The annexin II2p112 complex is the major protein component of the Triton X-100-insoluble low-density fraction prepared from MDCK cells in the presence of Ca2+

Actively motile cells from a cured strain of Crithidia deanei released proteins in phosphate buffer (pH 7.4). The molecular mass of the released polypeptides, which included some proteinases, ranged from 19 to 116 kDa. One of the major protein bands was purified to homogeneity by a combination of anion-exchange and gel filtration chromatographs. The apparent molecular mass of this protein was estimated to be 62 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE). The incorporation of gelatin into SDSPAGE showed that the purified protein presented proteolytic activity in a position corresponding to a molecular mass of 60 kDa. The enzyme was optimally active at 37 °C and pH 6.0 and showed 25% of residual activity at 28 °C for 30 min. The proteinase was inhibited by 1,10-phenanthroline and EDTA, showing that it belonged to the metalloproteinase class. A polyclonal antibody to the leishmanial gp63 reacted strongly with the released C. deanei protease. After Triton X-114 extraction, an enzyme similar to the purified metalloproteinase was detected in aqueous and detergent-rich phases. The detection of an extracellular metalloproteinase produced by C. deanei and some other Crithidia species suggests a potential role of this released enzyme in substrate degradation that may be relevant to the survival of trypanosomatids in the host.Key words: endosymbiont, trypanosomatid, extracellular, proteinase.

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Characterization of a novel glycosylphosphatidylinositol (GPI)-anchored protein in a triton insoluble low density fraction (TIF) from rat brain

Neuroscience Research ◽

10.1016/s0168-0102(98)82431-0 ◽

1998 ◽

Vol 31 ◽

pp. S313

Author(s):

Nobuo Funatsu ◽

Haruko Kumanogoh ◽

Shohei Maekawa

Keyword(s):

Rat Brain ◽

Low Density ◽

Density Fraction

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The outer membrane of Haemophilus influenzae type b: cell envelope associations of major proteins

Canadian Journal of Microbiology ◽

10.1139/m83-046 ◽

1983 ◽

Vol 29 (2) ◽

pp. 280-287 ◽

Cited By ~ 6

Author(s):

J. W. Coulton ◽

D. T. F. Wan

Keyword(s):

Molecular Weight ◽

Sodium Dodecyl Sulphate ◽

Cell Envelope ◽

Sodium Dodecyl ◽

Haemophilus Influenzae Type ◽

Major Protein ◽

Haemophilus Influenzae Type B ◽

Type B ◽

Molecular Weights ◽

Triton X

Membrane proteins fom the cell envelope of Haemophilus influenzae type b ATCC 9795 were examined by sodium dodecyl sulphate – polyacrylamide gel electrophoresis. When envelopes were extracted with a phosphate-based buffer containing 2% Triton X-100, a major protein of molecular weight 43 000 was detected in fractions containing cytoplasmic membrane proteins. The cell wall material which was Triton X-100 insoluble contained six major proteins of molecular weights 46 000, 40 000, 36 000, 30 000, 27 000, and 16 000. One of these proteins showed a shift in molecular weight from 27 000 to 36 000 when it was heated over a temperature range from 50 °C to 100 °C in buffer containing 2% sodium dodecyl sulphate, 5% 2-mercaptoethanol. This alteration in mobility could be demonstrated either by the membrane-bound form of the protein or by a detergent-soluble form of the protein. Enriched preparations of the 36 000 molecular weight form were obtained by a series of purification steps. Extraction of the Triton X-100 insoluble material with buffer containing 2% Triton X-100, 5.0 mM EDTA yielded chiefly one major protein molecular weight 30 000 and many minor protein species. Pretreatment of the Triton X-100 insoluble fraction with lysozyme followed by extraction with buffer containing 2% Triton X-100, 5.0 mM EDTA released two proteins of molecular weights 16 000 and 27 000 and few minor proteins. By these operational manipulations, the proteins of molecular weights 16 000 and 27 000 may be considered as peptidoglycan-associated proteins.

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LIPID EXTRACTION AND DISTRIBUTION STUDIES OF EGG YOLK LIPOPROTEINS

Canadian Journal of Biochemistry and Physiology ◽

10.1139/y63-078 ◽

1963 ◽

Vol 41 (1) ◽

pp. 657-666 ◽

Cited By ~ 4

Author(s):

W. G. Martin ◽

N. H. Tattrie ◽

W. H. Cook

Keyword(s):

Fatty Acid ◽

Neutral Lipids ◽

Lipid Extraction ◽

Egg Yolk ◽

Low Density ◽

Cholesterol Esters ◽

Density Fraction ◽

Distribution Studies ◽

Egg Yolk Lipoproteins ◽

Fatty Acid Compositions

The three lipoproteins of egg yolk, α- and β-lipovitellin and the low-density fraction (LDF), have been isolated and their lipid compositions determined. α- and β-lipovitellin comprise 22 to 26% lipid, of which 61% is phospholipid, 35% is triglyceride, and 4% is cholesterol and its esters. LDF contains about 89% lipid having 27% phospholipid, 69% triglyceride, and 4% cholesterol and cholesterol esters. The phospholipids of the three lipoproteins are similar, i.e., 74% lecithins, 18% cephalins, and 8% minor phospholipids. The fatty acid compositions of the neutral lipids, lecithins, and cephalins of the α- and β-lipovitellins were also similar, with only minor differences.Gentle extraction of the LDF solutions with ethyl ether readily removes about 85% of the total lipid and 55% of the phospholipid, while subsequent changes are slow. The lipoprotein residue contains 52% lipid which is mostly phospholipid; when the residual ether is removed, five sedimenting components are observed in the ultracentrifuge.

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Association of Engrailed homeoproteins with vesicles presenting caveolae-like properties

Development ◽

10.1242/dev.124.10.1865 ◽

1997 ◽

Vol 124 (10) ◽

pp. 1865-1875 ◽

Cited By ~ 4

Author(s):

A. Joliot ◽

A. Trembleau ◽

G. Raposo ◽

S. Calvet ◽

M. Volovitch ◽

...

Keyword(s):

Binding Sites ◽

Cell Adhesion Molecule ◽

Proteolytic Enzymes ◽

Subcellular Fractionation ◽

Low Density ◽

Toxin Binding ◽

Neural Cell Adhesion ◽

Neural Tissues ◽

Triton X ◽

Specific Membrane

We report here that the homeoproteins Engrailed-1 and Engrailed-2 are present in specific non-nuclear subcellular compartments. Using electron microscopy, we observed that chick-Engrailed-2 expressed in COS-7 cells associates with membrane fractions that are characterized as caveolae. This characterization is based on morphological, biochemical and immunological criteria such as, in particular, the absence of clathrin coat and the presence of caveolin and cholera toxin-binding sites. These data are fully confirmed by subcellular fractionation experiments, which demonstrate that transfected chick-Engrailed-2 is present in low density membrane fractions that are resistant to Triton X-100, enriched in caveolin and solubilized by the addition of a cholesterol-binding detergent, a set of properties highly characteristic of caveolae. The association of Engrailed-2 with specific membrane fractions observed after transfection in COS-7 cells is also observed for endogenous Engrailed-1 and Engrailed-2 expressed at late embryonic stages in the cerebellum and posterior mesencephalon of the rodent. Indeed, the two proteins are present in membrane fractions that bear all the characteristics of microdomains or caveolae-like domains, i.e. Triton X-100 resistance, saponin solubilization, low density on sucrose gradients, enrichment in glycosphingolipid GM1, absence of transmembrane Neural Cell Adhesion Molecule, presence of the glypiated (GPI-anchored) glycoprotein F3/F11 and of the acylated growth-associated protein GAP-43. Finally we demonstrate that part of the membrane-associated Engrailed, either expressed in COS-7 cells or endogenously present in neural tissues, is not accessible to proteolytic enzymes unless the membranes have been permeabilized with detergent. This study suggests that, in addition to their well-known presence in the nucleus, Engrailed proteins are also associated with caveolae-like vesicles that are primarily transported anterogradely into the axon, and that they can get access to a compartment compatible with secretion.

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