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P Ott

Publications and source records attributed to P Ott.

135 records · Page 8Linked to original sources
PubMed1982

A rapid and sensitive assay for determination of cholesterol in membrane lipid extracts.

A commercially available enzymatic assay (Boehringer Monotest) was modified to allow a rapid and sensitive determination of cholesterol in membrane lipid extracts. This was achieved by adding 0.5% Triton X-100 to the reagent solution. The detergent did not interfere with the assay. The relationship between the amount of cholesterol per assay and the absorbance at 500 nm was linear up to 100 micrograms. The recovery in the assay was better than 95%. The assay was applied to the determination of cholesterol in erythrocyte membrane lipid extracts.

Cholesterol↗
PubMed1982

Quantitative composition and characterization of the proteins in membrane vesicles released from erythrocytes by dimyristoylphosphatidylcholine. A membrane system without cytoskeleton.

Membrane vesicles were prepared by incubation of human erythrocytes with dimyristoylphosphatidylcholine [3] and isolated by isopycnic centrifugation on Dextran density gradients. Protein analyses were carried out with crossed immunoelectrophoresis and dodecylsulfate polyacrylamide gel electrophoresis. The right-side-out-oriented membrane vesicles contained membrane and cytoplasmic proteins of the erythrocyte but lacked cytoskeletal components. Comparison of proteins in vesicles and erythrocyte membranes showed that acetylcholinesterase was enriched two to six times in the vesicles relative to both membrane-spanning proteins, band 3, and glycophorin. Two further, hitherto unidentified, sialic acid-containing membrane antigens were found in the vesicles. Both faced the outside of the membranes and were enriched two to seven times. Ankyrin was not present in the membrane vesicles and spectrin could not be detected by dodecylsulfate polyacrylamide gel electrophoresis. We suggest that the redistribution of proteins in the vesicles reflects differences in their interactions with other membrane components and their relative mobility within the erythrocyte membrane.

Dimyristoylphosphatidylcholine↗
PubMed1981

Effect of dimyristoyl phosphatidylcholine on intact erythrocytes. Release of spectrin-free vesicles without ATP depletion.

Incubation of human erythrocytes with suspensions of sonicated dimyristoyl phosphatidylcholine resulted in dramatic morphological changes of the cells and release of membrane vesicles. The shedding of membrane vesicles was not preceded by ATP depletion and only occurred at temperatures of incubation that were above the phase transition temperature of dimyristoyl phosphatidylcholine. Membrane vesicles were separated from intact erythrocytes and exogenous dimyristoyl phosphatidylcholine by a series of centrifugation steps. The lipid composition of the membrane vesicles was similar to that of the native erythrocyte, and the predominant membrane proteins were band 3, glycophorin and acetylcholinesterase. Spectrin was not detected. Freeze-fracture electron microscopy showed vesicles (150 nm in diameter) with protein particles embedded in the lipid bilayer.

Acetylcholinesterase↗
PubMed1980

Reconstitution of human erythrocyte membrane acetylcholinesterase in phospholipid vesicles. Analysis of the molecular forms by cross-linking studies.

Unilamellar lipid vesicles were formed upon removal of Triton X-100 with Amberlite XAD-2 from a mixture of egg phosphatidylcholine and Triton-solubilized pure human erythrocyte membrane acetylcholinesterase. A majority of large (230 nm diameter) vesicles together with a minor population of smaller (30 nm diameter) strictures were observed in freeze-fracture electron micrographs. Reconstitution experiments performed with [phenyl-3H(n)]-Triton X-100 showed that only one detergent molecule per 600 lipid molecules was present in the vesicles. Density gradient centrifugation showed co-sedimentation of acetylcholinesterase with the lipid vesicles. About 60% of the incorporated enzyme was directed towards the vesicle exterior and could be partially degraded by papain. Mainly dimeric acetylcholinesterase was found when the reconstituted or, alternatively, the lipid-free but Triton-solubilized enzyme were cross-linked with glutaraldehyde. Aggregates were observed when the detergent-depleted oligomeric forms of the enzyme were cross-linked. The results thus indicate that mainly the dimeric enzyme form is present in a phospholipid environment.

Acetylcholinesterase↗
PubMed1980

Lipid-protein interactions in human erythrocyte-membrane acetylcholinesterase. Modulation of enzyme activity by lipids.

Purified human erythrocyte membrane acetylcholinesterase was incorporated into vesicles of various lipid compositions. The activities of the free and the lipid-associated enzyme were assayed at temperatures between 4 degrees C and 40 degrees C and the results were visualized as plots of log v versus 1/T (Arrhenius plots). For the purified, detergent-depleted enzyme a linear relation was obtained. If Triton X-100 was added to the assay medium a curved plot resulted. For acetylcholinesterase incorporated into dimyristoylphosphatidylcholine vesicles a clear break in the plot was observed at the phase transition temperature of the lipid. With lipids not experiencing a phase transition within the temperature range investigated, again a linear relation was obtained. These results show that the activity of human erythrocyte membrane acetylcholinesterase is strongly modulated by its hydrophobic environment.

Acetylcholinesterase↗
PubMed1978

Multiple molecular forms of acetylcholinesterase from human erythrocyte membranes. Interconversion and subunit composition of forms separated by density gradient centrifugation in a zonal rotor.

Quantitative separation of eight multiple molecular forms of acetylcholinesterase isolated from human erythrocyte membranes was accomplished by sucrose density gradient centrifugation in a zonal rotor. Hydrodynamic properties of the four most abundant oligomers were investigated by analytical ultracentrifugation. The S20, w values obtained for these components ranged from 12.5 S to 19.0 S with corresponding molecular weights between 500 000 and 1 131 000. These oligomers are built up of 6-14 subunits. Analysis of each isolated homogenous form after storage for 6 months at 4 degrees C revealed interconversion into the original eight components. Upon addition of Triton X-100 the oligomers disaggregate to yield a single 7-S form, which reaggregates upon removal of the detergent. Such reaggregation can be prevented by chaotropic ions indicating that hydrophobic interactions are important in the formation of these oligomeric forms.

Acetylcholinesterase↗
PubMed1975

Multiple molecular forms of purified human erythrocyte acetylcholinesterase.

1. Human erythrocyte acetylcholinesterase was solubilized by Triton X-100 and purified by affinity chromatography to a specific activity of 3800 IU/mg of protein. The yield of the purified enzyme was 25--45%. 2. Gel filtration on Sepharose 4-B in the presence of Triton X-100 revealed one peak of enzyme activity with a Stokes' radius of 8.7 nm. Density gradient centrifugation in 0.1% Triton X-100 showed one peak of enzyme activity with an S4 value of 6.3S. 3. Isoelectric focusing in Triton X-100 resolved the enzyme into five molecular forms with isoelectric points of 4.55, 4.68, 4.81, 4.98 and 5.18. Upon incubation with neuraminidase the enzyme activity in the first four forms was decreased with a concommitant increase in activity in the form with the higher isoelectric point. 4. After removal of excess Triton X-100 on Bio-Gel HTP, polyacrylamide gel electrophoresis showed seven bands of protein and corresponding bands of enzyme activity. Density gradient centrifugation of the detergent-depleted enzyme at high ionic strength revealed five multiple molecular forms with S4 values of 6.3 S, 10.2 S, 12.2 S, 14.2 S and 16.3 S. At low ionic strength, higher aggregates were observed in addition to the other forms. Dodecylsulfate-polyacrylamide gel electrophoresis gave one subunit only with an apparent molecular weight of 80 000. 5. These results suggest that human erythrocyte acetylcholinesterase, solubilized by Triton X-100, exists in various forms differing in net charge but of apparently similar molecular dimensions. After removal of the detergent, forms with different molecular sizes are observed.

Acetylcholinesterase↗