Influence of associated lipid on the properties of purified bovine erythrocyte acetylcholinesterase

Acetylcholinesterase has been isolated from bovine erythrocyte membranes by affinity chromatography using a m-trimethylammonium ligand. The purified enzyme had hydrophobic properties by the criterion of phase partitioning into Triton X-114. The activity of the hydrophobic enzyme was seen as a slow-moving band in nondenaturing polyacrylamide gels. After treatment with phosphatidylinositol-specific phospholipase C, another form of active enzyme was produced that migrated more rapidly toward the anode in these gels. This form of the enzyme partitioned into the aqueous phase in Triton X-114 phase separation experiments and was therefore hydrophilic. The hydrophobic form bound to concanavalin A in the absence of Triton X-100. As this binding was partially prevented by detergent, but not by α-methyl mannoside, D-glucose, or myo-inositol, it is in part hydrophobic. Erythrocyte cell membranes showed acetylcholinesterase activity present as a major form, which was hydrophobic by Triton X-114 phase separation and in nondenaturing gel electrophoresis moved at the same rate as the purified enzyme. In the membrane the enzyme was more thermostable than when purified in detergent. The hydrophobic enzyme isolated, therefore, represents a native form of the acetylcholinesterase present in the bovine erythrocyte cell membrane, but in isolation its stability becomes dependent on amphiphile concentration. Its hydrophobic properties and lectin binding are attributable to the association with the protein of a lipid with the characteristics of a phosphatidylinositol.Key words: acetylcholinesterase, bovine erythrocytes, phosphatidylinositol-specific phospholipase C, phase separation, affinity chromatography.

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The efficiency of various detergents for extraction and stabilization of acetylcholinesterase from bovine erythrocytes

Biochemistry and Cell Biology ◽

10.1139/o87-002 ◽

1987 ◽

Vol 65 (1) ◽

pp. 8-18 ◽

Cited By ~ 3

Author(s):

Rex K. M. Wong ◽

Christine P. Nichol ◽

M. Chandra Sekar ◽

Basil D. Roufogalis

Keyword(s):

Chain Length ◽

Membrane Lipids ◽

Acetylcholinesterase Activity ◽

Exchange Chromatography ◽

Erythrocyte Membranes ◽

Tween 20 ◽

Bovine Erythrocyte ◽

Critical Micelle Concentrations ◽

Triton X ◽

Nonionic Detergents

The efficiency of several nonionic detergents and a homologous series of zwitterionic detergents for the extraction of acetylcholinesterase (EC 3.1.1.7) from bovine erythrocyte membranes was examined. Of the nonionic detergents examined, the polyoxyethylene-based Tweens were the least effective solubilizing agents. Within this series, increasing the length of the saturated fatty acid chain progressively decreased the efficiency of enzyme recovery, while unsaturation in the side chain reversed this trend. In the Lubrol detergents, where the chain length of the alcohol group is variable, an increase in the length of the polyoxyethylene glycol group decreased the recovery of acetylcholinesterase in the solubilized state, without affecting the efficiency of extraction of total erythrocyte protein. As with the other nonionic detergents examined, Triton X-100 and octy1 β-D-glucoside were maximally effective in solubilizing acetylcholinesterase activity at concentrations greater than their respective critical micelle concentrations. In the sulfobetaine (N-alkyldimethylaminopropane sulphonate) zwitterionic detergent series, the longer alkyl chain zwittergents Z 316 and Z 314 were more efficient than the shorter chain length members of the series (Z 310 and Z 312). In contrast to the higher chain length compounds, short chain analogs were maximally effective at or below their critical micelle concentrations. After purification by ion-exchange chromatography and affinity chromatography, the enzyme extracted with the various detergents gave sedimentation coefficients between 6.8S and 7.6S, consistent with a dimeric structure. Acetylcholinesterase could also be efficiently released by 0.2 mM EDTA or 0.5 M NaCl from bovine erythrocyte membranes previously depleted of 70–80% of the membrane lipids by butanol. Nonlinear Arrhenius plots of enzyme activity were found whether acetylcholinesterase was solubilized with Tween 20, Lubrol PX, or Triton X-100. The present work confirms that bovine erythrocyte acetylcholinesterase requires detergents to solubilize it from membranes and that its activity depends on the structure of the amphiphiles used to solubilize the enzyme.

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The hyaluronate synthase from a eukaryotic cell line

Biochemical Journal ◽

10.1042/bj2900791 ◽

1993 ◽

Vol 290 (3) ◽

pp. 791-795 ◽

Cited By ~ 25

Author(s):

L Klewes ◽

E A Turley ◽

P Prehm

Keyword(s):

Monoclonal Antibodies ◽

Phase Separation ◽

Affinity Chromatography ◽

Cell Line ◽

Aqueous Phase ◽

Plasma Membranes ◽

Eukaryotic Cell ◽

Molecular Masses ◽

Triton X ◽

Hyaluronate Synthase

The hyaluronate synthase complex was identified in plasma membranes from B6 cells. It contained two subunits of molecular masses 52 kDa and 60 kDa which bound the precursor UDP-GlcA in digitonin solution and partitioned into the aqueous phase, together with nascent hyaluronate upon Triton X-114 phase separation. The 52 kDa protein cross-reacted with poly- and monoclonal antibodies raised against the streptococcal hyaluronate synthase and the 60 kDa protein was recognized by monoclonal antibodies raised against a hyaluronate receptor. The 52 kDa protein was purified to homogeneity by affinity chromatography with monoclonal anti-hyaluronate synthase.

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The inhibitory effects of polyoxyethylene detergents on human erythrocyte acetylcholinesterase and Ca2+ + Mg2+ ATPase

Biochemistry and Cell Biology ◽

10.1139/o89-021 ◽

1989 ◽

Vol 67 (2-3) ◽

pp. 137-146 ◽

Cited By ~ 5

Author(s):

R. Blaine Moore ◽

J. F. Manery ◽

J. Still ◽

V. N. Mankad

Keyword(s):

Human Erythrocyte ◽

Acetylcholinesterase Activity ◽

Significant Inhibition ◽

Erythrocyte Membranes ◽

Head Group ◽

Polar Head Group ◽

Detergent Concentration ◽

Human Erythrocyte Membranes ◽

Hydrophobic Tail ◽

Triton X

The activities of acetylcholinesterase and Ca2+ + Mg2+ ATPase were measured following treatment of human erythrocyte membranes with nonsolubilizing and solubilizing concentrations of Triton X-100. A concentration of 0.1% (v/v) Triton X-100 caused a significant inhibition of both enzymes. The inhibition appears to be caused by perturbations in the membrane induced by Triton X-100 incorporation. No acetylcholinesterase activity and little Ca2+ + Mg2+ ATPase activity were detected in the supernatant at 0.05% Triton X-100 although this same detergent concentration induced changes in the turbidity of the membrane suspension. Also, no inhibition of soluble acetylcholinesterase was observed over the entire detergent concentration range. The inhibition of these enzymes at 0.1% Triton X-100 was present over an eightfold range of membrane protein in the assay indicating an independence of the protein/detergent ratio. The losses in activities of these two enzymes could be prevented by either including phosphatidylserine in the Triton X-100 suspension or using Brij 96 which has the same polyoxyethylene polar head group but an oleyl hydrophobic tail instead of the p-tert-octylphenol group of Triton X-100. The results are discussed in regard to the differential recovery of enzyme activities over the entire detergent concentration range.Key words: Triton X-100, erythrocyte membranes, acetylcholinesterase, Ca2+ + Mg2+ ATPase, polyoxyethylene detergents.

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Endogenous glycosylphosphatidylinositol-specific phospholipase C releases renal dipeptidase from kidney proximal tubules in vitro

Biochemical Journal ◽

10.1042/bj3530339 ◽

2001 ◽

Vol 353 (2) ◽

pp. 339-344 ◽

Cited By ~ 11

Author(s):

Sung Wook PARK ◽

Kyong CHOI ◽

Cheol KIM ◽

Hwang Hee Blaise LEE ◽

Nigel M. HOOPER ◽

...

Keyword(s):

Enzyme Activity ◽

Phase Separation ◽

Phospholipase C ◽

Aqueous Phase ◽

Proximal Tubules ◽

Gpi Anchor ◽

Ph Optimum ◽

Hydrophilic Nature ◽

Triton X

Spontaneous enzymic release of renal dipeptidase (RDPase; EC 3.4.13.19), a glycosylphosphatidylinositol (GPI)-linked ectoenzyme, was observed in vitro during incubation of porcine proximal tubules at 37°C. Triton X-114 phase separation of the released RDPase showed that the majority of the enzyme activity partitioned into the aqueous phase, indicating its hydrophilic nature. Immunoblot analyses using an antibody against the cross-reacting determinant (CRD) inositol 1,2-cyclic monophosphate, the epitope formed by phospholipase C (PLC) cleavage of the GPI anchor on a protein, detected the released RDPase. Reprobing the immunoblot with an anti-RDPase serum showed the RDPase band co-migrating with the CRD band. The release of RDPase from the proximal tubules was a Ca2+-dependent process and had a pH optimum of 9.0. These results indicate that RDPase is released from the proximal tubules by the action of a distinct endogenous GPI-specific PLC.

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Renal dipeptidase is one of the membrane proteins released by phosphatidylinositol-specific phospholipase C.

Biochemical Journal ◽

10.1042/bj2440465 ◽

1987 ◽

Vol 244 (2) ◽

pp. 465-469 ◽

Cited By ~ 63

Author(s):

N M Hooper ◽

M G Low ◽

A J Turner

Keyword(s):

Staphylococcus Aureus ◽

Alkaline Phosphatase ◽

Phase Separation ◽

Bacillus Thuringiensis ◽

Bacillus Cereus ◽

Phospholipase C ◽

Membrane Fraction ◽

Similar Extent ◽

Pig Kidney ◽

Triton X

Renal dipeptidase (dehydropeptidase-I, EC 3.4.13.11) was released from pig kidney membrane preparations by treatment with phosphatidylinositol-specific phospholipase C from Staphylococcus aureus and Bacillus thuringiensis and a phospholipase C preparation from Bacillus cereus to a similar extent as alkaline phosphatase. Endopeptidase-24.11 and aminopeptidase N were not released by this treatment. After treatment of the membrane fraction with the S. aureus phospholipase C the dipeptidase was converted from an amphipathic to a hydrophilic form, as deduced from phase-separation experiments in Triton X-114. It is concluded that renal dipeptidase is anchored to the microvillar membrane by covalently attached phosphatidylinositol.

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In resting conditions, the pancreatic granule membrane protein GP-2 is secreted by cleavage of its glycosylphosphatidylinositol anchor

Biochemical Journal ◽

10.1042/bj2770879 ◽

1991 ◽

Vol 277 (3) ◽

pp. 879-881 ◽

Cited By ~ 14

Author(s):

E Paul ◽

F A Leblond ◽

D LeBel

Keyword(s):

Membrane Protein ◽

Phospholipase C ◽

Exocrine Pancreas ◽

Hydrophobic Properties ◽

Glycosylphosphatidylinositol Anchor ◽

Phosphodiester Bond ◽

Granule Membrane ◽

Anaesthetized Rats ◽

Triton X ◽

Hydrolysis Of

GP-2 is the major membrane protein of the exocrine pancreatic secretory granule. It is an integral protein which is anchored by a phosphatidylinositolglycan. In addition to being present in the soluble contents of the granule, GP-2 is also actively secreted by the pancreas. Although 93% of the GP-2 in the resting secretions of anaesthetized rats could be pelleted, Triton X-114 phase extraction showed that 70% of this GP-2 had lost its hydrophobic properties. Proteases have been postulated to release GP-2 from the membrane, but phospholipases also have the capacity to release the protein from the membrane by hydrolysis of its peculiar glycosylphosphatidylinositol membrane anchor. These studies show the presence of inositol 1,2-(cyclic)monophosphate on the secreted hydrophilic GP-2, confirming the involvement of an endogenous phospholipase C in the solubilization of GP-2 by the exocrine pancreas. It is therefore concluded that most of the GP-2 secreted by the pancreas of anaesthetized rats under resting conditions is released from the membrane by a phospholipase C which hydrolyses the phosphodiester bond linking GP-2 to its diradylglycerol anchor.

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Calmodulin affinity chromatography yields a functional purified erythrocyte (Ca2+ + Mg2+)-dependent adenosine triphosphatase

Biochemical Journal ◽

10.1042/bj1890081 ◽

1980 ◽

Vol 189 (1) ◽

pp. 81-88 ◽

Cited By ~ 74

Author(s):

K Gietzen ◽

M Tejcka ◽

H U Wolf

Keyword(s):

Affinity Chromatography ◽

Adenosine Triphosphatase ◽

Specific Activity ◽

Significant Loss ◽

Erythrocyte Membranes ◽

Purification Method ◽

Major Band ◽

Highly Sensitive ◽

Human Erythrocyte Membranes ◽

Triton X

The (Ca2+ + Mg2+)-dependent ATPase of human erythrocyte membranes was solubilized with deoxycholate and purified by calmodulin affinity chromatography to yield a functional enzyme. The method gave an enzyme purified 207-fold as compared with that of the erythrocyte membranes. The molecular weight of the ATPase was in the range 135 000-150 000, as revealed by a single major band after electrophoresis on dodecyl sulphate/polyacrylamide gels. The isolated enzyme was highly sensitive to calmodulin, since the activity was increased about 9-fold. At 37 degrees C and in the presence of calmodulin the purified ATPase had a specific activity of 10.1 mumol/min per mg of protein. Triton X-100 or deoxycholate stimulated the calmodulin-deficient enzyme in a concentration-dependent fashion whereby the calmodulin-sensitivity was lost. The purification method is suitable for studying the lipid-sensitivity of the ATPase, since the lipids can easily be exchanged without a significant loss of activity. A purification procedure described by Niggli, Penniston & Carafoli [(1979) J. Biol. Chem. 254, 9955-9958] resulted in an enzyme that indeed was pure but was lacking a predominant feature, namely the modulation by calmodulin.

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