Maize coleoptile microsomal vesicles showing calmodulin-stimulated ATPase activity were isolated from 4.5 day old dark grown seedlings. Calmodulin-stiirmlated ATPase activity was maximal (8 nmoles min<sup>-1</sup> (mg protein)<sup>-1</sup>) at 0.35 μM, inhibited by orthovanadate (Iso=20 μM), and specific for ATP. Calmodulin affinity chromatography was used to purify this ATPase after solubilisation with Triton X-100. Calmodulin-stimulated ATPase activity was present in the purified fraction, maximal stimulation (340 nmoles min<sup>-1</sup> (mg protein)<sup>-1</sup>) occurring at 0.3 μM calmodulin. After reconstitution into asolectin liposomes, maximal calmodulinstimulated ATPase activity (500 nmoles min<sup>-1</sup> (mg protein)<sup>-1</sup>) occurred at 0.025 μM. Affinity chromatography using buffers containing asolectin produced true basal activities; maximal calmodulin stimulation was at 0.01 μM (100 nmoles min<sup>-1</sup> (mg protein)<sup>-1</sup>). These results suggest that a calmodulin-stimulated ATPase was purified from the microsomal fraction. Inclusion of protease inhibitors (PMSF, chymostatin) during purification and electrophoresis yielded a polypeptide of 140,000 M<sub>r</sub>, similar to the M<sub>r</sub> of erythrocyte calmodulin-stimulated, calcium-pumping ATPase (CSCPA). Polypeptides of M<sub>r</sub> 91,000, 77-69,000, 51,000, and 40,000 were also present. A monospecific polyclonal antibody raised against erythrocyte CSCPA recognised the 140,000 M<sub>r</sub> polypeptide from maize, giving strong evidence that maize cells may contain a polypeptide similar to erythrocyte CSCPA. The reaction mechanism of the proposed maize CSCPA was investigated. After purification in the presence of PMSF phosphorylation was present at 140,000 M<sub>r</sub>; this turned over rapidly, was sensitive to hydroxylamine, dependent on calcium, inhibited by lanthanum and stimulated by calmodulin. This was consistent with formation of an acyl-phosphate intermediate, indicating that maize CSCPA is a P-type ATPase, having a reaction mechanism similar to that of the erythrocyte CSCPA. A monoclonal antibody (EA6) was raised to maize CSCPA purified without PMSF; this antibody recognised intact maize CSCPA and inhibited calmodulin-stimulated ATPase activity in microsomal fractions. This antibody also bound to other polypeptides present in microsomal and purified fractions, permitting tentative identification of proteolysis products.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:252998 |
| Date | January 1989 |
| Creators | Briars, Sally-Anne |
| Contributors | Dewey, F. M. |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:afa92f78-633d-4ae5-8cf5-37ac077acab2 |
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