Protein kinase activities in tonoplast and plasmalemma membranes from corn roots

Uri S. Ladror, Raymond E. Zielinski

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Protein kinase and phosphatase activities were studied in plasmalemma and tonoplast membrane fractions from corn (Zea mays L.) roots in order to test the hypothesis that the tonoplast H+-ATPase is regulated by intrinsic protein phosphorylation (G Zocchi, SA Rogers, JB Hanson 1983 Plant Sci Lett 31: 215-221), and to facilitate future purification of kinase activities from these membranes. Kinase activity in the plasmalemma was about three-fold higher than in the tonoplast, and displayed Michaelis Menten-type behavior with a Km value for MgATP2- of about 50 micromolar. Both activities were optimal at 3 millimolar free Mg2+ and had pH optima at 6.6 and 7.0 for the plasmalemma and tonoplast, respectively. Kinase activities in both fractions were stimulated by 1 micromolar free Ca2+, but calmodulin had no stimulatory effect, and chlorpromazine was inhibitory only at high concentrations. The pattern of phosphopeptides on SDS polyacrylamide gel electrophoresis was similar in both fractions except for one band of 50 kilodaltons that was present only in the tonoplast. A partially purified H+-ATPase fraction was prepared from tonoplast membranes, incubated under conditions optimal for protein phosphorylation. The three polypeptides (of 67, 57, and 36 kilodaltons), enriched in this fraction, did not become phosphorylated, suggesting that this protein is not regulated by endogenous protein phosphorylation. Protein phosphatase activity was detected only in the plasmalemma fraction. These results indicate that a regulatory cycle of protein phosphorylation and dephosphorylation may operate in the plasmalemma. The activity in the tonoplast appears to originate from plasmalemma contamination.

Original languageEnglish (US)
Pages (from-to)151-158
Number of pages8
JournalPlant physiology
Issue number1
StatePublished - 1989

ASJC Scopus subject areas

  • Physiology
  • Genetics
  • Plant Science

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