scholarly journals The effect of Triton WR-1339 on the subcellular distribution of Trypan Blue and 125I-labelled albumin in rat liver

1973 ◽  
Vol 136 (1) ◽  
pp. 57-65 ◽  
Author(s):  
Malcolm Davies
Keyword(s):  
Acid Phosphatase ◽  
Density Gradient ◽  
Cathepsin D ◽  
Trypan Blue ◽  
Gradient Centrifugation ◽  
Distribution Patterns ◽  
Equilibrium Density ◽  
Sucrose Density Gradient Centrifugation ◽  
Ionic Detergent ◽  
Triton Wr 1339

1. The density-gradient distribution patterns of acid phosphatase, Trypan Blue and denatured 125I-labelled albumin were studied by discontinuous sucrose- and isopycnic sucrose-density-gradient centrifugation on combined heavy and light mitochondrial (M+L) fractions of liver isolated from normal rats and from rats injected with Triton WR-1339. 2. The results obtained from the subfractionation of the M+L pellet of normal animals indicate that the equilibrium density of Trypan Blue and acid-insoluble radioactivity is the same as that for acid phosphatase, which suggests they are bound by a common membrane to form a distinct subcellular population of lysosomal nature. 3. In contrast, the analysis of the isopycnic gradients obtained on subfractionation of M+L pellets of liver isolated from rats treated with Triton WR-1339 show that the acid-insoluble radioactivity has an equilibrium density around 1.21, whereas the acid hydrolases, including cathepsin D, show the characteristic shift to an equilibrium density of around 1.12. Trypan Blue is distributed along the gradient with distinct peaks at densities 1.22 and 1.12. 4. Similar equilibrium-density distribution patterns were obtained with M+L pellets isolated from rats pretreated with Triton WR-1339 but not injected with Trypan Blue. 5. Treatment of the rats with Triton WR-1339 does not affect albumin digestion of isolated intact lysosomes despite the fact that most of the cathepsin D and the albumin ingested by phagocytosis are located in different vacuoles. 6. It is concluded from these experiments that in the liver of animals treated with Triton WR-1339 125I-labelled albumin is located within heterophagosomes which do not fuse with heterolysosomes containing the non-ionic detergent Triton WR-1339. The inability of these two lysosomal populations to fuse is not due to Trypan Blue.

Triton WR-1339 injected into rats enters renal renin granules

1988 ◽  
Vol 255 (3) ◽  
pp. E353-E356
Author(s):  
B. J. Morris
Keyword(s):  
Plasma Proteins ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Extracellular Fluid ◽  
Sucrose Density Gradient ◽  
Equilibrium Density ◽  
Juxtaglomerular Cells ◽  
Sucrose Density Gradient Centrifugation ◽  
Triton Wr 1339 ◽  
Extracellular Substances

To test directly the possibility that substances in extracellular fluid can gain access to renin storage granules in renal juxtaglomerular cells, rats were injected with Triton WR-1339, which binds to plasma proteins. A heavy granule fraction was prepared, and isopycnic sucrose density gradient centrifugation was performed. The renin granule peak was found to be altered from a mean equilibrium density of 1.202 g/ml in control rats to 1.196 g/ml for rats injected with Triton WR-1339 (P less than 0.005). The distribution of angiotensinogen, which is bound in kidney granules having a different buoyant density, was also examined and found to be unaltered. After injection, Triton WR-1339 binds to circulating plasma proteins. The results for renin support the possibility of pinocytotic uptake of protein-Triton WR-1339 complexes by the juxtaglomerular cells with subsequent fusion of the endocytotic lysosomal vacuoles with renin granules accounting for the translocation of ingested substances into the granule matrix. If so, the potential would therefore exist for interaction(s) of ingested extracellular substances with renin or other components in the granules. The present study has therefore demonstrated directly that endogenous extracellular substances may enter renin granules.

scholarly journals LYSOSOMES IN SKELETAL MUSCLE TISSUE

1970 ◽  
Vol 45 (2) ◽  
pp. 321-333 ◽  
Author(s):  
Peter G. Canonico ◽  
John W. C. Bird
Keyword(s):  
Skeletal Muscle ◽  
Acid Phosphatase ◽  
Connective Tissue ◽  
Phosphatase Activity ◽  
Cathepsin D ◽  
Acid Phosphatase Activity ◽  
Equilibrium Density ◽  
Acid Hydrolases ◽  
Triton Wr 1339 ◽  
Arylsulfatase Activity

Postnuclear supernates from homogenates of skeletal muscle from rats subjected to starvation, injections of Triton WR-1339, dextran-500, and dextran + corticosterone were fractionated by means of rate and isopycnic zonal centrifugation in sucrose—0.02 M KCl gradients. Zonal fractions were analyzed for protein, RNA, cytochrome oxidase, and up to six acid hydrolases. The results indicate the presence of two groups of lysosome-like particles. One group contributes approximately 95% of the cathepsin D and acid phosphatase activity and 75% of the acid ribonuclease, ß-glucuronidase, and arylsulfatase activity in muscle. It is characterized by a modal equilibrium density of 1.18 that is decreased by starvation, but is not shifted by dextran-500 or Triton WR-1339. The second group has a higher proportion of acid ribonuclease, ß-glucuronidase, and arylsulftase; the equilibrium density can be shifted by dextran-500 and Triton WR-1339. It is suggested that this group of lysosomes is derived from macrophages and other connective tissue cells, whereas the former group represents lysosome-like particles from muscle cells.

Purification and properties of boticin P produced by Clostridium botulinum

1974 ◽  
Vol 20 (3) ◽  
pp. 385-390 ◽  
Author(s):  
A. H. S. Lau ◽  
R. Z. Hawirko ◽  
C. T. Chow
Keyword(s):  
Density Gradient ◽  
Clostridium Botulinum ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Electron Microscopic Examination ◽  
Equilibrium Density ◽  
Type B ◽  
Electron Microscopic ◽  
Sucrose Density Gradient Centrifugation

A bacteriocin produced spontaneously by a nontoxigenic strain of Clostridium botulinum, type E, PM-15, has been isolated and designated boticin P. It was purified by ammonium sulfate precipitation, gel filtration on Sephadex G-100, sucrose density gradient centrifugation, and cesium chloride equilibrium density gradient centrifugation. Boticin P is composed mainly of proteins (98.8%) with a trace amount of carbohydrates (0.4%), and has an apparent molecular weight in excess of 4 × 106 daltons as estimated by gel filtration on Sepharose 2B. Electron microscopic examination of boticin P reveals a phage tail-like structure of 100 nm in length.Boticin P exerted a static effect on vegetative growth and spore outgrowth but not on the initial events of germination. The boticin was active on 10/12 toxigenic and 3/6 nontoxigenic type E and 2/2 nonproteolytic type B strains of C. botulinum. The activity spectrum on 27 strains supports the proposal that type E and the nonproteolytic type B strains belong to the same taxosubspecies.

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 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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