Atpase and Protein Kinase in Membranes From Corn Roots: Regulation of Tonoplast Atpase by Calmodulin or by Protein Kinase
Ladror, Uri Shlomo
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https://hdl.handle.net/2142/77663
Description
Title
Atpase and Protein Kinase in Membranes From Corn Roots: Regulation of Tonoplast Atpase by Calmodulin or by Protein Kinase
Author(s)
Ladror, Uri Shlomo
Issue Date
1986
Department of Study
Plant Biology
Discipline
Botany
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Plant Physiology
Language
eng
Abstract
A study was conducted to find if microsomal H('+)-ATPases from corn roots are regulated by Ca('2+)- or calmodulin (CaM)-dependent protein phosphorylation. This work included: (a) identifying the microsomal H('+)-ATPases; (b) characterizing protein kinase and phosphatase activities in microsomal fractions; (c) finding whether conditions that lead to protein phosphorylation inhibit H('+)-ATPase activities; and (d) purifying the H('+)-ATPase in order to find whether it becomes phosphorylated by a protein kinase.
Nitrate-sensitive and anion-stimulated ATPase activity, enriched primarily in light microsomal membranes, was identified as tonoplast-type (Tp). Vanadate-sensitive and cation-stimulated activity, associated with heavy microsomes, was identified as plasmalemma-type (Pm). Proton pumping activity, measured as uncoupler-stimulated ATPase activity or as quinacrine fluorescence quench, was enriched in the Tp, but was poor in the Pm. It appeared that both ATPase activities are electrogenic, but poor sealing of Pm vesicles prevented a reliable assay of proton pumping.
Protein kinase activity was detected in both fractions, and was 3 fold higher in the Pm than in the Tp. Both activities were optimal at 3 mM free Mg('2+) and were stimulated by 1 (mu)M free Ca('2+). Optimal pH values were 6.6 and 7.0 for the Pm and Tp, respectively. CaM did not stimulate kinase activities and chlorpromazine was inhibitory only at high concentrations, indicating that CaM-dependent activity was not detectable. Protein phosphatase activity was detected only in the Pm.
At 10 (mu)M, Ca('2+) had no effect on Tp-ATPase, and inhibited proton pumping only slightly. CaM stimulated the Tp-ATPase activity in the absence of an uncoupler, but not in its presence. Thus, in the presence of CaM, the uncoupler-dependent increment was smaller. CaM inhibited the formation of a proton gradient across Tp vesicles about 20% with no time lag, and stimulated the dissipation of pre-formed proton gradient by 40%, after the H('+)-ATPase was inhibited with nitrate. Thus, CaM does not affect the Tp-ATPase directly, but acts as an uncoupler. CaM had no effect on Pm-ATPase.
Three polypeptides, of 67, 57 and 36 kD, were enriched in partially purified Tp-ATPase fractions. None of these polypeptides became phosphorylated by protein kinase activity, indicating that the Tp-ATPase is not regulated by protein phosphorylation.
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