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.

scholarly journals Expression of Aleutian Mink Disease Parvovirus Capsid Proteins in a Baculovirus Expression System for Potential Diagnostic Use

1994 ◽  
Vol 6 (1) ◽  
pp. 23-29 ◽  
Author(s):  
Wai-Hong Wu ◽  
Marshall E. Bloom ◽  
Bradley D. Berry ◽  
Michael J. McGinley ◽  
Kenneth B. Platt
Keyword(s):  
Microscopic Examination ◽  
Expression System ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Electron Microscopic Examination ◽  
Baculovirus Expression System ◽  
Immunoblot Analysis ◽  
Capsid Proteins ◽  
Electron Microscopic ◽  
Nuclear Polyhedrosis

A 2.3-kb cDNA clone encoding Aleutian mink disease parvovirus (ADV) structural proteins VP1 and VP2 was inserted into the polyhedron gene of Autographa calijbmica nuclear polyhedrosis virus (AcNPV) and expressed by the recombinant virus, AcADV-1, in Spodoptera frugiperda-9 cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western immunoblot analysis (WIA) indicated that synthesis of both VP1 and VP2 was being directed by AcADV-1. Fluorescence microscopic examination of AcADV-1 -infected S. frugiperda-9 cells indicated that the recombinant protein was present within the nucleus of the cells, and electron microscopic examination of these cells revealed the presence of small particles 23–25 nm in diameter. Structures resembling empty ADV capsids could be purified on CsCl density gradients, thus indicating that the ADV proteins were self-assembling. The antigenicity of recombinant VP1 and VP2 was evaluated by WIA. Sera collected from 16 mink prior to infection with ADV did not react with VP1 and VP2. Ten sera collected from mink with counter current immunoelectrophoresis (CIE) titers greater than 4 (log2) reacted with VP1 and VP2 in WIA. Two of 6 sera with CIE titers of 4 and 1 of 14 sera with CIE titers <4 reacted with the recombinant proteins. These results suggest that baculovirus recombinant ADV capsid proteins may be useful as diagnostic antigens.

Electrophoretic and electron microscopic examination of the adductor and diductor muscles of an articulate brachiopod, Terebratalia transversa

1982 ◽  
Vol 60 (4) ◽  
pp. 550-559 ◽  
Author(s):  
William P. Eshleman ◽  
Jerrel L. Wilkens ◽  
Michael J. Cavey
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Electron Microscopic Examination ◽  
Light Chains ◽  
Electron Microscopic ◽  
Electrophoretic Patterns ◽  
Transmission Electron ◽  
Adductor Muscles ◽  
Regulation Of Contraction

The proteins of the striated adductor muscles, smooth adductor muscles, and diductor muscles of the articulate brachiopod Terebratalia transversa have been examined by sodium dodecyl sulfate – polyacrylamide gel electrophoresis. Electrophoretic patterns indicate the presence of paramyosin in all of these valve muscles. Tentative identification has also been made of the proteins responsible for actin and for myosin regulation of contraction (troponin–tropomyosin and myosin light chains, respectively). The myofilaments of the striated adductor cells, smooth adductor cells, and diductor cells have been characterized by transmission electron microscopy. The smooth adductor cells and the diductor cells exhibit very thick myofilaments which are fusiform in shape, exceptionally long, and axially banded. Morphological features of these thick myofilaments are consistent with those of paramyosin filaments found in other muscles and myoepithelia. Although the striated adductor cells contain paramyosin, it is not manifest in the thick myofilaments.

scholarly journals Characterization of the yrbA Gene ofBacillus subtilis, Involved in Resistance and Germination of Spores

1999 ◽  
Vol 181 (16) ◽  
pp. 4986-4994 ◽  
Author(s):  
Hiromu Takamatsu ◽  
Takeko Kodama ◽  
Tatsuo Nakayama ◽  
Kazuhito Watabe
Keyword(s):  
Optical Density ◽  
Protein Profile ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Electron Microscopic Examination ◽  
Coat Proteins ◽  
Electron Microscopic ◽  
Consensus Sequences ◽  
Insertional Inactivation ◽  
Coat Layer

ABSTRACT Insertional inactivation of the yrbA gene ofBacillus subtilis reduced the resistance of the mutant spores to lysozyme. The yrbA mutant spores lost their optical density at the same rate as the wild-type spores upon incubation with l-alanine but became only phase gray and did not swell. The response of the mutant spores to a combination of asparagine, glucose, fructose, and KCl was also extremely poor; in this medium yrbA spores exhibited only a small loss in optical density and gave a mixture of phase-bright, -gray, and -dark spores. Northern blot analysis of yrbA transcripts in varioussig mutants indicated that yrbA was transcribed by RNA polymerase with ςE beginning at 2 h after the start of sporulation. The yrbA promoter was localized by primer extension analysis, and the sequences of the −35 (TCATAAC) and −10 (CATATGT) regions were similar to the consensus sequences of genes recognized by ςE. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of proteins solubilized from intact yrbA mutant spores showed an alteration in the protein profile, as 31- and 36-kDa proteins, identified as YrbA and CotG, respectively, were absent, along with some other minor changes. Electron microscopic examination ofyrbA spores revealed changes in the spore coat, including a reduction in the density and thickness of the outer layer and the appearance of an inner coat layer-like structure around the outside of the coat. This abnormal coat structure was also observed on the outside of the developing forespores of the yrbA mutant. These results suggest that YrbA is involved in assembly of some coat proteins which have roles in both spore lysozyme resistance and germination.

scholarly journals A 13-mer peptide straddling the leucine33/proline33 polymorphism in glycoprotein IIIa does not define the PLA1 epitope

Blood ◽  
1991 ◽  
Vol 77 (9) ◽  
pp. 1964-1969 ◽  
Author(s):  
F Flug ◽  
R Espinola ◽  
LX Liu ◽  
C SinQuee ◽  
R DaRosso ◽  
...  
Keyword(s):  
Amino Acid ◽  
Polyclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Adenosine Diphosphate ◽  
Enzyme Linked Immunosorbent Assay ◽  
Amino Acid Polymorphism ◽  
Glycoprotein Iiia ◽  
Polymerase Chain ◽  
Induced Aggregation

Abstract We confirm the recent report (J Clin Invest 83:1778, 1989) of a polymorphism at amino acid 33 of platelet GPIIIa associated with the PLA1/PLA2 phenotype by using the polymerase chain reaction on cDNA derived from platelet RNA, using the base-pair primers 105–129 and 452- 428. Platelet cDNA from three PLA2-homozygous individuals, when digested with Nci I, gave two bands of 256 bp and 91 bp, whereas eight PLA1 cDNAs gave a single band of 347 bp. Two 13-mer amino acid peptides straddling the amino acid polymorphism: SDEALP (L/P) GSPRCD were synthesized for epitope studies. Two mouse polyclonal antibodies were raised: one against the PLA1-associated peptide, the other against the PLA2 peptide. Both antibodies react with either peptide, as well as with both PLA1 and PLA2 platelets. The PLA1 peptide did not block the binding of two different human anti-PLA1 antibodies to the 100-Kd GPIIIa band on immunoblot of platelet extracts; neither did it block the binding of the same antibodies to PLA1-platelet extracts in an enzyme-linked immunosorbent assay. Further studies were performed on the PLA1 epitope following subtilisin digestion of purified GPIIIa. A 55-Kd fragment was obtained that retained the PLA1 epitope as well as the first 13 N-terminal amino acids of GPIIIa. Reduction of the 55-Kd fragment resulted in loss of the PLA1 epitope with production of a 67- Kd, 21-Kd, and 10-Kd band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The 55-Kd band does not react with LK-2, a monoclonal antibody versus GPIIIa that inhibits adenosine diphosphate, collagen, epinephrine, and thrombin-induced aggregation. Thus, the PLA1 epitope is conformation-induced, resides on an N-terminal 55-Kd fragment composed of two or more peptides held together by -SH bonds, and is not required for platelet aggregation.

Plant NAD-Dependent Glutamate Dehydrogenase. Purification, Molecular Properties and Metal Ion Activation of the Enzymes from Lemna minor and Pisum sativum

1980 ◽  
Vol 35 (3-4) ◽  
pp. 213-221 ◽  
Author(s):  
Heinz-Walter Scheid ◽  
Adelheid Ehmke ◽  
Thomas Hartmann
Keyword(s):  
Amino Acid ◽  
Pisum Sativum ◽  
Glutamate Dehydrogenase ◽  
Gel Electrophoresis ◽  
Metal Ion ◽  
Lemna Minor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Equilibrium ◽  
Electron Microscopic

Abstract Glutamate dehydrogenase (ʟ-glutamate: NAD+ oxidoreductase (deaminating) EC 1.4.1.2) has been purified to homogeneity from Lemna minor and seeds of Pisum sativum. As established by polyacrylamide gel electrophoresis the Pisum-enzyme constitutes a multiple pattern of seven char­ge isoenzymes whereas the Lemna enzyme shows one single protein band. Molecular weights of 230 000 were calculated for both enzymes by sedimentation equilibrium measurements (Pisum-enzyme) and comparative gel filtration (Lemna-enzyme). Sodium dodecyl sulfate gel electrophoresis and electron microscopic observations revealed that both enzymes are composed of four identical subunits (molecular weight 58 500) arranged in a tetraedric structure. The amino acid compositions of both enzymes are similar to those of various hexameric glutamate dehydrogenases. The N-terminal amino acid of the Pisum-enzyme is alanine. Both enzymes require Ca2+ for maximal catalytic activity. For the Lemna-enzyme the K0.5 values for Ca2+ are 22 µᴍ (NADH-dependent reaction) and 4 µᴍ (NAD+ -dependent reaction), respectively. Ca2+ which to some extent can be replaced by Zn2+ does not affect the enzyme aggregation but seems to govern a reversible equilibrium between catalytically active and inactive enzyme forms.

scholarly journals Isolation of the intercellular glycoproteins of desmosomes.

1981 ◽  
Vol 90 (1) ◽  
pp. 243-248 ◽  
Author(s):  
G Gorbsky ◽  
M S Steinberg
Keyword(s):  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Insoluble Residue ◽  
Differential Centrifugation ◽  
Electron Microscopic ◽  
Nonionic Detergent ◽  
Cell Layers ◽  
Membrane Attachment

To characterize the desmosome components that mediate intercellular adhesion and cytoskeletal-plasma membrane attachment, we prepared whole desmosomes and isolated desmosomal intercellular regions (desmosomal "cores") from the living cell layers of bovine muzzle epidermis. The tissue was disrupted in a nonionic detergent at low pH, sonicated, and the insoluble residue fractionated by differential centrifugation and metrizamide gradient centrifugation. Transmission electron microscopic analyses reveal that a fraction obtained after differential centrifugation is greatly enriched in whole desmosomes that possess intracellular plaques. Metrizamide gradient centrifugation removes most of the plaque material, leaving the intercellular components and the adjoining plasma membranes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis coupled with methods that reveal carbohydrate-containing moieties on gels demonstrate that certain proteins present in whole desmosomes are glycosylated. These glycoproteins are specifically and greatly enriched in the desmosome cores of which they are the principal protein constituents, and thus may function as the intercellular adhesive of the desmosome.

scholarly journals Phosphorylation of vimentin in mitotically selected cells. In vitro cyclic AMP-independent kinase and calcium-stimulated phosphatase activities.

1989 ◽  
Vol 108 (1) ◽  
pp. 67-78 ◽  
Author(s):  
R M Evans
Keyword(s):  
Phosphatase Activity ◽  
Kinase Activity ◽  
Electron Microscopic Examination ◽  
Intermediate Filament Protein ◽  
Electron Microscopic ◽  
Intact Cells ◽  
Activity Peak ◽  
Casein Kinases ◽  
Phosphopeptide Mapping

The phosphorylation of the intermediate filament protein vimentin was examined under in vitro conditions. Cell cytosol and Triton-insoluble cytoskeleton preparations from nonmitotic and mitotically selected mouse L-929 cells exhibited vimentin kinase activity that is apparently cAMP and Ca2+ independent. The level of vimentin kinase activity was greater in preparations from mitotically selected cells than nonmitotic cells. Addition of Ca2+ to mitotic cytosol decreased net vimentin phosphorylation. Dephosphorylation experiments indicated that there is phosphatase activity in these preparations which is stimulated by addition of Ca2+. Fractionation of cytosol from nonmitotic cells on DEAE-Sephacel and phosphocellulose revealed a single major vimentin kinase activity (peak I). Fractionation of cytosol from mitotically selected cells yielded a similar activity (peak I) and an additional vimentin kinase activity (peak II) that was not found in nonmitotic preparations. Based on substrate specificity and lack of inhibition to characteristic inhibitors, the semipurified peak I and II vimentin kinase activities appear to be cAMP-independent enzymes that are distinct from casein kinases I and II. Phosphopeptide mapping studies indicated that both peak I and peak II vimentin kinases phosphorylate tryptic peptides in the NH2-terminal region of vimentin that are phosphorylated in intact cells. Electron microscopic examination of reconstituted vimentin filaments phosphorylated with both semipurified kinases indicated that phosphorylation induced filament disassembly. These experiments indicate that the increased phosphorylation of vimentin during mitosis may be catalyzed by a discrete cAMP-independent protein kinase. In addition, preparations from mitotic cells exhibited a Ca2+-stimulated phosphatase activity, suggesting that Ca2+ may play a regulatory role in vimentin dephosphorylation during mitosis.

Purification of infectious pancreatic necrosis (IPN) virus and comparison of polypeptide composition of different isolates

1978 ◽  
Vol 24 (1) ◽  
pp. 19-27 ◽  
Author(s):  
N. Chang ◽  
R. D. MacDonald ◽  
T. Yamamoto
Keyword(s):  
Gel Electrophoresis ◽  
Pancreatic Necrosis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Buoyant Density ◽  
Sodium Dodecyl ◽  
Particle Diameter ◽  
Virus Infectivity ◽  
Electron Microscopic ◽  
Infectious Pancreatic Necrosis ◽  
Virus Isolates

Infectious pancreatic necrosis (IPN) virus was partially purified by freon extraction of infected CHSE-214 cells and concentrated by polyethylene glycol (PEG) precipitation of virus from the medium. Both methods resulted in virus concentrates that could be further purified by two CsCl gradient centrifugations with little loss of infectivity. A recovery of 80 to 100% of the virus infectivity was obtained and over 100-fold concentration of viral infectivity was achieved by these methods. This purification was used to compare 10 isolates of IPN virus with regard to their physiochemical properties by electron microscopy, buoyant density in CsCl, and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the purified virions. Electron-microscopic observations showed that the virus isolates were identical in that they were isometric, hexagonal in profile, and had a particle diameter of 71 nm. The buoyant densities of the virus isolates in CsCl were found to be 1.33 g/ml. SDS-gel electrophoresis of the virus isolates revealed the presence of three polypeptides of molecular weight 50, 30, and 27 × 103 daltons designated as VP50, VP30, and VP27, respectively.

scholarly journals Enzymatic evidence for differences in the placement of Rh antigens within the red cell membrane

Blood ◽  
1990 ◽  
Vol 75 (1) ◽  
pp. 255-260 ◽  
Author(s):  
K Suyama ◽  
J Goldstein
Keyword(s):  
Phospholipase A2 ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cellular Membrane ◽  
Cytoplasmic Domain ◽  
Red Cell Membrane ◽  
Intact Cells ◽  
Antibody Preparation ◽  
D Cells ◽  
Papain Digestion

Abstract Intact erythrocytes of different Rh genotypes were subjected to various enzyme treatments, the effects of which were monitored by separating the membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and performing Western blotting using an antibody preparation that recognizes only Rh-related polypeptides. We found that treatment of intact cells with either phospholipase A2 or proteases such as papain did not alter the size of Rh antigen-containing polypeptides. In contrast, phospholipase A2 treatment followed by papain digestion cleaved a fraction of these polypeptides. This cleavage appears, from such digestions of Rh(D) positive and negative cells of different genotypes, to occur solely at the extracellular domain of Rh(D) polypeptide, while the extracellular domains of other Rh antigen-containing polypeptides are unaffected. Digestion of red blood cell ghosts and inside-out vesicles with trypsin showed that Rh(D), (C/c), and (E/e) antigen-containing polypeptides span the lipid bilayer having cytoplasmic domains susceptible to the action of proteases. The size of the cleavage products at the cytoplasmic domain of -D-/-D- cells was found to differ from that of other Rh(D) positive genotypes, due possibly to a difference in folding of Rh(D) polypeptide at its cytoplasmic domain and within the cellular membrane of these cells.

The Oligomeric Arrangement of the Light-Harvesting Chlorophyll a/6-Protein Complex of Photosystem II

1994 ◽  
Vol 49 (5-6) ◽  
pp. 337-342 ◽  
Author(s):  
Grzegorz Jackowski ◽  
Ewa Kluck
Keyword(s):  
Photosystem Ii ◽  
Chlorophyll A ◽  
Protein Complex ◽  
Light Harvesting ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Dianthus Caryophyllus ◽  
Lhc Ii ◽  
Induced Aggregation ◽  
Oligomeric Forms

Abstract The light-harvesting chlorophyll a/b-protein complex of photosystem II (LHC II) was isolated from carnation (Dianthus caryophyllus L.) leaves by K+-induced aggregation of n-hep-tylthioglucoside-treated photosystem II particles. When solubilized with a mixture of lithium docedyl sulphate, octyl-β-D-glucopyranoside and dodecyl-β-D-maltoside the LHC II was re­ solved by mild sodium dodecyl sulphate-polyacrylamide gel electrophoresis into four oligo­meric forms and a monomeric one. LHC II contained five major polypeptides only two of which (27 and 26 kDa) were found to be its authentic components. The oligomeric forms of LHC II were found to differ in the stoichiometric ratios of the polypeptides present. The 26 kD a polypeptide was enriched in the largest oligomeric forms while the 27 kDa polypep­tide tended to form a monomer or to assemble as lower oligomeric states of LHC II.

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