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.

Isolation and characterization of a novel dynein that contains C and A heavy chains from sea urchin sperm flagellar axonemes

1994 ◽  
Vol 107 (2) ◽  
pp. 345-351 ◽  
Author(s):  
E. Yokota ◽  
I. Mabuchi
Keyword(s):  
Electron Microscopy ◽  
Sea Urchin ◽  
Heavy Chain ◽  
Gradient Centrifugation ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Heavy Chains ◽  
Isolation And Characterization ◽  
Sea Urchin Sperm

A novel dynein (C/A dynein), which is composed of C and A heavy chains, two intermediate chains and several light chains, was isolated from sea urchin sperm flagella. The C/A dynein was released by the treatment with 0.7 M NaCl plus 5 mM ATP from the axonemes depleted of outer arm 21 S dynein. Sedimentation coefficient of this dynein was estimated by sucrose density gradient centrifugation to be 22–23 S. The C/A dynein particle appeared to be composed of three distinct domains; two globular head domains and one rod domain as seen by negative staining electron microscopy. The mobility of ‘A’ heavy chain of C/A dynein on SDS-gel electrophoresis was similar to that of A heavy chains (A alpha and A beta) of 21 S dynein. However, UV-cleavage patterns of C and A heavy chains of C/A dynein were different from those of A heavy chains of 21 S dynein. Furthermore, an antiserum raised against A heavy chain of C/A dynein did not crossreact with A heavy chains of 21 S dynein. Under the conditions in which the C/A dynein was released, some of inner arms were removed concomitantly from axonemes as observed by electron microscopy. These results suggested that C/A dynein is a component of the inner arms.

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.

Density Gradient Centrifugation and Electron Microscopic Characterization of Subcellular Fractions from Human Blood Platelets

1971 ◽  
Vol 25 (02) ◽  
pp. 252-267 ◽  
Author(s):  
A Siegel ◽  
P. H Burri ◽  
E. R Weibel ◽  
M Bettex-Galland ◽  
E. F Lüscher
Keyword(s):  
Electron Microscopy ◽  
Human Blood ◽  
Density Gradient Centrifugation ◽  
Blood Platelets ◽  
Gradient Centrifugation ◽  
Electron Microscopic ◽  
Osmotic Effect ◽  
Cellular Components ◽  
Human Blood Platelets

SummaryHomogenized human blood platelets have been fractionated by centrifugation in Ficoll and sucrose density gradients. The different fractions were examined by electron microscopy.Although Ficoll allows for the separation of very distinct zones, its ability to form complexes with cellular components made sucrose the preferable gradient. Sucrose, in spite of its unfavorable osmotic effect, allows for an acceptable fractionation of platelet components.

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 Chymotryptic digestion of Tetrahymena 22S dynein. I. Decomposition of three-headed 22S dynein to one- and two-headed particles.

1987 ◽  
Vol 105 (2) ◽  
pp. 887-895 ◽  
Author(s):  
Y Y Toyoshima
Keyword(s):  
Electron Microscopy ◽  
Gel Electrophoresis ◽  
Gradient Centrifugation ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Heavy Chains ◽  
Time Courses ◽  
The One ◽  
Dynein Heavy Chains

Molecular composition of Tetrahymena ciliary dynein has been examined by electron microscopy and gel electrophoresis. SDS-urea gel electrophoresis revealed that Tetrahymena 22S dynein contains three (A alpha, A beta, and A gamma) heavy chains whereas 14S dynein contains only one. The molecular masses of 22S and 14S dynein heavy chains were estimated to be approximately 490 and 460 kD, respectively. Electron microscopy of negatively stained specimens showed 22S dynein has three globular heads and thin stalks, whereas 14S dynein consists of a single head. Chymotrypsin digested each of the three 22S dynein heavy chains into large fragments with different time courses. Sucrose density gradient centrifugation separated the digestion products as two peaks. The one with a larger sedimentation coefficient mainly consisted of two-headed particles having binding ability to doublet microtubules, whereas the other with a smaller sedimentation coefficient consisted of only isolated globular particles. Both fractions had ATPase activities. Thus, one active head of 22S dynein can be isolated by chymotrypsin digestion.

Acetylcholinesterase: a useful marker for the isolation of sarcolemma from the bivalve (Modiolus demissus demissus) myocardium

1978 ◽  
Vol 34 (1) ◽  
pp. 193-208
Author(s):  
J.A. Watts ◽  
S.K. Pierce
Keyword(s):  
Cell Surface ◽  
Reductase Activity ◽  
Gradient Centrifugation ◽  
Surface Membrane ◽  
Sucrose Density Gradient ◽  
Electron Microscopic ◽  
Sucrose Density Gradient Centrifugation ◽  
Cardiac Muscle Cells ◽  
Succinate Cytochrome C Reductase ◽  
Enzymic Assay

The presence of cholinesterase activity in M. demissus hearts was demonstrated by light- and electron-microscopic histochemistry and by enzymic assay. The enzyme proved to be acetylcholinesterase (AChE) since acetylthiocholine was the preferred substrate, and eserine or BW284C5I inhibited the enzyme activity, while isoOMPA was without effect. The AChE was localized and uniformly distributed along the cell surface membranes of the cardiac muscle cells. A fraction 8-fold enriched in AChE was isolated from pooled ventricles by a combination of differential and sucrose density gradient centrifugation. This sarcolemmal fraction contained little mitochondrial contamination as determined by electron microscopy and by succinate cytochrome c reductase activity. In addition, this fraction stained uniformly for AChE, indicating that it was free of other membrane types (for example sarcoplasmic reticulum which did not stain for AChE). Therefore, this fraction contained purified cell surface membrane free of contamination by other membranous organelles.

Immunocytochemistry. A Review of Methodology for Electron Microscopic Applicaiton

1974 ◽  
Vol 32 ◽  
pp. 328-329
Author(s):  
Joseph E. Mazurkiewicz
Keyword(s):  
Electron Microscopy ◽  
Light Microscopy ◽  
Ultrastructural Level ◽  
Electron Microscopic ◽  
Biological Interest ◽  
Investigative Approach ◽  
Immunologic Activity ◽  
Dense Label

Immunocytochemistry is a powerful investigative approach in which one of the most exacting examples of specificity, that of the reaction of an antibody with its antigen, isused to localize tissue and cell specific molecules in situ. Following the introduction of fluorescent labeled antibodies in T950, a large number of molecules of biological interest had been studied with light microscopy, especially antigens involved in the pathogenesis of some diseases. However, with advances in electron microscopy, newer methods were needed which could reveal these reactions at the ultrastructural level. An electron dense label that could be coupled to an antibody without the loss of immunologic activity was desired.

mRNA localization by Electron microscopic in situ hybridization

1991 ◽  
Vol 49 ◽  
pp. 430-431
Author(s):  
J. A. Pollock ◽  
M. Martone ◽  
T. Deerinck ◽  
M. H. Ellisman
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Nucleic Acids ◽  
Recombinant Dna ◽  
Light And Electron Microscopy ◽  
Electron Microscopic ◽  
High Voltage Electron Microscopy ◽  
Functional Sites ◽  
Voltage Electron

Localization of specific proteins in cells by both light and electron microscopy has been facilitate by the availability of antibodies that recognize unique features of these proteins. High resolution localization studies conducted over the last 25 years have allowed biologists to study the synthesis, translocation and ultimate functional sites for many important classes of proteins. Recently, recombinant DNA techniques in molecular biology have allowed the production of specific probes for localization of nucleic acids by “in situ” hybridization. The availability of these probes potentially opens a new set of questions to experimental investigation regarding the subcellular distribution of specific DNA's and RNA's. Nucleic acids have a much lower “copy number” per cell than a typical protein, ranging from one copy to perhaps several thousand. Therefore, sensitive, high resolution techniques are required. There are several reasons why Intermediate Voltage Electron Microscopy (IVEM) and High Voltage Electron Microscopy (HVEM) are most useful for localization of nucleic acids in situ.

The Combining Ability of Glycoproteins IIb, IIIa and Ca2+ in EDTA-Treated Nonaggregable Platelets

1983 ◽  
Vol 50 (04) ◽  
pp. 848-851 ◽  
Author(s):  
Marjorie B Zucker ◽  
David Varon ◽  
Nicholas C Masiello ◽  
Simon Karpatkin
Keyword(s):  
Density Gradient ◽  
Combining Ability ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Electrophoretic Pattern ◽  
Sucrose Density Gradient ◽  
Irreversible Loss ◽  
Sucrose Density Gradient Centrifugation ◽  
Crossed Immunoelectrophoresis ◽  
Triton X

SummaryPlatelets deprived of calcium and incubated at 37° C for 10 min lose their ability to bind fibrinogen or aggregate with ADP when adequate concentrations of calcium are restored. Since the calcium complex of glycoproteins (GP) IIb and IIIa is the presumed receptor for fibrinogen, it seemed appropriate to examine the behavior of these glycoproteins in incubated non-aggregable platelets. No differences were noted in the electrophoretic pattern of nonaggregable EDTA-treated and aggregable control CaEDTA-treated platelets when SDS gels of Triton X- 114 fractions were stained with silver. GP IIb and IIIa were extracted from either nonaggregable EDTA-treated platelets or aggregable control platelets with calcium-Tris-Triton buffer and subjected to sucrose density gradient centrifugation or crossed immunoelectrophoresis. With both types of platelets, these glycoproteins formed a complex in the presence of calcium. If the glycoproteins were extracted with EDTA-Tris-Triton buffer, or if Triton-solubilized platelet membranes were incubated with EGTA at 37° C for 30 min, GP IIb and IIIa were unable to form a complex in the presence of calcium. We conclude that inability of extracted GP IIb and IIIa to combine in the presence of calcium is not responsible for the irreversible loss of aggregability that occurs when whole platelets are incubated with EDTA at 37° C.

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