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Title: Microsomal plasma membrane fragments
Author: Norris, Kenneth Allonby
ISNI:       0000 0001 3449 3665
Awarding Body: University of Surrey
Current Institution: University of Surrey
Date of Award: 1973
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Prior to the commencement of this study (in 1967), no methods had been published for the isolation from rat liver of the substantial proportion (up to 40%) of the plasma membrane fragments which sediment with the classical microsomal fraction, El Aaser et al. (1966a) and Fitzsimons (1969), using a B-IV zonal rotor, were unable to completely separate the plasma membrane fragments from the smooth endoplasmic reticulum by centrifugation in the presence or absence of Mg[2+] ions. Further attempts to purify the microsomal p.m. fragments by isopynic zonal centrifugation in the B-XIV and B-XV rotors are reported in this thesis. Centrifugation conditions (including the pH and Mg[2+] ion concentration of the gradient, and the use of flotation rather than sedimentation) were varied but failed to produce even an approximately pure p.m. fraction, as judged by the marker enzyme, AMPase. L-leucyl-beta-naphthylamidase was examined as a possible alternative p.m. marker enzyme but was found to be also present in lysosomes. The contribution of adsorbed soluble proteins, and of other subcellular entities, to the impurity of the p.m. was studied. A purified microsomal p.m. fraction was finally isolated by isopycnic centrifugation of a microsomal fraction pre-treated by incubation with lead ions and sonication. The concentration of lead ions during pre-incubation was found to be critical, 0.5mM being optimal for rat liver microsomes. The technique was used successfully for the isolation of p.m. fragments from rat hepatoma and dog liver microsomes, although a higher concentration of lead ions (1mM) was required in both cases. The submicrosomal distribution of various other enzymes, including alkaline and acid ribonucleases and phosphodiesterases, alkaline p-nitrophenylphosphatase and L-leucyl-beta-naphthylamidase was studied and the activities of these enzymes in microsomal p.m. compared to the activities in nuclear p.m. prepared by the technique of Hinton et al.(1970).
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available