The plasma membrane Ca2+-pump (PMCA) is a Ca2+-Mg2+- ATPase that expels
Ca2+ from cells to help them maintain low concentrations of cytosolic Ca2+. Four genes
(PMCAl to PMCA4) encode various isoforms ofthis pump. Caloxin is a novel peptide
that was selected for binding to the second putative extracellular domain of PMCAl.
Caloxin inhibits the Ca2+- Mg2+-ATPase in human erythrocyte leaky ghosts which
primarily contain PMCA4. The objectives of this thesis are to delineate the mechanism
of this inhibition and to determine its PMCA isoform selectivity.
Caloxin inhibition of the PMCA pump in erythrocyte ghosts is non-competitive
with respect to the substrates Ca2+ and ATP and the activator calmodulin. This was
expected because the high affinity binding site for Ca2+ and sites for ATP and calmodulin
are intracellular whereas caloxin is a peptide selected for binding to the second
extracellular domain of the pump. In the reaction cycle, PMCA forms a 140 kDa
acylphosphate enzyme intermediate from ATP (forward reaction) or orthophosphate
(reverse reaction). Caloxin inhibits the acylphosphate formation in the forward but not in
the reverse reaction. These results suggest that caloxin inhibits conformational changes
required during the reaction cycle of the pump.
In COS-M6 cells overexpressing PMCA4, caloxin inhibited the Ca2+ -Mg2+ATPase
but with a marginally lower affinity than in the erythrocyte ghosts. Caloxin
inhibition was also observed in insect cells overexpressing PMCA2. Several
unsuccessful attempts were made to overexpress functional PMCAl. The work on isoform selectivity would require high level of expression of various PMCA isoforms and
problems related to this requirement are discussed.
Caloxin appears to inhibit the PMCA pumps by binding to the second putative
extracellular domain and thus affecting the conformation of the protein. This is the first
identified role of an extracellular domain of a PMCA pump. / Thesis / Master of Science (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/22695 |
| Date | 08 1900 |
| Creators | Holmes, Melanie |
| Contributors | Grover, A.K, Biology |
| Source Sets | McMaster University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis |
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