Isolation and elucidation of structure of peptides from oat (Avena sativa) seedlings

The level of proteolytic activity in tissues of oat seedlings was characterized under acidic conditions, and the number and content of the main components in low-molecular-mass fractions of the extract were determined. The structures of the majority of predominant peptide components isolated from the extract were studied. The use of a database of protein structures helped suggest possible structures of protein precursors of the peptides isolated. Detailed information on a plant peptidome was obtained for the first time.     

A factor converting monoribosomes into polyribosomes during protein synthesis in vitro

An extract was prepared from rabbit reticulocyte ribosomes after treatment with potassium chloride as described previously (Miller, Hamada, Yang, Cohen & Schweet, 1967). The participation of the extract in cell-free protein synthesis was studied. Purified polyribosomes were isolated and converted into monoribosomes by incubation in the cell-free protein-synthesis system. The monoribosomes were isolated and found to be unable to synthesize protein in the cell-free system. The addition of the ribosomal extract to the system stimulated protein synthesis. This was accompanied by the conversion of some of the monoribosomes into polyribosomes. The active component or components of the extract were shown to be protein.     

The Formation of Stromules In Vitro from Chloroplasts Isolated from Nicotiana benthamiana

Stromules are stroma-containing tubules that have been observed to emanate from the main plastidic body in vivo. These structures have been shown to require cytoskeletal components for movement. Though numerous studies have shown a close association with the endoplasmic reticulum, nucleus, mitochondria, and other plastids, the mechanism of formation and their overall function remain unknown. A limiting factor in studying these structures has been the lack of a reconstituted system for in vitro stromule formation. In this study, stromule formation was induced in vitro by adding a plant extract fraction that is greater than 100 kDa to a population of isolated chloroplasts. Kinetic measurements show that stromule formation occurs within ~10 seconds after the addition of the plant extract fraction. Heat inactivation and apyrase treatment reveal that the stromule stimulating compound found in the extract fraction is a protein or protein complex 100 kDa or greater. The formation of the stromules in vitro with isolated chloroplasts and a concentrated fraction of cell extract opens an avenue for the biochemical dissection of this process that has heretofore been studied only in vivo.     

Cytotoxic withanolides from Acnistus arborescens

Three cytotoxic withanolides, two with new structures, were isolated from the leaves of Acnistus arborescens and their structures determined by a combination of 1D and 2D NMR, mass spectral, and molecular modeling studies. Dereplication analysis of the ethyl ether extract was useful for evaluating the components showing significant cytotoxic activity.     

Analysis of conformational variation in macromolecular structural models

Experimental conditions or the presence of interacting components can lead to variations in the structural models of macromolecules. However, the role of these factors in conformational selection is often omitted by in silico methods to extract dynamic information from protein structural models. Structures of small peptides, considered building blocks for larger macromolecular structural models, can substantially differ in the context of a larger protein. This limitation is more evident in the case of modeling large multi-subunit macromolecular complexes using structures of the individual protein components. Here we report an analysis of variations in structural models of proteins with high sequence similarity. These models were analyzed for sequence features of the protein, the role of scaffolding segments including interacting proteins or affinity tags and the chemical components in the experimental conditions. Conformational features in these structural models could be rationalized by conformational selection events, perhaps induced by experimental conditions. This analysis was performed on a non-redundant dataset of protein structures from different SCOP classes. The sequence-conformation correlations that we note here suggest additional features that could be incorporated by in silico methods to extract dynamic information from protein structural models.     

Isolation and characterization of cytoskeletons from cotton fiber cytoplasts

Summary Over the last 25 yr, success in characterizing the individual protein components of animal cytoskeletons was possible, in part, due to technical advances in the isolation and purification of anucleate cytoskeletons from animal cells. As a step towards characterizing protein components of the plant cytoskeleton, we have isolated cytoskeletons from cytoplasts (anucleate protoplasts) prepared from cotton fiber cells grown in ovule culture. Cytoplasts isolated into a hypertonic, Ca²⁺-free medium at pH 6.8 retained internal structures after extraction with the detergent, Triton X-100. These structures were shown to include microtubule and microfilament arrays by immunofluorescence and electron microscopy. Actin and tubulin were the only abundant proteins in these preparations, suggesting that microfilaments and microtubules were the major cytoskeleta elements in the isolated cytoskeletons. The absence of additional, relatively abundant proteins suggests that (a) other cytoskeletal arrays potentially present in fiber cells (e.g., intermediate filaments) were either lost during detergent extraction or were minor components of the fiber cell cytoskeleton; and (b) high ratios of individual cytoskeletal-associated proteins relative to actin and tubulin were not required to maintain microtubules and microfilaments in organized structures.     

Purification and characterization of allergens from Xanthium strumarium pollen

The allergenic components present in whole pollen extract of Xanthium strumarium were isolated by sequential ammonium sulphate precipitation, DEAE Sephadex A50 chromatography and gel filtration. The techniques of RAST inhibition and skin test were utilized to check the allergenicity of fractionated proteins revealing the presence of Xan Ib and Xan VIa as the important allergenic components. Xan Ib was found to be devoid of carbohydrate and had a molecular weight of 103,000 daltons. Xan VIa was a glycoprotein of molecular weight 17,000 daltons. The carbohydrate moiety of Xan VIa was found to be associated with allergenicity. The characteristic pattern of whole pollen extract on CIE and TLIEF showed 36 and 21 protein bands, respectively. The use of FPLC in isolation of partially purified allergens from Xanthium is discussed.     

In vitro packaging of DNA of the Bacillus subtilis bacteriophage SPP1

In vitro packaging of bacteriophage SPP1 DNA into procapsids is described and the requirements of this process were determined. Combination of proheads with an extract supplying terminase, DNA and phage tails yielded up to 10(7 )viable phages per milliliter of in vitro reaction under optimized conditions. The presence of neutral polymers and polyamines had a concentration and type dependent effect in the packaging reaction. The terminase donor extract lost rapidly activity at 30 degrees C in contrast to the stability of the prohead donor extract. Maturation to infective virions was observed using both procapsids assembled in SPP1 infected cells and procapsid-like structures assembled in Escherichia coli that overexpressed the SPP1 prohead gene clusters. Neither a majority of aberrant capsid-related structures present in the latter material nor procapsids lacking the portal protein inhibited DNA packaging. Addition of purified portal protein reduced DNA packaging activity in vitro only at concentrations 20-fold higher than those found in the SPP1 infected cell. The SPP1 DNA packaged in vitro originated exclusively from the terminase donor extract. This packaging selectivity was not observed in vivo during mixed infections. The data are compatible with a model for processive headful DNA packaging in which terminase and DNA co-produced in the same cell are tightly associated and can effectively discriminate the portal vertex of DNA packaging-proficient proheads from aberrant structures, from portal-less procapsids, and from isolated portal protein.     

Nuclear matrix, hnRNA, and snRNA in friend erythroleukemia nuclei depleted of chromatin by low ionic strength EDTA

A novel and rapid procedure is described for the preparation of chromatin-depleted nuclei (CDN) from Friend erythroleukemia cells under conditions that avoid the use of high salt concentrations. By this procedure isolated nuclei that had previously been incubated with DNase I to partially digest DNA were washed twice in 2 mM EDTA to extract the chromatin. The resulting structures contained 1% of DNA, 65% of total RNA, 60-80% of hnRNA, 74% of snRNA, 29% of protein, and 2% of histones contained in isolated nuclei. Electron microscopy revealed intact, spherical structures similar in diameter to isolated nuclei and consisting of dense networks of fibrils 50-100 A thick surrounded by distinct nuclear laminae. Although no morphological evidence was found for residual nucleoli, C23, a nucleolus-specific phospho-protein, remained centrally localized in CDN, while Sm antigen specific for snRNPs was diffusely localized but absent from central regions. Addition of 2 mM MgCl2 to CDN resulted in the reformation of morphologically distinguishable residual nucleoli. These studies suggest that nucleolar morphology is, in part, dependent upon divalent cations and components unique to the nucleolar matrix and demonstrate that little randomization of nuclear and nucleolar matrix fibrils occurs during CDN isolation in the absence of divalent cations.     

Modification of radiation response in mice by fractionated extracts of Panax ginseng

A water-soluble extract of the root of Panax ginseng, a plant native to northeastern China, was fractionated into three components: carbohydrate, protein, and saponin fractions. The fractions obtained were tested for their ability to protect against the lethal effects of 60Co gamma irradiation in C3H mice. The results were compared to the protective ability of the water-soluble fraction of whole ginseng. An experiment designed to test the optimum time of injection of whole ginseng showed that administration 24 h prior to irradiation was optimal. Ginseng extract or one of its three fractions was dose adjusted and injected intraperitoneally into mice that 24 h later were irradiated, whole body, with doses ranging from 7 to 11 Gy. The LD50 in 30 days was calculated using Probit analysis. The results indicated that the water soluble extract of whole ginseng gave the best protection against gamma radiation. The isolated protein and carbohydrate fractions gave less protection, while the saponin fraction did not protect.     

Cardiac effects of the extract and active components of Radix stephaniae tetrandrae. I. Electrically-induced intracellular calcium transient and protein release during the calcium paradox.

The present study was designed to compare the cardiac actions of the extract and individual components, tetrandrine (Tet) and fangchinoline (Fan), of Radix stephaniae tetrandrae (RST). We measured the electrically induced [Ca2+]i transient in single rat ventricular myocytes and protein release following perfusion with a Ca2+ free solution (the Ca2+ paradox) from the isolated perfused rat heart, both of which are known to relate to Ca2+ influx. We found that Tet inhibited both electrically induced [Ca2+]i transient and protein release during the Ca2+ paradox, while Fan had no significant effects. The RST extract containing 9% Tet and 6% Fan by weight also affected the [Ca2+]i transient, and was only slightly, though significantly, less effective/potent than Tet alone. On the other hand, RST extract had a significantly greater inhibitory effect on protein release during the Ca2+ paradox than Tet alone. The observations suggest that the RST extract, which contains a mixture of components, may have more potent effects in the heart than its main active component.     

Structures Containing Polyphosphate in Micrococcus lysodeikticus

Granular structures containing inorganic polyphosphate were found in Micrococcus lysodeikticus. These structures were isolated by fractionation of the bacterial extract obtained by lysing the organisms with lysozyme. The composition of the fraction which was enriched with these structures was found to be: protein, 24%; lipids, 30%; and polyphosphate, 27%. This fraction also contained small amounts of ribonucleic acids, carbohydrate, and polyvalent cations. The effect of different reagents and enzymes on the integrity of the granules was examined. It was noticed that they accumulate in the bacteria during the logarithmic phase of growth but disappear gradually during the stationary phase.     

Eucaryotic cell components that bind to chlamydial elementary bodies: the histones

HeLa-cell-membrane fractions isolated by sonication as used previously to identify chlamydial adhesins were examined by a blotting technique for binding chlamydial elementary bodies (EB). One HeLa cell protein with apparent molecular mass of 32 kDa was found to bind native EB. A monoclonal antibody (mAb) raised against this chlamydial binding host-cell protein reacted with eucaryotic histones. Histone fractions were capable of binding EB in an ELISA assay and histone H1 was identified as the chlamydial-binding host cell protein in the Hela cell membrane fraction. Probing with specific mAbs against histone H3 and DNA confirmed that chromatin components were present in the host-cell membrane extract. These data suggest that the HeLa-cell-binding chlamydial proteins were previously identified by their reaction with chromatin and not with membrane components.     

Changes in the protein composition of the synaptic structures of the brain of rats in tetanus intoxication]

As shown by the method of electrophoresis on polyacrylamide gel, the number of proteins with low electrophoretic mobility proved to be increased in the triton extract fractions of synaptic structures isolated from spinal cord of rats with local tetanus; no changes in the protein spectrum were revealed in the dodecyl-sulphate extract. In vitro tetanus toxin stimulated the lysin-H3 incorporation into the total proteins of synaptosomes of rat brain cortex.     

Purification and properties of the isolated honeybee Z-disc

Z-discs have been isolated from honeybee indirect flight muscle fibers with 0.43% lactic acid, and have been purified with differential and sucrose density-gradient centrifugation. In the light microscope, isolated Z-discs are pale, round, homogeneous structures with diameters ranging from 2 μm to 9 μm, depending on the nature of the suspending medium. In the electron microscope, small Z-discs (2 μm in diameter) examined on Formvar-coated grids are thick, dense and lacking in detail; swollen Z-discs (6 μm in diameter) have a reticular pattern with 3-fold symmetry. Sectioned isolated Z-discs show fine projections extending about 1300 Å from both surfaces. These projections may represent insoluble stubs of thin filaments or “C” filaments, which connect thick filaments to the Z-band.Although honeybee isolated Z-discs are very resistant structures that remain insoluble in a number of protein solvents and in solutions reported to extract Z-band material from vertebrate fibrils, it has been possible to solubilize them in 7 m-guanidine-HCl, 2.5 mm-dithiothreitol, 2.5 mm-EDTA (pH 7.5), and to resolve their components electrophoretically. Sodium dodecyl sulfate gel electrophoresis studies indicate that there are at least four polypeptides, with molecular weights of 87,000, 113,000, 158,000, and 175,000, localized in the isolated Z-disc. The Z-disc backbone contains no significant quantity of lipid as earlier reports have suggested. Total lipid extracted from Z-disc preparations with chloroform/ methanol comprises less than 1% of the Z-disc protein.     

Extremely thermostable elongation factor G from Aquifex aeolicus: cloning, expression, purification, and characterization in a heterologous translation system

The fus gene of the translation factor G (EF-G) from the hyperthermophilic bacterium Aquifex aeolicus was cloned under control of a phage promoter and overexpressed in Escherichia coli with the T7 RNA polymerase system. A heat denaturation step at 95 degrees C was used to purify the protein from the cell extract. This approach simplified the chromatographic procedures and decreased the protein loss since most of Escherichia coli proteins were denatured and precipitated. Ten milligrams of the highly purified protein was isolated from 4 liters of induced culture. The overproduced EF-G was active in ribosome-dependent GTP hydrolysis and a poly(U)-directed polyphenylalanine translation system with E. coli 70S ribosomes. The method presented here might facilitate functional and structural studies of important components of the protein biosynthesis system.     

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.

     

Gerronemins A-F, cytotoxic biscatechols from a Gerronema species

The gerronemins A-F (1-6) were isolated as the cytotoxic components of an extract of a Gerronema species detected in a screening for new cytotoxic metabolites from basidiomycetes. Their structures were elucidated by spectroscopic techniques, and they are composed of a C12-C16 alkane or alkene substituted at both ends by 2,3-dihydroxyphenyl groups. The gerronemins blocked the inducible expression of a hCOX-2 and iNOS promoter driven reporter gene with IC50-values of 1-5 microg/ml. In addition, cytotoxic activities were observed which were due the inhibition of cellular macromolecular syntheses.     

Preparative isoelectric focusing of immunologically reactive components of Aspergillus fumigatus mycelium

A water-soluble mycelial extract of Aspergillus fumigatus has been fractionated by preparative isoelectric focusing using carrier ampholytes in a layer of granulated gel. The separated components were located by staining paper prints from the gel. Within a narrow pH range of 2.5 units, multiple protein bands were visualized with Coomassie Brilliant Blue G. Periodate-Schiff-positive material was generally associated with the major protein zones. When these fractions were eluted the total recovery, calculated on the basis of protein and carbohydrate analyses of the isolated fractions, varied between 20 and 60% of the applied material. Low recoveries were associated with low recoveries of protein; recoveries of carbohydrate were higher and less variable. The immunological activity and specificity of the eluted fractions were assessed in an enzyme-linked immunosorbent assay for the detection of IgG antibodies to A. fumigatus.     

Immunochemical study of the antigenic structure of bacteria of genus Bordetella. IV. Fractionation of B. pertussis extracts and study of the immunochemical and biological properties of the isolated fractions]

Fractionation of an extract of pertussis microbes was carried out with the aid of gel-filtration on Sephadex G-100, ion-exchange chromatography on DEAE-cellulose, and preparative electrophoresis. Fractions differing in serological, immunogenic activity and the content of antigenic components were isolated. In using the method of gel-filtration of sefadex G-100 the greatest serological, immunogenic and histamine-sensitizing activity was possessed by the high-molecular fraction containing 8 of 11 antigenic components detected in the initial extract. The antigenic components were distributed into 5 fractions by the ion exchange chromatography on DEAE-cellulose. The greatest serological activity was possessed by fractions exiting from the column at the 0.01--0.04M interval of the phosphate buffer concentration. A method of preparative electrophoresis from the pertussis microbes extract was applied and two fractions were isolated from the anode and the cathode zones, each containing 4 antigenic components only, but possessing serological and immunogenic activity and having no histamine-sensitizing properties.