Acetylcholinesterase from human erythrocyte membranes: dimers as functional units

Ott, Peter; Lustig, Ariel; Brodbeck, Urs; Rosenbusch, Jürg P.

doi:10.1016/0014-5793(82)80437-7

Cited by 52 publications

(12 citation statements)

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“…The ultracentrifugation studies in MeOH at 24,000 rpm yielded an average molecular mass of 12,351, consistent with a dimeric state of PLN. The application of ultracentrifugation techniques to molecular mass determination of proteins in nonionic detergents has been described (Ludwig et al, 1982;Ott et al, 1982;Holzenburg et al, . CD spectra of the synthetic PLN.…”

Section: And Ultracentrifugation Studiesmentioning

confidence: 99%

Total synthesis and functional properties of the membrane‐intrinsic protein phospholamban

et al. 1993

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The membrane-intrinsic protein phospholamban (PLN), the regulatory protein of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, was chemically synthesized. The synthesis was accomplished by double couplings and efficient capping procedures, thus eliminating hydrophobic failure sequences. The crude peptide was purified by high-performance liquid chromatographic ion exchange and gel permeation chromatography in chloroform-methanol mixtures. Ion spray mass spectroscopy showed that the product had the correct molecular mass. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis runs produced the typical monomer-pentamer structural pattern. A predominantly helical CD spectrum was obtained in 0.075% C12E8 (67.9% helix, 1.8% beta, 12.2% turn, 18.1% random coil). Synthetic PLN was phosphorylated in detergent solutions by protein kinase A with a stoichiometry close to 1:1 (Pi to PLN monomer). Reconstitution of the isolated skeletal muscle SR Ca2+ ATPase in phosphatidylcholine membranes in the presence of PLN using the freezing and thawing technique yielded a preparation with lower Ca(2+)-dependent ATPase activity. The inhibition was mainly due to a decrease in the affinity (Km(Ca)) of the ATPase for Ca2+ and was partially reversed by PLN phosphorylation with protein kinase A. By contrast, addition of PLN to diluted intact SR vesicles uncoupled the Ca(2+)-transport reaction, suggesting an ionophoric effect of PLN. Because this effect was observed at very high PLN-to-SR vesicle ratios and was not influenced by PLN phosphorylation, its biological function is doubtful.

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Section: And Ultracentrifugation Studiesmentioning

confidence: 99%

Total synthesis and functional properties of the membrane‐intrinsic protein phospholamban

et al. 1993

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“…The basic functional form is a dimer of MW 160 kDa containing a single disulphide bond [3]. It has been estimated that there are 700-800 AChE dimers per erythrocyte [4], and these are manifest as at least five distinct molecular species [5].…”

Section: Introductionmentioning

confidence: 99%

Acetylcholinesterase in the human erythron. II. Biochemical assay

1988

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Acetylcholinesterase (AChE) is an integral erythrocyte membrane protein. A role for the enzyme in the developing human erythron is being explored. Assays of AchE by the standard Ellman technique overestimate the amount of enzyme by failing to account for the contribution of hemoglobin to the optical density of the reaction mixture. Furthermore, reliance on substrate selection alone for specificity is unsatisfactory. Incorporation of inhibitors of "true" AchE and of pseudocholinesterase confer greater ability to distinguish one enzyme from the other. In our experience, the inhibitor constant (Kl) for edrophonium, which is highly specific for AChE, is approximately 5 x 10(-5) M against adult human erythrocytes that contain significantly more total cholinesterase activity than do erythrocytes from umbilical cord blood. This consists of both "true" and "pseudo" enzyme, the former predominating and accounting for 0.75-1.65 (mean 1.02, median 0.87) femtomoles of substrate hydrolysed per min per cell in adult blood, with values of 0.15-1.04 (mean 0.71, median 0.73) obtained on cord blood. Moreover, the enzyme activity in neonatal erythrocytes has a rather different inhibitor profile from that of adult cells. AChE was also demonstrated in fresh (ALL) and cultured (K562 and HL60) human leukemic cells, as well as in primitive granulocyte-macrophage and erythroid cells cloned from normal human bone marrow. In the erythroid colonies the enzyme activity was 0-3.76 (mean 1.20, median 0.76) femtomoles per min per cell, apparently the first successful measurement of AChE in such cells.

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“…Proteins with alkylacyl glycosyl-PtdIns anchors in human erythrocytes include AChE (Roberts et al, 1989c), decay accelerating factor (Walter et al, 1990), and membrane inhibitor of reactive lysis . Their numbers of copies/cell have been reported to be 1500 molecules of AChE (Ott et al, 1982), 4000-4500 molecules of decay accelerating factor (Test et al, 1991) and 30000 molecules of membrane inhibitor of reactive lysis (S. T. Test, personal communication).…”

Section: Comparison Of Cellular Diradyl Gropins With Ache Glycosylptdmentioning

confidence: 99%

Alkylacyl glycerophosphoinositol in human and bovine erythrocytes

Bütikofer

Zollinger

Brodbeck

1992

European Journal of Biochemistry

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Glycosyl-inositolphospholipid (glycosyl-PtdIns) anchors of proteins in mammalian cells which have been analyzed so far are exclusively of the alkylacyl type. However, little is known about the putative precursor of glycosyl-Ptdlns, the alkylacyl derivative of glycerophosphoinositol (GroPIns), in these cells since it is generally believed that cellular GroPIns consists of diacyl-type molecular species only. In this report, we describe the isolation and identification of alkylacyl GroPIns molecular species in both human and bovine erythrocytes, and compare it with the molecular species compositions of the glycosyl-PtdIns anchors of human and bovine erythrocyte acetylcholinesterase. Diradyl GroPIns was isolated from lipid extracts of ghost membranes and treated with phospholipase C. Diradylglycerols of the glycosyl-PtdIns anchors of affinity-purified human and bovine erythrocyte acetylcholinesterase were generated by sequential treatment with glycoprotein phospholipase D and acidic phosphatase and by PtdIns-specific phospholipase C, respectively. Diradylglycerols were subsequently converted into benzoate derivatives and separated into diacyl, alkylacyl, and alkenylacylglycerol subclasses. The molecular species compositions were quantitated and determined by combined HPLC/mass spectrometry. We found that human and bovine erythrocyte membrane diradyl GroPIns consist of 1.5 -4.8% alkylacyl GroPIns. Molecular species analysis showed a heterogeneous species composition for both human and bovine erythrocyte alkylacyl GroPIns. Their compositions are distinctly different from those of human and bovine erythrocyte acetylcholinesterase glycosyl-PtdIns anchors. The number of alkylacyl GroPIns molecules/cell is roughly equal with the number of glycosyl-PtdIns-anchored proteins in human erythrocytes. Many proteins from a wide variety of eukaryotic cells are linked to membrane lipid bilayers by covalently attached glycosyl-inositolphospholipid (glycosyl-PtdIns) anchors (for reviews see Ferguson and Williams, 1988; Low, 1990;Cross, 1990). Glycosyl-PtdIns anchors share common structural features including linkage to the carboxyl terminus of the polypeptide via ethanolamine phosphate and the presence of three mannose residues connected to diradyl glycerophosphoinositol (GroPIns) by glucosamine. This core structure is susCorrespondence to P.

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Acetylcholinesterase from human erythrocyte membranes: dimers as functional units

Cited by 52 publications

References 11 publications

Total synthesis and functional properties of the membrane‐intrinsic protein phospholamban

Total synthesis and functional properties of the membrane‐intrinsic protein phospholamban

Acetylcholinesterase in the human erythron. II. Biochemical assay

Alkylacyl glycerophosphoinositol in human and bovine erythrocytes

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