scholarly journals Isolation and characterization of alkaline phosphatase-containing granules (phosphasomes) from human polymorphonuclear leucocytes

1985 ◽  
Vol 76 (1) ◽  
pp. 167-178
Author(s):  
G.P. Smith ◽  
G. Sharp ◽  
T.J. Peters
Keyword(s):  
Alkaline Phosphatase ◽  
Plasma Membrane ◽  
Sucrose Density Gradient ◽  
Subcellular Fractionation ◽  
Polymorphonuclear Leucocytes ◽  
Vesicle Membrane ◽  
Isolation And Characterization ◽  
Membrane Components ◽  
Counter Current

Human polymorphonuclear leucocytes were homogenized in isotonic sucrose and subjected to analytical subcellular fractionation by centrifugation on a discontinuous sucrose density gradient to produce a phosphasome-enriched fraction, contaminated primarily by plasma membrane. Experiments to separate these membrane fractions by centrifugation on gradients of Nycodenz or Percoll, or by toroidal coil counter current chromatography, were unsuccessful. Fractionation carried out on neutrophils previously suspended in isotonic sucrose containing a low concentration of digitonin resulted in the preparation of a highly purified phosphasome fraction, free of plasma membrane components. Electron-microscope cytochemistry of the purified fraction identified the phosphasomes as regular and irregular-shaped spheres and rods, the alkaline phosphatase being associated with the inner aspect of the vesicle membrane. These granule structures were very similar to phosphasomes observed in intact neutrophils. A proportion of the endoplasmic reticulum and Golgi membrane marker enzymes remained associated with the phosphasomes throughout the separation procedures.

scholarly journals Plasma membrane localization of alkaline phosphatase in HeLa cells.

1976 ◽  
Vol 24 (5) ◽  
pp. 659-667 ◽  
Author(s):  
C W Lin ◽  
M Sasaki ◽  
M L Orcutt ◽  
H Miyayama ◽  
R M Singer
Keyword(s):  
Alkaline Phosphatase ◽  
Plasma Membrane ◽  
Hela Cells ◽  
Dense Body ◽  
Enzyme Reaction ◽  
Subcellular Fractionation ◽  
Enzyme Characterization ◽  
Marker Enzyme ◽  
Electron Microscopic

The localization of alkaline phosphatase in HeLa cells was examined by electron microscopic histochemistry and subcellular fractionation techniques. Two monophenotypic sublines of HeLa cells which respectively produced Regan and non-Regan isoenzymes of alkaline phosphatase were used for this study. The electron microscopic histochemical results showed that in both sublines the major location of alkaline phosphatase is in the plasma membrane. The enzyme reaction was occasionally observed in some of the dense body lysosomes. This result was supported by data obtained from a subcellular fractionation study which showed that the microsomal fraction rich in plasma membrane fragments had the highest activity of alkaline phosphatase. The distribution of this enzyme among the subcellular fractions closely paralleled that of the 5'-nucleotidase, a plasma membrane marker enzyme. Characterization of the alkaline phosphatase present in each subcellular fraction showed identical enzyme properties, which suggests that a single isoenzyme exists among fractions obtained from each cell line. The results, therefore, confirm the reports suggesting that plasma membrane is the major site of alkaline phosphatase localization in HeLa cells. The absence of any enzyme reaction in the perimitochondrial space in these cultured tumor cells also indicates that the mitochondrial localization of the Regan isoenzyme reported in ovarian cancer may not be a common phenomenon in Regan-producing cancer cells.

Isolation and characterization of plasma membrane fromAcanthamoeba culbertsoni

1985 ◽  
Vol 7 (3-4) ◽  
pp. 365-373 ◽  
Author(s):  
Sanjay Kumar Mishra ◽  
N. K. Garg ◽  
A. M. Kidwai
Keyword(s):  
Plasma Membrane ◽  
Isolation And Characterization

scholarly journals Isolation and characterization of rabbit kidney brush borders

1972 ◽  
Vol 128 (5) ◽  
pp. 1319-1328 ◽  
Author(s):  
S. J. Quirk ◽  
G. B. Robinson
Keyword(s):  
Plasma Membranes ◽  
Sucrose Density Gradient ◽  
Molar Ratio ◽  
Rabbit Kidney ◽  
Kidney Cortex ◽  
Marker Enzymes ◽  
Isolation And Characterization ◽  
Adenosine Triphosphatases ◽  
Brush Borders

1. Brush borders were isolated from rabbit kidney-cortex homogenates by rate-zonal centrifugation through a sucrose density gradient in a B-XIV zonal rotor, followed by differential centrifugation. 2. The method of preparation gave brush borders of high purity with a reasonable yield. The morphological appearance supported the evidence from enzymic and chemical investigations, that the brush borders were only slightly contaminated with endoplasmic reticulum, mitochondria, lysosomes and nuclei. 3. The molar ratio of cholesterol to phospholipid lay within the range found in other plasma membranes, but the carbohydrate content was double that found in liver plasma membranes. 4. Alkaline phosphatase, maltase, trehalase and aminopeptidase were major enzymic constituents of the brush borders, and had an approximately equal yield and enrichment, but none of these enzymes fulfilled the criteria for marker enzymes. 5. Mg2+-dependent and Na+,K+-dependent adenosine triphosphatases, although found in brush borders, had low yields and low enrichments.

Isolation and Characterization of Plasma Membrane from Rat Mesenteric Arteries

1976 ◽  
Vol 13 (5) ◽  
pp. 279-292 ◽  
Author(s):  
Jiann-Wu Wei ◽  
Ronald A. Janis ◽  
Edwin E. Daniel
Keyword(s):  
Plasma Membrane ◽  
Mesenteric Arteries ◽  
Isolation And Characterization
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