dc.contributor.advisor |
Ralph, R.K. |
en |
dc.contributor.author |
Twigden, D. G. |
en |
dc.date.accessioned |
2007-08-28T09:40:54Z |
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dc.date.available |
2007-08-28T09:40:54Z |
en |
dc.date.issued |
1980 |
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dc.identifier |
THESIS 80-109 |
en |
dc.identifier.citation |
Thesis (PhD)--University of Auckland, 1980 |
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dc.identifier.uri |
http://hdl.handle.net/2292/1569 |
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dc.description |
Full text is available to authenticated members of The University of Auckland only. |
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dc.description.abstract |
Depriving P815 mouse mastocytoma cells of ca2+ ions by growing them with EGTA inhibited their growth. This effect was fully reversed by Ca2+ or partially reversed by Mg2+. Growth of mastocytoma cells was also inhibited by 10-4M DBcAMP (N6, 02'-dibutyryladenosine 3',5'-cyclic monophosphate) plus 10-3M theophylline, and the drug-treated cells were significantly more sensitive to fluctuations in extracellular [Ca2+] than untreated cells.
Early to mid-log phase cells had low Mg2+-ecto-ATPase activity, and ca2+-ecto-ATPase activity was not detected with these cells. However, both Mg2+-stimulated and Ca2+-stimulated ecto-ATPase activities increased in parallel during late-log and approaching stationary phase growth. The results were consistent with increased Mg2+- and Ca2+-stimulated ecto-ATPases being associated with inhibition of mastocytoma cell growth and they suggested that the activities might be the expression of one enzyme with different Vmax's when stimulated by Mg2+ or Ca2+. Magnesium was the more effective stimulatory cation at all stages of growth. A KME for Mg2+ of 0.5mM was determined for Mg2+-ATPase and a KME for Ca2+ of 0.65mM for Ca2+-ATPase of cells in late-log phase growth. A Km for ATP of 0.87mM was determined for both Mg2+- and Ca2+-stimulated ATP hydrolysis.
DBcAMP plus theophylline treatment of P815 mastocytoma cell cultures caused Mg2+-ecto-ATPase activity to increase 4.8 to 6.3-fold and Ca2+-ecto-ATPase activity to increase 6.8 to 9.1-fold. The Km for ATP of Mg2+- and Ca2+-stimulated ATP hydrolysis remained unchanged compared to untreated cells. However, the Ca2+-binding affinity of the ecto-ATPase increased significantly compared to untreated cells, and the Mg2+-binding affinity of the ecto-ATPase decreased simultaneously, giving a KME for Ca2+ of 0.20m.M and a KME for Mg2+ of 1.80mM for the Ca2+- or Mg2+-stimulatede ecto-ATPases. These results are consistent with several reports that there is an increase in Ca2+ bound to the plasma membrane of growth-arrested (eg. after serum deprivation, or DBcAMP treatment) or quiescent cells.
The effect of DBcAMP plus theophylline on cell growth and ecto-ATPase activity was reversed upon reseeding drug-treated cells in fresh medium without drug. The approximately 2-fold decrease in ecto-ATPase activity in a 7.5h period at the time of reinitiation of cell growth was consistent with an involvement of Ca2+- and Mg2+-stimulated ecto-ATPases in growth regulation of P815 mouse mastocytoma cells.
Phosphorylation of membranes by cAMP-dependent protein kinases is known to increase Ca2+ binding, and to decrease the permeability of membranes to Ca2+. DBcAMP plus theophylline-treated P815 mastocytoma cells develop increased amounts of cell surface-associated acidic polysaccharides (Davis and Ralph, 1975) and these complexes are known to be good Ca2+ ligands. Thus, a consequence of treating mastocytoma cells with DBcAMP plus theophylline may be to increase phosphorylation of their plasma membranes and the amount of associated acidic polysaccharide, thereby increasing the Ca2+ affinity of plasma membrane constituents (eg. the ecto-ATPase), reducing the ca2+ permeability of the plasma membrane, and decreasing growth. That some metabolic event such as cAMP-dependent phosphorylation was required to activate ecto-ATPase activity was shown by there being no increase in ecto-ATPase activity for ≥ 2h after drug addition to cultures. |
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dc.language.iso |
en |
en |
dc.publisher |
ResearchSpace@Auckland |
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dc.relation.ispartof |
PhD Thesis - University of Auckland |
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dc.relation.isreferencedby |
UoA9921872014002091 |
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dc.rights |
Restricted Item. Available to authenticated members of The University of Auckland |
en |
dc.rights |
Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated. |
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dc.rights.uri |
https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm |
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dc.title |
Cyclic AMP, ATPases and growth control of mastocytoma cells |
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dc.type |
Thesis |
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thesis.degree.grantor |
The University of Auckland |
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thesis.degree.level |
Doctoral |
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thesis.degree.name |
PhD |
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dc.rights.holder |
Copyright: The author |
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dc.identifier.wikidata |
Q112072983 |
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