The calmodulin stimulated ATPase of Zea mays L

Thesis



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^-1 (mg protein)^-1) 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^-1 (mg protein)^-1) occurring at 0.3 μM calmodulin. After reconstitution into asolectin liposomes, maximal calmodulinstimulated ATPase activity (500 nmoles min^-1 (mg protein)^-1) 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^-1 (mg protein)^-1). 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_r, similar to the M_r of erythrocyte calmodulin-stimulated, calcium-pumping ATPase (CSCPA). Polypeptides of M_r 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_r 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_r; 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.

Authors: Briars, S; Briars, Sally-Anne

• Publication date: 1989
• DOI: 10.5287/ora-kzg21v8xw
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Subjects: Botanical chemistry