scholarly journals The kinetics of incorporation in vivo of [14C]acetate and [14C]carbon dioxide into the fatty acids of glycerolipids in developing leaves

1975 ◽  
Vol 152 (2) ◽  
pp. 217-228 ◽  
Author(s):  
C. R. Slack ◽  
P. G. Roughan
Keyword(s):  
Fatty Acids ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Concomitant Increase ◽  
Sucrose Density Gradient Centrifugation ◽  
Plastid Membranes ◽  
Kinetics Of ◽  
Spinach Leaves

1. The patterns of incorporation of 14C into glycerolipid fatty acids of developing maize leaf lamina from supplied [1-14C]acetate and from 14CO2 during steady-state photosynthesis were similar. Oleate of phosphatidylcholine and palmitate of phosphatidylglycerol attained linear rates of labelling more rapidly than did other fatty acids, particularly the linoleate and linolenate of monogalactosyl diacylglycerol. 2. After the transfer of lamina from labelled to unlabelled acetate, there was a decrease in labelled oleate and linoleate of phosphatidylcholine and a concomitant increase in the amount of radioactivity in the linoleate and linolenate of monogalactosyl diacylglycerol. 3. The rapidly labelled phospholipids, phosphatidylcholine and phosphatidylglycerol, were shown by differential and sucrose-density-gradient centrifugation to be associated with different organelles, the former being mainly in a low-density membrane fraction, probably microsomal, and the latter mainly in chloroplasts. 4. During a 48h period after supplying spinach leaves with [14C]acetate, radioactivity was lost from the oleate of phosphatidylcholine present in fractions sedimented at 12000g and 105000g, and accumulated in the linolenate of monogalactosyl diacylglycerol of the chloroplast. 5. It is proposed that the phosphatidylcholine of some non-plastid membranes is intimately involved in the process of oleate desaturation and that this lipid serves as a donor of unsaturated C18 fatty acids to other lipids, principally monogalactosyl diacylglycerol, of the chloroplasts.

Detection of antibodies to rabies virus by immunoelectron microscopy

1978 ◽  
Vol 36 (2) ◽  
pp. 364-365
Author(s):  
R. K. Chaudhary ◽  
K. M. Charlton ◽  
M. T. Monette ◽  
A. E. Kelen
Keyword(s):  
Rabies Virus ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Standard Technique ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Live Virus ◽  
Human Diploid Cells ◽  
Diploid Cells

Immunization of humans and domesticated animals at risk of contracting rabies is common practice. The mouse neutralization test (MNT) is still the standard technique used for detecting and measuring antibody to rabies virus in sera of vacinees. However, it suffers from problems of reproducibility associated with tests performed in vivo. It also has the disadvantage of being expensive, time-consuming and hazardous. Hence, there has been a continuous search for a simple, rapid and safe test. In recent years, methods based on haemagglutination, haemadsorption, plaque reduction, immunofluorescence and radioimmunoassay have been developed, but none of them has eliminated the hazard involved in the use of live virus.With emphasis on laboratory safety, attempts were made to use inactivated rabies virus for antibody assay by immunoelectronmicroscopy (IEM), in comparison with the MNT.Inactivated rabies virus grown in human diploid cells was supplied by Connaught Laboratories Limited (CLL). The virus was purified by sucrose density gradient centrifugation and used at a concentration to give 35-50 particles per grid square.

Gastric and pancreatic intrinsic factor-mediated absorption of cobalamin in the dog

1989 ◽  
Vol 257 (3) ◽  
pp. G344-G349 ◽  
Author(s):  
R. M. Batt ◽  
N. U. Horadagoda
Keyword(s):  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Intrinsic Factor ◽  
Sucrose Density Gradient ◽  
Ileal Mucosa ◽  
Sucrose Density Gradient Centrifugation ◽  
Control Loops ◽  
Intrinsic Factors ◽  
Total Uptake

The effects of canine gastric and pancreatic intrinsic factors on uptake and subcellular localization of cobalamin have been investigated in vivo to determine whether these proteins could mediate the physiological absorption of cobalamin in the dog. Cyano [57Co]cobalamin was introduced into ileal loops in dogs under general anesthesia, either free (control) or bound to gastric or pancreatic intrinsic factor. At 2 h, total uptake of cobalamin by ileal mucosa was significantly enhanced after prior binding to either gastric or pancreatic intrinsic factor compared with controls. Displacement of receptor-bound cobalamin with EDTA showed that enhanced total uptake reflected increased internalization of cobalamin by both proteins. Findings after reorienting sucrose density gradient centrifugation of ileal mucosa from loops containing intrinsic factor-cobalamin complexes were consistent with a major lysosomal and perhaps endosomal localization of internalized cobalamin, in agreement with results after oral administration of cobalamin. In marked contrast, cobalamin was recovered predominantly in the soluble fractions and was not associated with particulate subcellular organelles in ileal mucosa from control loops. These findings suggest that both gastric and pancreatic intrinsic factors can promote the physiological absorption of cobalamin by receptor-mediated endocytosis in the dog.

Interaction of DNA with ribosomes in cell-free protein synthetizing systems of Chlorella pyrenoidosa

1969 ◽  
Vol 24 (3) ◽  
pp. 321-327 ◽  
Author(s):  
G. Galling
Keyword(s):  
Actinomycin D ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Chlorella Pyrenoidosa ◽  
Sucrose Density Gradient ◽  
Amino Acid Incorporation ◽  
Sucrose Density Gradient Centrifugation ◽  
Free Protein ◽  
Green Alga Chlorella ◽  
Kinetics Of

1. In cell-free protein synthetizing systems of the green alga Chlorella pyrenoidosa, DNA from various sources enhances the amino acid incorporation.2. This stimulation is neither inhibited by actinomycin D nor by chloramphenicol or cycloheximide (actidione).3. In the presence of ribonuclease, some precipitable polypeptide is formed with DNA, although the endogenous incorporation is completely inhibited by ribonuclease.4. After sucrose density gradient centrifugation, polysomal aggregates of ribosomes with DNA are found. Electron micrographs of such polysomes show a direct association of the DNA molecule with several ribosomes.5. In Chlorella, direct translation of DNA can be obtained also in the presence of neomycin. The kinetics of this reaction are different from those of endogenous m-RNA mediated and of DNA stimulated polypeptide synthesis.

scholarly journals Characterization of an oxygen-stable nitrogenase complex isolated from Azotobacter chroococcum

1979 ◽  
Vol 181 (3) ◽  
pp. 569-575 ◽  
Author(s):  
R L Robson
Keyword(s):  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Azotobacter Chroococcum ◽  
Sucrose Density Gradient Centrifugation ◽  
Molybdenum Iron Protein ◽  
Iron Protein ◽  
Protective Proteins

In crude cell-free extracts of Azotobacter chroococcum, nitrogenase was much less sensitive to irreversible inactivation by O2 than was the purified enzyme. When nitrogenase was partially purified by anaerobic discontinuous sucrose-density-gradient centrifugation, O2-tolerance was retained. This preparation was considerably enriched in four polypeptides, three of which were derived from the Mo-Fe(molybdenum-iron) protein and Fe (iron) protein of nitrogenase. The fourth was purified to homogeneity and shown to be an iron-sulphur protein (mol.wt. 14000) probably containing a 2Fe–2S centre. When this protein was added to purified nitrogenase, the enzyme was rendered O2-tolerant, through stabilization was Mg2+-dependent. The isolated O2-tolerant nitrogenase was an equimolar stoicheiometric complex between the MO–Fe, Fe and protective proteins. It is likely that the formation of this complex in vivo is the mechanism of ‘conformational protection’ in this organism.

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.

scholarly journals Alteration of hepatic glucocorticoid receptor stability and nuclear binding in vitro by citrate

1983 ◽  
Vol 210 (1) ◽  
pp. 259-263 ◽  
Author(s):  
J Hubbard ◽  
M Kalimi
Keyword(s):  
Glucocorticoid Receptor ◽  
Glucocorticoid Receptors ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Deae Cellulose ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Nuclear Binding ◽  
Wide Range

Citrate greatly stabilized rat hepatic unbound glucocorticoid receptors in cell-free conditions at 4 degrees C with optimal effectiveness at 5-15 mM. Control receptors were inactivated at 4 degrees C with a half-life of less than 12 h. However, in the presence of 10 mM-citrate, unbound receptors were almost completely stabilized for 48 h at 4 degrees C. Citrate at a concentration of 1-2 mM yielded half-maximal stabilization. The stabilizing effect of citrate was rather specific, as succinate, alpha-oxoglutarate, oxaloacetate, malate and pyruvate had no apparent stabilizing action. Citrate stabilized receptors over a wide range of H+ concentrations, with complete protection between pH 6.5 and 8.5. In addition, citrate appeared to have a significant effect on glucocorticoid-receptor complex activation into a nuclear binding form. Thus 5-10 mM-citrate enhanced nuclear binding, with optimal activation achieved at 10 mM concentration. As analysed by sucrose-density-gradient centrifugation and DEAE-cellulose chromatography, no apparent change was observed in the physical characteristics of the glucocorticoid receptor in the presence of citrate.

The centriolar complex isolated from starfish spermatozoa

1981 ◽  
Vol 49 (1) ◽  
pp. 33-49 ◽  
Author(s):  
R. Kuriyama ◽  
H. Kanatani
Keyword(s):  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Microtubule Assembly ◽  
High Concentration ◽  
Microtubule Organization ◽  
Sucrose Density Gradient Centrifugation ◽  
The Difference ◽  
Distal Centriole

Centrioles from spermatozoa of the starfish, Asterina pectinifera, were isolated and partially purified by solubilization of chromatin followed by sucrose density-gradient centrifugation. The ultrastructure of the isolated centriolar complex was investigated in whole mount preparations by electron microscopy. The complex unit was composed of a pair of centrioles and a pericentriolar structure, which associated with the distal end of the distal centriole by 9 spoke-like satellites extending radially to a marginal ring. Each satellite bifurcated at a dense node forming 2 fan-like shapes with a periodic striated pattern. The tubular structure of the centrioles easily disintegrated, leaving the pericentriolar structure or axonemal microtubules intact. The distal centriole in a spermatozoon served as an initiating site for flagellar microtubule assembly; that is, a number of “9 + 2′ axonemal tubules were observed adhering just beneath the distal end of the basal body. In experiments in vitro, polymerization of microtubule proteins purified from porcine brain was initiated by the structure at the ends of both proximal and distal centrioles, but not from the satellites or the marginal ring. Also, few if any microtubules were formed from the sides of each centriole, even in the presence of a high concentration of exogenous tubulin. On the other hand, centrioles of spermatozoa, when they were in mature ooplasm, could initiate the formation of sperm asters by microtubules. Therefore, centrioles in spermatozoa seem to be able to initiate microtubules in a 2 ways. A possible explanation of the difference between the 2 types of microtubule organization in vivo, i.e. in the sperm cell itself and in the ooplasm, it discussed.

3 ß-Hydroxysteroid Oxidoreductase in Suspension Cultures of Digitalis lanata EH RH

1990 ◽  
Vol 45 (9-10) ◽  
pp. 963-972 ◽  
Author(s):  
Hildegard Maria Warneck ◽  
Hanns Ulrich Seitz
Keyword(s):  
Incubation Temperature ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Cell Compartment ◽  
Sucrose Density Gradient Centrifugation ◽  
Ph Optimum ◽  
Digitalis Lanata ◽  
Substrate Kinetics ◽  
Soluble Part

Abstract A 3 β-hydroxysteroid oxidoreductase was isolated and characterized in the microsomes of Digitalis lanata cell cultures. The enzyme catalyzes the conversion of 5α-pregnane-3,20-dione to 5a-pregnan-3 β-ol-20-one and requires NAD(P)H2. The enzyme was found to have a pH optimum of 80. The reaction had an optimum incubation temperature of 25 °C with linear reduction for the first 4 h, reaching maximum enzyme activity after 7 h. Substrate kinetics for 5a-pregnane-3,20-dione and NADPH2 resulted in apparent Km-values of 18.5-20 (µM for 5a-pregnane-3,20-dione and 50-120 µM for the co-substrate NADPH2. In order to localize 3β-hydroxysteroid oxidoreductase differential centrifugation as well as linear sucrose density gradient centrifugation were performed. The results obtained lead to the conclusion that 3β-hydroxysteroid oxidoreductase is not associated with a single cell compartment, but consists of a major soluble part and a markedly smaller part of endoplasmic reticulum-associated activity

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