Membrane associated proteins of human erythrocytes

Pupkis, Mary Frances (1982) Membrane associated proteins of human erythrocytes. PhD thesis, University of Glasgow.

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Phosphatase activities of normal mature human erythrocytes have been measured using p-nitrophenyl phosphate and a radio-phosphorylated casein substrate. Most of the p-nitrophenyl phosphate phosphatase activity (99.9%) could be removed from the membranes by repeated washing. The small amount of activity remaining associated with the membrane had similar properties (acidic pH optimum, inhibition by Pi, F- and Ca2+, and activation by EDTA) when compared to the soluble enzyme. Lysis of red cells in the presence of calcium ions (0.1 to 1.0mM) increased the phosphatase activity associated with the membranes, suggesting that calcium potentiates the binding of cytoplasmic phosphatases to the membrane. Erythrocyte protease activities were measured using a radio-iodinated casein substrate. Approximately 18% of the total cellular proteolytic activity remained associated with the membranes after exhaustive washing of the ghosts. Of the membrane associated activity about 10% was detected on intact cells and is presumably located on the cell exterior. Proteolytic activities associated with the membrane and cytosolic fractions both showed two pH optima in the regions of pH 2.5 and 8.0. The activities measured at neutral pH, using the membrane and cytosolic fractions, were both inhibited by each of the following, PMSF, PCMB, DTT and EDTA, but to different degrees with respect to each inhibitor. Lysis of the cells in the presence of calcium ions (0.1 to 1.0mM) increased the level of the proteolytic activity associated with the membranes suggesting that calcium also potentiates the binding of cytoplasmic proteases to the membrane. Changes in the binding pattern of erythrocyte membrane proteins were examined, using SDS-polyacrylamide gel electrophoresis, as an additional means of detecting proteolytic activity. Characteristic changes in the protein binding pattern were observed after the incubation of membrane preparations. These changes were accelerated by lysis of red cells in the presence of calcium and could largely be inhibited by the addition of PMSF (1mM). Phosphatase and protease assays were performed on erythrocytes from a patient with spur cell anaemia. These samples showed elevated phosphatase and protease activities associated with the membranes when assayed using p-nitrophenyl phosphate and 125I-casein substrates respectively. Rapid degradation of the membrane proteins isolated from spur cells occurred following their isolation and could largely be inhibited by the addition of PMSF (1mM) at lysis. The results obtained suggest that elevated intracellular calcium might be an important factor in the degradation of erythrocyte proteins. A possible role for elevated intracellular calcium in spur cell anaemia and in other disorders associated with altered erythrocyte morphology is discussed. The possible contribution of contaminating leucocyte enzymes to the observed enzyme activites of erythrocytes was also examined. Leucocyte contamination had little effect on phosphatase assays, however the complete removal of white cells was shown to be an essential prerequisite for the assessment of erythrocyte membrane protease activites.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: J G Beeley
Keywords: Biochemistry
Date of Award: 1982
Depositing User: Enlighten Team
Unique ID: glathesis:1982-71997
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 17 May 2019 13:28
Last Modified: 17 May 2019 13:28

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