Glycoproteins: Biosynthesis and location in the nuclear membranes of BHK cell lines

McClure, Myra (1978) Glycoproteins: Biosynthesis and location in the nuclear membranes of BHK cell lines. MSc(R) thesis, University of Glasgow.

Abstract

The nuclear membrane glycoproteins of BHK cells were studied in several ways. Hydroxyurea and cytosine arabinoside are inhibitors of DNA synthesis and of [3H]-glucosaniine incorporation into intracellular glycoproteins, These two inhibitors with a third, 2-deoxy-D-glucose, were used as a potential means of elucidating the steps by which membrane glycoproteins are transported and secreted extracellularly, 2-Deoxyglucose proved a powerful inhibitor of [3H]-glucosamine uptake by BHK cells after only a short period of incubation, while allowing protein synthesis to occur at a reduced rate, 2-Deoxyglucose was found to exert no influence on the secretion of acid perceptible [3H]-glucosamine into the growth medium. The nature of the labelled extracellular glycosylated material was found to be, glycoprotein hyaluronic acid and choindroitin sulphate. In order to study the glycoproteins of the nuclear membrane, the isolation of nuclei, uncontaminated by other cellular components was necessary, A method of preparation is described which effects this without removal of the nuclear membrane. The possibility of preferential extraction of the glycoproteins from nuclear membrane was investigated using non-ionic detergents, although these proved to be non-selective in their solubility effects. The nuclear membrane from [3H]-glucosamine labelled BHK C13 cells and from their polyoma transformed counterparts (PYY), was isolated by two methods. One method involved the polyanion, heparin; the other was sonication. Recovery of the [3H]-glucosamine label was higher using the heparin method. Two bands of nuclear membrane material were consistently isolated on a sucrose step gradient at the 1,22/1,20 g ml-1 and 1,20/1,18 g ml-1 sucrose interface, In addition, a high proportion of the crude nuclear membrane material applied to the sucrose density gradient remained at the top of the gradient. Endoplasmic reticulum was isolated on 8-n identical sucrose step gradient. Most of the material was found at the top of the gradient and in the pelleted material. It is suggested that the detergent Tween 80 removed endoplasmic reticulum contaminated nuclei. Citric acid treatment of nuclei is reported to remove the outer nuclear membrane, A nuclear membrane preparation from citrate-washed BM nuclei predominantly reduced the level of [3H]-glucosamine at the 1,20/1,18 g ml-1 interface, suggesting that this fraction was enriched in the outer nuclear membrane. The final nature of the [3H]-glucosamine label introduced to the cell growth medium was found in the nuclear membrane - as glucosamine, galactoseamine (not in PYY nuclear membrane) and sialic acid. Nuclear membrane glycoprotein components isolated from BHK cell lines were show to have a binding affinity for the (lentil) lectin, Lens culinaris when the latter was coupled to Sepharose 4B. In this respect PTT membrane Band 2 components behaved anomalously in that a high proportion of the binding to the lectin was irreversible. Differences in the glycoprotein SDS polyaorylamide gel profile of nuclear membranes from both CIJ and TYI were observed.

• Item Type: Thesis (MSc(R))
• Qualification Level: Masters
• Additional Information: Adviser: J G Beeley
• Keywords: Cellular biology
• Date of Award: 1978
• Depositing User: Enlighten Team
• Unique ID: glathesis:1978-73743
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 14 Jun 2019 08:56
• Last Modified: 14 Jun 2019 08:56
• URI: https://theses.gla.ac.uk/id/eprint/73743