Isolation and characterization of a small intestinal surfactant-like particle containing alkaline phosphatase and other digestive enzymes

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.

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Rat small intestinal mucin: Isolation and characterization of a water-soluble mucin fraction

Archives of Biochemistry and Biophysics ◽

10.1016/0003-9861(72)90086-0 ◽

1972 ◽

Vol 150 (2) ◽

pp. 679-689 ◽

Cited By ~ 29

Author(s):

Agustin Bella ◽

Young S. Kim

Keyword(s):

Water Soluble ◽

Isolation And Characterization ◽

Intestinal Mucin ◽

Small Intestinal

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Characterization of a Highly Thermostable Alkaline Phosphatase from the Euryarchaeon Pyrococcus abyssi

Applied and Environmental Microbiology ◽

10.1128/aem.67.10.4504-4511.2001 ◽

2001 ◽

Vol 67 (10) ◽

pp. 4504-4511 ◽

Cited By ~ 52

Author(s):

Sébastien Zappa ◽

Jean-Luc Rolland ◽

Didier Flament ◽

Yannick Gueguen ◽

Joseph Boudrant ◽

...

Keyword(s):

Alkaline Phosphatase ◽

Disulfide Bonds ◽

Metal Ion ◽

Divalent Cations ◽

E Coli ◽

Isolation And Characterization ◽

Pyrococcus Abyssi ◽

Increased Activity ◽

Metal Ion Chelators

ABSTRACT This work reports the first isolation and characterization of an alkaline phosphatase (AP) from a hyperthermophilic archaeon. An AP gene from Pyrococcus abyssi, a euryarchaeon isolated from a deep-sea hydrothermal vent, was cloned and the enzyme expressed in Escherichia coli. Analysis of the sequence showed conservation of the active site and structural elements of theE. coli AP. The recombinant AP was purified and characterized. Monomeric and homodimeric active forms were detected, with a monomer molecular mass of 54 kDa. Apparent optimum pH and temperature were estimated at 11.0 and 70°C, respectively. Thus far,P. abyssi AP has been demonstrated to be the most thermostable AP, with half-lives at 100 and 105°C of 18 and 5 h, respectively. Enzyme activity was found to be dependent on divalent cations: metal ion chelators inhibited activity, whereas the addition of exogenous Mg(II), Zn(II), and Co(II) increased activity. The enzyme was inhibited by inorganic phosphate, but not by molybdate and vanadate. Strong inhibitory effects were observed in the presence of thiol-reducing agents, although cysteine residues of the P. abyssi AP were not found to be incorporated within intra- or interchain disulfide bonds. In addition,P. abyssi AP was demonstrated to dephosphorylate linear DNA fragments with dephosphorylation efficiencies of 93.8 and 84.1% with regard to cohesive and blunt ends, respectively.

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