Acetylcholinesterase from Human Erythrocytes as a Surrogate Biomarker of Lead Induced Neurotoxicity

Lead induced neurotoxicity in the people engaged in different occupations has received wide attention but very little studies have been carried out to monitor occupational neurotoxicity directly due to lead exposure using biochemical methods. In the present paper an endeavour has been made in order to assess the lead mediated neurotoxicity by in vitro assay of the activity of acetylcholinesterase (AChE) from human erythrocytes in presence of different concentrations of lead. The results suggested that the activity of this enzyme was localized in membrane bound fraction and it was found to be highly stable up to 30 days when stored at −20°C in phosphate buffer (50 mM, pH 7.4) containing 0.2% Triton X-100. The erythrocyte’s AChE exhibited Km for acetylcholinesterase to be 0.1 mM. Lead caused sharp inhibition of the enzyme and its IC50 value was computed to be 1.34 mM. The inhibition of the enzyme by lead was found to be of uncompetitive type (Ki value, 3.6 mM) which negatively influenced both the Vmax and the enzyme-substrate binding affinity. Taken together, these results indicate that AChE from human erythrocytes could be exploited as a surrogate biomarker of lead induced neurotoxicity particularly in the people occupationally exposed to lead.

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In Vitro Incorporation of GPI-Anchored Proteins Into Human Erythrocytes and Their Fate in the Membrane

Blood ◽

10.1182/blood.v91.5.1784 ◽

1998 ◽

Vol 91 (5) ◽

pp. 1784-1792 ◽

Cited By ~ 33

Author(s):

Gianluca Civenni ◽

Samuel T. Test ◽

Urs Brodbeck ◽

Peter Bütikofer

Keyword(s):

Membrane Vesicle ◽

Human Erythrocytes ◽

Membrane Extraction ◽

Membrane Microdomains ◽

Membrane Insertion ◽

Vesicle Formation ◽

Calcium Loading ◽

Lipid Moiety ◽

Triton X

Abstract In many different cells, glycosylphosphatidylinositol (GPI)-anchored molecules are clustered in membrane microdomains that resist extraction by detergents at 4°C. In this report, we identified the presence of such domains in human erythrocytes and examined the ability of exogenously-added GPI-anchored molecules to colocalize with the endogenous GPI-anchored proteins in these detergent-insoluble complexes. We found that the addition to human erythrocytes of three purified GPI-anchored proteins having different GPI lipid moieties resulted in their efficient and correct incorporation into the membrane. The extent of membrane insertion was dependent on the intactness of the GPI lipid moiety. However, unlike the endogenous GPI-anchored proteins, the in vitro incorporated GPI molecules were not resistant to membrane extraction by Triton X-100 at 4°C. In addition, in contrast to the endogenous GPI-anchored proteins, they were not preferentially released from erythrocytes during vesiculation induced by calcium loading of the cells. These results suggest that in vitro incorporated GPI-linked molecules are excluded from pre-existing GPI-enriched membrane areas in human erythrocytes and that these microdomains may represent the sites of membrane vesicle formation.

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Phosphoinositides regulate force-independent interactions between talin, vinculin, and actin

eLife ◽

10.7554/elife.56110 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 1

Author(s):

Charlotte F Kelley ◽

Thomas Litschel ◽

Stephanie Schumacher ◽

Dirk Dedden ◽

Petra Schwille ◽

...

Keyword(s):

Network Formation ◽

Focal Adhesions ◽

Membrane Composition ◽

Macromolecular Assemblies ◽

In Vitro Reconstitution ◽

Membrane Bound ◽

Underlying Mechanisms ◽

Higher Order Networks ◽

Made In

Focal adhesions (FA) are large macromolecular assemblies which help transmit mechanical forces and regulatory signals between the extracellular matrix and an interacting cell. Two key proteins talin and vinculin connecting integrin to actomyosin networks in the cell. Both proteins bind to F-actin and each other, providing a foundation for network formation within FAs. However, the underlying mechanisms regulating their engagement remain unclear. Here, we report on the results of in vitro reconstitution of talin-vinculin-actin assemblies using synthetic membrane systems. We find that neither talin nor vinculin alone recruit actin filaments to the membrane. In contrast, phosphoinositide-rich membranes recruit and activate talin, and the membrane-bound talin then activates vinculin. Together, the two proteins then link actin to the membrane. Encapsulation of these components within vesicles reorganized actin into higher-order networks. Notably, these observations were made in the absence of applied force, whereby we infer that the initial assembly stage of FAs is force independent. Our findings demonstrate that the local membrane composition plays a key role in controlling the stepwise recruitment, activation, and engagement of proteins within FAs.

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Lithium-induced modiphication of the physicochemical state of membrane proteins and lipids in human erythrocytes

Proceedings of the National Academy of Sciences of Belarus Biological Series ◽

10.29235/1029-8940-2021-66-3-295-301 ◽

2021 ◽

Vol 66 (3) ◽

pp. 295-301

Author(s):

G. P. Zubritskaya ◽

E. I. Slobozhanina

Keyword(s):

Lithium Salt ◽

Test System ◽

Human Erythrocytes ◽

Lithium Ions ◽

Physicochemical State ◽

A Cell ◽

Cell Test ◽

Membrane Bound ◽

Toxic Concentrations

The effect of various concentrations of lithium sulfate on human erythrocytes in vitro has been studied. It has been shown that the effect of lithium salt in maximum pharmacological and toxic concentrations on cells leads to a modification of the physicochemical state of membrane-bound proteins and lipids. It was found that in human erythrocytes exposed to lithium ions, there is a decrease in the activity of membrane-bound acetylcholinesterase and methgemoglobin reductase, as well as a change in the microviscosity of the lipid bilayer of membranes. The results obtained can be used to create a cell test system for assessing the toxicity of lithium compounds.

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Multiple forms of acetylcholinesterase from human erythrocytes

Biochemical Journal ◽

10.1042/bj1330521 ◽

1973 ◽

Vol 133 (3) ◽

pp. 521-527 ◽

Cited By ~ 44

Author(s):

David L. Wright ◽

David T. Plummer

Keyword(s):

Enzyme Activity ◽

Electrophoretic Pattern ◽

Human Erythrocytes ◽

Serum Cholinesterase ◽

Main Peak ◽

Multiple Forms ◽

Molecular Weights ◽

Enzyme Substrate ◽

Triton X ◽

Naphthyl Acetate

1. Acetylcholinesterase from human erythrocytes was solubilized with Triton X-100 in strong salt solution and partially purified by (NH4)2SO4 fractionation. This preparation showed three main bands of enzyme activity after electrophoresis on polyacrylamide gel and incubation with either α-naphthyl acetate or acetylthiocholine as enzyme substrate. Two of the multiple forms were completely inhibited by 10μm-eserine and one only partially. Treatment with neuraminidase had no effect on the electrophoretic pattern; therefore sialic acid does not appear to determine or affect the ratios of the acetylcholinesterase multiple forms, unlike those of the serum cholinesterase. 2. Chromatography of the preparation on Sephadex G-200 revealed one major peak of enzyme activity and a suggestion of two minor zones of mol.wt. 546000, 184000 and 93000 (i.e. in the proportion 6:2:1). The main peak was almost completely separated from the Triton X-100 and the overall purification was about 600-fold. Further attempts to purify the enzyme by absorption on calcium phosphate gels were unsuccessful. 3. Electrophoresis of the enzyme preparation on a polyacrylamide gradient for 24h revealed three main bands that corresponded to the three values for molecular weights obtained by column chromatography. After 70h of electrophoresis a further three zones of activity developed making six molecular entities, the molecular weights of which were simple multiples of a monomer, thus resembling the cholinesterase found in serum.

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Direct desaturation of intact galactolipids by a desaturase solubilized from spinach (Spinacia oleracea) chloroplast envelopes

Biochemical Journal ◽

10.1042/bj2890777 ◽

1993 ◽

Vol 289 (3) ◽

pp. 777-782 ◽

Cited By ~ 39

Author(s):

H Schmidt ◽

E Heinz

Keyword(s):

Double Bond ◽

Spinacia Oleracea ◽

Membrane Lipids ◽

Fatty Acyl ◽

Acyl Residue ◽

Solubilized Enzyme ◽

Membrane Bound ◽

Triton X ◽

Envelope Membranes

In plants, polyenoic fatty acids are synthesized by desaturase enzymes which use acyl groups of membrane lipids as substrates. To provide direct ‘in vitro’ evidence for this reaction, we solubilized envelope membranes from spinach (Spinacia oleracea) chloroplasts with Triton X-100 to release a membrane-bound n-6 desaturase. In the presence of oxygen and reduced ferredoxin, the solubilized enzyme desaturated a variety of substrates, such as free oleic acid, free erucic acid, 1-oleoyl-sn-glycerol 3-phosphate and the three galactolipids 1-oleoyl-2-(7′-cis-hexadecenoyl)-3-beta-D-galactopyranosyl-sn-glycerol, 1,2-dioleoyl-3-beta-D-galactopyranosyl-sn-glycerol and the ether analogue 1,2-di-(9′-cis-octadecenyl)-3-beta-D-galactopyranosyl-sn- glycerol. The in vitro desaturation of these exogenously added complex lipids with ester- and ether-linked substrate chains is unambiguous evidence for lipid-linked desaturation. The enzyme measures the insertion of the new double bond from the methyl end and the existing (n-9)-cis-double bond of an appropriate acyl or alkyl chain. The distal part of the substrate group, normally the carboxy end of a fatty acyl residue, is of less importance and, in particular, its activation in thioester form is not required.

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Phosphatidylglycerol as biosynthetic precursor for the poly(glycerol phosphate) backbone of bifidobacterial lipoteichoic acid

Biochemical Journal ◽

10.1042/bj2280683 ◽

1985 ◽

Vol 228 (3) ◽

pp. 683-688 ◽

Cited By ~ 7

Author(s):

H J Op den Camp ◽

A Oosterhof ◽

J H Veerkamp

Keyword(s):

Enzyme System ◽

Lipoteichoic Acid ◽

Bifidobacterium Bifidum ◽

Glycerol Phosphate ◽

Phosphate Backbone ◽

Membrane Bound ◽

Triton X ◽

Chloroform Methanol ◽

Lipoteichoic Acids

Phosphatidylglycerol functions as donor of the sn-glycerol 1-phosphate units in the synthesis in vitro of the 1,2-phosphodiester-linked glycerol phosphate backbone of the lipoteichoic acids of Bifidobacterium bifidum subsp. pennsylvanicum. The incorporation was catalysed by a membrane-bound enzyme system. After addition of chloroform/methanol the product formed coprecipitated with protein. The material was phenol-extractable and was co-eluted with purified lipoteichoic acid on Sepharose 6B. The reaction was stimulated by Triton X-100, UDP-glucose and UDP-galactose, but Mg2+ ions had no effect. The apparent values for Km and Vmax. of the phosphatidylglycerol incorporation were 1.4 mM and 3.1 nmol/h per mg of membrane protein, respectively. Labelled UDP-glucose and UDP-galactose were not incorporated into the lipoteichoic acid fraction by the particulate membrane preparation.

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In Vitro Incorporation of GPI-Anchored Proteins Into Human Erythrocytes and Their Fate in the Membrane

Blood ◽

10.1182/blood.v91.5.1784.1784_1784_1792 ◽

1998 ◽

Vol 91 (5) ◽

pp. 1784-1792 ◽

Cited By ~ 1

Author(s):

Gianluca Civenni ◽

Samuel T. Test ◽

Urs Brodbeck ◽

Peter Bütikofer

Keyword(s):

Membrane Vesicle ◽

Human Erythrocytes ◽

Membrane Extraction ◽

Membrane Microdomains ◽

Membrane Insertion ◽

Vesicle Formation ◽

Calcium Loading ◽

Lipid Moiety ◽

Triton X

In many different cells, glycosylphosphatidylinositol (GPI)-anchored molecules are clustered in membrane microdomains that resist extraction by detergents at 4°C. In this report, we identified the presence of such domains in human erythrocytes and examined the ability of exogenously-added GPI-anchored molecules to colocalize with the endogenous GPI-anchored proteins in these detergent-insoluble complexes. We found that the addition to human erythrocytes of three purified GPI-anchored proteins having different GPI lipid moieties resulted in their efficient and correct incorporation into the membrane. The extent of membrane insertion was dependent on the intactness of the GPI lipid moiety. However, unlike the endogenous GPI-anchored proteins, the in vitro incorporated GPI molecules were not resistant to membrane extraction by Triton X-100 at 4°C. In addition, in contrast to the endogenous GPI-anchored proteins, they were not preferentially released from erythrocytes during vesiculation induced by calcium loading of the cells. These results suggest that in vitro incorporated GPI-linked molecules are excluded from pre-existing GPI-enriched membrane areas in human erythrocytes and that these microdomains may represent the sites of membrane vesicle formation.

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Initiation factors in protein synthesis by free and membrane-bound polyribosomes of liver and hepatoma

Biochemical Journal ◽

10.1042/bj1520143 ◽

1975 ◽

Vol 152 (1) ◽

pp. 143-151 ◽

Cited By ~ 11

Author(s):

C N Murty ◽

E Verney ◽

H Sidransky

Keyword(s):

Protein Synthesis ◽

Initiation Factors ◽

Ionic Detergent ◽

Membrane Bound ◽

Triton X ◽

Stimulation Of

The activity of initiation factors obtained from free and membrane-bound polyribosomes of liver and of transplantable H5123 hepatoma of rats was investigated by using an assay of protein synthesis in vitro in which poly (U)-directed polyphenylalanine synthesis was measured. Initiation factors of membrane-bound polyribosomes prepared by using the anionic detergent deoxycholate exhibited less activity in incorporating [14C]phenylalanyltRNA into polypetides than did initiation factors of free polyribosomes. However, when membrane-bound polyribosomes were prepared after using the non-ionic detergent Triton X-100, no significant differences in activities in polyphenylalanine synthesis were observed between the initiation factors of free and membrane-bound polyribosomes. These results suggest that Triton X-100 is preferable to deoxycholate in the isolation of of initiation factors from polyribosomes. Initiation factors, prepared by using Triton X-100, of free polyribosomes of hepatoma exhibited greater activity in the stimulation of polyphenylalanine synthesis than did the initiation factors of free or membrane-bound polyribosomes of host livers or of membrane-bound polyribosomes of hepatomas.

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UPTAKE OF CORTICOSTEROIDS, RELATED STEROIDS AND THEIR WATER SOLUBLE ESTERS BY HUMAN ERYTHROCYTES

Acta Endocrinologica ◽

10.1530/acta.0.047s037 ◽

1964 ◽

Vol 47 (3_Suppl) ◽

pp. S37-S52 ◽

Cited By ~ 2

Author(s):

K. N. Agarwal ◽

H. Carstensen

Keyword(s):

Phosphate Buffer ◽

Human Erythrocytes ◽

Water Soluble ◽

List Type ◽

Extraction Separation ◽

Per Se

ABSTRACT The influence of different factors upon the affinity of human erythrocytes for corticosteroids was studied in vitro in a phosphate buffer. Properties connected with the erythrocytes per se were found to be of importance for the binding of the steroids. Methods were developed for extraction, separation and estimation of water soluble esters of corticosteroids.

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Hydrolysis of membrane-bound liver alkaline phosphatase by GPI-PLD requires bile salts

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.1996.271.4.g655 ◽

1996 ◽

Vol 271 (4) ◽

pp. G655-G663 ◽

Cited By ~ 7

Author(s):

J. T. Deng ◽

M. F. Hoylaerts ◽

M. E. De Broe ◽

V. O. van Hoof

Keyword(s):

Alkaline Phosphatase ◽

Bile Salt ◽

Bile Salts ◽

Small Range ◽

Liver Sinusoid ◽

Gpi Anchor ◽

Low Concentrations ◽

Membrane Bound ◽

Triton X

Circulating liver plasma membrane fragments (LPMF) were purified from human serum by means of a monoclonal antileucine aminopeptidase antibody, AD-1. This was done by immunoaffinity chromatography or by incubating the sera with AD-1-coated nitrocellulose disks. Alkaline phosphatase (ALP, EC 3.1.3.1) is bound to these LPMF through a glycosylphosphatidylinositol (GPI) anchor and is referred to as membrane-bound liver ALP (Mem-LiALP). Low concentrations of Triton X-100 or high bile salt concentrations released GPI anchor-bearing LiALP (Anch-LiALP) from purified LPMF; once released, Anch-LiALP was slowly and progressively converted to hydrophilic dimeric LiALP [soluble LiALP (Sol-LiALP)], free from its GPI anchor. Low levels of GPI-specific phospholipase D (GPI-PLD) activity were measured in the pure LPMF. Apparently, this membrane-associated GPI-PLD was released by the action of detergents and contributed to the spontaneous conversion of Anch-LiALP to Sol-LiALP. In the absence of detergents, GPI-PLD had little effect on Mem-LiALP, both in purified form as well as in serum. In vitro, isolated Anch-LiALP was converted to Sol-LiALP by both GPI-specific phospholipase C and GPI-PLD. Sol-LiALP in serum, however, appeared to be the product of GPI-PLD activity only. Five- to tenfold higher concentrations of Triton X-100 were needed to release Anch-LiALP from LPMF in serum, compared with those required in a solution of purified LPMF. In serum, as well as in purified conditions, only a small range of detergent of bile salt concentrations permitted the conversion of Mem-LiALP to Sol-LiALP. A model is proposed for the release in the circulation of Mem-LiALP, Anch-LiALP, and Sol-LiALP, involving both LPMF-associated GPI-PLD and liver sinusoid bile salts.

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