scholarly journals Long-chain acyl-coenzyme A synthetase activity of spinach chloroplasts is concentrated in the envelope

1977 ◽  
Vol 162 (2) ◽  
pp. 457-459 ◽  
Author(s):  
P G Roughan ◽  
C R Slack
Keyword(s):  
Phospholipase D ◽  
Specific Activity ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Synthetase Activity ◽  
Sucrose Density Gradient Centrifugation ◽  
Chain Acyl ◽  
Acyl Coenzyme A ◽  
Long Chain

Purified chloroplasts were disrupted and then fractionated by discontinuous sucrose-density-gradient centrifugation. Envelopes contained long-chain acyl-CoA synthetase at a specific activity 80 times the activity in the lamellae or the stroma. Acetyl-CoA synthetase was concentrated in the stroma, and chlorophyll was confined to the lamellae membranes. Phospholipase D was not detected in any fraction.

scholarly journals Aminoacyl-tRNA synthetase complex in Saccharomyces cerevisiae

1995 ◽  
Vol 309 (1) ◽  
pp. 321-324 ◽  
Author(s):  
C L Harris ◽  
C J Kolanko
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Trna Synthetase ◽  
Synthetase Activity ◽  
Aminoacyl Trna Synthetase ◽  
Sucrose Density Gradient Centrifugation ◽  
Cell Extracts ◽  
Molecular Masses

The size distribution of aminoacyl-tRNA synthetase activity was investigated in cell extracts prepared from Saccharomyces cerevisiae. Bio-Gel A-5M chromatography of 105,000 g supernatants separated isoleucyl-tRNA synthetase activity into three peaks, with apparent molecular masses (Da) of about 100,000, 350,000 and 10(6) or greater. Similar results were obtained with synthetases specific for glutamic acid, serine and tyrosine. Sucrose-density-gradient centrifugation of yeast supernatants also provided evidence for the existence of synthetase complexes. These data provide the first evidence for the existence of a high-molecular-mass aminoacyl-tRNA synthetase complex in yeast, perhaps similar to those reported in higher eukaryotes.

scholarly journals Purification, properties and assay of d-ribulose 5-phosphate 3-epimerase from human erythrocytes

1983 ◽  
Vol 211 (3) ◽  
pp. 617-623 ◽  
Author(s):  
A Karmali ◽  
A F Drake ◽  
N Spencer
Keyword(s):  
Density Gradient ◽  
Specific Activity ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sodium Dodecyl ◽  
Minor Component ◽  
Sucrose Density Gradient ◽  
Human Erythrocytes ◽  
Sucrose Density Gradient Centrifugation ◽  
Dimeric Form

A direct assay procedure is described for D-ribulose 5-phosphate 3-epimerase (EC 5.1.3.1) which exploits differences in the c.d. spectra of substrate and product. The enzyme has been purified from human erythrocytes and was resolved by gel filtration and sucrose-density-gradient centrifugation into a major component of apparent Mr 45 000 and a minor component of Mr 23 000. Electrophoresis in sodium dodecyl sulphate gave a single component corresponding to Mr 23 000. Kinetic and sucrose-density-gradient centrifugation data indicate dissociation of the dimeric form of the enzyme into monomers of low specific activity; substrate favours the active dimeric form of the enzyme. At concentrations of the enzyme where both forms of the enzyme are present initial velocity data yielded a Hill plot with an interaction coefficient of approx. 2.0, indicating co-operative binding of substrate under these conditions.

The subcellular localization of transglutaminase in normal liver and in glucagon-treated and partial hepatectomized rats

1983 ◽  
Vol 3 (12) ◽  
pp. 1085-1090 ◽  
Author(s):  
Sally E. Bruce ◽  
Timothy J. Peters
Keyword(s):  
Plasma Membrane ◽  
Subcellular Distribution ◽  
Specific Activity ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Normal Liver ◽  
Sucrose Density Gradient ◽  
Subcellular Fractionation ◽  
Sucrose Density Gradient Centrifugation ◽  
Significant Difference

Rat liver was homogenized in isotonic sucrose and subjected to analytical subcellular fractionation by sucrose density-gradient centrifugation. Transglutaminase, when assayed with putrescine and dimethylcasein as substrates, showed three distinct localizations, cytosol (73%), plasma membrane (20%), and nuclei (7%). The distribution was unaffected by homogenization in the presence of potassium chloride, indicating that the particulate activity was not due to adsorbed cytosolic enzyme. The specific activity and subcellular distribution of transglutaminase in rats which had received intra-peritoneal glucagon, stimulating endocytosis; or which had been subjected to sub-total hepatectomy 2, 16, or 32 h previously, showed no significant difference from control animals.

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.

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

scholarly journals Purification of Torpedo californica post-synaptic membranes and fractionation of their constituent proteins

1980 ◽  
Vol 185 (3) ◽  
pp. 667-677 ◽  
Author(s):  
J Elliott ◽  
S G Blanchard ◽  
W Wu ◽  
J Miller ◽  
C D Strader ◽  
...  
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Selective Extraction ◽  
Synaptic Membranes ◽  
Detergent Solution ◽  
Major Step ◽  
Sucrose Density Gradient Centrifugation ◽  
Torpedo Californica

A rapid methof for preparation of membrane fractions highly enriched in nicotinic acetylcholine receptor from Torpedo californica electroplax is described. The major step in this purification involves sucrose-density-gradient centrifugation in a reorienting rotor. Further purification of these membranes can be achieved by selective extraction of proteins by use of alkaline pH or by treatment with solutions of lithium di-idosalicylate. The alkali-treated membranes retain functional characteristics of the untreated membranes and in addition contain essentially only the four polypeptides (mol.wts. 40000, 50000, 60000 and 65000) characteristic of the receptor purified by affinity chromatography. Dissolution of the purified membranes or of the alkali-treated purified membranes in sodium cholate solution followed by sucrose-density-gradient centrifugation in the same detergent solution yields solubilized receptor preparations comparable with the most highly purified protein obtained by affinity-chromatographic procedures.

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