The effects of some enzymes on intercellular adhesion

Vicker, Michael Gordon (1971) The effects of some enzymes on intercellular adhesion. PhD thesis, University of Glasgow.

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Abstract

The formation of adhesions by cells of a permanent line derived from baby hamster kidney tissue (BHK/clone 13) has been investigated by aggregating the cells in suspension in a medium composed of glucose end salts. The initial single cell suspensions were produced by the use of trypsin to disperse cells grown in monolayer culture. The kinetics of aggregation was found to fit a rate equation in which the total particle concentration decayed exponentially from its initial value to a stable final value. This limiting final population contained both single cells end clusters of various sizes. Aggregation was followed by using the Coulter counter to measure the particle concentration during the process. Best fit curves and statistical information were obtained from the data using a KDF-9 computer. The aggregation of any cell suspension could be described by two parameters, the rote constant and final extent of aggregation. The effect of trypsin on adhesion was examined by treating coil cultures, end fresh suspensions with different concentrations of the enzyme before aggregation. In addition, trypsin was applied to pre-formed cell clusters. C-13 aggregation was progressively reduced by the application of increasing amounts of trypsin. Trypsin and pronase were found to be similarly effective in dispersing C-I3 clusters but collagenase, phospholipase C and EDTA (at pH 7.2) had no detectable effect on C-13 aggregation. It is concluded that protein or glycoprotein elements of the cell surface are important for adhesion in this system. The effect of the removal of cell surface neuraminic acid was studied either by 1) adding neureminidase to suspensions of pre-formed aggregates or fresh cell suspensions as they began aggregating; or 2) by pre-treating cell cultures before aggregation. The activity of the neureminidase was tested by a fluorimetric assay in which the neuraminic acid released from the cells by the enzyme was treated with noureminic acid aldolese, to yield pyruvate. The pyruvate formed was measured by using it to oxidize MADH in the presence of lactate dohydrogenase. The results showed that neuraminidase treatment increased aggregation by as much as 50% above the controls. This effect may be understood in terms of a decrease in the cell surface not negative charge or by a requirement for the newly exposed terminal sugar groups (formerly bound to nouraminate) in cell adhesion. It was found that complex media (e.g. Eagles medium) also caused an increase in the rate and extent of cell aggregation. The kinetics of the effect was different from that mentioned above. The components of Eagles medium were tested for their activity and it was found that only the amino acid L-glutamine caused aggregation. The effect was inhibited by NaF and by the specific analogue of L-glutamine, szaserine. L-glutamine is thought to function by supplying amino groups to cell surface amino sugars which may be directly required for intercellular adhesion. The results are discussed in terms of the known molecular composition, structure and metabolism of the cell surface.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: John Edwards
Keywords: Cellular biology
Date of Award: 1971
Depositing User: Enlighten Team
Unique ID: glathesis:1971-73079
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: http://theses.gla.ac.uk/id/eprint/73079

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