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.     

Isolation and characterization of oat aleurone and starchy endosperm protein bodies

To compare oat (Avena sativa L. cv Froker) aleurone protein bodies with those of the starchy endosperm, methods were developed to isolate these tissues from mature seeds. Aleurone protoplasts were prepared by enzymic digestion and filtration of groat (caryopsis) slices, and starchy endosperm tissue was separated from the aleurone layer by squeezing slices of imbibed groats followed by filtration. Protein bodies were isolated from each tissue by sucrose density gradient centrifugation. Ultrastructure of the isolated protein bodies was not identical to that of the intact organelles, suggesting modification during isolation or fixation. Both aleurone and starchy endosperm protein bodies contained globulin and prolamin storage protein, but minor differences in the protein-banding pattern by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were evident. The amino acid compositions of the protein body fractions were similar and resembled that of oat globulin. The aleurone protein bodies contained phytic acid and protease activity, which were absent in starchy endosperm protein bodies.     

The high molecular weight proteins released from cultured cells.

Studies have been performed with the serum-free culture medium taken from several fibroblast monolayer culture lines. A high molecular weight protein fraction was separated from the concentrated medium by sucrose density gradient centrifugation. Polyacrylamide gel electrophoresis was used to assess the degree of purification obtained. In the electron microscope the negatively stained high molecular weight proteins were found to closely resemble the alpha2-macroglobulins. The suggestion that these proteins from cultured cells resemble the cylindrical protein complex isolated from mammalian erythrocyte ghosts is not supported by this study. The results are discussed in the light of the extensive literature now available on the electron microscopy of high molecular weight proteins.     

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.     

Suborganellar localization of proteinase catalyzing the limited hydrolysis of pumpkin globulin

Protein bodies were prepared from the cotyledons of pumpkin (Cucurbita sp.) seeds by employing a nonaqueous isolation method. Both light micrographic examination and the marker enzyme assays have shown that the isolated protein bodies were intact and contamination with other cell organelles or cytoplasmic components was negligible. A proteolytic enzyme catalyzing the limited hydrolysis of carboxymethylated γ′ chain of globulin was found to be present in the protein bodies. The specific activity in the protein body (18 units per milligram protein) was higher than that in the whole cell extract (13 units per milligram protein), indicating that the limited proteolytic enzyme was localized in the protein body.

After lysis of the protein bodies using hypotonic buffer solution, the suborganellar components (matrix, membranes, and crystalloids) were separated by sucrose density gradient centrifugation. The crystalloid was composed of only globulin, a major seed protein. The major proteins of matrix and membrane fractions were shown to have mol wt of approximately 10,000. About 90% of the limited proteolytic activity was found in the matrix region.

     

Studies on the Proteinaceous Subcellular Particles in Rice Endosperm: Electron-Microscopy and Isolation

Electron-microscopic examination of rice endosperm revealed the existence of protein-aceous subcellular particles, 1 to 4 µ in diameter and spherical or oval in shape. Isolation of the particles was effected by differential centrifugation in density gradient medium after mechanical or enzymic disintegration of endosperm cells. The isolated particles were predominantly composed of protein, and residual constituents were mainly lipid and carbohydrate. Their shape and behaviors were similar to those found in the endosperm. These facts show that the subcellular particles concerned are “protein bodies” There seemed to be several kinds of protein bodies different with respect to their protein and lipid contents.     

Properties of reformed prolamellar bodies from illuminated and redarkened etiolated wheat plants

Prolamellar bodies were isolated from etiolated leaves of wheat ( Triticum aestivum L. cv. Walde, Weibull), which were illuminated for 4 h and then grown in darkness for 16 h. The inner etiochloroplast membranes were isolated by differential centrifugation, and prolamellar bodies and thylakoids were separated on a 10–50% continuous sucrose density gradient. The reformed prolamellar bodies contained phototransformable protochlorophyllide as the main pigment as shown by low temperature fluorescence spectra and high performance liquid chromatography. After illumination with 3 flashes of white light almost all of the protochlorophyllide was transformed to chlorophyllide. In the thylakoids, however, most of the protochlorophyllide was not phototransformed. The reformed prolamellar bodies and the thylakoids showed a fluorescence emission ratio 657/633 nm of 5.6 and 0.5, respectively. Both membrane systems contained also chlorophyllide and chlorophyll synthesized during the illumination. Polyacrylamide gel electrophoresis showed the main chlorophyllide oxidoreductasse.
Teransmission and scanning electron micrographs indicated that the reformed prolamellar bodies are mainly of the "narrow" type and that the prolamellar body fraction had only a minor contamination with thylakoid membranes.
The results obtained showed that reformed prolamellar bodies isolated from illuminated redarkened etiolated wheat leaves had features very similar to the prolamellar bodies isolated from etiolated leaves. This provides support for the idea that prolamellar bodies are an important natural membrane system which plays a dynamic role in the development of the etio-chloroplasts in light.     

The purification and characterization of Dictyostelium discoideum plasma membranes.

A new procedure for the purification of plasma membranes of Dictyostelium discoideum is described. Cells are broken by vigorously stirring in the presence of glass beads, and plasma membranes are isolated by equilibrium sucrose density centrifugation. The purified membranes are considerably enriched in alkaline phosphatase and 5'-nucleotidase and contain very low levels of succinate dehydrogenase and NADPH-cytochrome c reductase. The purified membranes contain relatively high levels of phospholipid, sterol and carbohydrate. They appear as a relatively homogeneous population of membrane vesicles in the electron microscope. This new method of purification is compared to previously published procedures which have been found to be unsuitable for our purposes.     

Isolation of myelin bodies from the kidney cortex of gentamicin-treated rats

Myelin bodies have been isolated from the kidney cortex of gentamicin-treated rats (100 mg gentamicin sulfate/kg body weight i.p. twice daily for 3 days) by a simple procedure involving differential centrifugation followed by equilibrium density centrifugation on a discontinuous sucrose gradient. Electron microscopy and assay of acid phosphatase suggest that the myelin bodies were obtained in virtually quantitative yield, essentially uncontaminated by other cellular structures and relatively intact. The method developed here may also prove applicable for the isolation of myelin bodies arising in connection with other drug treatments and may provide information on a number of toxic side-effects of clinical importance.     

Evaluation of isolation methods and culture conditions for rat bone marrow mesenchymal stem cells

Bone marrow mesenchymal stem cells (bMSCs) are multipotent and preferred for cell therapy. However, the content of bMSCs is very low. To propagate a large number of primary bMSCs rapidly has become a prerequisite for bMSC study and application. Different methods of isolating and culturing bMSC were used and compared among groups: bMSCs of group A are isolated using direct adherence method and cultured by conventional medium changing; of group B are isolated using direct adherence method and cultured by low volume medium changing; of group C are isolated using density gradient centrifugation and cultured by conventional medium changing; of group D are isolated using density gradient centrifugation and cultured by low volume medium changing. The average population doubling time (PDT), average generation time and the cumulative cell doubling level were calculated for every group. bMSCs cultured with complete medium containing 10, 11 and 15 % FBS were allocated into group a, b and c separatedly. Cell numbers were counted everyday under a microscope, the population doubling level curve was plotted and PDT was calculated. The growth curve of bMSC in group a, b and c was made. Both density gradient centrifugation and direct adherence methods obtained relatively pure bMSCs. A larger quantity of primary bMSCs were obtained by direct adherence. bMSC proliferation was faster when cultured via the low volume medium changing method at a serum concentration of 11 % than the other methods. Isolating bMSC by direct adherence and culturing by low volume medium changing at a serum concentration of 11 % is preferential for bMSC propagation.     

Isolation of protein bodies on sucrose gradients

Summary Storage protein bodies from sunflower cotyledons during early stages of seed germination were isolated on sucrose density gradients by isopycnic centrifugation. The density of this organelle on the gradients ranged between 1.26 and 1.36 g cm^-3. A proteinase with a pH optimum of 5.2 was associated with this organelle, and is probably responsible for degradation of storage protein. A NADH-dependent cytochrome-c reductase, a membrane marker enzyme with a pH optimum of 8.4, was also present in this organelle fraction.Abbreviations LPA for l-lysine-p-nitroanilide - LPAase for the peptidase which hydrolyzes this peptide This work was supported in part by the National Science Foundation Grant GB-17543, and published as Journal Article No. 5736 of the Michigan Agricultural Experiment Station.Supported by a Deutsche Forschungsgemeinschaft Fellowship.     

Expression of Sporophytic Storage Proteins in the Corm of the Quillwort (Isoetes echinospora Dur.)

Parenchyma cells from the corm tissue of the aquatic lycopod Isoetes echinospora Dur. were shown by electron microscopy to be packed with amyloplasts, lipid bodies, and protein bodies. The protein bodies are morphologically similar to those identified in seeds and certain vegetative tissues of higher plants. Globoid-containing protein bodies (1-10 [mu]m) isolated in a sucrose gradient possessed a buoyant density of 1.28 g/mL and contained globulin (salt-soluble) proteins. Sucrose gradient centrifugation of crude globulins revealed only two components with mean sedimentation coefficients of approximately 2S and 11S. The 2S component, designated VSP-IsA, was composed of a 15.7-kD polypeptide. The 11S component, designated VSP-IsB, had a molecular mass of 215 kD as estimated by gel filtration and was composed of 39- to 42-kD polypeptides. Two-dimensional gel electrophoresis showed constituent polypeptides distinguished by differences in net charge and molecular mass. Affinity-purified antibodies against VSP-IsA and VSP-IsB prepared and used as probes on immunoblots cross-react only with their specific antigens, suggesting that the proteins are not immunologically related. Indirect immunolocalization studies confirmed that VSP-IsB is deposited in protein bodies. These globulin proteins, like those from some seeds, form the principal storage reserves of the corm tissue.     

Chromoplasts of Tropaeolum majus L.: Isolation and characterization of lipoprotein elements

Summary Chromoplasts of unfolding petals of Tropaeolum majus contain large amounts of filaments (which, in sections, appear as tubules), and unevenshaped, isodiametric to elongated bodies (IBs). These structural elements are the major sites of the chromoplast pigments. They were freed from isolated chromoplasts and subjected to sucrose density gradient centrifugation. At a density of 1.080 g cm^-3 a distinct orange band contained almost exclusively fine filaments of 15–20 nm in diameter as shown after negative staining. Other filaments and most of the IBs were heterogeneous in size, shape, and density and were collected in two fractions of buoyant densities of 1.025 and 1.055 g cm^-3. The three fractions thus obtained comprise 15–33% protein, large amounts of carotenoids and their esters, glyco- and phospholipids, as well as minor amounts of tocopherols. A higher buoyant density of particles is correlated with a higher relative content of protein and glyco- and phospholipids and a lower relative content of carotenoids. The polypeptide pattern, as shown by SDS-polyacrylamide gel electrophoresis, is very similar in all three fractions. There is one main polypeptide, with a MW of about 30,000, accounting for up to 80% of the protein of each fraction.Abbreviations IBs uneven-shaped, isodiametric to elongated bodies     

Protein bodies of the soybean

Some microscope observations of the protein bodies of the cotyledon cells of the soybean (Glycine max) are described, together with changes in their appearance which occur on germination. Density gradient centrifugation permits the isolation of protein bodies from soymeal. They contain about 70% of the protein of the bean. Only 1 protein could be detected in them: glycinin, the major soybean protein.

The protein bodies were fractionated to light and heavy fractions. The former contained 97.5% protein, the latter 78.5%. RNA, phytic acid and lipids were also present. The 2 fractions probably differ only in the extent of contamination by other cell fragments.

     

Coated Vesicles From Locust Oocytes: Isolation and Characterization

Summary

This report demonstrates for the first time the isolation of coated vesicles from insect oocytes. Coated vesicles were purified from oocytes of Locusta migratoria by differential centrifugation and sucrose density centrifugation. The coated vesicles were characterized by electron microscopy, SDS-PAGE and scanning densitometry. Like coated vesicles isolated from pig brain and chicken oocytes, the coated vesicles from locust oocytes contained clathrin as the major protein component. Apart from clathrin, another major protein characteristic of coated vesicles had a molecular weight of about 115,000, and in addition, several minor unidentified bands were identified.     

Oar, a 115-kilodalton membrane protein required for development of Myxococcus xanthus.

Myxococcus xanthus is a developmental gram-negative bacterium which forms multicellular fruiting bodies upon nutrient starvation. This bacterium was found to contain a 115-kDa membrane protein which separated with the inner membrane fraction by sucrose density gradient centrifugation. The gene for this protein was cloned, and its DNA sequence was determined. The deduced amino acid sequence consists of 1,061 residues. This protein contains a putative signal sequence and many short segments, found scattered throughout the entire protein, that have sequence similarities with OmpA, a major outer membrane protein of Escherichia coli. Thus, the gene was designated oar (OmpA-related protein). A second open reading frame was found 36 bases downstream of the oar termination codon. This open reading frame encodes a protein of 236 residues and contains a putative lipoprotein signal sequence. An aor disruption mutation (delta oar) showed no effect on vegetative growth but caused abnormal morphogenesis during development and reduced myxospore formation. When examined with a light microscope, delta oar cells were unable to aggregate on developmental agar, indicating that Oar is required for cellular adhesiveness during development.     

The purification and characterization of Dictyostelium discoideum plasma membranes

A new procedure for the purification of plasma membranes of Dictyostelium discoideum is described. Cells are broken by vigorously stirring in the presence of glass beads, and plasma membranes are isolated by equilibrium sucrose density centrifugation. The purified membranes are considerably enriched in alkaline phosphatase and 5′-nucleotidase and contain very low levels of succinate dehydrogenase and NADPH-cytochrome $\text{c}$ reductase. The purified membranes contain relatively high levels of phospholipid, sterol and carbohydrate. They appear as a relatively homogeneous population of membrane vesicles in the electron microscope. This new method of purification is compared to previously published procedures which have been found to be unsuitable for our purposes.     

Localization and Solubilization of the Respiratory Nitrate Reductase of Bacillus stearothermophilus

Membranes were isolated from Bacillus stearothermophilus 2184D by lysozyme digestion of the cell wall and subsequent differential centrifugation. Observations with the electron microscope indicate that such membranes are relatively intact and have a typical membrane appearance. Nitrate will preferentially oxidize the cytochrome b of such membranes. Approximately 80% of the total respiratory nitrate reductase activity of whole cells can be localized in the washed membrane fraction and the process of membrane isolation results in a sixfold purification of this enzyme. Of several detergents tested, sodium dodecyl sulfate, Triton 114, and Triton X-100 are most effective in converting reduced methyl viologen-nitrate reductase to a form which will not pellet at 130,000 x g. Density gradient analysis reveals that such detergent-mediated solubilization converts virtually all membrane protein to a form of lighter density.     

Isolation and purification of a granulosis virus from infected larvae of the Indian meal moth, Plodia interpunctella.

A procedure was developed for purification of a granulosis virus inclusion body produced in vivo in the Indian meal moth, Plodia interpunctella (Hübner). Purification was accomplished by differential centrifugation, treatment with sodium deoxycholate, and velocity sedimentation in sucrose gradients. The adequacy of the procedure was confirmed by mixing experiments in which uninfected, radioactively labeled larvae were mixed with infected, unlabeled larvae. After purification, the virus was shown to be free of host tissue, to retain its physical integrity, and to be highly infectious per os. Preparations of purified virus consisted of homogeneous populations of intact inclusion bodies (210 by 380 nm) whose buoyant density was 1.271 g/cm3 when centrifuged to equilibrium in sucrose gradients. Electron microscopy of thin-sectioned virus or of virus sequentially disrupted on electron microscope grids demonstrated three components: protein matrix, envelope, and nucleocapsid.     

Isolation and purification of a granulosis virus from infected larvae of the Indian meal moth, Plodia interpunctella.

A procedure was developed for purification of a granulosis virus inclusion body produced in vivo in the Indian meal moth, Plodia interpunctella (Hübner). Purification was accomplished by differential centrifugation, treatment with sodium deoxycholate, and velocity sedimentation in sucrose gradients. The adequacy of the procedure was confirmed by mixing experiments in which uninfected, radioactively labeled larvae were mixed with infected, unlabeled larvae. After purification, the virus was shown to be free of host tissue, to retain its physical integrity, and to be highly infectious per os. Preparations of purified virus consisted of homogeneous populations of intact inclusion bodies (210 by 380 nm) whose buoyant density was 1.271 g/cm3 when centrifuged to equilibrium in sucrose gradients. Electron microscopy of thin-sectioned virus or of virus sequentially disrupted on electron microscope grids demonstrated three components: protein matrix, envelope, and nucleocapsid.