Isolation of intramitochondrial bodies in bovine adrenocortical cells by density gradient centrifugation.

Two method for isolating the intramitochondrial bodies from bovine adrenocortical cells are proposed. Electron microscopic examination shows that discontinuous sucrose density gradient centrifugation can separate the fraction rich in intramitochondrial bodies, but some indistinguishable fragments remain among them. Continuous sucrose density gradient centrifugation is probably superior to the former method in obtaining a highly purified fraction of the bodies. The amido black positive granules, presumed to be intramitochondrial bodies, are collected in the fractions of the sucrose density of around 1.27 (1.23--1.30), which lack cytochrome c oxidase activity.     

Characterization of prolamellar bodies and prothylakoids fractionated from wheat etioplasts

Prolamellar bodies and prothylakoids from etioplasts of wheat ( Triticum aestivum L. cv. Starke II, Weibull) were separated by sucrose density gradient centrifugation. Top-loaded and bottom-loaded sucrose gradients were compared. As a consequence of avoiding long time exposure of the membranes to low sucrose concentrations, separation in bottom-loaded gradients, as compared to separation in top-loaded gradients, resulted in a sharper and more narrow band of prothylakoids, and in better preservation of phototransformable protochlorophyllide, especially in the prothylakoids. In bottom-loaded gradients, the prothylakoids were found concentrated in a band at a density of 1.20 g'ml⁻¹. The prolamellar bodies were found at a density of 1.17 g'ml⁻¹. In top-loaded gradients the prothylakoids were found at a lower density than the prolamellar bodies. The prothylakoid fraction contained about 60% of the recovered protochlorophyllide and about 85% of the recovered protein. Absorption and fluorescence emission spectra revealed a higher amount of phototransformable protochlorophyllide, in relation to non-phototransformable, in the prolamellar body fraction than in the prothylakoid fraction. Polyacrylamide gel electrophoresis indicated a high proportion of protochlorophyllide reductase in the prolamellar bodies. Chloroplast ATPase (CF₁) was found predominantly in the prothylakoid fraction. Thus, our results strongly indicate the presence of phototransformable protochlorophyllide in the prolamellar bodies proper, while the main bulk of proteins are located in the prothylakoids.     

Subcellular distribution of β-amylase in developing barley endosperm

Protein bodies were isolated from maturing barley (Hordeum vulgare L. cv. Menuet) endosperm using sucrose density gradient centrifugation. Isolated protein bodies were not contaminated by other organelles such as mitochondria and microbodies, and contained a large amount of the barley storage protein, hordein, indicating that the protein bodies were prepared in high purity and were intact. Enzymatic and immunochemical analysis showed that β-amylase, which was previously thought to be localized in protein bodies, was not detected in the protein body fraction, but was found only in the cytosol fraction. The results show that β-amylase is synthesized as a cytosolic protein in maturing barley endosperm.     

The isolation of lamellar bodies and their membranous content from rat lung, lamb tracheal fluid and human amniotic fluid.

A simple one-step procedure is described for the isolation of lamellar bodies and their membranous content in high yield from rat lung. A linear sucrose gradient, ranging from 0.9M – 0.2M sucrose, is poured over a sample of homogenate in IM sucrose and the lamellar body fraction if floated to isopycnic equilibrium by high speed centrifugation. When the same procedure is applied to fluid drained from the lungs of newborn lambs or to human amniotic fluid collected late in pregnancy, a fraction is obtained which appears to consist of the membranous content of lamellar bodies in the process of unravelling.     

Polyhedral bodies and ribulose 1,5-diphosphate carboxylase of the blue-green alga Anabaena cylindrica

Summary The ribulose 1,5-diphosphate carboxylase (RUDP Case E.C. 4.1.1.39) activity of late log phase Anabaena cylindrica Lemm. was measured in vitro in fractions obtained by sucrose density gradient centrifugation. Two peaks of enzymic activity were obtained. One, accounting for about 80% of the total measurable activity occurred at the top of the gradient and appeared to be soluble activity; the second showed maximum activity in the 55% (w/w) sucrose fraction and represented 20% of the total activity. When the distribution of RUDP Case was assayed by immunoprecipitation using antiserum to RUDP Case from Euglena gracilis, the corresponding values were 59% and 41%. Electron microscopy of the various fractions showed that polyhedral bodies, which are sites of RUDP Case activity in other autotrophic prokaryotes, were also most abundant in the 55% (w/w) sucrose fraction.     

The localization of magnesium-protoporphyrin and protochlorophyllide in separated prolamellar bodies and prothylakoids of wheat treated with 8-hydroxyquinoline and δ-aminolevulinic acid

Dark-grown leaves of wheat ( Triticum aestivum L. cv. Starke II, Weibull) were treated in darkness with 8-hydroxyquinoline and δ-aminolevulinic acid in order to accumulate magnesium-protoporphyrin and/or magnesium-protoporphyrin monomethylester. Prolamellar bodies and prothylakoids were separated from the treated leaves by sucrose density gradient centrifugation. About 90% of the recovered magnesium-protoporphyrin/magnesium-protoporphyrin monomethylester and about 75% of the recovered protochlorophyllide was found in the prothlakoid fraction. The significance of the distribution pattern of the chlorophyll precursors between the prolamellar bodies and the prothylakoids is discussed. The results indicate that the prothylakoids are the site for synthesis of membrane-bound chlorophyll precursors and that phototransformable protochlorophyllide is a constituent of prolamellar bodies as well as of prothylakoids.     

Isolation and characterization of outer and inner envelope membranes of cyanelles from a glaucocystophyte, Cyanophora paradoxa

Envelope membranes were isolated by sucrose density gradient floatation centrifugation from the homogenate of cyanelles prepared from Cyanophora paradoxa. Two yellow bands were separated after 40 h of centrifugation. The buoyant density of one of the two fractions (fraction Y2) coincided with that of inner envelope membranes of spinach or plasma membranes of cyanobacteria. The other yellow fraction (fraction Y1) migrated to top of sucrose-gradient even at 0% sucrose. Pigment analysis revealed that the heavy yellow fraction was rich in zeaxanthin while the light fraction was rich in β-carotene, and the both fractions contained practically no chlorophylls. Another yellow fraction (fraction Y3) was isolated from the phycobiliprotein fraction, which was the position where the sample was placed for gradient centrifugation. Its buoyant density and absorption spectra were similar to outer membranes of cyanobacteria. We have assigned fractions Y2 and Y3 as inner and outer envelope membrane fractions of cyanelles, respectively. Protein compositions were rather different between the two envelope membranes indicating little cross-contamination among the fractions. H. Koike and Y. Ikeda contributed equally.     

The Use of Density Gradient Centrifugation for the Separation of Germinated from Nongerminated Spores

S ummary : The density gradient centrifugation of a suspension of spores of B. subtilis 8057 on both sucrose and renografin gradients gave 2 distinct fractions. Germination evidence suggested that the heavier fraction consisted of dormant spores and the less dense fraction, germinated spores. It is concluded that density gradient centrifugation may provide a useful technique for the separation of germinated from nongerminated spores.     

Die subzelluläre Lokalisation von Enzymen des Phenylpropanoidstoffwechsels im Antherentapetum: Phenylalanin-Ammonium-Lyase,Zimtsäure 4-Hydroxylase,SAM: Kaffeesäure 3-0-Methyltransferase

Summary After separation of the contents of anthers into pollen and tapetum fractions, the subcellular localization of tapetum enzymes involved in phenylpropanoid metabolism have been studied by differential centrifugation and by sucrose density gradient centrifugation.The experiments showed that nearly all the activity of both phenylalanine ammonia-lyase and an O-methyltransferase was in the soluble fraction. In contrast the cinnamic acid 4-hydroxylase activity is associated with the 12,000×g and 100,000×g pellet. After fractionation of the tapetum fraction by sucrose density gradient centrifugation, activity of cinnamic acid 4-hydroxylase was highest in those fractions with maximum NADH-Cytochrome c-reductase activity. Combination of the hydroxylase with ER is suggested.The results are discussed with special regard to the secretory function of the tapetum cells.     

Purification of plasma membranes from roots of barley: specificity of the phosphotungstic Acid-chromic Acid stain

Plasma membrane vesicles from roots of barley (Hordeum vulgare L., var. Arivat) had an equilibrium density in sucrose of about 1.16 grams per cubic centimeter, but could not be purified satisfactorily with the procedure developed for roots of other plant species. The reported procedure involving differential centrifugation to remove mitochondria (peak density of 1.18 grams per cubic centimeter) and subsequent density gradient centrifugation to purify plasma membrane vesicles was modified to include a narrower differential centrifugation fraction (13,000 to 40,000g instead of 13,000 to 80,000g) and a narrower density range in the sucrose gradient (1.15 to 1.18 grams per cubic centimeter instead of 1.15 to 1.20 grams per cubic centimeter). The fraction obtained by the modified procedure was between 60 and 70% pure as determined by staining with the phosphotungstic acid-chromic acid procedure, which was judged to be reliable for identifying plasma membrane vesicles in subcellular fractions from barley roots. The plasma membrane fraction was enriched in K⁺-stimulated ATPase activity at pH 6.5. The presence of nonspecific ATP-hydrolyzing activity in the plasma membrane fraction made it difficult to determine if the ATPase had properties in common with those reported for cation absorption in barley roots.     

EVIDENCE FOR A COMMON EVOLUTIONARY ORIGIN OF LIGHT-HARVESTING FUCOXANTHIN CHLOROPHYLL a/c-PROTEIN COMPLEXES OF PAVLOVA GYRANS (PRYMNESIOPHYCEAE) AND PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

A fucoxanthin-chlorophyll a/c-protein complex has been isolated from the prymnesiophyte Pavlova gyrans. Thylakoid membranes were treated with the mild anionic detergent sodium taurodeoxycholate followed by sucrose density gradient centrifugation. The brown fraction produced by this procedure was treated with Triton X-100 followed by a second sucrose density gradient centrifugation. A brown fraction isolated from this gradient was shown to be a light-harvesting complex nearly identical to that which is present in the diatom Phaeodactylum tricornutum. The complexes from the two organisms have nearly identical absorption and flourescence spectra, both complexes contain fucoxanthin and two other carotenoids, both contain four polypeptides of similar molecular weights, and polypeptides from both complexes cross react with antibodies raised to polypeptides of the Phaeodactylum tricornutum complex. Results suggest a common evolutionary origin for these light-harvesting complexes, in apparent contrast to the great differences in cell structure between prymnesiophytes and diatoms.     

Heterogeneity of mitochondrial particles in fresh and wounded tissues of sweet potato roots

A mitochondrial fraction prepared from fresh tissue of sweetpotato root was subjected to sucrose density gradient centrifugation.The distribution of cytochrome oxidase activity, after the centrifugation,showed the presence of at least three kinds of mitochondrialparticles which differed in their sedimentation velocity. Byrepeating the sucrose density gradient centrifugation, it wasdemonstrated that they are not interconvertible. There seemedto be no difference in the distribution between cytochrome andsuccinate oxidase activities. In the case of malate or succinatedehydrogenase activity, however, the greater the sedimentationvelocity of the particle, the greater was the dehydrogenaseactivity per unit of cytochrome oxidase activity. Some changesin the distribution of cytochrome oxidase activity in responseto aging of the tissue slices were observed. ¹This paper constitutes Part 62 of the Phytopathological Chemistryof Sweet Potato with Black Rot.     

The isolation and characterization of a plasma membrane and a myelin fraction derived from oligodendroglia of calf brain.

—A method is described for the fractionation of bulk isolated oligodendroglial cells from calf brain to produce both a plasma membrane and an attached myelin fraction. The cells are homogenized in a sucrose solution containing Mg²⁺ and K+ at a pH of 6·5. Crude membrane fractions are obtained from this homogenate by discontinuous sucrose density gradient centrifugation. After being subjected to osmotic shock, these fractions are purified by continuous sucrose density gradient centrifugation. The plasma membrane fraction, which bands at 1·0 m -sucrose, was identified by its morphology and enzyme content. Electron microscopy showed it to be a homogeneous preparation of vesicles composed, for the most part, of smooth trilaminar membranes. Enzymatic analysis revealed the presence of high specific activities of Na+, K+-ATPase, 5′-nucleotidase and 2′,3′-cyclic AMPase. Lipid analysis showed a higher galactolipid and lower phospholipid content than has been reported for neuronal and synaptic membranes. The attached myelin fraction, which bands at 0·7 m -sucrose has the typical multilamellar appearance of myelin, but differs considerably from normal myelin in having high concentrations of plasma membrane marker enzymes, and a lipid composition intermediate between normal myelin and the plasma membrane fraction. The ganglioside content and protein patterns of these fractions have also been examined.     

Ribulose 1,5-bisphosphate carboxylase and polyhedral bodies of Chlorogloeopsis fritschii

Ribulose 1,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39) activity was approximately equally distributed between supernatant and pellet fractions produced by differential centrifugation of disrupted cells of Chlorogloeopsis fritschii. Low ionic strength buffer favoured the recovery of particulate RuBP carboxylase. Density gradient centrifugation of resuspended cell-free particulate material produced a single band of RuBP carboxylase activity, which was associated with the polyhedral body fraction, rather than with the thylakoids or other observable particles. Isolated polyhedral body stability was improved by density gradient centrifugation through gradients of Percoll plus sucrose in buffer, which yielded apparently intact polyhedral bodies. These were 100 to 150 nm in diameter and contained ring-shaped, 12 nm diameter particles. It is inferred that the C. fritschii polyhedral bodies are carboxysomes. Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis of SDS-dissociated polyhedral bodies revealed 8 major polypeptides. The most abundant, with molecular weights of 52,000 and 13,000, correspond with the large and small subunits, respectively, of RuBP carboxylase.Abbreviations RuBP ribulose 1,5-bisphosphate - Ru5P ribulose 5-phosphate - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - EDTA ethylenediamine tetraacetic acid - Tris tris (hydroxymethyl) methylamine - IB isolation buffer - TCA trichloroacetic acid     

Protein Bodies from the Endosperm of Castor Bean: Subfractionation, Protein Components, Lectins, and Changes during Germination

Protein bodies from the storage endosperm of dry castor bean (Ricinus communis L.) were isolated by successive nonaqueous linear density gradient centrifugation. The isolated protein bodies were lysed by the addition of water, and the various structural components of the organelles were separated by sucrose gradient centrifugation. The matrix protein remained at the top of the gradient while the membrane, the crystalloids, and the globoids migrated to densities 1.15 g/cm³, 1.30 g/cm³, and > 1.46 g/cm³, respectively. The protein of the protein bodies was distributed evenly between the crystalloids and the matrix, and little protein was present in the globoids or the membrane.     

Thyrotropin (TSH) binding activities in bovine thyroid tissue: possible role of adenosine 3',5'-monophosphate dependent phosphorylation of thyroid plasma membranes in TSH receptor degradation

The relationship between thyroid plasma membrane phosphorylation and thyrotropin (TSH) receptor degradation was investigated by using bovine thyroid tissues. By fractionation of thyroid cytosol (105,000 X g supernatant of thyroid homogenate) in a continuous sucrose density gradient centrifugation, three different TSH binding activities were separated. During the incubation of thyroid plasma membranes, TSH binding activities were spontaneously released in vitro. By fractionation of the fraction containing released TSH binding activities in the same sucrose density gradient centrifugation, three different TSH binding activities were isolated. These peaks of TSH binding activity corresponded to the peaks of TSH binding activity obtained in cytosol fraction. Adenosine 3',5'-monophosphate (cyclic AMP) enhanced the release of TSH binding activities from the plasma membranes in vitro. After fractionation on a sucrose density gradient centrifugation of the supernatant of the plasma membranes which were preincubated with cyclic AMP, three different peaks of TSH binding activity were identified. These peaks corresponded to the peaks obtained in spontaneously released TSH binding activity. In this case, however, the amount of small molecule TSH binding activities was predominant compared to that of large molecule TSH binding activity. During the incubation of the plasma membranes with [r-32P]-ATP and with cyclic AMP, phosphorylated soluble proteins were released. The profile of the phosphorylated soluble proteins in the sucrose density gradient centrifugation showed three different peaks which corresponded to the peaks of binding activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and partial characterization of the rat liver ligandosome fraction

The subcellular distribution in rat liver of non-latent and latent NADH pyrophosphatase was determined by analytical sucrose density gradient centrifugation. Non-latent NADH pyrophosphatase activity was distributed similarly to the plasma membrane marker, 5′-nucleotidase. However, latent NADH pyrophosphatase was found at the low density region of the gradient, similar to the distribution of galactosyl transferase, a Golgi marker. A population of membranes, corresponding to those from the low density region, was prepared by discontinuous sucrose gradient centrifugation. Radiolabelled insulin was used, to monitor the involvement of these membranes in ligand internalization. The membrane perturbant, digitonin, was used to effect a partial separation between membranes bearing NADH pyrophosphatase and those bearing galactosyl transferase. The mechanism by which this separation is effected has been investigated and it was shown that, although digitonin caused a loss of enzyme latency, the density shift was not due to this effect. The partially purified ligandosome-rich fraction was characterized by enzymic and ultrastructural analysis. A novel EM cytochemical stain for NADH pyrophosphatase identified a vesicular fraction distinct from Golgi lamellae.     

Adsorption of local anesthetics on phospholipid membranes

The subcellular distribution in rat liver of non-latent and latent NADH pyrophosphatase was determined by analytical sucrose density gradient centrifugation. Non-latent NADH pyrophosphatase activity was distributed similarly to the plasma membrane marker, 5'-nucleotidase. However, latent NADH pyrophosphatase was found at the low density region of the gradient, similar to the distribution of galactosyl transferase, a Golgi marker. A population of membranes, corresponding to those from the low density region, was prepared by discontinuous sucrose gradient centrifugation. Radiolabelled insulin was used, to monitor the involvement of these membranes in ligand internalization. The membrane perturbant, digitonin, was used to effect a partial separation between membranes bearing NADH pyrophosphatase and those bearing galactosyl transferase. The mechanism by which this separation is effected has been investigated and it was shown that, although digitonin caused a loss of enzyme latency, the density shift was not due to this effect. The partially purified ligandosome-rich fraction was characterized by enzymic and ultrastructural analysis. A novel EM cytochemical stain for NADH pyrophosphatase identified a vesicular fraction distinct from Golgi lamellae.     

Phospholipid composition of lamellar bodies formed by fetal rabbit lung type II cells in organ culture

Lung tissue obtained from fetal rabbits of 23 days gestational age was maintained in organ culture to study the in vitro formation of lamellar body phospholipids. During the culture period, the epithelium of the prealveolar ducts of the explants differentiated to form type II pneumonocytes. After 8 days in culture, the explants were harvested, homogenized, and two lamellar body fractions were isolated by sucrose density gradient centrifugation. The lamellar body fraction which best retained the distinct multilamellar structure was recovered at the interface between a solution of buffer without sucrose and buffer containing 0.41 m sucrose. The phospholipid compositions of both lamellar body fractions were similar to those reported for lamellar bodies and surfactant isolated from fetal rabbit lung, with the exception of a slightly higher phosphatidylethanolamine content. The disaturated phosphatidylcholine content of the lamellar body fractions, expressed as a percentage of total lipid phosphorus, was not influenced by the presence of palmitate in the medium.     

Purification and initial characterization of lipase from the scutella of corn seedlings

The lipase from the scutella of corn (Zea mays) MO-17 seedlings was purified 272-fold to apparent homogeneity as evidenced by sodium dodecyl sulfate polyacrylamide gel electrophoresis and double immunodiffusion. The procedure involved isolation of the lipid bodies, extraction with diethyl ether, DE-52 ion exchange chromatography, and sucrose density gradient centrifugation. The enzyme had an approximate molecular weight of 270,000 daltons after sucrose density gradient centrifugation, and 65,000 daltons after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The lipase contained no cysteine and its molecular weight in sodium dodecyl sulfate was not reduced by β-mercaptoethanol. The amino acid composition as well as a biphasic partition using Triton X-114 revealed the enzyme to be a hydrophobic protein. Rabbit γ-globulin containing antibodies raised against the purified lipase formed one precipitin line with the lipase in a double diffusion test, and precipitated all the lipase activity from a solution.