Protein Kinase Activities in Tonoplast and Plasmalemma Membranes from Corn Roots

Abstract

Protein kinase and phosphatase activities were studied in plasmalemma and tonoplast membrane fractions from com (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 threefold higher than in the tonoplast, and displayed Michaelis Mententype 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 I micromolar free Ca2 , but calmodulin had no stimulatory effect, and chlorpromazine was inhibitory only at high concentrations. The pattem 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.It is well established that protein phosphorylation is a major mechanism by which second messengers produce their physiological responses in a variety of animal tissues (2,19,24 (3,13,20,26,27), and microsomes (8,23,26). Of these enzymes, only histone kinase (13), LHCP,,-kinase (1), and cytokinin-binding protein kinase (20) have been characterized with respect to their substrates. ' Present address:

Effect of Ca2+ and Calmodulin on ΔpH Formation in Tonoplast Vesicles from Corn Roots

“…To address this question, we studied the effect of CaM on the dissipation of ApH after H+-pumping was stopped by inhibiting the H+-ATPase or by depleting the ATP. As shown in Figure 2A (12).…”

“…These membranes were enriched in tonoplast-type ATPase activity that was inhibited strongly by N03-and less by VO43+, and stimulated by anions and not cations (1,12,16). ATP-dependent formation of ApH across these membrane vesicles, measured by fluorescence quench of quinacrine (1), was abolished by 20 mM N03-, whereas 100 MM V043+ had no effect (US Ladror, RE Zielinski, not shown).…”

“…The microsomal membranes used in this study were isolated from the 2.5 to 7% interface of a discontinuous gradient of dextran, as described previously (12). These membranes were enriched in tonoplast-type ATPase activity that was inhibited strongly by N03-and less by VO43+, and stimulated by anions and not cations (1,12,16).…”

“…Furthermore, a microsomal H+-ATPase from corn roots has been suggested recently to be regulated by Ca2+-CaM-dependent protein phosphorylation (21). These microsomal vesicles were isolated from a 0 to 10% interface of discontinuous gradients of dextran and were later found to originate from the tonoplast (1,12,16,19 …”

Effects of Deuterium Oxide on Growth, Proton Extrusion, Potassium Influx, and in Vitro Plasma Membrane Activities in Maize Root Segments

“…The inhibiting effect on H+ pumping and ATPase did not appear to be due to a nonspecific action of 2H20 on plasma membrane; NADH-ferricyanide reductase and 1,3-#-glucan synthase, two other plasma membrane activities, were slightly (less than 14%) inhibited by 2H20 (Table IV). Measurements of the ATPase activities present in the tonoplast-enriched fraction (19) and submitochondrial particles showed that they were not, or were very slightly, inhibited by 2H20 (data not shown).…”