scholarly journals Soluble low-Km 5′-nucleotidase from electric-ray (Torpedo marmorata) electric organ and bovine cerebral cortex is derived from the glycosyl-phosphatidylinositol-anchored ectoenzyme by phospholipase C cleavage

1992 ◽  
Vol 284 (3) ◽  
pp. 621-624 ◽  
Author(s):  
M Vogel ◽  
H Kowalewski ◽  
H Zimmermann ◽  
N M Hooper ◽  
A J Turner
Keyword(s):  
Cerebral Cortex ◽  
Phospholipase C ◽  
High Speed ◽  
Electric Organ ◽  
Bovine Brain ◽  
Soluble Enzyme ◽  
Torpedo Marmorata ◽  
Gpi Anchor ◽  
Glycosyl Phosphatidylinositol ◽  
Membrane Bound

Soluble and membrane-bound low-Km 5′-nucleotidase was isolated from high-speed supernatants and membrane fractions derived from the electric organ of the electric ray (Torpedo marmorata) or from bovine brain cerebral cortex. Purification of both enzymes included chromatography on concanavalin A-Sepharose and AMP-Sepharose. The contribution to the total of soluble enzyme activity was lower in electric organ (1.6%) than in bovine cerebral cortex (27.9%). Membrane-bound and soluble forms have very similar Km values for AMP and are inhibited by micromolar concentrations of ATP. Both forms cross-react with, and are inhibited by, an antibody against the membrane-bound surface-located (ecto-) 5′-nucleotidase from electric organ. The HNK-1 carbohydrate epitope is present on both forms of the Torpedo enzyme, but is entirely absent from bovine cerebral-cortex 5′-nucleotidase. An antibody specific for the inositol 1,2-(cyclic)monophosphate that is formed on phospholipase C cleavage of an intact glycosyl-phosphatidylinositol (GPI) anchor binds to the soluble, but not to the membrane-bound, form of the enzyme from both sources. Our results suggest that soluble low-Km 5′-nucleotidase in both electric organ and bovine brain is derived from the membrane-bound GPI-anchored form of the enzyme by the action of a phospholipase C and is not a soluble cytoplasmic enzyme.

scholarly journals Insulin stimulates the release of the glycosyl phosphatidylinositol-anchored membrane dipeptidase from 3T3-L1 adipocytes through the action of a phospholipase C

1997 ◽  
Vol 326 (2) ◽  
pp. 531-537 ◽  
Author(s):  
Sara MOVAHEDI ◽  
Nigel M. HOOPER
Keyword(s):  
Cell Surface ◽  
Phospholipase C ◽  
Blot Analysis ◽  
Enzymic Activity ◽  
Time Dependent ◽  
Gpi Anchor ◽  
Glycosyl Phosphatidylinositol ◽  
Cyclic Monophosphate ◽  
Glut 4

Membrane dipeptidase (MDP; EC 3.4.13.19) enzymic activity that was inhibited by cilastatin has been detected on the surface of 3T3-L1 cells. On differentiation of the cells from fibroblasts to adipocytes the activity of MDP increased 12-fold. Immunoelectrophoretic blot analysis indicated that on adipogenesis the increase in the amount of MDP preceded the appearance of GLUT-4. MDP on 3T3-L1 adipocytes was anchored in the bilayer by a glycosyl phosphatidylinositol (GPI) moiety as evidenced by its release into the medium in a hydrophilic form on treatment of the cells with bacterial phosphatidylinositol-specific phospholipase C and the appearance of the inositol 1,2-cyclic monophosphate cross-reacting determinant. Incubation of 3T3-L1 adipocytes with either insulin or the sulphonylurea glimepiride led to a rapid concentration- and time-dependent release of MDP from the cell surface. The hydrophilic form of MDP released from the cells on stimulation with insulin was recognized by antibodies against the inositol 1,2-cyclic monophosphate cross-reacting determinant, indicating that it had been generated by cleavage of its GPI anchor through the action of a phospholipase C.

scholarly journals Arrhenius plots of acetylcholinesterase activity in mammalian erythrocytes and in Torpedo electric organ. Effect of solubilization by proteinases and by a phosphatidylinositol-specific phospholipase C

1985 ◽  
Vol 231 (1) ◽  
pp. 237-240 ◽  
Author(s):  
P L Barton ◽  
A H Futerman ◽  
I Silman
Keyword(s):  
Phospholipase C ◽  
Electric Organ ◽  
Acetylcholinesterase Activity ◽  
Arrhenius Plots ◽  
Before And After ◽  
Break Points ◽  
Membrane Bound ◽  
Torpedo Electric Organ ◽  
Erythrocyte Ache ◽  
Lipid Environment

The temperature-dependence of the catalytic activity of acetylcholinesterase (AChE) from rat erythrocyte-ghost membranes and from Torpedo electric-organ membranes was examined. In the case of rat erythrocyte AChE, a non-linear Arrhenius plot was observed both before and after solubilization by a phosphatidylinositol-specific phospholipase C or by proteinase treatment. Similarly, no significant differences were observed in Arrhenius plots of Torpedo electric-organ AChE before or after solubilization. These results support our suggestion that the catalytic subunit of AChE does not penetrate deeply into the lipid bilayer of the plasma membrane and also suggest that care must be taken in ascribing break points in Arrhenius plots of membrane-bound enzymes to changes in their lipid environment.

scholarly journals Structure of the glycosyl-phosphatidylinositol membrane anchor of acetylcholinesterase from the electric organ of the electric-fish, Torpedo californica

1993 ◽  
Vol 296 (2) ◽  
pp. 473-479 ◽  
Author(s):  
A Mehlert ◽  
L Varon ◽  
I Silman ◽  
S W Homans ◽  
M A J Ferguson
Keyword(s):  
Electric Fish ◽  
Electric Organ ◽  
Methylation Analysis ◽  
Membrane Anchor ◽  
Gpi Anchor ◽  
Torpedo Californica ◽  
Glycosyl Phosphatidylinositol ◽  
Anchor Structure

The structure of the glycan moiety of the glycosyl-phosphatidylinositol (GPI) membrane anchor from Torpedo californica (electric fish) electric-organ acetylcholinesterase was solved using n.m.r., methylation analysis and chemical and enzymic micro-sequencing. Two structures were found to be present: Glc alpha 1-2Man alpha 1-2Man alpha 1-6Man alpha 1-4GlcN alpha 1-6myo-inositol and Glc alpha 1-2Man alpha 1-2Man alpha 1-6(GalNAc beta 1-4)Man alpha 1-4GlcN alpha 1-6myo-inositol. The presence of glucose in this GPI anchor structure is a novel feature. The anchor was also shown to contain 2.3 residues of ethanolamine per molecule.

scholarly journals The soluble ‘low-Km’ 5′-nucleotidase of rat kidney represents solubilized ecto-5′-nucleotidase

1991 ◽  
Vol 273 (2) ◽  
pp. 409-413 ◽  
Author(s):  
G Piec ◽  
M Le Hir
Keyword(s):  
Phospholipase C ◽  
High Speed ◽  
Catalytic Properties ◽  
Rat Kidney ◽  
Gel Filtration ◽  
Main Peak ◽  
Soluble Enzyme ◽  
Renal Brush Border Membranes ◽  
Triton X ◽  
Renal Brush Border

A soluble ‘low-Km’ 5′-nucleotidase has been described previously in several organs. It has been presumed to be of cytosolic origin and thus to play a role in the intracellular production of adenosine. Its catalytic properties are similar to those of the ecto-5′-nucleotidase of cell membranes. In the present study we compared molecular properties of the two enzymes in the kidney of the rat. The Mr of the main peak of soluble ‘low-Km‘ 5′-nucleotidase in gel-filtration chromatography was similar to that of the ecto-5′-nucleotidase solubilized by a phosphatidylinositol-specific phospholipase C from renal brush-border membranes. In phase-partition experiments using Triton X-114, the soluble enzyme appeared to be hydrophobic. Its hydrophobicity was decreased on treatment with a phosphatidylinositol-specific phospholipase C, suggesting that the soluble ‘low-Km’ 5′-nucleotidase contains the phosphatidylinositol anchor which is characteristic for the ecto-enzyme. An anti-ecto-5′-nucleotidase antiserum provoked an almost complete inhibition of the soluble enzyme. Immunoblotting using anti-ecto-5′-nucleotidase antiserum revealed in the high-speed supernatants a polypeptide with a similar Mr to the subunit of the ecto-5′-nucleotidase. The soluble ‘low-Km’ 5′-nucleotidase, like the ecto-5′-nucleotidase, bound specifically to concanavalin A. We conclude that the soluble ‘low-Km’ 5′-nucleotidase is not a cytosolic enzyme, but that it most probably originates from the solubilization of the ecto-5′-nucleotidase, and that it therefore cannot participate in the intracellular production of adenosine.

Intracellular localization of the glycosyl-phosphatidylinositol-specific phospholipase C of Trypanosoma brucei

1989 ◽  
Vol 93 (2) ◽  
pp. 233-240
Author(s):  
R. Bolow ◽  
G. Griffiths ◽  
P. Webster ◽  
Y.D. Stierhof ◽  
F.R. Opperdoes ◽  
...  
Keyword(s):  
Phospholipase C ◽  
Trypanosoma Brucei ◽  
Cellular Localization ◽  
Intracellular Localization ◽  
Surface Glycoprotein ◽  
Glycosyl Phosphatidylinositol ◽  
Membrane Bound ◽  
Cyclic Phosphate ◽  
Inositol Ring ◽  
Cytoplasmic Side

Glycosyl-phosphatidylinositol-specific phospholipase C (GPI-PLC) is a membrane-bound enzyme of bloodstream forms of Trypanosoma brucei, which cleaves the GPI-membrane anchor of the variant surface glycoprotein forming diacylglycerol and 1,2-cyclic phosphate on the inositol ring. The cellular localization of the enzyme was studied by fractionation of sub-cellular organelles and immunofluorescence microscopy and was found to be primarily cytoplasmic. This was confirmed by immuno-electron microscopy using cryo-sections, which showed that the labelling was predominantly on the cytoplasmic side of intracellular membranes but was absent from the plasma membrane including the region lining the flagellar pocket. The significance of these results for the possible function of the phospholipase is discussed.

scholarly journals Activation of the glycosyl-phosphatidylinositol-anchored membrane dipeptidase upon release from pig kidney membranes by phospholipase C

1994 ◽  
Vol 303 (2) ◽  
pp. 633-638 ◽  
Author(s):  
I A Brewis ◽  
A J Turner ◽  
N M Hooper
Keyword(s):  
Staphylococcus Aureus ◽  
Phospholipase C ◽  
Active Site ◽  
Enzymic Activity ◽  
Polyclonal Antiserum ◽  
Gpi Anchor ◽  
Glycosyl Phosphatidylinositol ◽  
Pig Kidney ◽  
Conformational Constraints ◽  
Maximal Activation

Incubation of pig kidney microvillar membranes with Bacillus thuringiensis or Staphylococcus aureus phosphatidylinositol-specific phospholipase C (PI-PLC) resulted in the release of a number of glycosyl-phosphatidylinositol (GPI)-anchored hydrolases, including alkaline phosphatase (EC 3.1.3.1), amino-peptidase P (EC 3.4.11.9), membrane dipeptidase (EC 3.4.13.19), 5′-nucleotidase (EC 3.1.3.5) and trehalase (EC 3.2.1.28). Of these five ectoenzymes only for membrane dipeptidase was there a significant (approx. 100%) increase in enzymic activity upon release from the membrane. Maximal activation occurred at a PI-PLC concentration 10-fold less than that required for maximal release. In contrast solubilization of the membranes with n-octyl beta-D-glucopyranoside had no effect on the enzymic activity of membrane dipeptidase. A competitive e.l.i.s.a. with a polyclonal antiserum to membrane dipeptidase indicated that the increase in enzymic activity was not due to an increase in the amount of membrane dipeptidase protein. Although PI-PLC cleaved the GPI anchor of the affinity-purified amphipathic form of pig membrane dipeptidase there was no concurrent increase in enzymic activity. In the absence of PI-PLC, membrane dipeptidase in the microvillar membranes hydrolysed Gly-D-Phe with a Km of 0.77 mM and a Vmax. of 602 nmol/min per mg of protein. However, in the presence of a concentration of PI-PLC which caused maximal release from the membrane and maximal activation of membrane dipeptidase the Km was decreased to 0.07 mM while the Vmax. remained essentially unchanged at 624 nmol/min per mg of protein. Overall these results suggest that cleavage by PI-PLC of the GPI anchor on membrane dipeptidase may relax conformational constraints on the active site of the enzyme which exist when it is anchored in the lipid bilayer, thus resulting in an increase in the affinity of the active site for substrate.

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