sodium cholate. The LDM fragments, which contained a portion of the electron transport system and the membrane adenosine triphosphatase complex, were purified by chromatography with Sepharose 6B. The LDM fragment preparations contained 0.10 +/- 0.02 mumol of lipid phosphorus per mg of protein, compared with 0.54 +/- 0.05 mumol of lipid phosphorus per mg of protein for washed membranes. The phospholipid associated with the LDM fragments consisted of 78 +/- 4% cardiolipin, 7 +/- 1% phosphatidylglycerol, and 15 +/- 3% phosphatidylethanolamine. Changes in the total membrane lipid composition (produced by culture conditions) did not alter the phospholipid composition of the LDM fragments. The adenosine triphosphate complex was separated from the other components of the LDM fragments by suspension of the fragments in 1% Triton X-100 and precipitation with antibody specific for the F(1) component of the adenosine triphosphatase complex. The phospholipid isolated with the adenosine triphosphatase complex consisted of 86% cardiolipin, 8% phosphatidylglycerol, and 6% phosphatidylethanolamine. In pulse-chase experiments with (32)P and [2-(3)H]glycerol, the labeling patterns of the phosphatididylglycerol and phosphatidylethanolamine associated with the LDM fragments were different from those of the bulk membrane phosphatidylglycerol and phosphatidylethanolamine. It was concluded that at least a portion of the phospholipid isolated with the LDM fragments was part of a native lipid-protein complex.     

Penicillium mycelium waste as protein supplement in animals

Dried Penicillium mycelium served as a protein source in animal diet when it was supplemented at 7.5% protein level along with 7.5% protein level from peanut meal. Under these conditions, the food consumption was optimal, and the rat growth response was comparable with 15% casein diet. The role of peanut meal appears to be twofold; it makes the mycelium diet more palatable and it supplies protein. The amino acids, lysine and threonine, which are found to be limiting in peanut meal, are reported to be present in the Penicillium mycelium. This type of formulation affords considerable economic advantage because both the peanut meal and the Penicillium mycelium are by-products and, therefore, are inexpensive sources of protein.     

Apolipoprotein C-III/sphingomyelin recombinants: formation, isolation, and characterization

The association of apolipoprotein C-III (apoC-III) from human very low density lipoprotein with sphingomyelin from egg yolk (EYSM) has been studied at the transition temperature (Tc) of the phospholipid. Upon incubation of aliquots of the apoprotein with increasing amounts of sphingomyelin, the alpha-helical content of the apoprotein increased from 20% in the absence of EYSM to a limiting value of 67% at a protein:lipid molar ratio of 1:200. The tryptophan fluorescence spectrum of the apoprotein exhibited a gradual blue shift from 356 nm in the absence of EYSM to 348 nm when the protein:lipid ratio in the complex had reached 1:50. Gel filtration chromatography of complexes formed by incubating the apoprotein and phospholipid at differing apoC-III:EYSM ratios demonstrated a disintegration of sphingomyelin vesicles into particles of decreasing size with increasing proportion of protein. This effect was confirmed by sedimentation velocity experiments in which the observed sedimentation coefficient of EYSM decreased from 14.0 S (for vesicles) to a limiting value of 7.0 S when the apoprotein:phospholipid ratio reached 1:50 in the complex. Electron micrographs of negatively stained EYSM vesicles showed spherical particles of 380-A diameter. Addition of apoC-III led to the formation of disk-shaped structures whose diameter decreased to a limiting value of 204 +/- 34 A at a protein:lipid ratio of 1:50. In contrast, the disk thickness was relatively constant at 51 +/- 2 A for all isolated complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipid droplet formation during vitellogenesis in the cecropia moth

Lipid droplets isolated by flotation from the ovary of Hyalophora cecropia were shown to constitute about 12 per cent of the dry weight of the egg, and about 60 per cent of extractable egg lipids. A second form of lipid reserve, conjugated lipoproteins in the protein yolk spheres, presumably makes up much of the remaining 40 per cent. The lipid droplets were 82 to 85% triglyceride and 3 to 8% phospholipid. Total egg lipids by contrast were 63 to 70% triglyceride and 24 to 31% phospholipid.When ³H-palmitate was injected into the haemocoel, lipid droplets from vitellogenic follicles were found to be heavily labelled. After 24 hr, over 70 per cent of the total follicular radioactivity was found in the isolated lipid droplets, 90 per cent of that being in the form of triglycerides. Autoradiograms confirmed that the deposition of lipid droplets like that of the protein yolk spheres occurs in the peripherial cytoplasm of the oöcyte, adjacent to the follicular epithelium.     

Does Gibberellic Acid Induce the Transfer of Lipase from Protein Bodies to Lipid Bodies in Barley Aleurone Cells?

We have examined the effect of gibberellic acid (GA₃) on the distribution of the enzyme responsible for mobilizing storage triacylglycerol in aleurone cells of Hordeum vulgare L. cv Himalaya. Using cellular fractionation techniques, we find that, in cells that have not been exposed to hormone, neutral lipase activity is principally associated with a pellet containing the membranes of protein bodies. If the cells are exposed to GA₃ for at least 1 hour, the majority of the lipase activity becomes associated with the lipid body fraction. The nature of the in vivo association between lipid bodies and protein bodies was examined using ultrarapid freezing followed by freeze-fracture electron microscopy. Our analysis indicates that the phospholipid monolayer surrounding the lipid body is directly continuous with the outer leaflet of the bilayer surrounding the protein body. Based on our data, we propose that lipase can be transferred from protein bodies (storage form) to lipid bodies (active form) by lateral diffusion within the plane of the fused phospholipid monolayer, and that the transfer can be controlled by gibberellic acid by an unknown mechanism.     

THE SPHEROSOMES AND THE RESERVE FAT IN PLANT CELLS

The deposition of reserve fat is studied in plants with a high, medium, or low lipid content, and is contrasted with the spherosomes of the same cells. In storage tissue with a high lipid content the reserve triglycerides have the form of comparatively large globules which are quite distinct from the spherosomes. In plants with a medium lipid content the reserve fat appears in the form of granules, globules, or oil-plasm, and as a homogeneous, interstitial deposition between the protein bodies. Plants with a low lipid content contain a very large number of spherosomes and only very few small sudanophil globules. The spherosomes and the reserve fat represent distinctly separate entities.     

Spherosomes of Castor Bean Endosperm: MEMBRANE COMPONENTS, FORMATION, AND DEGRADATION

The membrane components of the castor bean spherosomes were characterized. The storage triacylglycerols of isolated spherosomes were extracted with diethyl ether, and the membrane was isolated by sucrose gradient centrifugation. It had an apparent equilibrium density of 1.12 grams per cubic centimeter, and possessed an antimycin A-insensitive NADH cytochrome c reductase and an acid lipase. Phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol in roughly equal amounts were the major phospholipids. The membrane proteins were resolved into several major and minor protein bands of molecular weights ranging from 10,000 to 70,000 by acrylamide gel electrophoresis, and the protein pattern in the gel was different from those of the endoplasmic reticulum, mitochondrial, and glyoxysomal membranes.     

Lipid composition of a plasma membrane enriched fraction of maize roots

The lipid composition of a plasma membrane enriched fraction isolated from corn (Zea mays) roots was examined. On a wt basis, the lipid: protein ratio was 1.11. Phospholipids comprised 60% of total lipids with the major phospholipids being phosphatidylcholine (62%) and phosphatidylethanolamine (21%). Free sterol was the major neutral lipid. The sterol:phospholipid molar ratio was 0.31. The fatty acid composition of the membrane was predominantly linoleic (60%) and palmitic (30%).     

Phospholipid asymmetry in the isolated sarcoplasmic reticulum membrane

The total phospholipid content and distribution of phospholipid species between the outer and inner monolayers of the isolated sarcoplasmic reticulum membrane was measured by phospholipase A2 activities and neutron diffraction. Phospholipase measurements showed that specific phospholipid species were asymmetric in their distribution between the outer and inner monolayers of the sarcoplasmic reticulum lipid bilayer; phosphatidylcholine (PC) was distributed 48/52 +/- 2% between the outer and inner monolayer of the sarcoplasmic reticulum bilayer, 69% of the phosphatidyl-ethanolamine (PE) resided mainly in the outer monolayer of the bilayer, 85% of the phosphatidylserine (PS) and 88% of the phosphatidylinositol (PI) were localized predominantly in the inner monolayer. The total phospholipid distribution determined by these measurements was 48/52 +/- 2% for the outer/inner monolayer of the sarcoplasmic reticulum lipid bilayer. Sarcoplasmic reticulum phospholipids were biosynthetically deuterated and exchanged into isolated vesicles with both a specific lecithin and a general exchange protein. Neutron diffraction measurements directly provided lipid distribution profiles for both PC and the total lipid content in the intact sarcoplasmic reticulum membrane. The outer/inner monolayer distribution for PC was 47/53 +/- 1%, in agreement with phospholipase measurements, while that for the total lipid was 46/54 +/- 1%, similar to the phospholipase measurements. These neutron diffraction results regarding the sarcoplasmic reticulum membrane bilayer were used in model calculations for decomposing the electron-density profile structure (10 A resolution) of isolated sarcoplasmic reticulum previously determined by X-ray diffraction into structures for the separate membrane components. These structure studies showed that the protein profile structure within the membrane lipid bilayer was asymmetric, complementary to the asymmetric lipid structure. Thus, the total phospholipid asymmetry obtained by two independent methods was small but consistent with a complementary asymmetric protein structure, and may be related to the highly vectorial functional properties of the calcium pump ATPase protein in the sarcoplasmic reticulum membrane.     

Phospholipids and protein adaptation of Pseudomonas sp. to the xenoestrogen tributyltin chloride (TBT)

A tributyltin (TBT)-resistant strain of Pseudomonas sp. isolated from an overworked car filter was tested for its adaptation to TBT. The isolate was checked for organotin degradation ability, as well as membrane lipid and cellular protein composition in the presence of TBT. The phospholipid profiles of bacteria, grown with and without increased amounts of TBT, were characterized using liquid chromatography/electrospray ionization/mass spectrometry. The strain reacted to the biocide by changing the composition of its phospholipids. TBT induced a twofold decline in the amounts of many molecular species of phosphatidylglycerol and an increase in the levels of phosphatidic acid (by 58 %) and phosphatidylethanolamine (by 70 %). An increase in the degree of saturation of phospholipid fatty acids of TBT exposed Pseudomonas sp. was observed. These changes in the phospholipid composition and concentration reflect the mechanisms which support optimal lipid ordering in the presence of toxic xenobiotic. In the presence of TBT the abundances of 16 proteins, including TonB-dependent receptors, porins and peroxidases were modified, which could indicate a contribution of some enzymes to TBT resistance.     

The lipid composition of flight muscle mitochondria isolated from the blowfly, Phormia regina.

The role of lipids in membrane structure and function was studied by measuring the major lipid classes in mitochondria isolated from flight muscle of the blowfly, Phormia regina. Approximately 98% of the total lipid is phospholipid. Neutral lipid constitutes the remaining 2% of the total. Phosphatidylethanolamine accounts for 55–60% of the phospholipid. A molecular ratio of 4:1:1 is found for phosphatidylethanolamine, phosphatidylcholine, and cardiolipin (diphosphatidylglycerol). The neutral lipids include cholesterol, about 20%, and quinone, 40–45% of the total. The free fatty acid content of the neutral lipid fraction is variable, apparently being generated by endogenous phospholipase activity. The fatty acids of the neutral and phospholipid classes are predominantly 14–18 carbon acids; long-chain fatty acids of 20 and 22 carbons are essentially absent. The neutral lipid fraction contains 43% saturated and 51% monoenoic fatty acids. More than 65% of the phospholipid fatty acids are unsaturated. The principal fatty acids are palmitic, palmitoleic, oleic, linoleic, and linolenic. No trace of α- or β-tocopherol is detected. As vitamin E is considered an important naturally occuring antioxidant that prevents lipid peroxidation, the apparent absence of α- and β-tocopherol in these mitochondria coupled with intense oxidative activity of the mitochondria leads to the suggestion that blowfly flight muscle mitochondria may be particularly susceptible to peroxidative damage.