scholarly journals Synthesis of the major oil-body membrane protein in developing rapeseed (Brassica napus) embryos. Integration with storage-lipid and storage-protein synthesis and implications for the mechanism of oil-body formation

1989 ◽  
Vol 258 (1) ◽  
pp. 285-293 ◽  
Author(s):  
D J Murphy ◽  
I Cummins ◽  
A S Kang
Keyword(s):  
Brassica Napus ◽  
Seed Development ◽  
Storage Proteins ◽  
Gradient Centrifugation ◽  
Major Protein ◽  
Oil Body ◽  
Oil Bodies ◽  
Electron Microscopic ◽  
Sucrose Density Gradient Centrifugation ◽  
Body Protein

The synthesis of the major protein and lipid storage reserves during embryogenesis in oilseed rape (Brassica napus L., cv. Mikado) has been examined by biochemical, immunological and immunocytochemical techniques. The mature seeds contained about 45% (w/w) storage oil and 25% (w/w) protein. There were three major seed protein components, i.e. about 40-50% total protein was cruciferin, 20% was napin and 20% was a 18 kDa hydrophobic polypeptide associated with the proteinaceous membrane surrounding the storage oil bodies. Embryogenesis was divided into four overlapping stages with regard to the synthesis of these storage components: (1) for the first 3 weeks after flowering, little, if any, synthesis of storage components was observed; (2) storage-oil synthesis began at about week 3, and maximal rates were from weeks 4 to 7; (3) synthesis of the soluble storage proteins cruciferin and napin started at week 6 and rates were maximal between weeks 8 and 11; (4) the final stage was the synthesis of the 19 kDa oil-body polypeptide, which started at weeks 8-10 and was at a maximal rate between weeks 10 and 12. The synthesis of the 19 kDa oil-body protein therefore occurred independently of the synthesis of the soluble seed storage proteins. This former synthesis did not occur until shortly before the insertion of the 19 kDa polypeptide into the oil-body membrane. No evidence was found, either from sucrose-density-gradient-centrifugation experiments or from immunogold-labelling studies, for its prior accumulation in the endoplasmic reticulum. Conventional and immunogold-electron-microscopic studies showed that oil bodies were synthesized in the early to middle stages of seed development without a strongly electron-dense membrane. Such a membrane was only found at later stages of seed development, concomitantly with the synthesis of the 19 kDa protein. It is proposed that, in rapeseed embryos, oil bodies are initially formed with no proteinaceous membrane. Such a membrane is formed later in development after insertion by ribosomes of the hydrophobic 19 kDa polypeptide directly into the oil bodies.

scholarly journals Purification and characterization of oil-bodies (oleosomes) and oil-body boundary proteins (oleosins) from the developing cotyledons of sunflower (Helianthus annuus L.)

1996 ◽  
Vol 314 (1) ◽  
pp. 333-337 ◽  
Author(s):  
Mark MILLICHIP ◽  
Arthur S. TATHAM ◽  
Frances JACKSON ◽  
Gareth GRIFFITHS ◽  
Peter R. SHEWRY ◽  
...  
Keyword(s):  
Helianthus Annuus ◽  
Storage Proteins ◽  
Phospholipid Content ◽  
Major Protein ◽  
Oil Body ◽  
Oil Bodies ◽  
Helianthus Annuus L ◽  
Novel Structure ◽  
Body Boundary ◽  
Helical Proteins

Oil-bodies, from the immature cotyledons of sunflower (Helianthus annuus L.), were difficult to purify to homogeneity using conventional techniques. The major protein contaminants were albumin and globulin storage proteins. A protocol has been developed, therefore, based upon the stringent washing of the oil-body fraction in 9 M urea, which effectively removed almost all the contaminating protein as judged by SDS/PAGE. The urea-washed oil-bodies were enriched in two major proteins of Mr 19000 and 20000. These proteins were oleosins as demonstrated by their amino acid compositions and the sequence analysis of peptides produced by CNBr cleavage. Far-UV CD spectra of the oleosins in trifluoroethanol, trifluoroethanol/water mixtures and as mixed micelles in SDS, were typical of α-helical proteins with α-helical contents of some 55%. The phospholipid content of the urea-washed preparations was less than 0.1% of that required to form a half-unit membrane surrounding the oil-body. The oil-body surface therefore appears to be an unusual and novel structure, covered largely by an oleosin protein coat or pellicle rather than a conventional fluid membrane, half-unit or otherwise.

Biochemical and Structural Changes in Mitochondria and Other Cellular Components of Pea Cotyledons During Germination

1972 ◽  
Vol 50 (7) ◽  
pp. 725-737 ◽  
Author(s):  
T. Solomos ◽  
S. S. Malhotra ◽  
S. Prasad ◽  
S. K. Malhotra ◽  
Mary Spencer
Keyword(s):  
Electron Microscopy ◽  
Structural Changes ◽  
Gradient Centrifugation ◽  
Microscopic Analysis ◽  
Sucrose Density Gradient ◽  
Electron Microscopic ◽  
Sucrose Density Gradient Centrifugation ◽  
Cellular Components ◽  
Further Development

Integrated studies comprising biochemical and electron microscopic analysis suggested that the increase in respiratory activity of pea cotyledon mitochondria during germination results from further development of the original mitochondria present in dormant seeds. Electron microscopy of isolated mitochondria as well as mitochondria in situ has revealed that membranes are scarce in the mitochondria present in dormant seeds. Mitochondrial cristae become well developed during the initial stages of germination. Crude mitochondrial preparations from pea cotyledons were fractionated by sucrose density gradient centrifugation and analyzed through electron microscopy. These studies showed that, at all stages of germination, "peroxisome"-like structures were present in the fractions of higher sucrose densities than that containing mitochondria. Biochemical studies revealed that the activities of catalase (H2O2:H2O2 oxidoreductase, EC 1.11.1.6) and peroxidase (guaicol:H2O2 oxidoreductase, EC 1.11.1.7) were associated mainly with these fractions and their activities increased during germination.

Oil body proteins sequentially accumulate throughout seed development in Brassica napus

2011 ◽  
Vol 168 (17) ◽  
pp. 2015-2020 ◽  
Author(s):  
Pascale Jolivet ◽  
Céline Boulard ◽  
Annick Bellamy ◽  
Benoît Valot ◽  
Sabine d’Andréa ◽  
...  
Keyword(s):  
Brassica Napus ◽  
Seed Development ◽  
Oil Body

INTERMICROSOMAL DISTRIBUTION OF AROMATIZING ENZYME SYSTEM IN EQUINE TESTICULAR TISSUE

1973 ◽  
Vol 72 (2) ◽  
pp. 366-375 ◽  
Author(s):  
Ran Oh ◽  
Bun-ichi Tamaoki
Keyword(s):  
Microsomal Fraction ◽  
Density Gradient Centrifugation ◽  
Enzyme System ◽  
Gradient Centrifugation ◽  
Electron Microscopic Examination ◽  
Sucrose Density Gradient ◽  
Testicular Tissue ◽  
Electron Microscopic ◽  
Sucrose Density Gradient Centrifugation ◽  
Cytochrome P 450

ABSTRACT The microsomal fraction (10 000–105 000 × g precipitate) of equine testes was fractionated into the smooth- and the rough-surfaced microsomal subfractions by a sucrose density-gradient centrifugation in the presence of CsCl. The validity of this fractionating procedure was confirmed by electron microscopic examination and also by chemical analysis of the RNA contents in these subfractions. The aromatizing enzyme system (19-hydroxylase and aromatase) which was concentrated in the microsomal fractions among the organellae was found to be localized in the smoothsurfaced microsomal fraction. The cytochrome P-450 which was also involved in the process of enzymatic aromatization was detected exclusively in the smooth-surfaced microsomal fraction. The distribution of the aromatizing system between the two microsomal subfractions of equine testes was discussed in comparison with that in human full term placentae.

scholarly journals SEDIMENTATION PROPERTIES OF RAT LIVER MITOCHONDRIA

1970 ◽  
Vol 46 (1) ◽  
pp. 17-26 ◽  
Author(s):  
John N. Loeb ◽  
Daniel V. Kimberg
Keyword(s):  
Rat Liver ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Electron Microscopic ◽  
Sucrose Density Gradient Centrifugation ◽  
Sedimentation Behavior ◽  
Sucrose Medium ◽  
Mitochondrial Volume

A prediction of the velocity of sedimentation of rat liver mitochondria in sucrose gradients is made on the basis of recent measurements of the size of isolated mitochondria suspended in sucrose medium and the model proposed by Bentzel and Solomon to describe the osmotic behavior of mitochondria. The experimentally observed velocity is extremely close to the predicted value and confirms by a different approach the estimate of mitochondrial volume made by Baudhuin and Berthet on the basis of electron microscopic measurements. Because cortisone treatment of rats is known to result in a marked increase in mitochondrial size as observed under the electron microscope, mitochondria were co-isolated from livers of control and cortisone-treated animals, and the sedimentation behavior of the mixtures was examined by sucrose density gradient centrifugation. Mitochondria from cortisone-treated animals were found to sediment 1.4 times as rapidly as those from control animals, indicating that their increased size cannot entirely be due to an increased imbibition of fluid from the surrounding sucrose medium, and that the change in size must at least in part be due to a change in content of nondiffusible mitochondrial components. Although the increase in sedimentation velocity of mitochondria from cortisone-treated animals is striking, it is less than that predicted solely on the basis of their size relative to that of control mitochondria. It is concluded that the increases in mitochondrial size and content of nondiffusible components produced by cortisone treatment are accompanied by alterations in mitochondrial composition as well.

scholarly journals Some studies on the composition and surface properties of oil bodies from the seed cotyledons of safflower (Carthamus tinctorius) and linseed (Linum ustatissimum)

1980 ◽  
Vol 190 (3) ◽  
pp. 551-561 ◽  
Author(s):  
C R Slack ◽  
W S Bertaud ◽  
B D Shaw ◽  
R Holland ◽  
J Browse ◽  
...  
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Electron Microscopic Examination ◽  
Carthamus Tinctorius ◽  
Oil Body ◽  
Oil Bodies ◽  
Electron Microscopic ◽  
Intact Cells ◽  
Glycerolipid Composition ◽  
Induced Aggregation

1. The average oil-body diameter in intact cells of developing linseed (Linum usitatissimum) and safflower (Carthamus tinctorius) cotyledons was similar (about 1.4 micrometer), and there was little change in size after oil bodies were isolated and repeatedly washed. 2. The glycerolipid composition of washed oil bodies from both developing and mature cotyledons of the two species was similar; oil bodies from ten different batches of cotyledons contained 4.3 +/- 0.16 mumol of 3-sn-phosphatidylcholine and 25.2 +/- 1.7 mumol of diacylglycerol per 1000 mumol of triacylglycerol. During four successive washings of a once-washed oil-body preparation, the proportion of diacylglycerol to triacylglycerol remained constant and that of 3-sn-phosphatidylcholine to triacylglycerol decreased by only 20%. 3. The protein content of thrice-washed oil bodies from the two species was similar, about 2.4% of the weight of glycerolipids, and appeared to be independent of the stage of cotyledon maturity. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated that the protein of purified oil bodies from the two species consisted mainly of only four polypeptides and that two of the polypeptides from each species had apparent mol.wts. of 17500 and 15500. Similar patterns of polypeptides were obtained after the hydrolysis of the 15500-mol.wt. polypeptides from linseed and safflower oil bodies by Staphylococcus aureus V8 proteinase, whereas the proteolysis of the 17500-mol.wt. polypeptides from the two species produced different patterns of polypeptides. 4. The 3-sn-phosphatidylcholine in oil-body preparations was hydrolysed about 85% by bee-venom phospholipase A2 without any apparent coalescence of the oil bodies. Incubation with lipase from Rhizopus arrhizus caused rapid coalescence of the oil bodies, and this lipase appeared to initially hydrolyse diacylglycerols in preference to triacylglycerol. 5. Oil bodies from both species were almost completely dispersed in suspensions of pH between 7.1 and 8.3, but formed large aggregates at pH values between 6.7 and 3.9; pH-induced aggregation caused no coalescence. Aggregates formed under acidic conditions were dispersed by re-adjusting the pH of suspensions to 8.3. 6. A freeze-etch electron-microscopic examination of isolated oil bodies indicated that these organelles were bounded by some form of membrane with a particle-free outer surface.

A Na+-Ca2+ exchange process in isolated sarcolemmal membranes of mesenteric arteries from WKY and SHR rats

1985 ◽  
Vol 249 (1) ◽  
pp. C166-C172 ◽  
Author(s):  
M. A. Matlib ◽  
A. Schwartz ◽  
Y. Yamori
Keyword(s):  
Cell Membrane ◽  
Membrane Fraction ◽  
Gradient Centrifugation ◽  
Exchange Process ◽  
Mesenteric Arteries ◽  
Shr Rats ◽  
Electron Microscopic ◽  
Sucrose Density Gradient Centrifugation ◽  
High Potassium ◽  
Wky Rats

The existence of a Na+-Ca2+ exchange process in cell membrane vesicles isolated from mesenteric arteries of Wistar-Kyoto normotensive (WKY) and spontaneously hypertensive (SHR) rats was investigated. Membranes from cleaned mesenteric arteries were isolated by sucrose density gradient centrifugation, which yielded three distinct membrane fractions. The lighter membrane fraction of both WKY and SHR rats was enriched in 5'-nucleotidase activity, a marker for cell membrane, by about 10-fold, based on the activity in the homogenate, and was higher in membranes of SHR compared with WKY rats. Ouabain-sensitive Na+-K+-ATPase activity, another marker for cell membrane, was also concentrated in the lighter membrane fraction and was lower in the membranes of SHR compared with WKY rats. Higher activities of 5'-nucleotidase and Na+-K+-ATPase of both WKY and SHR rats was taken as evidence that the lighter membrane fraction was enriched in plasma membrane. Electron microscopic examination indicated that the membranes were in vesicular form. When the vesicles were loaded with Na+, a time-dependent uptake of Ca2+ was observed if the assay was carried out in high potassium to create a Na+ concentration gradient across the membrane of the vesicles. Very little Ca2+ uptake was observed when the vesicles were loaded with K+ or when the uptake of Ca2+ was carried out under conditions in which the Na+ gradient across the vesicle membranes was reduced. Ca2+ uptake in Na+-loaded vesicles of SHR rats was only slightly increased compared with WKY rats. The data indicate that a Na+-Ca2+ exchange process exists in the cell membrane of rat mesenteric arteries.

Purification and properties of boticin P produced by Clostridium botulinum

1974 ◽  
Vol 20 (3) ◽  
pp. 385-390 ◽  
Author(s):  
A. H. S. Lau ◽  
R. Z. Hawirko ◽  
C. T. Chow
Keyword(s):  
Density Gradient ◽  
Clostridium Botulinum ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Electron Microscopic Examination ◽  
Equilibrium Density ◽  
Type B ◽  
Electron Microscopic ◽  
Sucrose Density Gradient Centrifugation

A bacteriocin produced spontaneously by a nontoxigenic strain of Clostridium botulinum, type E, PM-15, has been isolated and designated boticin P. It was purified by ammonium sulfate precipitation, gel filtration on Sephadex G-100, sucrose density gradient centrifugation, and cesium chloride equilibrium density gradient centrifugation. Boticin P is composed mainly of proteins (98.8%) with a trace amount of carbohydrates (0.4%), and has an apparent molecular weight in excess of 4 × 106 daltons as estimated by gel filtration on Sepharose 2B. Electron microscopic examination of boticin P reveals a phage tail-like structure of 100 nm in length.Boticin P exerted a static effect on vegetative growth and spore outgrowth but not on the initial events of germination. The boticin was active on 10/12 toxigenic and 3/6 nontoxigenic type E and 2/2 nonproteolytic type B strains of C. botulinum. The activity spectrum on 27 strains supports the proposal that type E and the nonproteolytic type B strains belong to the same taxosubspecies.

Iron oxidation by cell envelopes of Thiobacillus ferrooxidans

1974 ◽  
Vol 20 (12) ◽  
pp. 1647-1652 ◽  
Author(s):  
Carl Bodo ◽  
D. G. Lundgren
Keyword(s):  
Thiobacillus Ferrooxidans ◽  
Gradient Centrifugation ◽  
Iron Oxidation ◽  
Broad Band ◽  
Major Protein ◽  
Sucrose Density Gradient Centrifugation ◽  
Ph Optimum ◽  
Oxidation Activity ◽  
Whole Cells ◽  
Cell Envelopes

The parameters for iron oxidation by isolated cell envelopes of Thiobacillus ferrooxidans were studied using a Clark oxygen electrode. Envelopes were obtained by breaking thoroughly washed cells in a French pressure cell and by sedimenting the envelope fraction by ultracentrifugation. The envelopes were partially purified by sucrose density-gradient centrifugation. A broad band was separated at a specific gravity of about 1.21 g/cm3 which coincided with the major protein peak and iron oxidation activity. Electron micrographs confirmed that the active preparations were cell envelopes consisting of the cytoplasmic membrane, peptidoglycan layer, and the outer lipopolysaccharide–lipoprotein double track layer of gram-negative bacteria. Numerous membrane vesicles were also present. Lysozyme had little effect upon iron oxidation activity whereas treating cell envelopes with Triton X-100 destroyed activity. Iron oxidation by cell envelopes was completely inhibited by heating in boiling water or by adding 1% trichloroacetic acid to the reaction cuvette.Kinetic analysis of iron oxidation by envelopes in β-alanine-SO42− buffer (pH 3.5) showed an apparent Km of 5.4 × 10−3 M. The apparent Km with whole cells was 2.1 × 10−3 M. The pH optimum for iron oxidation by cell envelopes was between pH 3.0 and 3.5 while for whole cells the optimum was pH 2.0–2.5.Spectrophotometric studies identified the membrane-bound cytochromes as cytochromes c and a; cytochrome b was also present but its function is unknown at this time.

Changes in the nitrogenous components of maturing rapeseed (Brassica napus)

1971 ◽  
Vol 49 (9) ◽  
pp. 1733-1735 ◽  
Author(s):  
A. J. Finlayson ◽  
C. M. Christ
Keyword(s):  
Brassica Napus ◽  
Nitrogen Content ◽  
Protein Content ◽  
Seed Development ◽  
Seed Weight ◽  
Storage Proteins ◽  
Mature Seed ◽  
Early Stages ◽  
Seed Growth

Changes in seed nitrogen content, seed weight, precipitatable nitrogen (protein) content of maturing rapeseed show that there is a greater increase in precipitatable nitrogen during the early stages of seed development than at the later stages. Proteins found in the mature seed, however, are not detected until the last 3 weeks of seed growth. The deposition or completion of synthesis of the storage proteins apparently occurs over a 14-day period in rape.

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