scholarly journals Ectoenzymes of the kidney microvillar membrane. Differential solubilization by detergents can predict a glycosyl-phosphatidylinositol membrane anchor

1988 ◽  
Vol 250 (3) ◽  
pp. 865-869 ◽  
Author(s):  
N M Hooper ◽  
A J Turner
Keyword(s):  
Membrane Proteins ◽  
Phospholipase C ◽  
Transmembrane Proteins ◽  
Sodium Deoxycholate ◽  
Membrane Anchor ◽  
Glycosyl Phosphatidylinositol ◽  
Octyl Glucoside

The pattern of solubilization of nine kidney microvillar ectoenzymes by a range of detergents distinguished two classes of membrane proteins: those released from the membrane by bacterial phosphatidylinositol-specific phospholipase C and those not so released. The latter group of transmembrane proteins were solubilized efficiently (greater than 80%) by all the detergents examined. In contrast, proteins released by phosphatidylinositol-specific phospholipase C were solubilized effectively only by octyl glucoside, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate and sodium deoxycholate. Octyl glucoside solubilized the amphipathic forms of the ectoenzymes examined, suggesting that this may be a useful detergent in the purification of glycosyl-phosphatidylinositol-anchored ectoenzymes.

scholarly journals Erythrocyte membrane inhibitor of reactive lysis: effects of phosphatidylinositol-specific phospholipase C on the isolated and cell- associated protein

Blood ◽  
1990 ◽  
Vol 75 (1) ◽  
pp. 284-289 ◽  
Author(s):  
MH Holguin ◽  
LA Wilcox ◽  
NJ Bernshaw ◽  
WF Rosse ◽  
CJ Parker
Keyword(s):  
Membrane Proteins ◽  
Phospholipase C ◽  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Membrane Attack Complex ◽  
Human Erythrocyte Membrane ◽  
Glycosyl Phosphatidylinositol ◽  
Normal Human ◽  
Erythrocyte Membrane Proteins

Abstract The erythrocyte membrane inhibitor of reactive lysis (MIRL) is an 18-Kd protein that controls complement-mediated hemolysis by restricting the activity of the membrane attack complex. MIRL expression on the erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH) is abnormally low, and the greater susceptibility of PNH erythrocytes to complement is causally related to this deficiency. Inasmuch as other proteins that are deficient in PNH are anchored to the membrane through a glycosyl phosphatidylinositol moiety, studies were undertaken to determine if MIRL shares this structural feature. Normal human erythrocytes that had been radiolabeled with 125I were incubated with phosphatidylinositol- specific phospholipase C (PIPLC), and the supernate and the solubilized membrane proteins were immunoprecipitated using anti-MIRL antiserum. The MIRL that was specifically released into the supernate had an Mr of 19 Kd, while the MIRL that remained bound to the membrane had an Mr of 18 Kd. A quantitative assay showed that approximately 10% of erythrocyte MIRL was susceptible to PIPLC; however, treatment with PIPLC had no effect on either the electrophoretic mobility or the functional activity of purified MIRL. These studies show that the effects of PIPLC on MIRL are similar to those observed for other human erythrocyte membrane proteins that are anchored by a glycosyl phosphatidylinositol moiety.

Erythrocyte membrane inhibitor of reactive lysis: effects of phosphatidylinositol-specific phospholipase C on the isolated and cell- associated protein

Blood ◽  
1990 ◽  
Vol 75 (1) ◽  
pp. 284-289
Author(s):  
MH Holguin ◽  
LA Wilcox ◽  
NJ Bernshaw ◽  
WF Rosse ◽  
CJ Parker
Keyword(s):  
Membrane Proteins ◽  
Phospholipase C ◽  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Membrane Attack Complex ◽  
Human Erythrocyte Membrane ◽  
Glycosyl Phosphatidylinositol ◽  
Normal Human ◽  
Erythrocyte Membrane Proteins

The erythrocyte membrane inhibitor of reactive lysis (MIRL) is an 18-Kd protein that controls complement-mediated hemolysis by restricting the activity of the membrane attack complex. MIRL expression on the erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH) is abnormally low, and the greater susceptibility of PNH erythrocytes to complement is causally related to this deficiency. Inasmuch as other proteins that are deficient in PNH are anchored to the membrane through a glycosyl phosphatidylinositol moiety, studies were undertaken to determine if MIRL shares this structural feature. Normal human erythrocytes that had been radiolabeled with 125I were incubated with phosphatidylinositol- specific phospholipase C (PIPLC), and the supernate and the solubilized membrane proteins were immunoprecipitated using anti-MIRL antiserum. The MIRL that was specifically released into the supernate had an Mr of 19 Kd, while the MIRL that remained bound to the membrane had an Mr of 18 Kd. A quantitative assay showed that approximately 10% of erythrocyte MIRL was susceptible to PIPLC; however, treatment with PIPLC had no effect on either the electrophoretic mobility or the functional activity of purified MIRL. These studies show that the effects of PIPLC on MIRL are similar to those observed for other human erythrocyte membrane proteins that are anchored by a glycosyl phosphatidylinositol moiety.

scholarly journals Site of palmitoylation of a phospholipase C-resistant glycosylphosphatidylinositol membrane anchor

1992 ◽  
Vol 284 (2) ◽  
pp. 297-300 ◽  
Author(s):  
M A J Ferguson
Keyword(s):  
Phospholipase C ◽  
Trypanosoma Brucei ◽  
Periodate Oxidation ◽  
Membrane Anchor ◽  
Glycosyl Phosphatidylinositol ◽  
Oxidation Analysis ◽  
Inositol Ring ◽  
Repetitive Protein

The site of palmitoylation of the phosphatidylinositol moiety of the glycosyl-phosphatidylinositol membrane anchor of Trypanosoma brucei procyclic acidic repetitive protein was studied by using periodate oxidation. Analysis of the products by g.c.-m.s. allowed the assignment of 40 and 60% of the palmitate to the 2-position and the 3-position respectively of the myo-inositol ring.

scholarly journals Structure of the glycosyl-phosphatidylinositol membrane anchor of the Leishmania major promastigote surface protease.

1990 ◽  
Vol 265 (28) ◽  
pp. 16955-16964 ◽  
Author(s):  
P Schneider ◽  
M A Ferguson ◽  
M J McConville ◽  
A Mehlert ◽  
S W Homans ◽  
...  
Keyword(s):  
Leishmania Major ◽  
Membrane Anchor ◽  
Glycosyl Phosphatidylinositol

scholarly journals Glycosyl-phosphatidylinositol-anchored membrane proteins can be distinguished from transmembrane polypeptide-anchored proteins by differential solubilization and temperature-induced phase separation in Triton X-114

1991 ◽  
Vol 280 (3) ◽  
pp. 745-751 ◽  
Author(s):  
N M Hooper ◽  
A Bashir
Keyword(s):  
Phase Separation ◽  
Membrane Proteins ◽  
Aqueous Phase ◽  
Hydrophobic Membrane ◽  
Rich Phase ◽  
Glycosyl Phosphatidylinositol ◽  
Ionic Detergent ◽  
Membrane Spanning ◽  
Triton X ◽  
Insoluble Pellet

Treatment of kidney microvillar membranes with the non-ionic detergent Triton X-114 at 0 degrees C, followed by low-speed centrifugation, generated a detergent-insoluble pellet and a detergent-soluble supernatant. The supernatant was further fractionated by phase separation at 30 degrees C into a detergent-rich phase and a detergent-depleted or aqueous phase. Those ectoenzymes with a covalently attached glycosyl-phosphatidylinositol (G-PI) membrane anchor were recovered predominantly (greater than 73%) in the detergent-insoluble pellet. In contrast, those ectoenzymes anchored by a single membrane-spanning polypeptide were recovered predominantly (greater than 62%) in the detergent-rich phase. Removal of the hydrophobic membrane-anchoring domain from either class of ectoenzyme resulted in the proteins being recovered predominantly (greater than 70%) in the aqueous phase. This technique was also applied to other membrane types, including pig and human erythrocyte ghosts, where, in both cases, the G-PI-anchored acetylcholinesterase partitioned predominantly (greater than 69%) into the detergent-insoluble pellet. When the microvillar membranes were subjected only to differential solubilization with Triton X-114 at 0 degrees C, the G-PI-anchored ectoenzymes were recovered predominantly (greater than 63%) in the detergent-insoluble pellet, whereas the transmembrane-polypeptide-anchored ectoenzymes were recovered predominantly (greater than 95%) in the detergent-solubilized supernatant. Thus differential solubilization and temperature-induced phase separation in Triton X-114 distinguished between G-PI-anchored membrane proteins, transmembrane-polypeptide-anchored proteins and soluble, hydrophilic proteins. This technique may be more useful and reliable than susceptibility to release by phospholipases as a means of identifying a G-PI anchor on an unpurified membrane protein.

Evidence for glycosyl-phosphatidylinositol anchoring of Toxoplasma gondii major surface antigens

1989 ◽  
Vol 9 (10) ◽  
pp. 4576-4580
Author(s):  
S Tomavo ◽  
R T Schwarz ◽  
J F Dubremetz
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Toxoplasma Gondii ◽  
Phospholipase C ◽  
Surface Antigens ◽  
Water Soluble ◽  
Radioactive Label ◽  
Glycosyl Phosphatidylinositol ◽  
Major Surface

The four major surface antigens of Toxoplasma gondii tachyzoites (P43, P35, P30, and P22) were made water soluble by phosphatidylinositol-specific phospholipase C (PI-PLC). These antigens were biosynthetically labeled with 3H-fatty acids, [3H]ethanolamine, and [3H]carbohydrates. Treatment of 3H-fatty-acid-labeled parasite lysates with PI-PLC removed the radioactive label from these antigens. A cross-reacting determinant was exposed on these antigens after PI-PLC treatment.

Inter-residue interactions in alpha-helical transmembrane proteins

2018 ◽  
Vol 35 (15) ◽  
pp. 2578-2584 ◽  
Author(s):  
Eduardo Mayol ◽  
Mercedes Campillo ◽  
Arnau Cordomí ◽  
Mireia Olivella
Keyword(s):  
Membrane Proteins ◽  
Present Report ◽  
Protein Structures ◽  
Transmembrane Proteins ◽  
Globular Proteins ◽  
Supplementary Information ◽  
Backbone Carbonyl ◽  
Composition Characteristic ◽  
Almost All ◽  
Residue Composition

Abstract Motivation The number of available membrane protein structures has markedly increased in the last years and, in parallel, the reliability of the methods to detect transmembrane (TM) segments. In the present report, we characterized inter-residue interactions in α-helical membrane proteins using a dataset of 3462 TM helices from 430 proteins. This is by far the largest analysis published to date. Results Our analysis of residue–residue interactions in TM segments of membrane proteins shows that almost all interactions involve aliphatic residues and Phe. There is lack of polar–polar, polar–charged and charged–charged interactions except for those between Thr or Ser sidechains and the backbone carbonyl of aliphatic and Phe residues. The results are discussed in the context of the preferences of amino acids to be in the protein core or exposed to the lipid bilayer and to occupy specific positions along the TM segment. Comparison to datasets of β-barrel membrane proteins and of α-helical globular proteins unveils the specific patterns of interactions and residue composition characteristic of α-helical membrane proteins that are the clue to understanding their structure. Availability and implementation Results data and datasets used are available at http://lmc.uab.cat/TMalphaDB/interactions.php. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals A simple purification of procyclic acidic repetitive protein and demonstration of a sialylated glycosyl-phosphatidylinositol membrane anchor

1993 ◽  
Vol 291 (1) ◽  
pp. 51-55 ◽  
Author(s):  
M A Ferguson ◽  
P Murray ◽  
H Rutherford ◽  
M J McConville
Keyword(s):  
Hydrophobic Interaction Chromatography ◽  
Glycosylation Site ◽  
Surface Glycoprotein ◽  
Membrane Anchor ◽  
Cell Surface Glycoprotein ◽  
African Trypanosomes ◽  
Glycosyl Phosphatidylinositol ◽  
Repeat Domain ◽  
Rapid Purification ◽  
Repetitive Protein

The procyclic acidic repetitive protein is the major cell-surface glycoprotein of the insect-dwelling procyclic forms of the Trypanosoma brucei species of African trypanosomes. The glycoprotein contains an acidic Glu-Pro repeat domain, a glycosyl-phosphatidylinositol membrane anchor and a putative asparagine glycosylation site. In this paper we describe a rapid purification scheme for this glycoprotein, using solvent extraction and hydrophobic interaction chromatography, and a partial characterization of the glycosylphosphatidylinositol membrane anchor. The carbohydrate composition of the anchor is extremely unusual; it contains on average nine GlcNAc, nine Gal, and five sialic acid residues. This is the first description of such a heavily substituted and negatively charged anchor. A comparison between the trypanosome procyclic surface and the Leishmania promastigote surface is also presented.

Incorporation of human liver and placental alkaline phosphatases into liposomes and membranes is via phosphatidylinositol

1990 ◽  
Vol 68 (9) ◽  
pp. 1112-1118 ◽  
Author(s):  
Lee Kihn ◽  
Dorothy Rutkowski ◽  
Robert A. Stinson
Keyword(s):  
Alkaline Phosphatase ◽  
Phospholipase C ◽  
Human Liver ◽  
Red Cell ◽  
Osteosarcoma Cells ◽  
Alkaline Phosphatases ◽  
Membrane Anchor ◽  
Gradient Gel Electrophoresis ◽  
Triton X ◽  
Phosphatidylinositol Phospholipase C

As assessed by incorporation into liposomes and by adsorption to octyl-Sepharose, the integrity of the membrane anchor for the purified tetrameric forms of alkaline phosphatase from human liver and placenta was intact. Any treatment that resulted in a dimeric enzyme precluded incorporation and adsorption. An intact anchor also allowed incorporation into red cell ghosts. The addition of hydrophobic proteins inhibited incorporation into liposomes to varying degrees. Alkaline phosphatase was 100% releasable from liposomes and red cell ghosts by a phospholipase C specific for phosphatidylinositol. There was no appreciable difference in the rates of release of placental and liver alkaline phosphatases, although both were approximately 250 × slower in liposomes and 100 × slower in red cell ghosts than the enzyme's release from a suspension of cultured osteosarcoma cells. Both enzymes were released by phosphatidylinositol phospholipase C as dimers and would not reincorporate or adsorb to octyl-Sepharose. However, the enzyme incorporated, resolubilized by Triton X-100, and cleansed of the detergent by butanol treatment was tetrameric by gradient gel electrophoresis, was hydrophobic, and could reincorporate into fresh liposomes. A monoclonal antibody to liver alkaline phosphatase inhibited the enzyme's incorporation into liposomes, and abolished its release from liposomes and its conversion to dimers by phosphatidylinositol phospholipase C.Key words: alkaline phosphatase, liposome, phosphatidylinositol, membrane anchor.

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